_ - — _ LIBRARY Michigan State University .1 :PIIIIBPORY. Mill ‘ ABSTRACT SODIUM: A FACTOR IN GROWTH OF BLUE-GREEN ALGAE BY Amelia Kay Ward The response of heterocystous blue-green algae to varying concentrations of sodium was examined in axenic culture as well as in situ studies. Laboratory cultures of Anabaena cylindrica were treated with four concentrations of sodium and three concentrations of nitrate to determine the response in terms of rates of acetylene reduction, l4C assimilation, excretion of organic carbon, concentration of chlorophyll a, particulate organic carbon, particulate organic nitrogen, and heterocyst and filament numbers. Natural populations of blue-green algae, predominantly Aphanizomenon sp., were used for sodium enrichment studies in which carbon-14 assimilation and acetylene reduction provided a measurement of comparative photosynthetic carbon fixation and elemental nitrogen fixation, respectively. Cultures of Anabaena cylindrica with no Na+ added suffered from decreased rates of acetylene reduction) l4C assimilation, excretion of organic carbon as well as lower (grab Amelia Kay Ward O; a” concentrations of chlorophyll a and particulate organic carbon compared to cultures supplied with 5, 10, and 50 mg Na+ liter-l. Sodium deficient cultures released a higher percentage of previously fixed carbon as organic carbon. No differences in any area measured were demonstrable among cultures grown with 5, 10, and 50 mg Na+ liter-1. High nitrate concentrations (20 mg NO liter-l) resulted in 3 decreased rates of acetylene reduction and heterocyst numbers in sodium sufficient and sodium deficient cultures; however, decreased particulate organic nitrogen content at high nitrate levels occurred only in sodium deficient cul- tures. Higher percentages of excreted organic carbon occurred with increasing nitrate concentrations in sodium deficient cultures. Sodium enrichment of natural phyto- plankton pOpulations indicated increased photosynthetic carbon fixation with small additions of sodium (5 mg Na+ liter-l), whereas higher concentrations (50, 100, and 200 mg Na+ liter-l) elicited neither a stimulatory nor an inhibitory response. No increase in in situ acetylene reduction rates occurred with additions of sodium. SODIUM: A FACTOR IN GROWTH OF BLUE-GREEN ALGAE BY Amelia Kay Ward A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1974 L: “(f-E5 ' .3— fl ACKNOWLEDGMENTS I would like to express my sincere appreciation to Dr. Robert G. Wetzel for his continued guidance and support throughout the investigatory aspects of this study as well as in the preparation of the manuscript. Appreciation is also extended to Drs. Wetzel, Michael J. Klug, and Peter G. Murphy for their critical evaluation of the manuscript. The use of gas chromatographic equipment, generously made available by Dr. Klug, is gratefully acknowledged. Invalu- able discussions with Gordon L. Godshalk, Donna K. King, Kelton R. McKinley, and Grover M. Ward as well as the technical expertise of Jayashree Sonnad aided immensely in the completion of this project. This research was supported financially by ABC Grant AT-(ll-l)-1599, COO-1599-84. ii Ir‘f'c -.' - TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . iv LIST OF FIGURES. . . . . . . . . . . . v INTRODUCTION. . . . . . . . . . . . . . 1 MATERIALS AND METHODS. . . . . . . . . . . 5 Laboratory. . . . . . . . . . . . . . 5 Culturing Procedures. . . . . . . . . . Acetylene Reduction . . . . . . . . . . 7 Chlorophyll a . . . . . . . . . 8 Carbon Assimilation and Excreted Organic carbon 0 I O O O O O O I O O O 8 Particulate Organic Carbon. . . . . . . . 9 Particulate Organic Nitrogen . . . . . . . 9 Filament and Heterocyst Enumeration. . . . . 10 Sodium Analysis . . . . . . . . . . . 10 Field Procedures. . . . . . . . . . . . lO Carbon-l4 Sodium Enrichment Bioassays . . . . 10 Acetylene Reduction Enrichment Bioassays . . . ll Enumeration of Algal Cells and Heterocysts . . 12 RESULTS AND DISCUSSION . . . . . . . . . . 13 Response of Anabaena cylindrica to Sodium . . . 13 Response of Anabaena cylindrica to Sodium and Nitrate . . . . . . . . . . . . 18 Response of Natural Populations of Blue- -green Algae to Sodium Enrichment. . . . . . . . 28 SUMMARY AND CONCLUSIONS . . . . . . . . . . 35 BIBLIOGRAPHY. . . . . . . . . . . . . 37 iii :7? I. ~ ' _| ‘I '. LIST OF TABLES Table Page 1. Results of two-way analysis of variance on data from axenic cultures of Anabaena cylindrica in response to sodium and nitrate, including levels of signifi- cance: **, Pi0°013 *, P:0.05; n.s., not significant . . . . . . . . . . 21 2. Major groups of algae present on 11 July 74 at 0.5 meters in Wintergreen Lake . . . . 33 iv q‘fin—fi ‘i.”‘ i l Figure 1. LIST OF FIGURES Page Concentration of chlorophyll a and rates of acetylene reduction of axenic Anabaena cylindrica over time at four concen- trations of sodium and three concen- trations of nitrate. Standard deviations are given for acetylene reduction unless one standard deviation is less than the diameter of the point. Sodium concen- trations (mg liter' ): 0 °---'; 5 °———' 10 '-'-'; SO ""'. . . . . Rates of 14C assimilation and excretion of organic carbon (: S.D.) of axenic Anabaena cylindrica . . . . . . . . l6 O ‘0 Excreted organic carbon as percentage carbon assimilated (: S.D.) of axenic Anabaena cylindrica on day 14 . . . . . l7 Concentration of particulate organic carbon (: S.D.) of axenic Anabaena cylindrica on day 14 . . . . . . . . l9 Concentration of chlorophyll a and rates of acetylene reduction of Anabaena cylindrica over time at two concentra- tions of sodium and three concentrations of nitrate. Standard deviations are included unless one standard deviation was less than the diameter of the point. . 22 1) Density of filaments and heterocysts (ml- on day 14 of axenic Anabaena cylindrica in response to variations in sodium and nitrate. Standard deviations are included unless one standard deviation was less than the width of the line . . . 