THESIb IIIIIIIIIIII III I IIIIIIIIIIII 293I 01072 8099 This is to certify that the thesis entitled ROLE OF LIGHT, CARBON DIOXIDE, AND NITROGEN IN REGULATION OF BUOYANCY AND GROWTH OF BLUE- GREEN ALGAE presented by Craig Nelson Spencer has been accepted towards fulfillment of the requirements for Ph .0. degree in Fisheriele [4deer K 1.7%”; Major professor Date 2/10/84 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution MSU LIBRARIES RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES wilt be charged if book is returned after the date stamped beIow. ROLE OF LIGHT, CARBON DIOXIDE, AND NITROGEN IN REGULATION OF BUOYANCY AND GROWTH OF BLUE-GREEN ALGAE by Craig Nelson Spencer A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Ph.D. Department of Fisheries and Wildlife 1984 (:DCOPYRIGHT BY CRAIG NELSON SPENCER 198A ABSTRACT ROLE OF LIGHT, CARBON DIOXIDE, AND NITROGEN IN REGULATION OF BUOYANCY AND GROWTH OF BLUE-GREEN ALGAE BY Craig Nelson Spencer A three-way interaction between light, carbon dioxide, and inorganic nitrogen was demonstrated to control the growth and buoyancy of Anabaena flos-aquae, a nitrogen fixing, gas vacuolate blue-green alga. At high light intensities (>25 uE/mz/sec) cell growth rates were high, and the algae were non—buoyant regardless of the availability of carbon dioxide and/or inorganic nitrogen. The mechanism responsible for this lack of buoyancy appeared to involve elevated cell turgor pressures which resulted in collapse of pressure sensitive gas vacuoles. At lower light intensities (<25 uE/mz/sec), growth rates were reduced and cell buoyancy was controlled by the availability of carbon dioxide and inorganic nitrogen. These two parameters had opposite effects on cell buoyancy; carbon dioxide»limitation increased buoyancy» while reduced inorganic nitrogen availability reduced buoyancy. The mechanism responsible for buoyancy regulation at low light intensities appeared to involve alteration of the rate of synthesis of gas vacuoles as controlled by the cellular carbon/nitrogen ratio. Cell buoyancy and synthesis of nitrogen-rich gas vacuoles appeared to be inversely related to the cellular carbon/ nitrogen ratio. 2 A model was developed for predicting conditions favorable for the development of nuisance blooms of Anabaena flos-aquae and other blue-green algae in lakes. Nutrient enrichment of lakes leading to increased photosynthetic activity creates conditions favorable for the formation of surface blue-green algal blooms. This is due to a general decrease in light penetration and reduced carbon dioxide concentrations, both of which tend to increase blue-green algal buoyancyu IAlthough nitrogen depletion is commonly associated with the appearance of nitrogen-fixing, blue-green algae in lakes, nitrogen limitation reduces blue-green algal buoyancy. Therefore, increased availability of inorganic nitrogenwwill increase the likelihood of the appearance of nuisance blue-green algal blooms. Results and implications are discussed in an ecological and management context. ACKNOWLEDGMENTS I would like to thank Dr. Darrell King, my major professor, for devoting untold hours of guidance, encouragement, dis- cussion, and support to me throughout my graduate career. Thanks go to committee members Dr. Donald Hall and Dr. Michael Klug for helpful discussions and ideas concerning my research, and to Dr. Niles Kevern for serving on my committee and provid- ing me with teaching and other opportunities in the department. I appreciate the technical help of Dr. Marylou Guerinot,and of Dr. Peter Wolk for advice and use of his laboratory and equipment. I want to thank fellow graduate students Chuck Annett, Jane Repko, and Steve Vanderlaan for providing technical, moral, and diversionary support during this work. Finally, thanks go to my wife, Suzy, for her understanding, support, and patience throughout the peaks and valleys of graduate school and this research. ii TABLE LIST OF TABLES . . . . . . LIST OF FIGURES . . . . . . INTRODUCTION . . . . . . . MATERIALS AND METHODS . . . EXPERIMENTAL APPROACH APPARATUS AND PROCEDURE Algal Microcosms Nutrient Media . Sampling Procedure Algal Cultures . ANALYTICAL METHODS . Temperature . . Alkalinity . . pH . . . . . . Ammonia-Nitrogen Carbon/Nitrogen Ra Nitrogen Fixation Cell Density . . Buoyancy . . . . Turgor Pressure . Carbon Dioxide . Growth Rate Calcul RESULTS 0 O O O O O O O O 0 PRELIMINARY EXPERIMENTS OF CONTENTS 8 tio a i t Buoyancy Regulation . . . Gas Vacuole Conten t and Buoyancy iii Page ANABAENA FLQS- -AQUAE MICROCOSM EXPERIMENTS 2 uE/mz/sec . . . . S uE/m2 /sec . . . 10 uE/mz/sec . . 20 uE/m2 2/sec . . 50 uE/m 2/sec . . EXPERIMENTAL REDUCTION ALGAL GROWTH RATES . . DISCUSSION C O O C C C O O O BUOYANCY . . PHYSIOLOGICAL GROWTH RATES AN INDEX TO SURFACE BLOOM FORMATION MECHANISMS FOR BUOYANCY REGULATION A BUOYANCY REGULATION MODEL PRELIMINARY EVIDENCE FOR BLUEGREEN ALGAE IN LAKES MANAGEMENT OF BLUE GREEN Chemical Control. . Biological Control CONCLUSIONS . . . . . . . . . APPENDICES . . . . . . . . . LITERATURE CITED . . . . . . ANABAENOPSIS OR P ALGAL iv BLOOMS RESSURE TABLE 1. LIST OF TABLES Selected data from repeated Anabaena flos-guae growth experiments at 10 pE/m‘/sec . . Mean cell turgor pressures and standard deviations for Anabaena flos-aquae grown at various light intensities . . . . . . . Percent positively buoyant Anabaena flos-aquae cells grown at S and 30 uE/m‘/sec, and with various sucrose concentrations . . . . Michaelis-Menton kinetic constants from Anabaena flos-aguae growth experiments Page 42 63 70 73 LIST OF FIGURES FIGURE 1. 2. Experimental microcosm and lighting arrange- ment 0 O I O O O O O O O O O O O C O O O O O 0 Typical pressure collapse curve used in calculation of cell turgor pressure . . . . . . Pressure collapse curves of various algal culture densities . . . . . . . . . . . . . . . Pressure collapse curves using various sucrose concentrations . . . . . . . . . . . . . . . . Buoyancy reSponge of (a) Anabaenopsis Elenkinii grown at S uE/m ésec and (b) Anabaena flos-aquae grown at 10 uE/m /sec. The cultures originally contained no inorganic nitrogen; however, the cultures were split into two replicates and NH4-N was added to one replicate as indicated by the arrow . . . . . . . . . . . . . . . . . (a) Pressure collapse curve from an Anabaena flos—aquae culture containing 89% positively buoyant cells. (b) Relationship between buoyancy of the same culture used in 6a, and the percent of original gas vacuoles collapsed through application of pressures determined from 6a . . . . . . . . . . . . . . . . . . . . Anabaena flos-aguae @ 2 uE/mz/sec, June 1 experiment: time course measurements of (a) cell concentration, (b) carbon dioxide, (c) heterocysts, (d) positively buoyant cells, (e) turgor pressure, (f) C/N ratio, and (g) nitrogen fixation . . . . . . . . . . . . . . . . . . . vi Page 19 21 22 29 31 34 FIGURE Page 8. 10. ll. 12. 13. 14. 15. 16. Anabaena flos-aquae @ 5 uE/mz/sec, February 12 experiment: time course measurements of (a) cell concentration, (b) carbon dioxide, (c) heterocysts, (d) positively buoyant cells, (e) nitrogen fixation, and (f) C/N ratio . . . . . 38 Anabaena flos-aquae @ 10 pE/mZ/sec, February lZexperiment: time course measurements of (a) cell concentration, (b) carbon dioxide, (c) heterocysts, (d) positively buoyant cells, (e) nitrogen fixation, and (f) C/N ratio . . . . . 4S Anabaena flos-aquae @ lO uE/mz/sec, June 1 experiment: time course measurements of (a) cell concentration, (b) carbon dioxide, (c) heterocysts, (d) positively buoyant cells, (e) turgor pressure, and (f) C/N ratio . . . . . . 46 Anabaena flos-aquae @ 10 uE/mz/sec, May 26 experiment: time course measurements of (a) cell concentration, (b) carbon dioxide, (c) heterocysts, (d) positively buoyant cells, (e) turgor pressure, and (f) C/N ratio . . . . . . 47 Anabaena flos-aquae @ 10 uE/mZ/sec, October 4 experiment: time course measurements of (a) cell concentration, (b) carbon dioxide, and (c) positively buoyant cells. 48 Anabaena flos-aquae @ 10 pE/mz/sec, May 12 experiment: time course measurements of (a) cell concentration, (b) carbon dioxide, (c) heterocysts, (d) positively buoyant cells, (e) turgor pressure, and (f) C/N ratio . . . . . . 49 Anabaena flos-aquae @ 20 uE/mz/sec, February 12 experiment: time course measurements of (a) cell concentration, (b) carbon dioxide, (c) heterocysts, (d) positively buoyant cells, (e) nitrogen fixation, and (f) C/N ratio . . . . . S8 Anabaena flos-aquae @ 20 uE/mz/sec, October 4 experiment: time course measurements of (a) cell concentration, (b) carbon dioxide, and (c) positively buoyant cells . . . . . . . . . 59 Anabaena flos-aquae @ 50 uE/mz/sec, February 12 experiment: time course measurements of (a) cell concentration, (b) carbon dioxide, (c) heterocysts, (d) positively buoyant cells, (e) nitrogen fixation, and (f) C/N ratio . . . . . 65 vii FIGURE 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. Page Sample relationship between specific growth rate and carbon dioxide concentration, and Michaelis-Menton kinetic constants . . . . . . 72 Buoyancy niche of Anabaena flos-aquae relative to light intensity and carbon dioxide concen- tration (a) grown without inorganic nitrogen and (b) grown with NH4-N . . . . . . . . . . . 76 Relationship between the percent positively buoyant cells, light intensity, and carbon dioxide concentration for Anabaena flos-aquae (a) grown without inorganic nitrogen and (b) grown with NH4-N . . . . . . . . . . . . . . . 79 Relationship between the specific algal growth rate, light intensity, and carbon dioxide con- centration for Anabaena flos-aquae (a) grown without inorganic nitrogen and (b) growth with NH4-N . . . . . . . . . . . . . . . . . . . . . 81 Relationship between the specific growth rate of positively buoyant algae, light intensity, and carbon dioxide concentration for Anabaena flos-aquae (a) grown without inorganic nitrogen and (b) grown with NH4-N . . . . . . . . . . . 83 Cell turgor pressure as a function of light intensity for Anabaena flos-aquae (i 95% confidence intervals) . . . . . . . . . . . . . 87 Percent positively buoyant cells as a function of the cellular C/N ratio for Anabaena flos- a ua at light intensities of 2, S, and 10 p3 m /sec . . . . . . . . . . . . . . . . . . . 89 Percent positively buoyant cells as a function of the cellular C/N ratio for Anabaena flos- a ua at light intensities of 20 and 50 uE m /sec . . . . . . . . . . . . . . . . . . 92 Buoyancy regulation model which illustrates interactions of light, carbon dioxide, and nitrogen with various cellular processes, materials, and regulators responsible for controlling algal buoyancy . . . . . . . . . . 94 Buoyancy niche of (a) Anabaenopsis Elenkinii and (b) Anabaena flos-aquae relative to light intensity and carbon dioxide concentration . . 100 viii FIGURE Page 27. Seasonal means of the density of non-blue- green and blue-green phytoplankton in Ponds 2 and 3 with sparse cladoceran zooplankton, and Ponds l and 4 with abundant cladoceran zooplankton O O O I O O O O O I O I O O O O O O 108 ix INTRODUCTION One of the most undesirable symptoms of lake eutrophication is the common shift in phytoplankton dominance from diatoms and green algae to nuisance blue-green algal blooms. While diatoms and green algae generally are passed readily up the food chain supporting fish production, blue- green algae commonly are not grazed by aquatic herbivores (Lund, 1965: Porter, 1973, 1977). Instead, the primary fate of blue-green algal biomass is decomposition by bacteria which may result in dissolved oxygen depletions leading to fish kills. Another undesirable attribute of most of the common bloom forming blue-green algae is the presence of gas vacuoles. Gas vacuoles may impart positive buoyancy to the algae, allowing formation of nuisance surface scums. Such accumulations reduce water clarity, foul beaches, produce taste and odor problems, clog intake filters, and occasionally release potent toxins. While the appearance of blue-green algal blooms generally has been correlated with nutrient enrichment and lake eutro- phication, there remains considerable speculation about the exact mechanisms responsible for the shift to blue-green algal l 2 dominance. Many different theories have been advanced, but none at present are adequate for predicting or understanding the mechanisms responsible for the appearance of blue-green algal blooms. Examples include suggestions that blue-green algae may have superior growth rates compared to other algae at reduced nitrogen levels (F099, 1975; Horne gt $1., 1972), reduced carbon dioxide levels (King, 1970, 1972; Shapiro, 1973), and low oxygen concentrations (Padan, 1977; Brock, 1973). IOther factors which have been proposed as contributors to the success of blue-green algae include increased organic content of the water (Singh, 1955: Horne and F099, 1970), increased temperatures (Jackson, 1965), increased monovalent cations (Wetzel, 1975) , selective grazing by zooplankton (Lund, 1965; Porter, 1973, 1977), and nutrient limitation (Schelske and Stoermer, 1971, 1972). While many of these factors may be of importance, to date, no method is available which allows accurate prediction of the onset of blue-green algal blooms from measures of physical, chemical, and biological parameters. A growing body of research has appeared recently concerning buoyancy regulating capacities of many blue-green algae through alteration of their cellular gas vacuole content. Gas vacuoles consist of many hollow, gas filled cylinders termed gas vesicles which may impart positive cell buoyancy to the algae. Among the algae, gas vacuoles are found exclusively in certain blue-green algal species. All of the common planktonic bloom forming species possess gas vacuoles, and the ability of these algae to regulate cell buoyancy may 3 result in a competative advantage over other algae under certain conditions (Lund, 1959; Walsby, 1970; F099 gt 31., 1973). Almost a century ago Klebahn (1895) demonstrated that gas vacuoles can provide positive cell buoyancy to blue-green algae. Since then a number of researchers have reported vertical migration and stratification of blue-green algal populations in lakes (Lund, 1959; F099 33 31., 1973; Reynolds and Walsby, 1975) . However, the impetus for research on buoyancy regulation of these algae stems from pioneering work by A.E. Walsby and co—workers over the last decade (Walsby, 1968, 1971, 1973; Dinsdale'and‘Walsby, 1972; Grant and Walsby, 1977; Walsby and Booker, 1980). Walsby discovered that the common blue-green algae Anabaena flos-aquae showed decreased gas vacuole content and reduced buoyancy when grown at elevated light intensities. At low light intensities, gas vacuole content and cell buoyancy in this alga were markedly increased. Walsby proposed that the loss of buoyancy at high light intensities may result from elevated cell turgor pressures causing collapse of pressure sensitive gas vacuoles. Cell turgor pressures are elevated under high light intensities due to rapid photosynthetic rates resulting in the accumulation of low molecular weight organic compounds, and potassium ions (Dinsdale and Walsby, 1972; Grant and Walsby, 1977; Allison and Walsby, 1981). Accumulation of these osmotically active substances results in an influx of water into the cells causing cell turgor pressures to increase. 4 More recently it has been found that changes in nutrient availability also may result in alteration of blue-green algal buoyancy. Reduced carbon dioxide levels appear to increase cell buoyancy in Anabaena flos-aquae and Oscillatoria spp. (Booker and Walsby, 1979; Paerl and Ustach, 1982; Klemer 33 31., 1982; Van Rijn and Shilo, 1983). Reduced inorganic nitrogen levels appear to reduce the buoyancy of Oscillatoria spp. and buoyancy may be increased with the addition of ammonia- nitrogen (Walsby and Klemer, 1974; Klemer 33 31., 1982; Van Rijn and Shilo, 1983). The availability of nitrogen may be important in regulating the rate of synthesis of gas vacuoles*which in turn affects cell buoyancy. Gas vacuoles are composed almost exclusively of protein (Jones and Jost, 1971, 1972;‘Walsby and Buckland, 1969) and algae grown under nitrogen limited 'conditions often show decreased cell protein levels (F099, 1959, 1975; Holm-Hanson 3t 3;. , 1959; Konopka and Schnur, 1981; Morris, 1981). Therefore, when the availability of nitrogen is reduced resulting in reduced protein synthesis, synthesis of protein rich gas vacuoles may be reduced as well (Klemer 33 33., 1982). It appears that the availability of light, carbon dioxide, and inorganic nitrogen may all be important in buoyancy regulation of blue-green algae. All three of these parameters tend to become reduced as a result of phosphorus enrichment of lakes (King, 1972, 1979, 1980). Light and carbon dioxide often are reduced due to increased algal photosynthesis and 5 larger algal standing crops associated with phosphorus enrichment. PhosPhorus enrichment also tends to accelerate the loss of inorganic nitrogen from lakes through plant uptake, ammonia volatilization, and denitrification (King, 1980) . Reduced light and carbon dioxide appear to increase the buoyancy of Anabaena and Oscillatoria thereby increasing the bloom— forming potential of these algae (Booker and Walsby, 1979; Walsby and Booker, 1980; Walsby and Klemer, 1974; Paerl and Ustach, 1982; Van Rijn and Shilo, 1983). However, reduced inorganic nitrogen availability reduces the buoyancy of Oscillatoria (Klemer 33.33., 1982; Van Rijn and Shilo, 1983). Depletion of inorganic nitrogen in phosphorus enriched lakes is believed widely to result in dominance of the phytoplankton by nitrogen-fixing blue-green algal blooms since the growth of other algae is limited under these conditions. However, I have found no reports in the literature relating nitrogen availability to buoyancy regulation of nitrogen- fixing blue-green algae. This information may be important since nitrogen fixation in blue-green algae is primarily driven by light energy, and light intensities often are reduced severely in eutrophic lakes. As such, the supply of cellular nitrogen to nitrogen-fixing blue-greens also may be reduced in eutrophic lakes, and this may affect their buoyuancy in a manner similar to that previously reported for the non-nitrogen fixing alga Oscillatoria. Much of the recent research concerning the formation of blue-green algal blooms in lakes has focused on alteration of 6 cell buoyancy in response to changing environmental conditions. However, the physiological growth rates of blue-green algae under these same environmental conditions also may be critical in determining whether or not a blue-green bloom will form. The availability of light, carbon dioxide, and inorganic nitrogen have all been reported to affect algal growth rates (Novak and King, 1973: Ward and Wetzel, 1980). The role of light, carbon dioxide, and nitrogen have been shown to be important individually in the regulation of the growth rate and buoyancy of a variety of algae. However, no model exists combining the limits imposed by all three of these environmental parameters concurrently on both the growth rate and buoyancy of blue-green algae. This research was designed to evaluate the interacting roles of light, carbon dioxide, and nitrogen in regulation of buoyancy and growth of the nitrogen fixing blue-green alga, Anabaena flos-aquae, under phosphorus-rich conditions. MATERIALS AND METHODS EXPERIMENTAL APPROACH The primary objective of this research was to study the three-way interaction between light, carbon dioxide, and inorganic nitrogen in the regulation of buoyancy and growth of nitrogen fixing blue-green algae. The bulk of the experimental results presented here come from laboratory growth experiments using the algae, Anabaena flos-aquae. .Additional results are presented for Anabaenopsis Elenkinii. The algae were grown in batch culture microcosms and the three environmental parameters of interest were varied as follows. Algal microcosms were set up at various light intensities. Within each microcosm, the carbon dioxide concentration declined from the beginning to the end of the experiment due to photosynthetic uptake of carbon dioxide. Companion microcosms were established at each light intensity with one microcosm containing no inorganic nitrogen so that the sole algal nitrogen source was from nitrogen fixation, and the other microcosm supplied with an abundance of ammonia- nitrogen. 8 The primary parameters monitored were the response of algal buoyancy and growth to the various conditions of light, carbon dioxide, and nitrogen. A number of additional parameters were measured in an attempt to elucidate causal mechanisms for the observed responses of algal growth and buoyancy. These included measures of the cellular carbon/nitrogen ratio, the rate of nitrogen fixation, heterocyst content, and cell turgor pressure. APPARATUS AND PROCEDURES Algal Microcosms Growth experiments were conducted in batch culture microcosms consisting of 600 m1 Corning polystyrene tissue culture flasks (Figure l) . These flat walled flasks permitted distortion-free light penetration and their narrow width (3.5 cm) minimized light attenuation from one side of the flask to the~other. Each flask was sealed with a rubber stopper fitted with two 15 gauge syringe needles. One syringe needle, through which samples were withdrawn, was stoppered. The other was attached to a latex tube stoppered with a 0.22 pm inline Millipore filter disc. The latter served as a vent tube which prevented pressure increases inside the microcosms due to photosynthetic oxygen production, while allowing minimal exchange of carbon dioxide with the atmosphere. Microcosms were gently mixed several times each day by inverting the flasks several times while clamping the vent tube. 1 OO LI / side View front view Figure 1. Experimental microcosm and lighting arrangement. 10 Microcosms were arranged on racks and continuously illuminated from one side with two 48 inch, 40 watt Sylvania “Gro—Lux" fluorescent lights (Figure IJ. Microcosms were placed approximately 20 cm away from the lights, and light intensities were adjusted by covering the lights with black nylon cloth mesh and/or fine mesh wire screen painted flat black to prevent alteration of the light spectrum (Luebbers and Parikh, 1966). Light intensity was measured on the front side of the microcosms with a LI-COR light meter (model LI-510 integrator and model LI-1928 sensor) which measures ”photosynthetically active radiation" (PAR), defined as those wavelengths between 400 and 700 nm. Light intensities are reported in units of microeinsteins per square meter per second (pE/mz/sec) and may be converted to other commonly reported units using Equation 1. These conversions are for "Gro-Lux" lights only and may be different for other light sources. 1.00 uE/mZ/sec = 4.00 foot candles = 43.04 lux (1) Nutrient Media The algal nutrient media used in this research is shown in Appendix 1, and was modified from Kevern and Ball (1965). This base medium is dominated by monovalent cations which reduces the potential for carbonate precipitation at high pH values. The Kevern and Ball media was modified to provide an excess of all nutrients required for maximal growth of blue- green algae with the exception of carbon dioxide and inorganic 11 nitrogen. Modifications included increasing the phosphorus concentration, addition of trace vitamins (Guillard and Lorenzen, 1972), and substitution of non-nitrogen containing micronutrients for those that originally contained nitrogen. Algae were grown in microcosms under conditions of no inorganic nitrogen or abundant inorganic nitrogen supplied as NH4C1. Initial nitrogen concentrations in microcosms supplied with inorganic nitrogen were normally 0.5 mg NH4-N/1. In some early experiments, higher NH4-N concentrations were used, up to 10 mg NH4-N/1; however, at high pH levels, this created potentially toxic conditions for the algae (Fogg and'Than-Tun, 1958: Stewart, 1964). As a result of using lower initial NH4- N levels, periodic additions were required as NH4-N levels declined during the experiments. Attempts were made to maintain the nitrogen concentration between 0.1 and 0.5 mg NH4-N/1. At high light intensities, NH4—N concentrations declined rapidly and several NH4-N additions were required each day, particularly during exponential growth. Sampligg Procedure Samples were withdrawn aseptically from the microcosms by inverting the flasks, removing the syringe needle plug, and gently bubbling nitrogen gas in through the vent tube to replace the sample volume as it was withdrawn. Extreme care was exercised during sample withdrawal and handling to minimize pressure changes capable of accidentally collapsing pressure sensitive gas vacuoles. 12 Algal Cultures The Anabaena flos-aquae culture used in this study was strain 1403/13f, obtained from the Culture Centre of Algae and Protozoa in Cambridge, England. This is the same algal strain used in research by A.E. Walsby (Walsby, 1971; Dinsdale and Walsby, 1972; Walsby and Booker, 1980). The Anabaenopsis Elenkinii culture was kindly provided by C.P. Wolk and was originally isolated from the Water Quality Management Facility ponds at Michigan State University. Both algal cultures contained bacteria when originally obtained. Ther cultures remained unialgal throughout the experiments, and bacteria were reduced in seed cultures through repeated transfers into media containing no inorganic nitrogen. Prior to inoculating microcosms, seed cultures generally were grown up to a density of approximately 2x106 cells/ml in aerated cultures without inorganic nitrogen at an incident light intensity of 25 uE/mz/sec. Due to the high density of algal cells, the average light intensity encountered by these cells was much lower than 25 uE/mz/sec. Algae grown in this manner were normally positively buoyant prior to inoculation of experiments. Non—buoyant seed algae were obtained from aerated seed cultures grown at 50 uE/mz/sec, without inorganic nitrogen. 13 ANALYTICAL METHODS Temperature A standard laboratory thermometer was inserted in a reference microcosm and the temperature of the media was recorded immediately prior to each sampling period. Alkalinity Titratable alkalinity was measured at the beginning of each experiment using a potentiometric procedure. Fifty ml samples were titrated with 0.02N H2804 and titratable alkalinity was calculated using the equivalence point taken to be the inflection point of the titration curve (Dick, 1973) . All alkalinity values reported represent the mean of two titrations. 93 Measurements of pH were made with an Orion model 901 digital microprocessor ionalyser equipped with an Orion Ross combination pH electrode. This unit was calibrated before each use with standard buffer solutions. Measurements of pH were made immediately after sample collection. Samples were placed in a temperature controlled water bath and mixed with a magnetic stirrer during pH measurement. Ammonia-Nitrogen Ammonia-nitrogen (NH4-N) was measured using the phenate method described by Solorzano (1969). The method is based on the indophenol-blue reaction between ammonia, phenol, and l4 hypochlorite. Final color development was measured with a Bausch and Lomb Spectronic 21 spectrophotometer. Steps were taken to remove algal filaments prior to the addition of chemical reagents since at high density they may interfere with color development measurements. Preliminary studies on samples filtered through 0.45 pm Millipore filters under low vacuum showed significant release of NH4-N from the algal cells into the filtrate. Consequently, the algae were removed by centrifugation. Carbon/Nitrogen Ratio (CZN RATIO) The total carbon to total nitrogen ratio (on a weight/ weight basis) in the algal cells was measured with a Perkin- Elmer model 24 elemental analyser following methods described by Sharp (1974). Algal cells were concentrated onto 24 um! diameter Whatman GF/C filters and dried in a vacuum dessicator. Filters were then inserted into small aluminum foil tubes and combusted in the elemental analyser. Nitrogen Fixation Nitrogen fixation. was estimated using the acetylene reduction technique similar to the procedure described by Flett 35 3;. (1976). Five ml sample aliquots were placed in 17 m1 Pyrex culture tubes and tightly stoppered with rubber serum caps. 'Tubes were injected with 1.7’m1 acetylene gas generated from calcium carbide placed in water. Tubes were then shaken and placed adjacent to the original microcosms. Tubes were incubated for 3 to 24 hours depending on the culture density 15 and light intensity, such that sufficient ethylene was produced to yield accurate determinations by gas chromatography. At the end of each incubation period, assay tubes were fixed with two drops of 10 percent trichloroacetic acid. Ethylene production was measured with a Varian Series 1200 flame ionization gas chromatograph equipped with a two meter stainless steel column packed with Porapak R. A standard curve was prepared and nitrogen fixation rates were calculated assuming 3.0 nmoles ethylene produced was equivalent to one nmole nitrogen fixed. Cell Density Cell densities, expressed as cells/ml, were measured using a membrane filtration technique similar to that described by McNabb (1960). Measured sample volumes were filtered through 0.45 pm Millipore filters. Filters were air dried and mounted on glass microscope slides using several drops of Cargille type A immersion oil which cleared the filters. Total cell numbers and number of heterocysts were enumerated in 40 randomly chosen microscope fields at 400x using a Leitz phase contrast microscope. Mean number of cells/ml and the percent of total cells which were heterocysts were calculated. Although cell densities are expressed as cells/m1, it should be noted that both Anabaena flos-aquae and Anabaenopsis Elenkinii grow in multicelled filaments, not as single cells. 16 Buoyancy Cell buoyancy was measured using methods described by Walsby and Booker (1980) . One ml samples were gently pipetted into Sedgwick Rafter counting cells and placed adjacent to the original microcosms for two hours. This allowed the algae to equilibrate their vertical position in the one millimeter deep chambers. The Sedgwick Rafter cells were then carefully transported to a microscope stage, where the number of floating, neutral, and negatively buoyant cells were enumerated by focusing down through the counting chamber. Floating, or positively buoyant, cells were taken to be those cells at the top of the chamber resting against the coverslip. Sinking, or negatively buoyant, cells were counted as those resting on the bottom of the chamber. Any cells not touching the top or bottom of the chamber were recorded as neutrally buoyant. At least 25 microscope fields at 100x were enumerated for each sample, from which the percentages of positively buoyant, neutral, and negatively buoyant cells were calculated. In preliminary experiments, a felt tipped marking pen was used to place a small mark on the bottom of the Sedgwick Rafter cell and the coverslip to facilitate visual establishment of the top and bottom of the chamber as viewed under the microscope. This procedure was quickly abandoned when it was discovered that the marking pen ink could cause gas vacuoles to collapse. Similarly, it was found that epoxy cement, which was used occasionally to reconstruct broken Sedgwick Rafter cells, could 17 also cause gas vacuoles to collapse unless it was allowed to completely dry for several weeks. Turgor Pressure Cell turgor pressure was measured using the procedure and apparatus developed by Walsby (1971, 1973) . In this procedure, turgor pressure is determined indirectly by measuring the difference in pressure required to collapse gas vacuoles in algae suspended in media verses algae suspended in a hypertonic sucrose solution. Collapse of gas vacuoles was measured indirectly using a Monitek model 21 nephelometer equipped with a glass cuvette which could be pressurized up to 150 pounds per square inch (psi) The pressure cuvette was constructed by annealing one end of a nine mm diameter Fisher Porter tube and securely clamping the other end to another nine mm Fisher Porter tube which was connected to a compressed nitrogen gas cylinder. The cuvette could be incrementally pressurized while in place in the nephelometer using a two stage high pressure regulator attached to the nitrogen tank. The procedure involved placing an algal sample suspended in media in the pmessure cuvette and recording changes in optical scattering (in units of NTU's) as pressure was applied in 5 psi increments up to 150 psi. Gas vacuoles are highly light refractory, rigid structures which collapse as a critical pressure is reached. As gas vacuoles collapse, optical scattering declines as measured with a nephelometer. The gas vacuoles do not all collapse at the same critical pressure; 18 and since the gas vacuole concentration is proportional to light scattered, the relative percentage of gas vacuoles collapsed can be calculated at each pressure applied using Equation 2. collapsed = x(100) (2) % gas vacuoles NTUinitial ' NTUx at x psi NTUinitial ' NTUfinal Where: NTUinitial = optical scattering of the algae with intact gas vacuoles NTUx = optical scattering of the algae pressurized to x psi NTUfinal = optical scattering of the algae with all gas vacuoles collapsed A typical pressure collapse curve for cells suspended in media is shown as the curve on the left in Figure 2. A second collapse curve was then made using a replicate algal sample to which sucrose was added yielding a hypertonic solution. This solution causes water to flow out of the cells by osmosis and results in elimination of the cell turgor pressure. With no cell turgor pressure, additional external pressure must be applied to yield collapse of gas vacuoles equivalent to those suspended in media. The resulting hypertonic pressure collapse curve is shown displaced to the right in Figure 2. The difference in pressure required to collapse 50 percent of the gas vacuoles in the two solutions is taken to be the original cell turgor pressure (Figure 2). Each curve represents the»mean of three separate pressure collapse measurements. In samples with low cell densities 19 0 IO- /rnedio / sucrose 33 zo- o 30- DJ 2 4o. 4 3‘ so- JE'E‘E-£LR_3§.