23 Figure 7. Concentrations (: S.D.) of particulate organic carbon and particulate organic nitrogen on day 14 of Anabaena cylin- drica in response to variations in sodium and nitrate . . . . . . . . 8. Rates of 14C assimilation and excretion of organic carbon (: S.D.) on day 14 of Anabaena cylindrica in response to varying sodium and nitrate . . . . . 9. Excreted organic carbon as percentage carbon assimilated (: S.D.) on day 14 of Anabaena cylindrica in response to varying sodium and nitrate . . . . . lO. Photosynthetic carbon fixation and excreted organic carbon of phytoplankton populations in response to sodium enrich- ment, 16 July 73, in Wintergreen Lake over a 12- hour incubation period. Lakewa er control °——— +50 mg NI+ liter- . "-- .; 10 mg Na+ liter 0 o o o o . 200 mg Na liter.- .-.-. o o o 0 11. (a) Photosynthetic carbon fixation, (b) rates of excretion of organic carbon, and (c) acetylene reduction of phyto- plankton populations in response to sodium enrichment, 11 July 74, in Winter— green Lake at 0.5 meters. 0 = lakewater control (i S.D.). . . . . . . . . Vi Page 25 26 27 30 32 H I"..- ’J.’ INTRODUCTION Several investigators have reported positive growth responses in blue-green algal cultures to the pres- ence of relatively high concentrations of sodium (Allen, 1952; Batterton & Van Baalan, 1971; Benecke, 1898; Emerson & Lewis, 1942; Gerloff, 33 31., 1952; and Kratz & Myers, 1955). Maximum growth was obtained at various levels ranging from one mg Na+ liter.1 and higher for Anacystis nidulans (Batterton & Van Baalan, 1971), 5 mg Na+ liter”1 and higher for Anabaena cylindrica (Allen & Arnon, 1955) to -1 40 mg Na+ liter for Anabaena variabilis and Anacystis nidulans (Kratz & Myers, 1955). Blue-green algae are among the few plants which have an absolute sodium requirement (Allen & Arnon, 1955). In studies with Anabaena gylindrica the criteria for an essential nutrient were defined as follows: (1) normal plant growth is absent without the element; (2) the deficiency symptoms caused by the absence of the element are removed on its addition to the medium; (3) the requirement is specific (the substitution of another nutrient for the element cannot be made); and (4) the function of the element or its direct effect on the metabolism of the plant is identified. The physiological function of sodium in blue—green algae has never been elucidated completely. Sodium has been shown to inhibit nitrate reductase activity in cul- tures of Anabaena cylindrica grown with nitrate (Brownell & Nicholas, 1967). Sodium deficient cultures resulted in enhanced nitrate reductase activity and accumulation of toxic levels of nitrate. Higher levels of sodium were required to avoid deficiency symptoms in cultures grown with nitrate (0.4 meq/l NaCl) as compared with cultures grown without nitrate (0.004 meq/l NaCl). The paucity of information regarding the physio- logical function of sodium is matched by the lack of information on the possible ecological effectsof sodium on natural populations of blue-green algae. Provasoli (1969) conjectured that increases in salinity in Lake Washington could have been a contributory factor to the ensuing blue-green algal bloom (Edmondson, gt 31., 1956). The presence of both high levels of sodium (50 mg Na+ liter-1) and dense populations of blue-green algae have been noted in hypereutrOphic Sylvan Lake in contrast to low levels of sodium (3 mg liter-l) and a lack of blue-green dominant populations in Goose Lake and hard-water lakes in general (Wetzel, 1966). The occurrence of the two phenomena may be unrelated, but the possibility of sodium contributing as one of several interrelated causal factors appears worthy of further investigation. Ecologically, nitrogen-fixing blue-green algae are of particular interest because of their potential to con- tribute simultaneously to the carbon and nitrogen input of certain water strata. Large populations of heterocystous blue—green algae and maximum nitrogen—fixing rates are generally associated, spatially and temporally, with minimal concentrations of combined inorganic nitrogen forms (Goering & Neess, 1964; Stewart, 33 31., 1970; Duong, 1972; Horne & Goldman, 1972; Horne, 3E 31., 1972). However, the presence of inorganic forms does not necessarily preclude elemental nitrogen fixation (Dugdale & Dugdale, 1965; Goering & Dugdale, 1966; Horne & Fogg, 1970). The under- standing of the effects of any factor which has the potential to influence directly the nitrogen metabolism of heterocystous blue-green algae would seem of major impor- tance in further clarifying the many complex interactions which result in the presence of natural populations of heterocystous blue-green algae. Sodium has been implicated as such a factor. The primary objective of the following study was to investigate the response of blue-green algae to various levels of sodium concentration. It was restricted to nitrogen—fixing members of the Cyanophyta with major emphasis on facets of carbon and nitrogen metabolism. The research followed two directions: Assays of the response of Anabaena cylindrica in axenic culture to varying concentrations of sodium and nitrate. 