“L°. C) a) . u so a‘ a 70- 2 > so- 00 < (9 END‘ Ioo . . . . r - o 25 so 75 IOO |25 I50 PRESSURE APPLIED (psi) Figure 2. Typical pressure collapse curve used in determining cell turgor pressure. 20 (>4x105 cells/ml), more precise pressure collapse curves were obtained if the algae were first concentrated. 'The algae were gently concentrated by partial filtration through 5 (pm Millipore filters. Examples of pressure collapse curves showing improved curve generation in more concentrated samples from the same culture are shown in Figure 3. Cell turgor pressures are reported in units of psi which can be converted to other commonly reported units using Equation 3. 1 psi = 6.8947 KN/m2 = 0.0689 bar ' (3) Although the procedure for measuring turgor pressure sounds relatively straightforward, numerous difficulties were encountered in carrying out the procedure. Many of these difficulties originated in obtaining suitable pressure collapse curves. Ideally, the curves as shown in Figure 2 should be parallel and show the maximum possible separation between the two curves. However, the shape and location of the curves could be changed by altering the sucrose concentration used, and by altering the length of time the algae were left in the sucrose solution before measurements were made. Preliminary experiments with Anabaenopsis Elenkinii suggested an optimal sucrose concentration of 0.4M with algae left in hypertonic solution for 5-15 minutes, after which measurements were made (Figure 4). This correlated well with conditions used by Walsby (1971, 1973) . However, a more recent 21 o 8.8 xlOscells/ml 0 4.4 xlOscells/ml A 0.88xl05cells/ml 07.000.000.0000086 A A o ‘2 o 9. 20. . A O O A DJ 0) CL S 404 . .l o o 8 CD A A O a) B so— 0 g; o A g 8 > o (I) 80- 0 O < o (.9 Q o o . 8 P o '00 r 1‘ A L1 ‘ ' AL. 0 20 4O 60 80 I00 PRESSURE APPLIED (psi) Figure 3. Pressure collapse curves of various algal culture densities. \ K‘\\ 0.2” \ \ 0.3M \ \ DAM" 20. \ \\ \ a \ \\ 05M E: \ \\ \ 0.6M w \ \\ \ ' a \\ \ \ ‘ 0.7M s \\ \ \ -’ \ (‘5‘; so. 0.0M \ \ \ g o.IM—— I \ \ g; 0.8hl §§ \\\\ g 30 - \\§\ 2’: \\\\ (9 \\.\\ Ioo , . . . Q . o 20 40 so so Ioo PRESSURE APPLIED (psi) Figure 4. Pressure collapse curves using various sucrose concentrations. 23 paper by Allison and Walsby (1981) recommended 0.7M sucrose for 1.7 minutes. Subsequent experimentation under these conditions did give superior results; therefore, turgor measurements made after May 25, 1983, were conducted under these conditions. Carbon Dioxide Free carbon dioxide concentrations were calculated from pH, alkalinity, and temperature data based on equations from Harvey (1957) and Park (1969), as shown in Equation 4. H2 C02 = a (4) K1(H + 2K2) Where: C02 = free carbon dioxide concentration, moles/l a = carbonate bicarbonate alkalinity, corrected for hydroxyl ion concentration, eq/l. H = hydrogen ion concentration, eq/l K1 = first dissociation constant for carbonic acid K2 = second dissociation constant for carbonic acid Growth Rate Calculations Algal biomass accural was assumed to follow the first order growth equation over the time interval between any two measurements (Equation 5). Mt = M0 ent (5) Where: Mt = mass at time (t) Mo = mass at time (0) u = specific growth rate t = time 24 The specific growth rate ()1) can be calculated for various time intervals using Equation 6. =1th-lnMo t (6) Specific growth rates were calculated in two different ways: substituting cell densities (cells/ml) for the mass terms in Equation 6, and substituting total organic carbon for the mass terms in Equation 6. Total organic carbon consists of organic carbon in the initial seed algae plus any carbon fixed by algae in the microcosm. The initial carbon in the algal seed was measured with a Perkin Elmer elemental analyser as described in the method section for C/N ratios. Organic carbon fixed by the algae was calculated from estimates. of changes in total inorganic carbon in the media which were calculated using pH, temperature, and alkalinity data based on equations derived by Harvey (1957), Park (1969), and King and Novak (1974). Total inorganic carbon ( ECOZ) was calculated using Equation 7 with terms defined in Equation 4. H2 2c02 = a (K1)+ H + K; a . 2x2 (7) As algae photosynthesize and take up carbon dioxide in closed microcosms, the pH rises and total inorganic carbon (JECOZI decreases as calculated using Equation 7. The amount 25 of carbon fixed by algal photosynthesis can.be«calcu1ated using Equation 8. c:fixed = AECOZ = ECOZ initial " 2(:02 final I8) The specific growth rate»ofIalgae can generally be related to the concentration of the limiting substrate by the Michaelis Menton or Monod equation shown below. ”*= “max(:—£L—') (9) KS 5 Where: u specific growth rate (time‘l) + “max = maximum specific growth rate (time-1) S = limiting substrate concentration Ks = limiting substrate concentration when u = 8 umax This equation can be expanded as shown below to include the threshold concentration of S, termed Sq, which represents the substrate level below which no net growth occurs (King, 1980). n = ”max (8 - Sq) (10) (KS - Sq) + (S - Sq) The primary limiting substrate in these experiments was carbon dioxide. The carbon dioxide threshold concentration (Sq) was taken to be the lowest carbon dioxide concentration measured during an experiment. Values for the kinetic constants Ks and ”max were obtained from linear transformation plots of (S-Sq)/p verses (S-Sq) (Dowd and Riggs, 1965). Estimates of 26 u from both methods (cell densities and total organic carbon) were combined to generate single estimates of KS and “max- RESULTS Preliminary Experiments Buoyancy Regulation Preliminary experiments were conducted to evaluate the buoyancy response of Anabaenopsis Elenkinii and Anabaena flos- m grown at various light intensities. These nitrogen fixing blue-green algae were grown under conditions similar to those used by Walsby and Booker (1980). The growth media contained no inorganic nitrogen, and carbon dioxide concentrations were maintained near equilibrium levels by aeration when necessary. Initial experiments conducted at elevated light intensities (50 uE/mz/sec) resulted in a non- buoyant condition for both algal types. Similar results were reported for Anabaena flos-aguae by Walsby and Booker (1980). Walsby and co—workers suggest the loss of buoyancy at high light intensities results from increased cell turgor pressures and collapse of gas vacuoles (Walsby, 1971; Dinsdale and Walsby, 1972; Grant.and Walsby, 1977: Walsby and Booker, 1980; Allison and Walsby, 1981). 27 28 At.low light intensities‘Walsby‘s model predicts Anabaena flos—aquae should be in a buoyant condition; however, the buoyancy response observed in my experiments was different than expected (Figure Sa). AnabaenOpsis Elenkinii was grown at 5 uE/mZ/sec with an initial algal seed containing 81 percent positively buoyant cells. The algae remained highly buoyant for three days, and then cell buoyancy gradually decreased. By day 8 less than one percent of the cells were positively buoyant. The algae showed positive growth and looked healthy throughout this period; however, cell buoyancy had clearly been lost. In order to test the hypothesis that a reduced cellular nitrogen supply may explain the loss of buoyancy of Anabaenopsis Elenkinii at low light intensity due to reduced nitrogen fixation rates, the culture was split into two microcosms on day nine. Ammonia-nitrogen was added to one of the microcosms yielding a concentration of 10 mg NH4-N/l. Nitrogen was not added to the other microcosm. As seen in Figure 5a, without nitrogen, the algae remained non-buoyant. However; within three days after addition of NH4-N, algae in the other microcosm showed a significant increase in buoyancy. By day 17, 95 percent of the cells in this culture were positively buoyant. A similar experiment was conducted with Anabaena flos- aguae (Figure 5b). The algae were grown without inorganic nitrogen at 10 uE/mz/sec, and by day two less than one percent of the cells were positively buoyant. Following the addition 29 ICN) (a) (”HIS (96) Positively Buoyant o 54' '63 I0I2I4I'6Iezozz t-t wan: NH4-N H Without NH4-N K30 8C): 6C>4 (:CHS (95) 4o- 20- Posiiively Buoyant DAYS Figure 5. Buoyancy response of (a) Anabaenopsis Elenkinii grown at 5 pE/mzsec and (b) Anabaena flos-3guae grown at 10 uE/mzsec. The cultures originally contained no inorganic nitrogen; however, the cultures were split into two replicates and NH4-N was added to one replicate as indicated by the arrow. 30 of NH4-N, algal buoyancy increased significantly, while algae with no nitrogen addition remained non-buoyant. It is clear from these preliminary experiments that the availability of nitrogen may be important in determining the buoyancy of Anabaenopsis Elenkinii and Anabaena flos-aquae. Although similar findings have been reported for the non- nitrogen fixer Oscillatoria (Walsby and Klemer, 1974; Klemer _3 _l., 1982; Van Rijn and Shilo, 1983), this appears to be the first study reporting the potential importance of the availability of nitrogen in buoyancy regulation of nitrogen fixing blue-green algae. Gas Vacuole Content and Buoyancy Although the density of gas vacuoles and cell buoyancy varied considerably depending on the experimental conditions, at least some gas vacuoles were visible by light microscopy in almost all algal cells throughout these experiments. In some cases, however, little or no visual difference in gas vacuole content could be detected between positively and negatively' buoyant cells. Therefore» an experiment ‘was conducted to determine how changes in gas vacuole content may affect cell buoyancy. A complete pressure collapse curve was generated for an Anabaena flos-aquae culture containing 89 percent positively buoyant cells (Figure 6a) . Various percentages of gas vacuoles were then collapsed in individual samples in 5 and 10 percent increments by pressurizing the samples to an appropriate degree 31 ... o as B 20. (a) a) o. S _.I 40~ o o 8 .I 60" CD 8 § 80- m <1 <9 IOO , i O 20 4O 60 80 IOO PRESSURE APPLIED (psi) mm :3 EL 80- (b) >- a) d j z» u; 6(Tj ;: o 8 s (L :5 '4CF C) :3 In 20‘ O . O 20 4O '60 80 IOO GAS VACUOLES COLLAPSED (°/o) Figure 6. (a) Pressure collapse curve from an Anabaena flos-aquae culture containing 89% positively buoyant cells, and(b) relationship between buoyancy of the same culture used in 63, and the percent of original gas vacuoles collapsed through application of pressures determined from 6a. 32 as determined from Figure 6a. The buoyancy of each of these samples was then measured (Figure 6b). It is apparent that relatively small changes in gas vacuole content may cause large changes in buoyancy. By collapsing only 15 percent of the gas vacuoles, the percent of buoyant cells declined from 89 percent to 19 percent. When 30 percent of the original gas vacuoles were collapsed, cell buoyancy was lost completely. ANABAENA FLOS-AQUAE MICROCOSM EXPERIMENTS Following preliminary studies, a set of experiments was designed to evaluate the three-way interaction between light, carbon dioxide, and nitrogen in regulation of growth and buoyancy of Anabaena flos-aquae. The basic experimental design was described earlier in the Materials and Methods Section. Two mechanisms were hypothesized to be important in regulating cell buoyancy. The first involves alteration of cell turgor pressure resulting in potential collapse of gas vacuoles (Walsby, 1971; Dinsdale and Walsby, 1972; Grant and Walsby, 197: Allison and Walsby, 1981). The second involves alteration of the rate of gas vacuole synthesis (Smith and Peat, 1967; Klemer 35 31., 1982; Konopka, 1983). In order to evaluate the importanceiof the first proposed regulating mechanism, cell turgor pressures were monitored under various environmental conditions. The second mechanism was evaluated indirectly since the actual rate of gas vacuole synthesis was not measured. Instead, cellular C/N ratios, 33 nitrogen fixation rates, and the abundance of heterocysts were monitored under various conditions. All of these latter parameters are related in some way to the cellular nitrogen supply which appears to be important in regulation of the synthesis of gas vacuoles. Results from microcosm growth experiments for Anabaena flos-aquae are presented for each light intensity in Figures 7 to 16. Growth experiments were conducted with Anabaena flos- aguae at light intensities of 2, 5, 10, 20, and 50 uE/mz/sec. In most experiments two microcosms were set up at each light intensity. One of these contained no NH4-N, the other was maintained with nitrogen concentrations generally between 0.1 and 0.5 mg NH4-N/l. 2 uE/mz/sec EXPERIMENTS Cell Density and Carbon Dioxide Growth of Anabaena flos-aquae»at 2,uE/m2/sec was marginal (Figure 7a). A¢.this low light intensity, without NH4-N, cell densities increased slightly from 1.69 x 105 to 2.11 x 103 cells/ml by the fifth day and then declined. Growth was slightly improved with NH4-N with a maximum density of 2.57 x 105 cells/m1 reached on day eight. Carbon dioxide concen- trations declined initially in both microcosms (Figure 7b); however, levels remained above saturation throughout the 20 day experiments due to respiration and low rates of photo- synthetic carbon dioxide uptake. CELLS/ml I: I05 o—oWiIhout NH -N o I l ‘ l 0 8 I2. IS 120 DAYS A I5- ES to "" IO- 93 § In 5 g.- in :t o I I I 1 IO 8 I2 K5 2C) 70— DAYS 7565-: 3 o: 3603 a: : I'55- so I I T I C) 8 I2 I63 2C) DAYS “3 9 l5« x 3 I0- 8 2 g 01 0 4 8 34 A"--'¢With NH -N 3 I0- 3 LD- N 8 01- b 0.0. r l I I ' o 4 8 I2 l6 20 ICC? INDS.BNKNflUWT CEHJJB «X» .b 9 $ ENNNS 20-» CI 0, u x u (3 4» 8 ”2 I6 2C) 81 (DANS 9. 7‘ 3... g 6- z \ U I 4' I T U r j ‘0 4' 8 I2 “5 20 NYS (2 I6 20 DAYS Figure 7. Anabaena flos-aquae @ 2 pE/mZ/sec, June 1 experiment: time course measurements of (a) cell concentration, (b) carbon dioxide, (c) heterocysts, (d) positively buoyant cells, (e) turgor pressure, (f) algal C/N ratio, and (g) nitrogen fixation. 35 Buoyancy The initial seed culture contained 80.8 percent positively buoyant cells. Without NH4-N, the algae remained highly buoyant through the fifth day and then began to lose buoyancy (Figure 7d). By day 11, less than three percent of the algae were positively buoyant, and the algae remained essentially non- buoyant for the remainder of the experiment. With NH4-N, the algae remained positively buoyant throughout the experiment, although in the last ten days cell buoyancy decreased slightly. Percent Heterocystsi Nitrogen Fixation, and 334:3 The percentage of heterocysts increased from 5.2 to about 10 percent in the microcosm without NH4-N, while decreasing to about two percent with NH4-N (Figure 7c). Nitrogen fixation rates were quite low at this low light intensity in the microcosm without NH4-N; while with NH4-N, nitrogen fixation was essentially not detectable. 'The concentration of NH4-N remained between 0.5 and 0.25 mg N/l in the culture with inorganic nitrogen. A single addition of NH4-N was made on day 13. Turgor Pressure! C/N Ratioy and Analysis Algal growth was slightly better with NH4-N than without nitrogen. However, the major difference between the two microcosms was that with NH4-N the algae were buoyant, while algae without NH4-N became non-buoyant after a period of time. The availability of the three external environmental variables, light, carbon dioxide, and nitrogen remained relatively 36 constant throughout the three week experiments due to minimal growth of algal biomass at this low light intensity. Therefore, any buoyancy changes observed should be attributed to equilibration to environmental conditions imposed when the microcosms were first initiated. Cell turgor pressure and cellular C/N ratio were the two major internal cell indicies studied as potentially important in regulation of gas vacuole Content and cell buoyancy; Cell turgor pressures remained low and declined in both microcosms throughout the experiments (Figure 7). In their studies, Walsby and co-workers reported that Anabaena flos-aquae grown at reduced light intensities had low turgor pressures which did not result in collapse of gas vacuoles (Walsby, 1971; Dinsdale and Walsby, 1972; Grant and Walsby, 1977; Walsby and Booker, 1980). Low turgor pressure which declined would not appear to explain the loss of cell buoyancy in the culture without NH4-N (Figure 7). Therefore, some other factor must be involved. Cell buoyancy at 2 pE/mz/sec appeared to be more closely related to the cellular C/N ratio (Figure 7). The C/N ratio of algae without NH4-N increased from an initial value of 4.72 in the buoyant seed culture to a value of 6.93 on day 11. As the C/N ratio approached a value of six on day eight, cell buoyancy had begun to decline,and by day 11 the algae were essentially all non—buoyant. The algal C/N ratio in the culture supplied with abundant NH4-N remained at a value of about 5.0 throughout the experiment, much lower than in the microcosm 37 without NH4-N. With this low C/N ratio, the algae were buoyant throughout the experiment. Without NH4—N, it appears that the cellular supply of nitrogen derived from nitrogen fixation was reduced as reflected by an increasing C/N ration Nitrogen fixation rates were quite low at this low light intensity; and as the relative cellular nitrogen supply declined, it appears that gas vacuole synthesis was reduced and the existing gas vacuoles were diluted out by cell growth. Consequently, cell buoyancy declined and the algae eventually became totally non-buoyant. With an abundant supply of inorganic nitrogen as NH4-Nr nitrogen assimilation relative to carbon fixation and assimilation was high as suggested by the continued low cellular C/N ratio. ‘Under these conditions, the algae remained buoyant apparently due to ongoing active synthesis of gas vacuoles. 5 pE/mz/sec EXPERIMENTS Cell Density and Carbon Dioxide Anabaena flos-aquae showed increased growth at 5 uE/mZ/sec (Figure 8) compared to 2 pE/mZ/sec. Cell densities without NH4-N reached a maximum of 6.51 x 105 cells/ml on day 23. Similar initial growth was observed with NH4-N; however, cell densities later increased more rapidly, and peaked sooner, reaching a maximum of 8.00 x 105 cells/ml on day 20. After maximum cell densities were reached in both cultures, the senescent algae deteriorated quickly with filaments falling 38 5 I HWIII‘IOUI NH4-N A—AWiih NI'h-N 0’ 1 CELLS/mi I: I05 f DAYS INN! HE TE ROCYSTS PI.) Pos BUOYANT CELLS (7.) q I q 1 q q d O - .\ I2 is 20 24 28 DAYS 6 4'8 I2 DAYS 5 to O I» .p n: O C .A a) N O I ‘P “P 9 8 Is- . E 9 7 aim 35- § . r; s- S 2 . so-m 4IT 7 ITVITT‘I71 O48|2¢5202423O48l26202428 DAs DAYS Figure 8. Anabaena flos-aguae @ 5 pE/mZ/sec, February 12_experiment: time course measurements of (a) cell concentration, (b)carbon dioxide, (c) heterocysts, (d) positively buoyant cells, (e) nitrogen fixation, and (f) algal C/N ratio. 39 apart and cell densities declining rapidly. Carbon dioxide concentrations increased slightly on the first day in both experiments and then showed steady declines due to photosynthetic uptake, reaching minimum threshold values on dates corresponding to maximum cell densities. Buoyancy The initial seed culture contained 99.9 percent positively buoyant cells. After the first day without NH4-N, cell buoyancy declined steadily; and by the sixth day, less than four percent of the cells were positively buoyant. Cell buoyancy then remained low; however, on day 10 as carbon dioxide levels declined below 5 nmoles/1, cell buoyancy began to increase. By day 14, 20.0 percent of the cells were positively buoyant. Near the end of the experiment, cell buoyancy again declined as the senescent culture deteriorated rapidly. In the microcosm with abundant NH4-N, the algae remained highly buoyant through day 14 and then buoyancy declined as the carbon dioxide threshold was approached and the culture deteriorated. Percent Heterocysts, Nitrogen Fixation, and 334:3 The percent heterocysts increased to nearly 10 percent without NH4-N, while decreasing to 2.0 to 3.0 percent with NH4-N. Nitrogen fixation rates without combined nitrogen remained near 2.0 x 10"5 ng‘N fixed/cell/hr until near the end of the experiment when the rate declined. Nitrogen fixation 40 rates with'NH4-N’were markedly lower and showed a maximum rate of 0.67 x 105 ng N fixed/cell/hr on day 16. Repeated additions of NH4-N were necessary to maintain the nitrogen concentrations between 0.1 and 0.5 mg NH4-N/l in the microcosm with inorganic nitrogen. Daily NH4-N additions were required during the exponential growth phase. Turgor Pressurei C/N Ratio, and Analysis The primary external environmental variable which showed significant change during the course of each experiment was the carbon dioxide concentration. Changes in carbon dioxide concentration in these experiments were correlated with changes in cell buoyancy. The presence or absence of NH4-N also clearly affected cell buoyancy. Cell turgor pressures were not measured in these 5 uE/mz/sec experiments; however, Walsby and co—workers have shown that at this low light intensity, insufficient turgor pressures are developed in Anabaena flos-aguae to effect a collapse of gas vacuoles (Walsby, 1971; Dinsdale and Walsby, 1972; Grant and Walsby, 1977; Walsby and Booker, 1980). The cellular mechanism responsible for regulation of buoyancy at 5 uE/mZ/sec appeared to involve the cellular C/N ratio in a manner similar to that described at 2 uE/mZ/sec. At high carbon dioxide levels, without NH4-N, the C/N ratio increased rapidly from 4.4 on the first day to a maximum value slightly above 8.0 on the sixth day. During this period, the algae apparently were fixing and accumulating carbon at a 41 faster relative rate than they were fixing and accumulating nitrogen. As a result, the C/N ratio increased. When the C/N ratio approached a value of 6.0 and above, cell buoyancy decreased, presumably due to reduced synthesis of gas vacuoles relative to cell growth in response to the low cell nitrogen supply. As the carbon dioxide concentration declined below 5.0 nmoles/l, the fixation and accumulation of carbon decreased and the C/N ratio began to decrease. Cell buoyancy remained low until the C/N ratio declined below a value of 6.0 on about the eleventh day. Cell buoyancy then increased. This buoyancy increase is attributed to increased synthesis of gas vacuoles relative to cell growth in response to an increased relative cell nitrogen supply as reflected by the decreasing C/N ratio. The decline in cell buoyancy at the end of the experiment does not appear to be related to the cell C/N ratio which remained below 6.0 during this period. The algae were highly stressed at this time by extremely low carbon dioxide levels and pH values approaching 10. It appeared that as the senescent algae deteriorated, the gas vacuoles deteriorated as well and cell buoyancy decreased. In the microcosm with abundant NH4-N, the cellular C/N ratio remained below 6.0 throughout the experiment and the algae remained buoyant. With an abundant cell nitrogen reserve resulting from high NH4—N availability and a C/N ratio below 6.0, it appears that gas vacuole synthesis kept pace with cell growth and the algae remained buoyant. 42 10 uE/mz/sec EXPERIMENTS The only significant replication of experiments occurred with Anabaena flos-aquae grown at 10 uE/mz/sec. Five growth experiments were conducted over five different time periods between October, 1982, and June, 1983. It is felt that the 'variability observed in these repeated experiments can be used as a guide to interpreting results from the other light intensities. Results from the 10 uE/mZ/sec experiments are presented in Figures 9 to 13. Selected measurements from these experiments are presented along with measures of variability in Table 1. Table 1. Selected data from repeated Anabaena flos-aquae growth experiments at 10 nE/mz/SeC-,& means & standard deviations. Date NBA-N ggzakfioint Eaximum ”max Sq w are ensity -1 begin to cells/ml (hr ) (”M 002/1) float(nM/l) x105 2-12-83 without 2.11 7.81 0.00994 0.036 6-1-83 without 6.72 6.28 0.00943 0.348 5-26-83 without 5.67 9.26 0.01203 0.076 10-4-82 without 4.65 10.27 0.01190 0.054 5-12-83 without never 9.20 0.00636 0.162 x=8.56 x=0.00993 x=0.135 9:1.56 s=0.0023l s=0.129 x*=4.79 x*=8.40 x*=0.01083 x*=0.129 s*=1.97 s*=1.74 s*=0.00133 s*=0 147 2-12-83 with >32.36 9.70 0.01175 0.044 6-1—83 with >56.05 7.92 0.01223 0.233 5-26-83 with >24.57 11.65 0.01482 0.046 5-12-83 with never 9.20 0.00846 0.092 x-9.62 x=0.01182 x=0.104 s=1.55 s=0.00261 s=0.089 x*=9.76 x*-0.01293 x*=0.108 s*=1.87 s*=0.00165 s*-0.108 A calculated without 5-12—83 43 Throughout this research, most growth exeriments were initially seeded with buoyant algae. However, it was decided that some experiments should be repeated with non-buoyant as well as buoyant initial seed cultures. By using initial cultures of differing buoyancy, it could then be determined if prior algal buoyancy might influence the buoyancy response observed. Highly buoyant seed algae were used to initiate 10 uE/mz/sec experiments on February 12, 1983 (Figure~9) andIJune l, 1983 (Figure 10) while low buoyancy seed cultures were used to initiate the experiments on May 26, 1983, October 4, 1982, and May 12, 1982 (Figures 11, 12, and 13). Cell Density and Carbon Dioxide The density of algae in microcosms with NH4-N increased slightly faster and peaked earlier at higher maximum cell densities than in microcosms without NH4-N (Figures 9 to 13). Maximum cell densities and corresponding minimum carbon dioxide threshold concentrations may be compared in Table l. The May 12 experiments had noticeably higher bacterial densities and the algal filaments did not appear as healthy as in the other experiments. This will be discussed in more detail later. Buoyancy Cell buoyancy decreased rapidly at high carbon dioxide levels in the experiments without.NH4-N and positively buoyant algal seed (Figures 9 and 10). Within three days, only 0.1 and 4.0 percent of the cells were positively buoyant in the February 12 and June 1 experiments, respectively. As carbon 44 dioxide levels declined below 2.1 and 6.7 nmoles/l, respectively, cell buoyancy in these cultures began to increase rapidly. At least 60 percent of the cells in both microcosms became positively buoyant before cell buoyancy declined again towards the end of each experiment. In the companion microcosms with NH4—N, cell buoyancy remained high until near the end of the experiments (Figures 9 and 10). In the May 26 and October 4 microcosms seeded with non- buoyant algae and without NH4-N, cell buoyancy remained low for a period of days (Figures 11 and 12). Cell buoyancy began to increase as carbon dioxide concentrations declined below 6.7 and 4.6 nmoles/l as shown in Figure 11 and Figure 12, respectively. Buoyancy increased to the point where approximately 50 percent of the cells were positively buoyant in both cultures before declining at the end of the experiments. In the May 26 experiment with NH4-N, the initially non- buoyant algae increased rapidly in buoyancy near the beginning of the experiment; and by the fourth day, over 60 percent of the cells were positively buoyant (Figure 11). Cell buoyancy remained high until the culture senescent culture deteriorated. The October 4 experiment did not contain a microcosm with NH4-N. The May 12 experiments were seeded initially with non- buoyant algae. The algae remained non-buoyant throughout the experiments, both with and without NH4-N (Figure 13). 45 lo- O—OWiIhouI NH4-N A——AWiih NH4-N ID 9 b x E (I) .J .J I.” O O! 'i rT "1I ' 1 f I ‘ f fiT 0.0I fir' I V I fit V TffifT O 2 ‘4 6 8 ND I2 F4 0' 2 4» 6 8 H3 (2 (4 DAYS DAYS ,.K30 ...... ‘ ‘ SE . a c a a) 0 50" EIO- "' 0 £401 C) at 8 I5 5 “’20-. u: 01 I E 00 * ér 4r ' é : é T T * ififil 0‘ I r I V I r I ffi IO‘ I2 I4. O 2 ‘4 6 DAYS DAM: IO I2 I4 ‘0 20- 19 8 I: I5‘ 0 z - \ F- =-; :0- g :5 z r: s- 3 z 20" 4111*IIIrTIIIITI 0 2 ‘I 6 8 K3 I2. L4 0 2 «4 6 8 H3 (2 N4 DANS DAYS Figure 9. Anabaena flos-3guae @ IOIME/mZ/sec, February 12 experiment: time course measurements of (a) cell concentration, (b) carbon dioxide, (c) heterocysts, (d) positively buoyant cells, (e) nitrogen fixation, and (f) algal C/N ratio. 46 10' HWiihouI NH-N HWEII‘I NH-N a 4 4 8- IO "9 V —~ b j: 6.- 2 I01 E g 4. 3 Lo- m N ‘9 <3 25 0 01" oTrrt'v'r"'t_' 0.0'oturrtfivviTI—I o 2 4 6 8 2 4 6 8 I0 I2 DAYS’ K) I2 [MNRS ,3IOO~ i: A I ‘n 80' t: 5' c a m o I- 'o. I- ? 5 o >- 40‘ 8 5 8 E m 20- % '2’ Of f I I I I fl I I l o ‘D ‘4 (5 8 (0 l2 I2 DAYS 70- TZEEffg C/N RATIO TURGOR (psi) 0| ‘1' ' I j j T I—fi i—i v I 02468I0I2 02488Iofi2 DAYS DAYS Figure 10. Anabaena flos-aguae @ 10 uE/mZ/sec, June 1 experiment: time course measurements of (a) cell concentration, (b) carbon dioXide, (c) heterocysts, (d) positively buoyant cells, (e) turgor pressure, and (f) algal C/N ratio. 47 I0 o—ownhoui NH4-N A—AWiIh NH4-N In 3" 9 ‘6‘ i :I“ a U U Z-I OT‘ f1 * ‘l'jfi o'246'elo I2 I4 DAYS ,.HDCF 3~° (D a..|5- S a Q 33 K)‘ P- 9 5 8 8 :2 5‘ a: «n E c 2 o . “A. O 468 IO l2 I4 (JAYS 7CP- 81 ii ‘ c) ‘7‘ I: 4 g o z I- 8 51f 50 fIvyv‘1 f'1lvfi 4TI'I'I' ‘I""' 02468lOI2l4 02458I-0I2I4 IJAYS DAYS Figure 11. Anabaena flos-3guae @ 10‘pE/m2/sec, may 26 experiment: time course measurements of (a) cell concentration, (b) carbon dioxide, (c) heterocysts, (d) positively buoyant cells, (e) turgor pressure, and (f) algal C/N ratio. 48 (0' In 5‘ Q " 64 E a 1’3 4« .I Lu 0 I 2.1 o '1 T T T T— O 2 4 6 E! K) (2 DAYS IO g (04 5 LO-I b N o 0 OJ‘ 0.0I f , I T If . (3 2 4. 8 K) l2 DAYS ,.NDCF 32 In 805 :I ‘8' .. 6"I c a... 8 ,é, 20w o fivfi 1‘ I I I ‘1 O 2 44 6 8 l0 I2 DAYS Figure 12. Anabaena flos-aquae @ 10 pE/mZ/sec, October 4 experiment: time course measurements of (a) cell concentration, (b) carbon dioxide, and (c) positively buoyant cells. 49 HWiIhoui NH4-N Z-‘t---'AWIII'I NH4-N (0' 9 c j 6- 3 Im E g 4-I 3 Lo- 3 .0" 2 a ‘9 01* b OFTI'IFTTT'TV'V‘Ooo. ffvovrrtfrrt'I 0 2 4 6i 8 "3 I2 I4 (3 2 .4 5 3 "3 (2 I4 DAYS (JAYS AIOOI 2‘3 .3 I5- 3 80" 2‘. w ‘9 EXP E IO- .