13 3133 studies designed to describe the response of natural pOpulations of heterocystous blue- green algae to sodium enrichment by means of carbon-14 radioassay as well as acetylene reduction techniques. MATERIALS AND METHODS Laboratory Culturing Procedures A culture of Anabaena cylindrica, generously supplied by Professor Akihiko Hattori, University of Tokyo, Tokyo, Japan, was maintained in axenic culture for labora- tory experiments. The medium used for experimental purposes, G4 (a modification of Gorham's medium), was of the following composition (mg liter—1): KZCO 26; 3! K3C6H507.H20, 235; K2 4, 39; M9504.7H20, 75; CaC12, ferric citrate, 6; nitrilotriacetic acid, 40. Potassium HPO 27; nitrate was used in medium containing nitrate. Sodium was variable and added as NaCl. Micronutrients were supplied as follows (mg liter-1): ZnSO4.7H20, 1; MnSO L1Cl, 0.5; COC12.6HZO, 0.5; (NH4)6MO7024.4H20, 0.1. The pH was adjusted to 7.0 with 0.5 N KOH before autoclaving; 4°H20’ 1; final pH after autoclaving was 7.9. The medium was dis- pensed in 300 m1 aliquots to 500 m1 polycarbonate flasks. Polycarbonate was used to prevent Na+ contamination from glass flasks. G4 medium with no NaCl added, autoclaved once in a polycarbonate flask and stored for 26 days was I ‘h'.. 6-:- found to have an absolute Na+ concentration of 0.236 mg Na+ liter-1 compared with 0.661 mg Na+ liter.l in medium autoclaved once in a glass flask and stored fOr the same period of time. Repeated autoclaving also increased the sodium concentration of the medium in a glass flask, whereas no increase in sodium concentration was observed in medium repeatedly autoclaved in a polycarbonate flask. Sodium concentrations in the text and figures refer to sodium added as NaCl, not the absolute sodium concentration of the medium. An aliquot from a fresh stock culture was used as an inoculum for experimental flasks. Twenty m1 of stock culture were transferred aseptically to 100 m1 G4 medium without sodium in a glass flask; twenty m1 from this flask were then transferred to 100 m1 G4 medium without sodium in a polycarbonate flask. A two m1 aliquot from the poly- carbonate flask was used as an inoculum for each experi- mental flask, thus, sufficiently diluting the sodium from the stock culture. Duplicate flasks of all treatments were prepared in each experiment. Experimental cultures were maintained in a Sherer growth chamber on a rotating platform at 23°C and at a light intensity of 1936 lux (Weston Illumination Meter, Model 756) supplied from fluorescent and incandescent light sources on a light regime of 16 hours light and 8 hours dark. 7‘?“ “TE—W ’1 Acetylene Reduction The acetylene reduction technique for determination of elemental nitrogen fixation was first developed (Dilworth, 1966; Schollhorn & Burris, 1966) and applied to blue-green algae in the mid-19605 (Stewart, 33 31., 1967, 1968). Since then it has been used extensively in both laboratory and 13 3133 studies. However, it is an indirect technique and not without problems (see discussion in Fogg, 33 31., 1973, and Stewart, 1973). In this study it was used as a reasonable estimate of the comparative Nz-fixing ability of heterocystous blue-green algae. Aliquots (5 ml) from each culture flask were transferred aseptically to a 15 m1 serum vial and sealed with serum bottle caps. Vials were flushed for two minutes with a nitrogen-free gas: CO 0.0395%; 0 23.60%, argon 2' 2’ balance (Matheson Gas Products, Joliet, Illinois), injected with 1.0 m1 purified acetylene (Matheson Gas Products), and incubated two hours under the same conditions as the culture flasks. The algae were killed with 0.2 m1 2% HgClz. Control vials were injected with HgCl immediately after 2 injection of acetylene and allowed to incubate under the same conditions as the experimental vials. Samples (1.0 m1) from the headspace were injected into a Packard 409 gas chromatograph equipped with a Hz-flame ionization detector. A stainless steel column (3 mm X 2 meters) packed with Porapak-N (80-100 Mesh) was used for all analyses. Oven temperature was 650C. Flow rates of gases 1 1 were as follows: He, 11 m1 minute- ; H2, 18 m1 minute- ; air, 240 m1 minute-l. Areas of ethylene peaks (height of peak X width at 1/2 peak height) were quantified by relating the areas of the peaks to a known series of standards of purified ethylene (Matheson Gas Products). Chlorophyll a Aliquots (5 or 10 ml) were withdrawn aseptically from each culture flask and filtered onto a Millipore AA filter. Filters were homogenized (Teflon homogenizer) in 90% basic aqueous acetone, centrifuged, and the absorption of the supernatant measured by a Hitachi-Perkin Elmer spectrophotometer (Model UV-VIS 139). Calculations were those from Parsons and Strickland (1963) modified by Westlake (1969). Corrected chlorophyll a and pheopigments were also measured using the equations of Parsons and Strickland (1963) modified by Wetzel and Westlake (1969). Carbon Assimilation and Excreted Organic Carbon The day an experiment was terminated, a 35 ml aliquot was withdrawn from_each experimental flask and placed in a sterile 100 ml erlenmeyer flask. Either 400 ml or 100 pl of KH14CO 3 (4.96 uCi ml-l) were injected into each flask. After a two hour incubation period under the same conditions as the experimental flasks, duplicate 5.0 m1 aliquots were filtered onto Millipore HA filters for Geiger-Muller radioassay (Nuclear-Chicago D—47 of known {TN'm-fl counting efficiency). Excreted organic carbon was measured from the same flasks. Ten m1 of culture sample were fil- tered onto a Millipore HA filter; the filtrate was acidified w1th 3% H3PO4, 2 pipetted onto planchets for radioassay as above. purged with N for two minutes, and two m1 Particulate Organic Carbon The day an experiment was terminated, duplicate 25 m1 samples from each experimental flask were filtered onto pre-combusted (45 minutes, 525°C) glass fiber filters (Reeve Angel, 984R). Filters were analyzed for carbon by the procedures of Strickland and Parsons (1968) in which wet oxidation by acid dichromate is used with dextrose (D-glucose) as a standard. Particulate Organic Nitrogen Samples to be analyzed for particulate organic nitrogen were filtered by the same procedure as those for particulate organic carbon. Filters were subjected to a modification of the Kjeldahl determination described by McKenzie and Wallace (1954). Filters were predigested overnight in four m1 of concentrated H2804 with 150 mg salicylic acid. After the addition of 300 mg Na .5H 0, 23203 2 3 9 K 80 and 150 mg HgO, samples were digested over heat 2 4' until clear and then refluxed for 45 minutes. Samples were steam distilled, using methyl red in dilute boric acid as an indicator, and titrated against 0.005 N HCl. An indi- cator blank was used for the determination of the endpoint. .12 *. vee..- 1‘ 10 Filament and Heterocyst Enumeration Samples from each culture flask were preserved with Lugol's solution. One ml was transferred to a Sedgewick- Rafter chamber for counting at 150x. Sodium Ana1ysis Sodium concentrations of culture media and filtered lakewater samples were measured with a Jarrell Ash atomic absorption spectrophotometer (Model 82-700). Field Procedures Carbon-14 Sodium Enrichment Bioassays Water samples were collected with a Van Dorn water sampler and distributed to 500 ml erlenmeyer flasks (summer, 1973) or 125 m1 bottles (summer, 1974). Samples were injected with NaCl from autoclaved stock solutions to yield a final known Na+ concentration. Three replicate light bottles and one dark bottle were used for each con— centration as well as for untreated lakewater controls. 14 Bottles were injected with 1.0 ml KH CO (4.96 uCi ml—l), 3 suspended at the depth from which the water was collected and allowed to incubate. After incubation, 25 m1 aliquots were filtered onto Millipore HA filters, fumed with HCl (Wetzel, 1965a) and analyzed by radioassay as described in Laboratory Procedures. ll Acetylene Reduction Enrichment Bioassayg Methods of 13 3133 acetylene reduction followed closely those described by Duong (1972). Water samples were collected and the algae concentrated by gently fil- tering one liter of lakewater through 30 um Nitex netting; algae were kept suspended in a small amount of filtrate during filtration to prevent disruption of cells and then rinsed from the netting into a beaker and resuspended in 100 m1 of lakewater filtrate. Aliquots (7 ml) from this sample were distributed to 15 m1 serum vials, injected with 0.5 ml of an appropriate NaCl solution, stoppered, flushed for two minutes with nitrogen-free gas (see Laboratory Procedures), injected with 1.0 m1 acetylene, resuspended at the depth from which the water was collected, and incubated for two hours. Three replicate vials were used for each sodium concentration as well as for untreated lakewater samples. The algae were killed with 0.2 m1 2% HgCl Controls were injected with HgCl immediately after 2' 2 injection with acetylene and incubated with experimental vials. Vials were brought to the laboratory and 1.0 m1 of headspace gas injected into a Varian Aerograph gas chromatograph (Model 500-D) with a flame ionization detector. A stainless steel column (3 mm X 2 meters) packed with Porapak-N (80-100 mesh) was used for all analyses. Flow rates of the gases were as follows: He, 18 ml minute-l; H 25 m1 minute-l; and air, 18 ml minute-1 2' 12 "WHW1 Column temperature was 500C. Ethylene was quantified as described in Laboratory Procedures. Enumeration of Algal Cells and’Heterocysts Phytoplankton samples were preserved with Lugol's solution, 5 ml placed in a settling chamber and counted at 200 X using a Wild inverted microscope. RESULTS AND DISCUSSION Response of Anabaena cylindrica to Sodium The first three experiments were designed to deter- mine if varying levels of sodium elicit different responses from the laboratory culture of Anabaena cylindrica. Four concentrations of sodium at one concentration of nitrate were used in each experiment, the nitrate level varying in each experiment. However, the exact values obtained in one experiment cannot be compared directly with values in other experiments because of variations in initial inoculum cell density. Therefore, only the responses to four con- centrations of sodium at a given level of nitrate were compared. The greatest difference in rates of acetylene reduction at each concentration of nitrate occurred between the levels of 0 and 5 mg Na+ liter”1 (Figure l). Nested one-way analyses of variance revealed a significant dif- ference in the acetylene reduction rates on day 14 among sodium treatments at the 1% level (P:0.01). Little dif- ference resulted from concentrations of sodium at 5, 10, and 50 mg Na+ liter—l. Increases in concentrations of 13 I, .m ”‘1?" . . l 1 I 'x1 O 14 ...... om u.l.l. 0H «.Ill. m “.1II. o "A aumufla may mcoflumuusmosoo Esfioom .usHom may mo Hmuwewflo on» swan mmma ma soHuMA>m© pumpcmum 0:0 mmwass cofluoscwu mswaaumom now sm>flm mum mGOADMA>wp oumosmum .mumnufis mo mcowwmuusmosoo owns» can ESHUOm mo m:0wumuusmosoo usOM #m mafia um>o moaupcflamo mammnmsd cacmxm mo cofluoscmu ocoamumom mo mmumu can m Hawsmouoano mo coaumnusmosoo <3 ‘0 (V N In g o ,Jw e TIAHdoamHofir' F21H .-Iw swam-113 Wu b b F b - 7mm»... noz 85 RE... "02 3m vamp... "oz 86 .H musmfim . 5.23 ‘."'. "‘ 15 (Z chlorophyll a, used as an estimate of increasing biomass, closely resembled increases in rates of acetylene reduction (Figure 1). The only exception to this was the treatment 1 in which cultures grown with 0 mg Na+ liter- and an initial concentration of 20 mg NO3 liter-l reached a maximum acetylene reduction rate on day 8 and decreased steadily to an almost negligible rate on day 14. However, concentra- tions of chlorOphyll a in the same treatment continued to increase over the same period of time. This was the only treatment in which the acetylene reduction rate decreased with time. Rates of 14C assimilation reflected a pattern similar to that of acetylene reduction; that is, carbon uptake in cultures without sodium was consistently one- third that of cultures grown at the three other concentra- tions of sodium (Figure 2). No marked differences were found among the higher sodium concentrations. In contrast to rates of 14C assimilation, rates of excretion of organic carbon in cultures with no added sodium were approximately one-half those of cultures with added sodium (Figure 2). The relationship between rates of excreted organic carbon and rates of 14 C assimilated can be seen more clearly when excreted organic carbon is expressed as per cent carbon assimilated (Figure 3). In all experiments a greater percentage of carbon assimilated (as inorganic 14C) was excreted as organic carbon in cultures with no added sodium. Although the actual percentages were small, this is a 16 f “c ASSIMILATED EXCRETED ORGANIC CARBON 0 mg N03 LlTER" I 0 mg No, man" I I no = I ;- + . 