- 8 g 40‘ 35 5- ago i- 20‘I m c 05 d I 2 0 v r ' I ' I fi fr r f r r i O O 24 6 8 I0|2I4 02 4 ~6 8IO I2 I4 DAYS DAYS 70- a. 365‘ o 7.! a: i: ’ geo- g 3 " ——A '- 5 e 5; EH i sorfT'I'l'I—TTTrjl 4fi‘rTrIIr1IrTII‘I 024680121402468i0l2l4 DAYS DAYS Figure 13.Anabaena flos-aquae @ 10 uE/mZ/sec, May 12 experimentztime course measurements of (a) cell concentration, (b) carbon dioxide, (c) heterocysts, (d) positively buoyant cells, (e) turgor pressure, and (f) algal C/N ratio. 50 Percent Heterocysts, Nitrogen Fixation, and 334:3 In cultures without inorganic nitrogen, the percent heterocysts equilibrated at levels from 8 to 10 percent; while with NH4-N, the percent heterocysts declined to two to four percent (Figures 9 to 13) . Ammonia-nitrogen was added periodically throughout the experiments to microcosms containing inorganic nitrogen to maintain the concentration between 0.1 and 0.5 mg NH4—N/l. The May 12 microcosm with NH4- N also was initiated with 2 mg NO3-N/l in an attempt to provide a more stable inorganic nitrogen supply. Nitrogen fixation was measured only during the February 12 experiment (Figure 9). The rate of nitrogen fixation without NH4—N increased to a maximum of just over 6.0 x 10’5 ng N fixed/cell/hour on day six and then generally declined throughout the remainder of the experiment. Nitrogen fixation rates were much lower in the microcosm with NH4-N. The maximum rate measured was 1.4 x 10'5 ng N fixed/cell/hour; and after the fifth day, nitrogen fixation rates declined to extremely low levels. Turgor Pressurei CZN Ratigy and Analysis Results from all of the 10 pE/mZ/sec experiments, except those initiated on May 12, are analysed collectively. The May 12 experiments are analysed separately later since the condition of the algal seed as well as other factors were different than in any of the other experiments. 51 The primary external environmental parameter which changed during the course of each experiment was the carbon dioxide concentration. Changes in carbon dioxide concentra- tions in addition to the presence or absence of NH4-N appeared to be related to the changes in cell buoyancy observed during these experiments. The buoyancy response of algae in microcosms initiated with buoyant seed cultures was generally the same as in those initiated with non-buoyanct seed cultures with the exception of an initial equilibration period of several days. With abundant NH4-N, Anabaena flos-aquae either remained buoyant or quickly became buoyant at high carbon dioxide levels. As the cultures grew in the presence of NH4-N, the algae remained buoyant until near the end of the experiments when the senescent cultures deteriorated. In cultures without NH4-N at high carbon dixoide levels, the algae either remained non-buoyant or quickly lost buoyancy. The algae remained in this condition until the carbon dioxide concentration declined to 4.79 +_ 1.97 nmoles/l. At this point, cell buoyancy began to increase and within three to four days at least 50 percent of the cells became positively buoyant. As the cultures deteriorated at the end of the experiments, cell buoyancy declined rapidly. The internal cellular processes responsible for alteration of cell buoyancy at 10 uE/mZ/sec did not appear to involve cell turgor pressures. Turgor pressure measurements were made only during the May 26 and June 1 experiments. Turgor pressures during the May 26 experiment without NH4-N remained 52 below 60 psi throughout the experiment and showed no significant change during the period from the sixth to the twelfth when cell buoyancy increased dramatically (Figure 11). With NH4- N, the cell turgor pressure declined as the algae initially became buoyant; however, the algae remained buoyant when the maximum turgor pressure of 62.5 psi was reached on the seventh day. These observations suggest that factors other than the cell turgor pressure were involved in determining cell buoyancy. Cell turgor pressures measured during the June 1 experiments (Figure 10) were slightly higher than during the May 26 experiments just described. However, they also do not appear to explain the buoyancy responses observed. Without NH4-N, cell turgor pressures gradually declined throughout the experiment with a maximum value of 64.0 psi recorded at the beginning of the experiment when the algae were buoyant. ‘With NH4—N, a similar turgor pressure of 64.2 psi was measured on day four and the algae remained highly buoyant. Cell buoyancy began to decline later in this experiment when the cell turgor pressure was between 59.0 and 63.0 psi as the culture deteriorated. Taken together, cell turgor pressures measured during the 10 uE/mz/sec experiments do not appear to explain the buoyancy response observed at this light intensity. The cellular C/N ratio appeared to correlate well with the cell buoyancy responses observed at this light intensity. Regulation of cell buoyancy at 10 uE/mz/sec appears to operate 53 in a manner similar to that previously described for the experiments at 5 and 2 pE/mz/sec. At high carbon dioxide levels without NH4-N, the C/N ratio in initially buoyant cultures increased to levels equal to or greater than 6.0, and cell buoyancy declined rapidly (Figures 9 and 10). Under these conditions, the algae were apparently fixing and accumulating carbon at a faster rate than nitrogen, causing the cellular C/N ratio to increase. With the resulting decrease in cell nitrogen reserves, synthesis of gas vacuoles apparently lagged behind cell growth and the algae lost buoyancy. In the culture with non—buoyant initial seed, the C/N ratio remained at or above 6.0 at high carbon dioxide levels, and the algae remained non-buoyant (Figures 11 and 12) . As the carbon dioxide concentration decreased in these experiments without NH4-N, the C/N ratio eventually began to decline and cell buoyancy increased (Figures 9 through 11). Although the nitrogen fixation rate generally decreased with declining carbon dioxide levels, it appears that the fixation and accumulation of carbon eventually decreased at a faster rate resulting in the observed decreases in C/N ratio. With the consequent increases in relative cell nitrogen supply, it appears that the rate of synthesis of gas vacuoles increased relative toicell growth causing the observed increases in cell buoyancy. The C/N ratio in microcosms with NH4-N was generally lower than in cultures without NH4-N. The C/N ratio remained less 54 than 6.0 throughout the May 26 and June 1 experiments with NH4-N (Figures 10 and 11). The C/N ratio in the February 12 experiment with NH4-N remained close to 6.0 throughout the experiment; however, values slightly above 6.0 were measured on several occasions (Figure 9). With lower C/N ratios resulting from an abundant external NH4-N supply, it appears that the relative internal cellular nitrogen supply was high in these cultures. Under these conditions, it appears that the rate of gas vacuole synthesis remained high relative to cell growth resulting in buoyant algae throughout the experiments. Cell buoyancy did eventually decline as the senescent cultures deteriorated. The preceeding analysis of buoyancy regulation at 10 pE/mz/sec does not include consideration of results from the May 12 experiments in which the algae never became buoyant (Figure 13). Cell turgor pressures in these cultures both with and without NH4-N, were in the same range as in the other 10 pE/mz/sec experiments. A maximum of 61.1 psi was recorded on day four in the microcosm with NH4-N (Figure 13). As such, cell turgor pressure does not appear to explain lack of buoyancy in the May 12 experiments. The algal C/N ratio in the microcosm without NH4-N remained above 6.0 for the entire experiment with a maximum of 7.11 measured on day four (Figure 13). In the other 10 pE/mZ/sec experiments, the C/N ratio without NH4-N declined to 6.0 or below as carbon dioxide levels declined. The algae then became 55 buoyant. It is possible that the high bacteria densities and less healthy seed culture in the May 12 experiment may have contributed to maintenance of a high C/N ratio which resulted in decreased gas vacuole synthesis and lack of buoyancy. However, in the May 12 experiment with NH4-N, the C/N ratio decreased from an initial value of 6.02 to 5.50 on day four, and 5.41 on day nine and the algae failed to become buoyant here either . Cell buoyancy would have been expected to increase at these relatively low C/N ratios. The lack of buoyancy in the May 12 experiments was not satisfactorily explained by analysing the cell turgor pressures or C/N ratios. However, there were a number of other factors which were unusual in these cultures. The seed algae for these microcosms came from an Iold seed culture well past the exponential growth phase. The algal filaments were slightly fragmented and bacterial densities were much higher than in other seed cultures. Algal growth rates and maximum cell densities in the May 12 experiments were comparable to growth in the other 10 uE/mZ/sec experiments (Table 1). However, the same May 12 seed was used to initiate experiments at 5 uE/mZ/sec, and no growth was observed. Within seven days at 5 uE/mz/sec, the algal filaments became highly fragmented and the culture deteriorated rapidly. Interpretation of the May 12 experiments are further complicated by the fact that in the microcosm provided with inorganic nitrogen, NO3-N was added in addition to NH4-N in 56 an attempt to provide a more stable nitrogen supply. This is the only experiment reported here where NO3-N was used. I have no reason to suspect that the presence of NO3-N contributed to the lack of buoyancy in this culture. Other studies with Anabaena flos-aquae have shown that cultures with NO3-N grow better than those without NH4-N, although not as well as with NH4—N (Ward and Wetzel, 1980; Rhee and Lederman, 1983). However, since the May 12 experiment was the only experiment whererNO3-N'was used, the possibility that it may have somehow affected cell buoyancy cannot be ruled out. The lack of cell buoyancy in the aberrant May 12 experiments is not completely explained. However, the presence of excessive bacteria, a fragmented seed culture which showed no growth at 5 uE/mz/sec, and the use of NO3-N, all may have been involved. The existence of these complicating factors, none of which were present in the other 10 ‘pE/mz/sec experiments, are grounds for weighing these results less heavily in subsequent discussion. 20 pE/mZ/sec EXPERIMENTS Growth experiments with Anabaena flos-aquae were conducted at 20 pE/mz/sec beginning on October 4, 1982, and also February 12, 1983. The first experiment was only run without NH44N. The February 12 experiments were conducted both with and without NH4-N. 57 Cell Density and Carbon Dioxide There was little difference in cell growth in microcosms with and without NH4-N in the February 12 experiments until near the end of the growth phase when a slightly higher maximum cell density was reached in the microcosm with NH4-N (Figure 14) . Carbon dioxide levels declined rapidly during these experiments, reaching minimum growth threshold concentrations on dates corresponding to maximum cell densities. The October 4 experiment (Figure 15) was seeded with a higher initial density and increased to a higher maximum cell density than the February 12 experiment. However, both experiments reached nearly identical carbon dioxide threshold concentrations of 0.046 and 0.045 nmoles/1, respectively, on day six. Buoyancy The initial algal seed in the February 12 experiments contained 99.9 percent positively buoyant cells. Without NH4-N, the algae remained highly buoyant for the first six hours; however, after 24 hours, less than 0.1 percent of the cells were positively buoyant (Figure 14). The algae then remained non-buoyant throughout the remainder of the experiment. With NH4-N, cell buoyancy decreased gradually at high carbon dioxide levels (Figure 14). By the third day, only 34 percent of the cells were positively buoyant. As carbon dioxide levels declined below 1.0 nmole/1, cell buoyancy began to increase, reaching a peak of 79 percent positively buoyant cells on the fifth day. Cell buoyancy then declined as the senescent culture deteriorated. 58 ,0. O—OWithout NH4-N A—-AWiih NH4-N In IOOH 2 a b I 2 I0- 5§ £5 -i 105 d N CD CD 0 Old 0.0I . i 1 o I 2 3 4 S 6 7 DAYS .ID 2% In 80‘ :3 '5' :I 9.. c m a, o 60‘ I- (o‘ I- 8 § 40- 8 8 In 5 in 2 I; vs °1 3 8 OI 1 I I I o I 2 3 4 5 6 2 DAYS DAYS m 20- 81 I9 6 I- : Io- g >< 2: r: 5- 3 5| 2 S O 4 0|23 4667 DAYS Figure 14. Anabaena flos-aguae @ ZolpE/mZ/sec, February 12 experiment: time course measurements of (a) cell concentration, (b) carbon dioxide, (c) heterocysts, (d) positively buoyant cells, (e) nitrogen fixation, and (f) algal C/N ratio. 59 l2- IO‘ 03 a l I m h l CELLS/ml x lo5 0 NJ as H a m «1 DAYS ICF 002 (HMS/l) MOCF 8 FKEiEMlmflUWT!CELLS 0%» m A ‘P 9 (3 OH 3‘55 5 6? Figure 15. Anabaena flos-aquae @ 20 pE/mz/sec, October 4 experiment: time course measurements of (a) cell concentration, (b) carbon dioxide, and (c) positively buoyant cells. 60 TheTOctober 4 experiment was seeded with algae containing 13 percent positively buoyant cells. Cell buoyancy in this microcosm without NH4-N remained low for the first four days (Figure 15) . However, as opposed to the February 12 experiment without NH4—N, algae in this microcosm increased in buoyancy at low carbon dioxide levels. As the carbon dioxide concentration declined below 0.1 umoles/l on the fifth day, cell buoyancy increased markedly. By day six, 61.5 percent of the cells were positively buoyant. Thereafter, cell buoyancy declined as the culture deteriorated. Percent Heterocystsy Nitrogen Fixation, and HEAZE Heterocysts, NH4-N, and nitrogen fixation 'were only measured at 20 uE/mZ/sec during the February 12 experiments. The percent heterocysts increased to nearly 10 percent in the microcosm without NH4-N, while decreasing to three to four percent with NH4-N (Figure 14). Nitrogen fixation rates were much higher in the microcosm without inorganic nitrogen than with NH4-N (Figure 14). After an initial equilibration period, a maximum rate of 9.1 x 10‘5 ng N fixed/cell/hour was measured between the first and second day. The rate of nitrogen fixation without NH4-N then generally declined during the remainder of the experiment correlating with decreasing carbon dioxide levels. By the sixth day, the rate had fallen to 0.75 x 10"5 ng N fixed/cell/hour. The maximum nitrogen fixation rate with NH4-N was 3.2 x 10"5 ng N fixed/cell/hour measured between day zero and day one (Figure 14). The rate then gradually declined for the 61 remainder of the experiment and by day six nitrogen fixation was not detectable. Ammonia-nitrogen was supplied at an initial concentration of 0.5 mg NH4-N/l. Additions of NH4—N were generally made twice a day in an attempt to maintain the concentration between 0.1 and 0.5 mg NH4-N/l. On several brief occasions the concentration fell below this range. Turgor Pressure, C/N Ratio, and Analysis The primary environmental variable which changed throughout the course of these experiments again was the carbon dioxide concentration. As in experiments at other light intensities, the buoyancy of Anabaena flos-aquae at 20 uE/mz/sec responded to changes in the carbon dioxide concentration as well as to the presence or absence of NH4-N. At high carbonmdioxide concentrations without.NH4-N, cell buoyancy remained low or decreased rapidly from a high initial value. 