1 .o. l a p- -( J 40 ; 1 Jam . l 10 2 mg No, LlTER" 2 mg No3 LlTER" T T :2 x I Tr *' = + ‘ :2 D I D Z > «u» E 2 2 9-) w L -I :2 5 . Z O 1 ‘ ’ - 200 8 O o §’° ’ ‘ ' c o 20 mg N0J LITER" 20 mg NDJJTER" , J so -+ 1 ‘ 1 K» 24 T F*—;- 1 so I 1 . 18 3 60 I2 ‘ r ' 40 6 # ~20 .4 0; 0 0 5 IO 50 O 5 IO 50 mg Na’ LITE)?" ADDED AS NaCI Figure 2. Rates of 14C assimilation and excretion of organic carbon (: S.D.) of axenic Anabaena cylindrica. 17 I? ."_L"f!i_n 1 J O 0 mg No3 LITER" 2.0 I 1 8 T E 1.0 l : .3. o g 2 mg N03 LITER" 2 2.0- I - O I g '0)- ; .1 < . 0 3° 0 (0 mm N03 LITER"; 4 20- + - O O M 1.0- I i -+ 0 o 5 10 50 mg Na‘ LITER" ADDED As NaCl Figure 3. Excreted organic carbon as percentage carbon assimilated (: S.D.) of axenic Anabaena cy1indrica on day 14. 18 probable indication of a less efficient system in terms of carbon utilization in the sodium deficient cultures. The quantity of particulate organic carbon present in cultures with added sodium was consistently twice that present in cultures without sodium (Figure 4). Since rates of carbon assimilation in cultures with added sodium were at least three times that of sodium deficient cultures, there appears to be a loss of carbon after assimilation in cultures with added sodium not entirely explicable in terms of excreted organic carbon. Possible explanations include: (1) Higher rates of CO2 evolution occurred in sodium sufficient cultures. (2) Rates of 14C assimilation in sodium sufficient cultures were less than three times that of sodium deficient cultures prior to day 14. Rates of 14C assimilation were measured only on the last day of the experiment; the particulate organic carbon represented an accumulation over a 14-day period. Both explanations are entirely speculative since no data are available on CO2 evolution or rates of 14C assimilation prior to the 14th day of the experiments. Response of Anabaena cylindrica to Sodium and Nitrate In order to investigate responses to nitrate and sodium, experiments were designed to compare two concen- trations of sodium (0 and 5 mg Na+ liter-l) at three concentrations of nitrate (0, 2, and 20 mg NO3 liter-1). The two concentrations of sodium were found to produce 19 PARTICULATE ORGANIC CARBON 0 mg N03 men" rmfl‘ ‘ 40 F‘“ 20 ”‘1" 1"- 0 2m N03 LIT ER" E 1 5 40 . , « m J. T I -* tr 3 20 2 I7 0 20 mg N03 LITER" HH 40 3 4 20 ‘ l 0 O 5 10 50 mg Na+ LITER" ADDED AS NaCl Figure 4. Concentration of particulate organic carbon (: S.D.) of axenic Anabaena gylindrica on day 14. 20 highly significant results (P30.01) in all areas for which measurements are available (Table l). Differing concen- trations of nitrate produced significant results in four areas: acetylene reduction (P30.01), heterocyst numbers (P:0.01), particulate organic nitrogen (P:0.05), and excreted organic carbon as percent 14C assimilated (P:0.01). Three of the four areas are associated closely with facets of nitrogen metabolism. The addition of 2 mg NO3 liter-l appeared to make little difference in rates of acetylene reduction at either concentration of sodium compared to rates at 0 mg NO3 liter.1 (Figure 5). However, the addition of 20 mg NO3 liter.1 decreased the rates of acetylene reduction compared to 0 and 2 mg NO liter-1 at both levels 3 of sodium. Cultures grown at 5 mg Na+ liter"1 continued to increase in rates of acetylene reduction through day 14, whereas cultures grown at 0 mg Na+ liter.1 and 20 mg NO3 liter.l reached a maximum on day 11 and decreased to a negligible rate by day 14. Heterocysts, generally accepted as the sites of nitrogen fixation under externally aerobic conditions (Wolk, 1973), decreased in numbers at higher concentrations of nitrate in both sodium sufficient and sodium deficient cultures. Cultures treated with the lowest sodium con- centration (0 mg Na+ liter-l) and highest nitrate concen- tration (20 mg nitrate liter-l) were the only ones having fewer heterocysts ml.l than filaments ml-l (Figure 6). 21 Table 1.--Results of two-way analysis of variance on data from axenic cultures of Anabaena cylindrica in response to sodium and nitrate, including levels of significance: **, P:0.01; *, P:0.05; n.s., not significant. Sodium Nitrate S X N Acetylene reduction ** ** n.s. Heterocyst numbers ** . ** * Particulate organic nitrogen ** * n.s. l4C assimilation ** n.s. n.s. Excreted organic carbon ** n.s. n.s. Particulate organic carbon ** n.s. n.s. % Excreted organic carbon ** ** * Chlorophyll a ** n.s. n.s. Filament numbers ** n.s. n.s. .usfiom map mo umumemfio on» can» mmma was coflumfl>mc pumocmum oco mmmacs pmosaocw mum mcoflumm>mp oumvcmum .mumuuwc mo msowumnucmocoo mmusu new Edwo0m mo msowumuucmocoo 03» um mafia um>o moauvsfiamo mswmnmcd mo cowuosomu msmamumom mo mmumu can a Hamsmouoano mo scaumnucmosou m>m© numncmum mco mmmacs owvsHosfl mum msoflumm>mo pumpsmum .mumuumc can Esfloom cm msowumwum> on mmcommmu cw mowucCAHNw mcwmnmc< oacmxm no «a amp so AHIHEV manhoouwumg can mucmfimHHm mo >ummcmo m. o n o 62 m2 9%onme HH +———¢ HH whzwz<£u o O N 3 8 .101 x .Jw SHSBINDN .m musmwm 24 Particulate organic nitrogen content of cultures grown with 5 mg Na+ liter-1 did not vary with increasing nitrate concentration (Figure 7), indicating that cultures grown at the higher nitrate concentrations, where acetylene reduction decreased, were efficiently supplementing ele- mental nitrogen fixation with reduction of nitrate. However, sodium deficient cultures decreased in particulate organic nitrogen with increasing nitrate concentration, indicating a lag in nitrogen assimilation at higher con- centrations of nitrate. Particulate organic carbon, rates of 14C assimi- lation, and rates of excretion of organic carbon (Figures 7 and 8) did not vary with increases in nitrate concentration. As in the other experiments, particulate organic carbon in sodium sufficient cultures (5 mg Na+ liter-l) was approximately twice that in sodium deficient cultures, whereas rates of 14C assimilation were approxi- mately three times greater. The percentage of 14C assimilated and then excreted as organic carbon increased with increasing nitrate concentration in sodium deficient cultures (Figure 9). In summary, when no sodium was added to cultures of Anabaena cylindrica, rates of acetylene reduction, 14C assimilation, and excretion of organic carbon were signifi— cantly reduced as well as concentrations of chlorophyll a and particulate organic carbon compared with cultures grown with 5, 10, and 50 mg Na+ liter—1. No marked differences Figure 7. 25 . PARTICULATE ORGANIC CARBON . —*a TI. 40» T T . E 1 2! . J O G) g 20 I I + I q 0 I on ~ . 3. O . PARTICULATE ORGANIC NITROGEN - E a » l I I - 2: Lu . . O O I E 4- - 2 - o I- q a. O Na” 0 5 o 5 o 5 N03 0 2 20 mg LITER.I Concentrations (: S.D.) of particulate organic carbon and particulate organic nitrogen on day 14 of Anabaena cylindrica in response to variations in sodium and nitrate. COUNTS MINUTE" 1000 COUNTS MINUTE" + Na N03 Figure 8. 26 [ EXCRETED ORGANIC CARBON 140 -I 100'- .I . I . 1 + 605- .. L . 20- - C “C ASSIMILATED 30- I T . I l . 20 4 I0 . _ -I 0’ 5 o 5 0 5 2 20 mg LITER" Rates of 14C assimilation and excretion of organic carbon (+ S.D.) on day 14 of Anabaena cylindriCa in response to varying sodium and nitrate. 27 EOC AS % CARBON ASSIMILATED I'I-I Na" 05 05 05 NO3 0 2 20 mg LITER’I Figure 9. Excreted organic carbon as percentage carbon assimilated (: S.D.) on day 14 of Anabaena cylindrica in response to varying sodium and nitrate. 28 w--——-——---~1| were found in cultures supplied with 5, 10, and 50 mg Na+ liter-1. Increases in nitrate concentrations resulted in a decrease in acetylene reduction and heterocyst number in both sodium deficient and sodium sufficient cultures; how- ever, a decrease in particulate organic nitrogen was Observed only in sodium deficient cultures as nitrate con- centrations were increased. An increase in the percentage organic carbon excreted was also apparent in sodium deficient cultures with increasing nitrate concentrations. The effects of high nitrate concentrations on sodium deficient cultures observed in this study are congruous with the results reported by Brownell and Nicholas (1967) in which toxic levels of nitrite accumulated when nitrate was present in sodium deficient cultures. That nitrite was accumulating in sodium deficient cultures in this study is a possibility; however, if so, it did not affect signif- icantly aspects of carbon metabolism (rates of 14C assimilation, excretion of organic carbon and particulate organic carbon) by day 14 of the incubation period. Highest nitrate concentrations used in this study (20 mg NO3 liter-l) were much lower than those used in the Brownell and Nicholas (1967) study (10 mM KNOB) and are closer to those found in natural aquatic systems. Response of Natural Populations of Blue-green Algae to Sodium Enrichment 13 situ investigations were initiated in order to describe the response of natural populations of 29 heterocytous blue-green algae to additions of sodium. Typically, the cation composition of hard-water, marl lakes of southwestern Michigan is completely dominated by calcium and magnesium. The preponderance of divalent cations and the consequent low monovalent:diva1ent cation ratio has been proposed as one factor, among several, which functions in a cyclical manner to suppress productivity in marl lakes (Wetzel, 1969). Enrichment studies were conducted in the summers of 1973 and 1974 in Wintergreen Lake, a eutrOphic hard- water system which receives significant amounts of allochthonous and autochthonous organic input. The sodium concentrations rarely deviate from approximately 5 mg Na+ liter-l, a concentration differing little from that of other more oligotrophic hard-water lake systems in the area. Extensive work has been done on the lake describing basic limnological parameters as well as studies with particular emphasis on carbon and nitrogen metabolism (Duong, 1972; Manny, 1972; and Wetzel, 33 31., 1974). Characteristically, a dense heterocystous blue-green population develops in the summer; it was during a period of such development that the enrichment experiments were conducted. Figure 10 illustrates the effects of sodium added in concentrations of 50, 100, and 200 mg Na+ liter”1 above background lake water (4.98 mg Na+ liter-1) at a depth of one meter. The predominant alga in the phytoplankton was Figure 10. BOOOF‘C ASSIMILATION In 4000 |.— D Z IE 0) p— O I . g _ EXCRETED ORGANIC CARBON o O 3O 1200 1600 2000 TIME (HOURS) Photosynthetic carbon fixation and excreted organic carbon of phytoplankton populations in response to sodium enrichment, 16 July 73, in Wintergreen Lake over a 12-hour incubation p riod. _Eakewater con- trol . .; 50 mg Na liter '--- I 100 mgl'N'aT+ liter'l ---;-; 200 mg Na liter” '-'-°. 