131theiFebruary'12 experiment, the algae remained non- buoyant for the rest of the experiment, even at very low carbon dioxide levels (0.05 pmoles/l). In the October 4 experiment without NH4-N, cell buoyancy increased significantly when the carbon dioxide level decreased below 0.1 pmoles/l. In both experiments, cell growth and carbon dioxide depletion were rapid. In the February 12 experiment, carbon dioxide levels decreased to 0.1 pmole/l in five days, and 24 hours later the carbon dioxide threshold was reached. In the October 4 experiment, the carbon dioxide concen— tration, where the algae became buoyant (0.1 pmoles/l) was so 62 near the growth threshold, that there was little time for cellular metabolic processes to respond and equilibrate to those low carbon dioxide levels. This may explain why in one experiment the algae became buoyant and in the other they did not. Unfortunately, further analysis of the October 4 experiment is not possible since the only parameters measured during this preliminary experiment were cell density, buoyancy, and carbon dioxide. Cellular C/N ratios measured during the February 12 experiments (Figure 14) did not correlate nearly as well with changes in cell buoyancy as in the lower light intensity experiments described previously. Without NH4—N, the C/N ratio increased to values above 6.0 at high carbon dioxide levels, and the algae lost buoyancy. However, the C/N ratio then declined to 5.8 on day two and 5.3 on day three, and the algae remained non-buoyant. lThereafter the C/N ratio remained close to 6.0. Fitmlprevious discussion, it might have been expected that the algae would become buoyant at these low C/N ratios, but they did not. With NH4-N, the C/N ratio remained less than 5.6 throughout the entire experiment (Figure 14). During the period of declining buoyancy in the early part of the experiment, the C/N ratio was measured as low as 3.9. Again, using the theory presented at lower light intensities, an increase in cell buoyancy would be predicted during this period of low C/N ratio instead of the observed decrease in buoyancy. 63 Walsby and Booker (1980) also found that the buoyancy of Anabaena flos-aquae was greatly reduced at light intensities above 20 pE/mz/sec. The cell turgor pressure may rise sufficiently to result in collapse of gas vacuoles and loss of cell buoyancy at these light intensities (Walsby, 1971; Dinsdale and Walsby, 1972; Grant and Walsby, 1977). Although cell turgor pressures were not measured during the 20 pE/mz/sec experiments shown in Figures 14 and 15, turgor pressures were measured at 20 pE/mz/sec on a number of other occasions. These measurements, together with turgor measurements made at other light intensities, are compiled in Table 2 which shows mean turgor pressures and standard deviations for each light intensity. These measurements come from a variety of different carbon dioxide and nitrogen levels. The mean turgor pressure at 20 pE/mz/sec was higher than at the lower light intensities, which supports Walsby and co—workers conclusions concerning the role of turgor pressure in buoyancy loss at elevated light intensities. Table 2. Mean cell turgor pressures and standard deviations for Anabaena-flos-aquae grown at various light intensities. Light (pE/mZ/sec) 2 5 10 20 50 Turgor x 58.78 59.12 59.96 64.50 68.70 pressure (S) (2.98) (2.15) (3.07) (1.00) (1.20) (psi) 64 50 pE/mZ/sec EXPERIMENTS Cell Density and Carbon Dioxide The presence of NH4-N did not enhance algal growth at 50 pE/mz/sec as it did at lower light intensities (Figure 16). Cell densities increased at similar rates in microcosms with and without NH4-N at 50 uE/mZ/sec; however, a slightly higher maximum cell density was reached without NH4-N. The concentration of carbon dioxide declined and reached minimum threshold concentrations on dates corresponding to maximum cell densities in both microcosms. Buoyancy The microcosms were seeded with 99.9 percent positively buoyant cells. Cell buoyancy declined rapidly both with and without NH4-N at 50 pE/mz/sec (Figure 16). Within 12 hours, less than 0.1 percent of the cells were positively buoyant in both microcosms. The algae then remained non-buoyant throughout the remainder of the growth phase. As the senescent culture deteriorated and cell concentrations declined, a small percentage of cells became buoyant in both microcosms on the fourth day. At this point the algal filaments were highly fragmented and most of the buoyant cells were single cells. By the next day, very few intact cells were found and none of these were buoyant. 6S )o- o—ownnout NH4-N a—mmn NH4-N B- a l0 “e .. 07 b .. 5. 3 l E ‘é (n 49 3 LG- j N U C) CD 2. 0 DJ- 01— 1 I T ' 00' u r I I o l 2 y 4 5 o jI 2 3 5 DAYS DAYS ,3Io i: 80 A (5- g d ES C 13 g: 5 8 § § 5 (n (n g 8 of . 5 o I 2 3 4 5 2 3 DAYS DAYS 0 20- '9 g (3- 2 g. :5 It» 3‘: :5 z (T. 5" B 2 g o v 3| I I I 1 o 2 3 4 o l 2 3 A 5 DAYS DAYS Figure 16. Anabaena flos-aquae @ SO yE/mZ/sec, February 12 experiment: Time course measurements of (a) cell concentration, (b) carbon dioxide, (c) heterocysts, (d) positively buoyant cells, (e) nitrogen fixation, and (f) algal C/N ratio. 66 Percent Heterocysts, Nitrogen Fixation, and E§42fl The percent heterocysts increased to about 10 percent in the microcosm without NH4-N and decreased to three to four percent with NH4-N (Figure 16). The rate of nitrogen fixation without NH4-N increased initially as the percent heterocysts equilibrated, and reached a maximum of 21.0 x 10"5 ng N fixed/cell/hour on the second day (Figure 16). The nitrogen fixation rate then declined as the carbon dioxide concentration declined. By day four nitrogen fixation was not detectable in this microcosm. With NH4-N, the rate of nitrogen fixation was much lower with a maximum rate measured at 8.13 x 10'5 ng N fixed/cell/hour on the second day (Figure 16). Thereafter the nitrogen fixation rate declined rapidly. The microcosm with NH4-N was provided with an initial concentration of 0.5 mg NH4-N/1. Subsequent additions of NH4- N were required in an attempt to maintain the concentration between 0.1 and 0.5 mg NH4-N. On several brief occasions the concentration dropped below this range. Following an initial equilibration period, the rate of nitrogen fixation at 50 pE/mZ/sec, as well as the other light intensities studied, tended to decrease as the carbon dioxide concentration declined. During the initial equilibration period, the percent heterocysts was adjusted to the prevailing nitrogen conditions: increasing in the absence of NH4-N, while decreasing in the presence of abundant.NH4-N. Similar results showing the inhibition of heterocyst production in the presence 67 of high levels of NH4-N have been reported previously (F099, 1949; Stewart 33 31., 1968; Ogawa and Carr, 1969). However, I have found no published studies in which the rate of nitrogen fixation was measured at various carbon dioxide concentrations. The general decline in the rate of nitrogen fixation in response to decreasing carbon dioxide levels was not unexpected since nitrogen fixation is dependent on photosynthesis for generation of reducing power and carbon skeletons (Lex and Stewart, 1973; ‘Winkenbach and Wolk, 1973). Therefore, as the rate of photosynthesis and carbon fixation decline in response to decreased carbon dioxide availability, the rate of nitrogen fixation would also be expected to decline. Turgor Pressure, C/N Ratio, and Analysis As in experiments at other light intensities, carbon dioxide was the primary external environmental parameter which changed during the course of the 50 pE/mZ/sec experiments. Cell buoyancy was lost rapidly at high carbon dixoide levels. The algae remained essentially non—buoyant throughout the rest of the growth phase in cultures both with and without NH4-N even at very low carbon dioxide levels. As the senescent cultures deteriorated and cell densities declined, there was a small increase in cell buoyancy. The cellular C/N ratios at 50 pE/mZ/sec, as with 20 pE/mz/sec, did not appear to correlate with cell buoyancy in the manner described for the lower light intensities (Figure 16). Without NH4-N, the C/N ratio increased initially to a maximum of 6.9 after 24 hours; and during this period, cell 68 buoyancy was lost. However, the C/N ratio declined to a value of 3.9 after 48 hours and remained less than 5.0 for the remainder of the experiment. With relatively high cell nitrogen reserves suggested by these low C/N ratios, an accumulation of gas vacuoles and increased cell buoyancy during this period might have been expected. With NH4-N, the cellular C/N ratio increased initially to 5.62 after 12 hours and cell buoyancy was lost. The C/N ratio then declined to 3.9 after 34 hours and remained below 4.5 throughout the growth period. Again, the algae remained non-buoyant under conditions seeming to favor increased gas vacuole synthesis. As suggested previously, development of high cell turgor pressures at this high light intensity may have resulted in a collapse of gas vacuoles and loss of cell buoyancy under these conditions. Although turgor pressures were not measured during the Feburary 12 experiments, the mean turgor pressure at 50 pE/mZ/sec calculated from measurements made on other occasions was elevated (Table 2). The slight increase in buoyancy which occurred as the algae fragmented and cell densities declined at the end of the experiments, may suggest that the algae did not have sufficient time to equilibrate to the low carbon dixoide levels during the growth phase which only lasted three days. Given additional time, more algae may have become buoyant under very low carbon dioxide conditions at 50 pE/mz/sec. While additional work would be required to investigate this possibility, conditions 69 of very low carbon dioxide levels co—occurring with elevated light intensities as created in the laboratory are probably uncommon in lakes. Low carbon dioxide concentrations in lakes are normally the result of high rates of photosynthesis associated with dense algal blooms. The presence of these dense algal blooms results in reduced light penetration and low light availability throughout much of the epilimnion. Experimental Reduction of Turgor Pressure It appears from my results and other studies (Walsby, 1971, 1973; Walsby and Booker, 1980; Grant and Walsby, 1977; Allison and Walsby, 1981) that cell buoyancy is lost at high light intensities owing to the buildup of high cell turgor pressures resulting in collapse of gas vacuoles. In order to further test this theory, attempts were made to artificially reduce cell turgor pressures under high light intensity and monitor changes in buoyancy. The role of turgor pressure in buoyancy loss at high light intensities could be further substantiated if the algae lost buoyancy with normal turgor pressures, but remained buoyant with reduced turgor pressures. Initial experiments in turgor pressure reduction involved adding sucrose to the external media. It was determined that a 0.1 M sucrose solution should cause a significant reduction of cell turgor pressure (Figure 4). An Anabaena flos—aquae culture containing 75.0 percent positively buoyant cells was seeded at 30 pE/mz/sec and 5 pE/mZ/sec, and sucrose was added 70 at 0.01 M and 0.1 M concentrations. Observed buoyancy responses are compared to controls with no sucrose addition in Table 3. Table 3. Percent positively buoyant Anabaena flos-aquae cells grown at 5 and 30 pE/mz/sec, and with various sucrose concentrations. Sucrose 5 (Elm /sec 30 uE/m /sec concentration % buoyant cells °/o buoyant cells 0.0M 77.0 0.0 0.0lM 78.2 “.0 o .l M 76.5 80. 6 After 12 hours, there was no significant change in buoyancy in any of the cultures at 5 pE/mz/sec. At 30 uE/mz/sec buoyancy was completely lost in the control while increasing sucrose concentrations noticeably reduced the loss of buoyancy. 'These results appear to support the theory that increased cell turgor pressures are the cause of buoyancy loss at high light intensity. However, there are several factors which complicate this interpretation. After 36 hours, bacterial densities in the cultures with sucrose addition increased tremendously. The algae became fragmented and the flasks became noticeably brown in coloration instead of green. A second complication lies in the fact that sucrose is not only metabolized by bacteria, but by many blue-green algae as well (Allison gt .31., 1937, 1953; Fay, 1965; watanabe and Yamamoto, 1967). Although the sucrose did not appear to affect buoyancy at low 71 light, the possiblity exists that algal metabolism of sucrose at high light intensity may have somehow affected cell buoyancy. In order to eliminate these complications, I attempted to find 21 sucrose replacement which was non—metabolizable, osmotically active, and non-toxic. 'The first candidate selected was mannitol. Unfortunately, it was found that 0.1 M concentrations of mannitol caused lysis of gas vacuoles. Polyethylene glycol of molecular weights 400 and 1000 also*was tried and rejected since it too resulted in reduced buoyancy presumably due to lysis of gas vacuoles. Further research along this avenue was terminated, though it may still merit future investigation. ALGAL GROWTH RATES Specific growth rates of Anabaena flos—aquae were cal- cuated as functions of carbon dioxide concentration in the microcosm growth experiments. Calculations were made using Michaelis—Menton kinetics, as described in the Methods section. A sample figure showing the relationship between the specific growth rate and the carbon dioxide concentration is shown in Figure 173 The kinetic constants Pmax' ks, and sq, frommwhich the curve was constructed, were calculated from the data points shown. Kinetic constants are compiled for individual experi— ments for each light intensity and nitrogen level in Table 4. 72 0.0 I 5 -' ”max= 0-0'0 K3 3 0.320 Sq =0.040 0.0I0- (hr") 0.0054 0.000- 00! 0.! LG l0 I00 Figure 17. Sample relationship between specific growth rate and carbon dioxide concentration, and Michaelis-Menton kinetic constants. 73 mmH.O OONN.H HOOQ0.0 suHB MOINHIN on mHH.O OOOO.H mva.O usonuHZ MOINHIN om va.O ONMH.O «OHN0.0 suHS MOINHIN ON va.O O¢H¢.O OMON0.0 usosuH3 MOINHIN ON O¢0.0 MON.O hme0.0 usonuH3 NOIQIOH ON Nm0.0 OOON.O ovOO0.0 suH3 MOINHIm OH mmN.O OBO0.0 MNNH0.0 suHS MOIHIO OH OO0.0 «mmH.O NO¢H0.0 nuH3 MOIONIm OH «V0.0 NmNH.O thH0.0 nuH3 MOINHIN OH NOH.O HNO0.0 OMOO0.0 usonuH3 MOINHlm OH O¢m.O MbH¢.O mva0.0 unocuHS MOIHIO OH Oh0.0 HHO0.0 MONH0.0 uzonuHS MOIONIm OH OM0.0 HNON.O «OOO0.0 usocuHS MOINHIN OH va.O NOmw.O OOHH0.0 usonuH3 NOIVIOH OH mNH.O HHmh.O ONmO0.0 suHB MOINHIN m mOm.O meh.O NNOO0.0 uaosuH3 MOINHIN m O.wm 0.0¢ mMNO0.0 nuH3 MOIHIO N O.mv m.HO OOHO0.0 usonqu MOIHIO N ANwUZnO Auoozav lean. zuemz xouooe .oou\~s\mnc m an ace: ucoEHuomxm usmfin mucoewuomxm susouo oaamanDHu unmanned scum muaauncou ofiuocfim cowuaozthHoanOHz .4 «Home DISCUSSION Most.of the common planktonic blue-green algae which form blooms in lakes possess gas vacuoles which allow considerable alteration of cell buoyancy (Lund, 1959; Walsby, 1970; F099 31 31., 1973; Reynolds and Walsby, 1975). Regulation of cell buoyancy has been used to explain the often observed vertical migration of these algae to form dense surface blooms, or in some cases, distinct stratified subsurface populations (Reynolds and Walsby, 1975; F099 31 31., 1973). Significant alteration of cell buoyancy may involve subtle changes in the gas vacuole content of blue-green algae. As was shown in this study, a 10 percent reduction in the gas vacuole content of a buoyant Anabaena flos-aguae culture resulted in a significant decrease in algal buoyancy (Figure 6b). Previous studies have reported that changes in the availability of light and nutrients may result in alteration of the gas vacuole content and changes in cell buoyancy in a number of different blue-green algae (Walsby, 1971; Klemer 31 31. , 1982; Paerl and Ustach, 1982; Van Rijn and Shilo, 1983) . In 74 75 this study, changes in the buoyancy of Anabaena flos-aquae were monitored in response to changes in the availability of light, carbon dioxide, and ammonia-nitrogen. Buoyancy Niche Data from the microcosm growth experiments were compiled to define the zone of light, carbon dioxide, and inorganic nitrogen where Anabaena flos-aquae appears to be buoyant (Figure 18). Buoyant conditions shown in this figure and tabulated in Appendix 2 were determined from careful examination of trends of cell buoyancy in Figures 7 to 16 described in the Results and Discussion section. Buoyant regions were determined from breakpoints in these figures where the algae showed distinct increases or decreases in buoyancy. In cases where multiple experiments were conducted under the same environmental conditions, mean values were used in Figure 18. The lower line in Figure 18 represents the growth threshold below which no growth occurred. The buoyant niche without NH4- N is quite small (Figure 18). As shown in Figure 18 and reported by others (Walsby, 1971; Walsby and Booker, 1980), Anabaena flos-aquae is not buoyant at elevated light intensities. In order for Anabaena flos-aquae to become buoyant without NH4-N, it appears that the light intensity must be reduced below 25 pE/mZ/sec and the carbon dioxide concentration must be reduced to 5 pmoles/l or less, well below atmospheric 76 31.6 ‘ atmos. equilibrium- [0.0 . 