31 Aphanizomenon sp. Although no marked enhancement of photo— synthetic carbon fixation was Observed over untreated lakewater controls, neither was there a marked suppression over a 12-hour incubation period, which supplies further evidence for the halotolerant nature of blue-green algae (Pillai, 1954, 1955; Batterton & Van Baalan, 1971). In contrast to these results were those illustrated in Figure 11 in which a two- to three-fold stimulation in photosynthetic carbon fixation was caused by the addition of 5 mg Na+ liter.l over background lakewater (4.99 mg Na+ liter-1) over a four hour incubation period. The addition of 50 mg Na+ liter.l caused highly erratic results. The same concentrations of sodium did not increase the rates of acetylene reduction (Figure 11). Again, the dominant phytoplankter was Aphanizomenon (Table 2). These prelimi- nary bioassays suggest that, whereas the addition of large quantities of sodium have no effect on photosynthetic carbon fixation in this system, the addition of small quantities of sodium does enhance carbon fixation. The develOpment and associated literature of bio- assay studies as a tool in understanding nutrient relation- ships in laboratory cultures and in natural populations as well as the advantages and disadvantages of such a technique have been discussed by Wetzel (1965b). Ideally, enrichment bioassays provide a rapid determination of nutrients which may be in short supply and, hence, limiting productivity among natural phytoplankton populations. Problems exist Figure 11. 32 I a 20 - . fl __ q 2 I x 10 I- I .4 2 Q. 0 - .. 0 200 - I b « E I 0 ‘00 I- A 1 ~ I I 'I' 0 a: :I: I T C '-‘ 0.8 - 1 I ~ V :I: (3‘ a4 - - 2 1 0 o 5 so mg Na+ LITER" ADDED AS NaCI (a) Photosynthetic carbon fixation, (b) rates of excretion of organic carbon, and (c) acetylene reduction of phytoplankton populations in response to sodium enrichment, 11 July 74, in Wintergreen Lake at 0.5 meters. 0 = lakewater control (: S.D.). 33 Table 2.--Major groups of algae present on 11 July 74 at 0.5 M in Wintergreen Lake. Genus Number liter-l Aphanizomenon sp. filaments 16,524,300 heterocysts 2,988,200 Anabaena sp. filaments 89,200 heterocysts 22,300 Microcystis sp. 22,300 Ceratium sp. 423,700 Cryptomonas sp. 178,400 Trachelomonas sp. 66,900 in interpretation in that the response of a population may vary not only among aquatic systems, but with time and at differing concentrations of the added nutrient due to changes in interacting factors within the system as well as the interaction of such factors with the added nutrient. The interpretation is compounded further when the physio- logical function of the compound in unknown. The initial bioassays performed in this study are certainly subject to all of the above problems of interpretation. Although sodium concentrations vary little within Wintergreen Lake throughout the year, components which may alter the response to sodium may vary. More specifically, the response of the Aphanizomenon population may vary within the period of the bloom. The pOpulation used in the 34 . ? I ‘.fi§.£‘£lfihd I A enrichments of 1974 was extraordinarily dense (16 million filaments liter-l) and crashed dramatically a few days after the bioassays. That the population was stressed during the period of the experiment is probable; that this "stressed" condition is related to the positive response to added sodium is speculative. Obviously, many questions remain with regard to the role of sodium in aquatic systems, particularly as it affects blue-green algal populations. Although direct extrapolation from axenic laboratory cultures is impossible, laboratory studies, by minimizing lack of control, make it possible, among other functions, to pinpoint areas of potential importance to natural populations. The response of sodium deficient cultures to nitrate in this study as well as the results reported by Brownell and Nicholas (1967) that sodium inhibits nitrate reductase suggest intriguing possibilities for the role of sodium in aquatic systems in terms of interactions between inorganic and elemental nitrogen assimilation. SUMMARY AND CONCLUS IONS The response of axenic cultures of Anabaena cylindrica to four concentrations of sodium and three con- centrations of nitrate were examined with special emphasis on aspects of nitrogen and carbon metabolism. 13 3133 sodium enrichment bioassays were employed to ascertain the response of natural pOpulations of heterocystous blue- green algae to additions of sodium. The acetylene reduction technique was used as a comparative measure of the N -fixing 2 ability of blue-green algae, and the assimilation of inorganic radioactive carbon provided a means of deter- mining rates of photosynthetic carbon fixation. The following conclusions can be drawn from this study: 1. Cultures with no NaCl added suffered from decreased rates of acetylene reduction, l4C assimilation, excretion of organic carbon as well as lower con- centrations of chlorophyll a and particulate organic carbon as compared to cultures supplied with 5, 10, and 50 mg Na+ liter-1. 2. Sodium deficient cultures released a higher per- centage of previously fixed carbon as organic 35 36 carbon, possibly indicating a less efficient system in terms of carbon utilization. No differences in any area measured were demon- strable among cultures grown with 5, 10, and 50 mg Na+ liter-1. High nitrate concentrations (20 mg NO liter-l) 3 resulted in decreased rates of acetylene reduction and heterocyst numbers in sodium sufficient and sodium deficient cultures; however, decreased particulate organic nitrogen content at high levels occurred only in sodium deficient cultures. Higher percentages of excreted organic carbon occurred with increasing nitrate concentrations in sodium deficient cultures. Preliminary sodium enrichment bioassays performed in Wintergreen Lake indicated increased photo- synthetic carbon fixation with small additions of sodium (5 mg Na+ liter-1 ). Higher concentrations (50, 100, and 200 mg Na+ liter-1) elicited neither a stimulatory nor an inhibitory response in carbon fixation over untreated lakewater controls. No increase in 13 3133 acetylene reduction rates occurred among natural populations with additions of sodium over the short incubation period utilized. BIBLIOGRAPHY BIBLIOGRAPHY Allen, M. B. 1952. The cultivation of Myxophyceae. Arch. Mikrobiol. 11:34-53. Allen, M. B., and D. I. Arnon. 1955. Studies on nitrogen- fixing blue-green algae. II. The sodium require- ment of Anabaena cylindrica. Physiologia Plantarum. 3:653-660. Batterton, J. C., and C. Van Baalan. 1971. Growth responses of blue-green algae to sodium chloride concentration. Arch. Mikrobiol. 13:151-165. Benecke, W. 1898. Uber Culturbedingungen einiger Algen. Bot. Zeit. 33:83. Brownell, P. F., and D. J. D. Nicholas. 1967. Some effects of sodium on nitrate assimilation and N2 fixation in Anabaena cylindrica. Plant Physiol. 33:915-921. Dilworth, M. 1966. Acetylene reduction by nitrogen-fixing preparation from Clostridium pasteurianum. Biochem. Biophys. Acta. 127:285—291. Dugdale, V. A., and R. C. Dugdale. 1965. Nitrogen metabo- lism in lakes. III. Tracer studies of the assimilation of inorganic nitrogen sources. Limnol. Oceanogr. 13:53-57. Duong, T. P. 1972. Nitrogen fixation and productivity in a eutrOphic hard-water lake: 13 situ and laboratory studies. Ph.D. dissertation, Michigan State University. 241 pp. Edmondson, W. T., G. C. Anderson, and D. R. Peterson. 1956. Artificial eutrophication of Lake Washington. Limnol. Oceanogr. 1:47-53. Emerson, R., and C. M. Lewis. 1942. The photosynthetic efficiency of phycocyanin in Chroococcus and the problem of carotenoid participation in photo- synthesis. J. Gen. Physio. 33:579. 37 38 F099, G. E., W. D. P. Stewart, P. Fay, and A. E. Walsby. 1973. The Blue-green Algae. Academic Press, New York. 459 pp. Gerloff, G. C., G. P. Fitzgerald, and F. Skoog. 1952. The mineral nutrition of Microcystis aeruginosa. Amer. J. Bot. 33:26-32. Goering, J. J., and R. C. Dugdale. 1966. Estimates of 13 situ rates of nitrogen uptake by Trichodesmium sp. in the tropical Atlantic Ocean. Limnol. Oceanogr. 11:614-620. Goering, J. J., and J. C. Neess. 1964. Nitrogen fixation in two Wisconsin lakes. Limnol. Oceanogr. 2:535-539. Horne, A. J., and G. E. Fogg. 1970. Nitrogen fixation in some English lakes. Proc. Roy. Soc. Lond. B. 175:351-366. Horne, A. J., and C. R. Goldman. 1972. Nitrogen fixation in Clear Lake, Calif. 1. Seasonal variation and the role of heterocysts. Limnol. Oceanogr. 11:678-692. Horne, A. J., J. E. Dillard, D. K. Fujita, and C. R. Goldman. 1972. Nitrogen fixation in Clear Lake, Calif. II. Synoptic studies on the autumn Anabaena bloom. Limnol. Oceanogr. 11:693-703. Kratz, W. A., and J. Myers. 1955. Nutrition and growth of several blue-green algae. Amer. J. Bot. 33:282-287. Manny, B. A. 1972. Seasonal changes in organic nitrogen content of net- and nannophytoplankton in two hard-water lakes. Arch. Hydrobiol. 11:103-123. McKenzie, H. A., and H. S. Wallace. 1954. The Kjeldahl determination of nitrogen: A critical study of digestion conditions--temperature, catalyst, and oxidizing agent. Aust. J. Chem. 1:55-70. Parsons, T. R., and J. D. H. Strickland. 1963. Discussion of spectrophotometric determination of marine plant pigments, with revised equations for ascertaining chlorophylls and carotenoids. J. Mar. Res. 11:155-163. 39 Pillai, V. K. 1954. Growth requirements of a halophilic blue-green alga, Phormidium tenue (Menegh). Indian J. Fish. 1:130-144. Pillai, V. K. 1955. Observations on the ionic composition of blue-green algae growing in saline lagoons. Proc. Nat. Inst. Sci. India, Part B. Biol. Sci. .31:90-102. Provasoli, L. 1969. Algal nutrition and eutrophication. 13 Eutrophication: Causes, Consequences, Correctives. National Academy of Sciences, Washington, D.C. pp. 574-593. SchOllhorn, R., and R. H. Burris. 1966. Study of inter- mediates in nitrogen fixation. Fed. Proc. 13:710. Stewart, W. D. P. 1973. Nitrogen Fixation. pp. 260-278. 13: N. G. Carr and B. A. Whitton, eds. The Biology of Blue-green Algae. University of California Press, Berkeley. Stewart, W. D. P., G. P. Fitzgerald, and R. H. Burris. 1967. 13 situ studies on N fixation using the acetylene reduction techniq e. Proc. Nat. Acad. Sci. U.S.A. 33:2071-2078. Stewart, W. D. P., G. P. Fitzgerald, and R. H. Burris. 1968. Acetylene reduction by nitrogen-fixing blue- green algae. Arch. Mikrobiol. 33:336-348. Stewart, W. D. P., T. Mague, G. P. Fitzgerald, and R. H. Burris. 1971. Nitrogenase activity in Wisconsin lakes of differing degrees of eutrophication. New Phytol. 13:497-509. Strickland, J. D. H., and T. R. Parsons. 1968. A Practical Handbook of Seawater Analysis. 2nd edition. Bull. Fish. Res. Ed. Canada, 167. 309 pp. Westlake, D. F. 1969. Estimating quantity and quality of biomass--Macrophytes. pp. 25-32. 13: R. A. Vollenweider, ed. A manual on methods for measuring primary production in aquatic environments. Blackwell Scientific Publication, Oxford. Wetzel, R. G. 1965a1 Necessity for decontamination Of filters in C measured rates of photosynthesis in fresh waters. Ecology. 33:540-542. Wetzel, Wetzel, Wetzel, Wetzel, Wetzel, 40 R. G. 1965b. Nutritional aspects of algal pro- ductivity in marl lakes with particular reference to enrichment bioassays and their interpretation. Mem. Ist. Ital. Idrobiol., 13 Supp1.:l37-157. R. G. 1966. Variations in productivity of Goose and hypereutrOphic Sylvan lakes, Indiana. Invest. Indiana Lakes and Streams. l(5):l47-l84. R. G. 1969. Factors influencing photosynthesis and excretion of dissolved organic matter by aquatic macrophytes in hard-water lakes. Verh. int. Ver. Limnol. 11:72-85. R. G., and D. F. Westlake. 1969. Estimating quantity and quality of biomass-~Periphyton. pp. 33-40. 13: R. A. Vollenweider, ed. A manual on methods for measuring primary production in aquatic environments. Blackwell Scientific Publication, Oxford. R. G., B. A. Manny, W. S. White, R. A. Hough, and K. R. McKinley. 1974. Wintergreen Lake: A study in hypereutrOphication. (In prep.). Wolk, C. P. 1973. Physiology and cytological chemistry of blue-green algae. Bacteriol. Rev. 31:32-101. IIIIIIII’IIIIIES 78 0368 ”'IIIIIIIIIIIM‘MM