3J6 ‘ LOO - (u moles/g) BUOYANT ZONE WITHOUT NH -N 0.32 ‘ 4 0.IO- 0.03- Growth Threshold 0.0! [ l I T 1—‘v—. 0 IO 20 30 40 6o VlfSOO un LIGHT INTENSITY sunlight (uE/mzsec) 3|.6. atmos milibrium-) BUOYANT ZONE IDO- T _ WI H NH4N 9‘ 5 I (b) (u moles/l ) 3 ? 0.32" 010'“ ca2 0.0 3-1 fl I'M ~- 9 l L '6 HT INTENSITY sunfiéht (uE/mzsec) Figure 18. Buoyancy niche of Anabaena flos—aguae relative to light intensity and carbon dioxide concentration (a) grown without NRA-N, and (b) grown with NH4—N. 77 equilibrium carbon dioxide levels (Figure 18). Within the buoyant zone, the higher the light intensity, the lower the carbon dioxide level must be before the algae become buoyant. Other researchers also have reported increased buoyancy with decreasing carbon dioxide availability (Booker and Walsby, 1979; Paerl and Ustach, 1982; Klemer 31_31., 1982). When NH4-N was available, the buoyant niche for Anabaena flos-aguae has 'markedly' expanded (Figure 18). At light intensities below 15 pE/mz/sec, the buoyant zone extended up to carbon dioxide concentrations in excess of atmospheric equilibrium (Figure 18). In order to become buoyant in the presence of abundant NH4-N, reduction of light intensities appear to be of primary importance. Five microcosm growth experiments were conducted at 10 pE/mZ/sec without NH4-N. In four of these experiments, the algae became buoyant at reduced carbon dioxide levels. The mean breakpoint concentration for algal buoyancy for these experiments was 4.79 3; 1.97 pmoles C02/l. In the fifth experiment (May 12), the algae did not become buoyant at any carbon dioxide concentration; however, this experiment was initiated with an unhealthy seed culture which had abnormally high bacterial densities. The buoyancy niche of Anabaena flos-aquae shown in Figure 18 outlines the buoyancy thresholds of the algae with respect to the availability of light, carbon dioxide, and ammonia- 78 nitrogen. Within the buoyant zones shown in Figure 18, the percentage of buoyant cells varied considerably. The buoyant niche of Anabaena flos-aquae therefore may be expanded to include the actual percentage of positively buoyant cells under various environmental conditions. This is shown in three dimensional form in Figures 19a and b. Again, in cases where multiple data were available, the mean values are shown in these figures. By comparing these two figures, it is clear that not only is the buoyant niche of Anabaena flos-aquae expanded in the presence of NH4-N, but the percentage of positively buoyant cells within this zone is greatly increased as well. Other researchers have reported increased buoyancy of Oscillatoria with the addition of NH4-N (Walsby and Klemer, 1974; Van Rijn and Shilo, 1983). One of the primary goals of this research project was to develop a model for predicting conditions favorable for the formation of blooms of Anabaena flos-aqgae and other blue- green algae. While knowledge of algal buoyancy under various environmental conditions as shown in Figures 18 and 19 may be of critical importance in predicting bloom formation, the physiological growth rate of the algae also is important in the development of blooms. For example, environmental conditions may develop where Anabaena flos-aquwe is highly buoyant, but the growth rate of the algae is minimal. Under these conditions, the potential for bloom formation may be reduced. On the other hand, conditions may be such that only 79 Without (a) _ NH4 N 00 388 Positively Buoyant Cells (9’6) 20 DC- a) 9 Positively Buoyant Cells ('l.) O o 40- \ \°é\°‘\‘ 20 l o‘ Figure 19. Relationship between the percent positively buoyant cells, light intensity, and carbon dioxide concentration for Anabaena flos- 3quae (a) grown without NBA-N and (b) grown with NH4-N. 80 a small percentage of .Anabaena flos-aqgae filaments are buoyant; however, if they are growing very rapidly, a significant algal bloom may potentially develop. Physiological Growth Rates In order to assess the role of algal growth rates in bloom development, the data presented previously in Table 4 were used to construct three-dimensional plots of the relationship between the specific growth rate and the availabilities of light and carbon dioxide. Separate figures were constructed for Anabaena flos-aguae with and without NH4-N (Figures 20a and b). Again, where multiple data were available for the same environmental conditions, mean values were used in these figures. The highest specific growth rates occurred at high light intensity and high carbon dioxide levels (Figure 20). As the light intensity and/or carbon dioxide concentration decreased, the specific growth rate decreased. The specific growth rates were slightly higher with NH4-N than without NH4-N except at 50 pE/mz/sec. Ward and Wetzel (1980) and Rhee and Lederman (1983) also report increased growth rates of Anabaena flos- 33333 with the addition of NH4-N. The slight depression of the specific growth rate at 50 pE/mz/sec with NH4-N (Table 4) may have resulted from potential toxic effects of ammonia in these rapidly growing cultures in which the pH also increased rapidly; Other studies have reported toxic affects of ammonia 81 (0) Without 11 (hr.') p (hrfl) Figure 20. Relationship between the specific algal growth rate, light intensity, and carbon dioxide concentration for Anabaena flos-aquae (a) grown without NRA-N and (b) grown with NH4-N. 82 on algae at high pH levels (Stewart, 1964; F099 and Than—Tun, 1958). While the presence of NH4-N had a slight effect on physiological growth rates of Anabaena flos-aquae, the presence of NH4-N had a large effect on cell buoyancy as shown previously in Figure 19. The specific growth rates shown in Figures 20a and b represent physiological maximum growth rates, since the algae were not allowed to sink out of the photic zone in the laboratory microcosms. The maximum physiological specific growth rate (pmax) at 50 uE/mZ/sec and 17 pmoles COZ/l was 0.046 (hr‘l) which is equivalent to a doubling time of 1.25 days, assuming 12 hours of light per day. It is apparent that under conditions of high light and high carbon dioxide, Anabaena flos-aquae has the capability of growing very rapidly. However, the algae are not buoyant at high light and high carbon dioxide levels (Figure 18); therefore, a surface bloom would not be expected under these conditions. An Index to Surface Bloom Formation Since both (cell buoyancy' and algal growth rate are important in the formation of blue-green algal blooms, these two parameters were combined to form a single estimator of the potential for bloom formation. The specific growth rates under various conditions of light, carbon dioxide, and ammonia- nitrogen shown in Figures 20a and 20b were multiplied by the percent of positively buoyant cells under those same conditions shown in Figures 19a and 19b. This product, which represents 83 (a) Without Nth-N u (hr‘) for BUOYANT ALGAE 0.0l 2 .0 O o a) u (hF') for BUOYAN T ALGAE 0.004 Figure 21.Relationship between the specific growth rate of positively buoyant algae, light intensity, and carbon dioxide concentration for Anabaena flos-aguae (a) grown without NH4-N and (b) grown with NH4-N. 84 the specific growth rate of positively buoyant cells, is plotted as a function of light and carbon dioxide in Figure 21a without NH4-N, and Figure 21b with NH4-N. The specific growth rate of positively buoyant cells, which takes into account both buoyancy and grwoth rate, may be used as an index to the potential for the formation of surface blooms of Anabaena flos- 33333. It is clear from Figures 21a and b that using this index the potential for the formation of surface blooms of Anabaena flos-aquae is much greater if NH4-N is available. The maximum specific growth rate of buoyant cells without NH4-N was only 0.0026 hr'1 which is equivalent to a doubling time of 22.2 days, assuming 12 hours of light per day; The maximum specific growth rate with NH4-N was approximately 0.0106 hr"1 which is equivalent to a doubling time of 5.4 days. The maximum specific growth rate of buoyant algae occurred at a light intensity of 10 pE/mz/sec both with and without NH4-N. The maximum rate with NH4-N occurred at carbon dioxide levels above atmospheric equilibrium; while without NH4-N, the maximum occurred at a reduced carbon dioxide concentration of approximately 5 nmoles/l. The large increase in the specific growth rate of buoyant cells with the addition of NH4-N was due primarily to increases in cell buoyancy as compared to those algae Without NH4-N as shown in Figures 19a and b, rather than differences in physiological growth rate (Figures 20a and b). 85 Figures 21a and 21b are presented to serve as a model for predicting the conditions favorable for the formation of blue- green algal blooms. The model probably underestimates the potential for surface bloom formation because the assumption is made that only positively buoyant algae will contribute to a bloom. However, algae which are negatively buoyant may sink in the water column and later encounter conditions such as reduced light intensity or increased nitrogen availability which may result in subsequent positive cell buoyancy. The model also ignores any resuspension of negatively buoyant algae by vertical mixing. Although vertical mixing tends to be reduced during summer stratification when blue-green algae commonly form surface blooms, it may be important in some water bodies such as shallow reservoirs subject to significant wind mixing throughout the summer. As can be seen in Figure 21, the most important environmental variable in regulating the potential formation of surface blooms of gas vacuolate blue-green algae appeared to be the light intensity. Anabaena flos-aquae was non-buoyant at light intensities above 25 pE/mz/sec regardless of the availability of carbon dioxide or nitrogen. Van Rijn and Shilo (1983) also report that among light, carbon dioxide, and nitrogen, light takes precedence in buoyancy regulation of Oscillatoria spp. In my research when the light intensity was less than 25 pE/mz/sec, the availability of carbon dioxide and NH4—N became important in buoyancy regulation. These two 86 parameters had opposite effects on buoyancy; carbon dioxide depletion generally increased buoyancy, while reduced NH4-N availability reduced buoyancy. Similar findings have been reported by Klemer 31 31. (1982) and Van Rijn and Shilo (1983) . Mechanisms for Buoyancy Regulation In the preceeding text, evidence was presented for regulation of buoyancy and growth of Anabaena flos-aguae as a function of a three-way interaction between light, carbon dioxide, and nitrogen. Little mention was made of mechanisms to explain how changes in these three environmental variables caused the observed buoyancy responses. Walsby and co-workers have shown that cell turgor pressure may control buoyancy of Anabaena flos-aquae through the collapse of pwessure sensitive gas vacuoles by high turgor pressures (Walsby, 1971; Dinsdale and Walsby, 1972; Grant and Walsby, 1977; Allison and walsby, 1981). Such high turgor pressures may develop under elevated light intensities due to the rapid accumulation of low molecular weight compounds. In my research, the turgor pressure of Anabaena flos- aguae tended to increase with increasing light intensities (Figure 22). Using these data together with evidence from Walsby and co—workers, the lack of positive cell buoyancy of Anabaena flos-aquae at light intensities greater than 25 )JE/mz/sec (Figure 21) may be explained by collapse of gas vacuoles by high turgor pressures. 87 75" (psi) 13 I TURGOR PRESSURE 50 1 F I 02 E IO 20 50 LlGHT INTENSITY (pE/mz/sec) Figure 22. Cell turgor pressure as a function of light intensity for Anabaena flos-aqgae (195% confidence intervals). 88 At low light intensities, turgor pressures were reduced (Figure 22) and, as discussed previously, Walsby has shown that gas vacuoles are not collapsed under these conditions. It has been proposed that algal buoyancy in this case is regulated by the rate of synthesis and accumulation of gas vacuoles (Walsby, 1970; Reynolds, 1972; Reynolds and Walsby, 1975; Konopka, 1983). In my research, cell buoyancy at light intensities of 10 pE/mz/sec or less appeared to be related with the cellular C/N ratio (Figures 7 to 11). Changes in C/N ratios in these experiments were related with changes in the availability of carbon dioxide and NH4-N. It appears that under low light intensities, the cellular C/N ratio, as controlled by the availability of carbon dioxide and NH4-N, is involved in regulating the rate of formation and accumulation of gas vacuoles. A number of recent studies have shown that the buoyancy of various blue-green algae may be controlled by the availabilty of carbon dioxide or inorganic nitrogen (Paerl and Ustach, 1982; Klemer e1 31., 1982; Van Rijn and Shilo, 1983). The availability of carbon dioxide and/or inorganic nitrogen affect algal C/N ratios, as reported or inferred from another set of studies on various blue-green and non-blue-green algae (Ward and Wetzel, 1980; Goldman e_t_a_1. , 1979; Galloway, 1980; Morris, 1981; Konopka and Schnur, 1981) . However, no one has suggested a causal relationship between cellular C/N ratios and blue- green algal buoyancy. 89 loo-1) . I O Q%@O O 0 o A O 0. 3° 80; M a (a .. 0 :j A‘3 UJ 0‘1) . U 60- ‘0, F- O 3 S ‘ ' C C E; ‘3 I 4()' >. .J C ">’ e E ‘ ‘ g; 0 0_ 2(3- ' I I C C C I IA ‘.l (7 ‘ I ' l ' I *7fl,‘h"-T'.b— I ' I ' I C/N RATIO Figure 23. Percent positively buoyant cells as a function of the cellular C/N ratio at low light Intensities. (O) 10 pE/mZ/sec without NH4-N, (O) 10 pE/mZ/sec with NH4-N, (I) 2SpE/mZ/sec without NH4-N, (0)5 pE/mi /sec with NH4-N, (A) 2 uE/m2 /sec without NH4-N, and (A) 2 pE/m2 /sec with NH4-N. 90 The percent of positively buoyant cells is plotted as a function of the cellular C/N ratio of Anabaena flos-aquae in Figure 23. Data in this figure comes from growth experiments conducted at 2, 5, and 10 pE/mz/sec, with and without NH4-N. Data collected during the period of algal deterioration at high pH and very low carbon dixoide levels occurring near the end of these experiments were not included in Figure 23. When the cellular C/N ratio was greater than about 6.0 to 6.5, cell buoyancy tended to be low (Figure 23) . AS described in.the Results and Discussion section, this generally occurred in microcosms without inorganic nitrogen when carbon dioxide concentrations were high. Under these conditions, the relative rate of carbon fixation and accumulation appeared to be greater than that for nitrogen relative to algal needs. 'This resulted in high cellular C/N ratios and reduced cell nitrogen concentration. In this condition it is probable that available cell nitrogen was directed more into cell growth than to synthesis of protein-rich gas vacuoles. Consequently, cell buoyancy was low. Conversely, when the cellular C/N ratio at low light intensities was less than 6 to 6.5, Anabaena flos-aquae tended to be buoyant (Figure 23). In microcosms without NH4-N, this generally occurred at low carbon dioxide concentrations. Under these conditions, it appears that the rate of carbon fixation and accumulation was high relative to that for nitrogen. ‘With abundant NH4—N, the cellular C/N ratio was generally low 91 throughout the low light experiments at all carbon dioxide levels. In either case, with low cellular C/N ratios, accumula- tion of nitrogen was evidently high relative to accumulation of cellular carbon. With relatively high cell nitrogen concentrations, synthesis of gas vacuoles could continue unimpeded by the demand for nitrogen for cell growth. As would be expected, cell buoyancy was elevated under these conditions. At light intensities of 20 and 50 pE/mZ/sec, the cellular C/N ratio did not appear to be related with cell buoyancy (Figure 24). While Anabaena flos-aquae cultures with C/N ratios above 6.0 were non-buoyant, there were actually more cases where the algae were non-buoyant with C/N ratios well below 6.0. From the preceeding discussion, gas vacuole syn- thesis would be expected to increase at these low'C/N ratios. While this may have occurred, increased cell turgor pressures at these elevated light intensities would prevent the accumula- tion of gas vacuoles by collapsing them. In summary, it appears that cell buoyancy is reduced at high light intensities by increased cell turgor pressures which cause collapse of gas vacuoles. At reduced light intensities, there is a three-way interaction between light, carbon dioxide, and nitrogen which appears to regulate the cellular C/N ratio which in turn controls gas vacuole synthesis and cell buoyancy. A Buoyancy Regulation Model In the preceeding discussion a case is presented for regulation of cell buoyancy by either cell turgor pressures 92 I00.' ,3 0(>- a 3‘ a) . .J _.| DJ 0 60- p. <2: 6 d 8 0 ° 443“ 5 “J 0 Z d '2 (D g: 2(7- .l ()fi 1 W—‘F j 2 3 4 5 6 7 8 9 IO C/N RATIO Figure 24. Percent positively buoyant cells as a function of the cellular C/N ratio at high light intensities. (O) 50 pE/mZ/sec without NHé-N, (O) 50 pE/mZ/sec with NH4-N, (I) 20 uE/mZ/sec without NH4-N, and (o) 20 pE/mZ/sec with NH4—N. 93 or cellular C/N ratios. Changes in these two regulators were correlated with changes in the availability of light, carbon dioxide, and NH4-N. An integrative mechanistic model shown in Figure 25 was constructed which illustrates how these three environmental parameters may interact to control the regulators which determine cell buoyancyu 'This conceptual model is based on results from this study together with existing published theories. As shown in Figure 25, light, carbon dioxide, and ammonia-nitrogen are involved in a number of metabolic pro- cesses which interact tolcontrol both the cell turgor pressure and C/N ratio. These two regulators then interact to control the rate of synthesis and potential collapse of gas vacuoles which together determine whether the algae are buoyant or non- buoyant. When the availability of light is high, photosynthesis proceeds rapidly. This leads to an accumulation of low mole- cular weight compounds (Figure 25), since at elevated light intensities these early photosynthetic products tend to be produced faster than they are metabolized (Grant and Walsby, 1977; Konopka and Schnur, 1980, 1981; Fay and Gibson, 1982). Potassium ions also accumulate intracellularly under these conditions due to a light-driven potassium ion pump (Allsion and Walsby, 1981). The accumulation of potassium ions and low molecular weight products, both of which are osmotically active, causes the cell turgor pressure to increase due to uptake of water by osmosis. Generation of high turgor pressure 94 .aocozosn Hmec ucwHHouucoo paw oHnHmcoomow muoumHawou can .wHoHuoumE .mommuoouo uonHHoo maoHua> :uH3 cowouuH: was .oonoHo cannon .ustH mo mcoHuoououcH moumIDmSHHH sows: Hapoe coaumstou xocmxosm .mm ouswwm 33¢ .0 3.2.60: .22:an 3.29:8 835 3.633 .8905 2395 3.5.5835 £92.25 2o30> woo wannabe. c3822 335:3 . 232$ 30 . .ASO ca: 9830.“. . : I . I, ’ 33:60 7 I 1 3.2.8) 30 I 62 0o. €62.60 .5, ngfiissiv 05¢qu 8300.02 30... . Abhfi. 83:8 6 L63: 3. U 8333 So 95 causes collapse of pressure sensitive gas vacuoles. As shown in my research (Figure 6), the loss of only 10 percent of the cells gas vacuoles may significantly reduce cell buoyancy. Under high light intensities, the turgor pressure regulator shown in Figure 25 is elevated resulting in collapse of gas vacuoles. In this condition, the algae will tend to be in a non-buoyant condition regardless of the cellular C/N ratio or the rate of gas vacuole synthesis (Figure 25). As the availabilities of light and carbon dioxide decline, the rate of photosynthesis and the production of low molecular weight compounds declines. Available photosynthate is anabol- ized into higher molecular weight compounds such as amino acids, proteins, and polysaccharides, and the pool of low mole- cular weight compounds decreases (Grant and Walsby, 1977; Konopka and Schnur, 1980, 1981). The rate of potassium ion uptake also declines (Allison and Walsby, 1981) . Taken together, these events cause cell turgor pressures to decline limiting the collapse of gas vacuoles. Turgor pressures may decrease to an extent where there is no significant collapse of gas vacuoles (Figure 25). At this point, cell buoyancy would appear to be determined largely by the rate of synthesis of gas vacuoles relative to cell growth, as controlled by the cellular C/N ratio regulator shown in Figure 25. The availability of nitrogen becomes important in this model, together with light and carbon dioxide, in determining the cellular C/N ratio. If NH4-N is abundantly available as 96 NH4-N, it is rapidly assimilated and incorporated into nitro- genous cellular materials such as proteins, drawing on the photosynthate pool for carbon skeletons. With available NH4- N, particularly at reduced light intensities, accumulation of nitrogenous compounds is elevated relative to the fixation and accumulation of carbon. This results in low cellular C/N ratios or high protein levels as reported in this study and others (Nihei 35 al., 1954; Ward and Wetzel, 1980; Morris, 1981; Konopka and Schnur, 1980, 1981). It appears that under these conditions, the rate of synthesis of protein rich gas vacuoles is increased relative to cell growth leading to an accumulation of gas vacuoles in the algal cells. Therefore, with abundant ammonia-nitrogen and reduced light intensities, the algae are buoyant as shown in Figure 19b due to low C/N ratios resulting in increased synthesis of gas vacuoles which are not collapsed since cell turgor pressures are low (Figure 25). If the availability of NH4-N is low, the majority of nitrogen supplied to nitrogen fixing blue-green algae must come from reduction of atmospheric nitrogen. This energeti- cally demanding reaction is normally driven by light energy, but also requires a source of reductant and a supply of carbon skeletons (Lex and Stewart, 1973; Winkenbach and Wolk, 1973). Both of these are supplied from the photosynthate pool (Figure 25). The rate of nitrogen fixation therefore is regulated by the availability of light for energy, and the availability of 97 light and carbon dioxide which control the generation of the required photosynthate. Under nitrogen fixing conditions, light and carbon dioxide, the two main environmental parameters which control nitrogen fixation, also control photosynthesis and carbon fixation (Figure 25). Since the rate of carbon and nitrogen fixation both are involved in determining the cellular C/N ratio, control of this buoyancy regulator under nitrogen fixing conditions is complex. Several studies have reported that blue-algae grown at elevated light intensities have high cellular C/N ratios (Ward and Wetzel, 1980; Peterson gt al., 1977). Ward and Wetzel (1980) found that nitrogen fixation saturates at lower light intensities than carbon fixation, which results in elevated cellular C/N ratios at high light intensities. However, in my study the cellular C/N ratios for Anabaena flos-aquae cultures grown at 20 and 50 uE/mz/sec did not follow this trend. The C/N ratios in these cultures were often quite low (Figurer 21). While elevated C/N ratios at high light intensities reported in the aforementioned studies would tend to decrease gas vacuole synthesis and reduce cell buoyancy, the algae would be non-buoyant anyway due to the development of high turgor pressures at elevated light intensities. In summary, the availability of light, carbon dioxide, and nitrogen interact to control a number of cellular processes which determine the cellular turgor pressure and C/N ratio as 98 shown in Figure 25. It is proposed that positive cell buoyancy can be achieved only when both the turgor pressure and C/N ratio are reduced. In all other cases, the algae are non- buoyant (Figure 25). This model is consistant with previous observations of reduced cell buoyancy at elevated light intensities (Figune l9), and high turgor pressures (Figure 22) . Likewise, the model fits evidence from low light intensities where turgor pressures were reduced (Figure 22) and cell buoyancy was correlated with cellular C/N ratios (Figure 23). With abundant NH4-N at low light intensities, C/N ratios were low and buoyancy was high regardless of the carbon dioxide concentration (Figures 19b and 23). Without NH4-N at low light intensities, carbon dioxide had to be reduced beforerC/N ratios were reduced sufficiently for algal buoyancy to increase (Figures 19a and 23). In addition to turgor pressures and the rate of gas vacuole synthesis, a third mechanism recently has been proposed as a potential regulator of blue-green algal buoyancy. This involves control of cell density through the accumulation of heavy ”ballast” molecules (Figure 25). Walsby gt El- (1983) and Oliver gt’ El- (1983) have reported that the alga Oscillatoria thiebaetii has extremely strong gas vacuoles which can withstand high cell turgor pressurs. They suggest from preliminary evidence that buoyancy in this algae may be controlled by the accumulation of heavy polysaccharides or other heavy materials. 99 Preliminary Evidence for Anabaenopsis The preceeding model and discussion have been based primarily on data collected for the alga Anabaena flos-aquae. Preliminary data were collected on another gas vacuolate, nitrogen-fixing alga, Anabaenopsis Elenkinii. 1x tentative buoyancy "niche" was constructed for this alga (Figure 26a) to allow comparison to the buoyancy niche of Anabaena flos- aquae shown in Figure 18 and again in Figure 26b. It is clear from Figure 26 that light, carbon dioxide, and ammonia-nitrogen interact to regulate the buoyancy'of.Anabaen9psis Elenkinii in a manner similar to that described for Anabaena flos-aquae although their respective buoyancy niches differ. Anabaenopsis Elenkinii appears to be buoyant under a wider range of conditions than Anabaena flos-aquae, as shown in Figure 26. However, Anabaena flos-aquae is a frequent dominant algae found in eutrophic lakes, while Anabaenopsis Elenkinii is less common in lakes although it may be found in abundance in sewage oxidation ponds (Marx, 1980; Spencer, 1981). From Figure 26, it appears that Anabaenopsis Elenkinii has a buoyancy advantage over Anabaena flos-aquae at elevated light intensities and low carbon dioxide concentrations. However, these conditions may not be found commonly in lakes. In general, if there is sufficient algal photosynthesis to significantly reduce carbon dioxide levels, light penetration is also likely to be reduced due to the presence of dense 100 BUOYANT ZONE 3| WITH ”Hi" BUOYANT ZONE .6 ‘ WITHOUT NH -N W \\ “ 3.l6- .$ ‘2 C; ""fl"”4:iu’n‘v"pr"r' 9' Iv S \ 9 9 .. 9‘9‘9‘9‘9.‘¢ 9 0.32‘ . “““‘.O.. ~ o‘.¢¢.o.o.oo 8 I 9‘ 9 9 9 9 9‘ «t‘k‘afig‘fi! 093‘ ANABAENQP§|§ _ — om ELENKINIIf j ' ' 1 0 IO 20 so 40 so LIGHT INTENSITY (uE/mzsec) SLGT mwm- uouNT zone l0.0d \ \ IIITH tug-N O‘. \ .. 1w l "’"‘ L 5 |\%’9 S '-°°‘ 999.0,. E . §$$$$v§ auouNT ZONE 3 0.32, wag; 9‘ wITHOUT all-I444 L \. ,____ O OIO- \“\ "" 0.03. ANABAgNA Em,“ F_LO§-AQUAE Threshold 0'0! 0 1 2'0 3'0 4'0 sir I30 LIGHT INTENSITY mm (uE/mzsec) Figure 26. Buoyancy niche of (a) Anabaenopsis Elenkinii and(b) Anabaena flos-aquae relative to light intensity and carbon dioxide concentration. lOl phytoplankton populations. Anabaena flos—aquae appears to have the buoyancy advantage under conditions of reduced light, carbon dioxide, and inorganic nitrogen, and these conditions may be found more frequently in eutrophic lakes. Blue-Green Algae in Lakes The buoyancy niches and models presented here may be applied in a broader context in explaining various field obser- vations of the location, timing, and physical and chemical conditions in lakes where blooms of various gas-vacuolate blue- green algae are found. If the light intensity is high in the upper epilimnetic waters of lakes, the potential for formation of surface blooms of blue-green algae should be greatly reduced as shown in Figure 21 due to collapse of gas vacuoles under high turgor pressure. Numerous field studies support this contention (Walsby and Klemer, 1974; Konopka, 1982; Lynch and Shapiro, 1981; Spencer, 1981). In general, light penetration is high inIoligotrophic lakes and surface blooms of blue-green algae generally are absent under these conditions. Of course, light intensities decline with depth and unless a lake is shallow, there will normally be some depth‘where the light intensity is reduced below 25 pE/mz/sec even in Oligo- trophic lakes. Therefore, in clear water, stratified lakes, if gas vaculate blue-green algae are present, they may be stratified well below the surface. Field reports of deep chlorophyll maxima are common in lakes with high light 102 penetration (Moll and Stoermer, 1982) and these populations often are dominated by blue-green algae such as Oscillatoria (Walsby and Klemer, 1974; Konopka, 1982; Saunders, 1972) . These studies report well defined populations of blue-green algae stratified at depths of six meters or more. If the algae are artifically removed from this zone, they tend to adjust their buoyancy to re-equilibrate within this preferred light zone (Konopka, 1982). I have found no reports of Anabaena flos-aquae forming well defined deep water maxima. Water temperatures in the deep water metalimnetic regions of lakes are cooler than near surface temperatures, and this may explain the absence of Anabaena in this region. .Anabaena and many other common bloom forming blue-green algae are reported to have a warm temperature growth optimum while Oscillatoria appears to grow well at low temperatures and has been reported to form occasional blooms in ice covered lakes (Hutchinson, 1967). While mid-summer blue-green populations may stratify at deep depths in less enriched lakes, the maximum phytoplankton standing crop in most oligotrophic and mesotrophic north temperate lakes normally occurs during the period of spring turnover (Hutchinson, 1967, F099, 1975). These algal populations commonly are dominated by diatoms in early spring followed by green algae, flagellates, and other non-blue-green algae (King, 1970; Moss, 1980; Wetzel, 1975). Decline in the spring and early summer phytoplankton populations normally is 103 associated with thermal stratification of the lake and decreased availability of phosphorus and occasionally other nutrients in the epilimnion (Lund, 1950, 1954, 1971; Fogg, 1975). Phosphorus enrichment leading to increased spring and early summer non-blue-green.algal.populations causes decreased light availability and tends to increase the potential for the formation of surface blue-green algal blooms by increasing cell buoyancy (Figure 21). Phosphorus enrichment may also stimulate algal photosynthesis sufficiently to cause depletion of carbon dioxide (King, 1970, 1972) which also may increase blue—green algal buoyancy and increase bloom potential (Figure 21). Previous studies'have hypothesized that blue-green algal dominance, commonly correlated with low carbon dioxide concentrations, may be explained by superior growth rates of blue-green algae under these conditions (King, 1970, 1972; Shapiro, 1973). However, comparison of physiological growth rates of a variety of algal types at low carbon dioxide levels generally reveal no clear advantage for blue-green algae (King, 1980; Repko and Spencer, unpublished data). The sinking rates of diatoms and green algae have been reported to increase with increased stress on the algae imposed by decreased availability of light and carbon dioxide (Jaworski st 31., 1981; Smayda, 1970; King and Hill, 1978; Sharp, 1977; Titman and Kilham, 1976; Kairesalo, 1980). In addition, merely 104 reducing the growth rate of negatively buoyant algae by reducing light and carbon dioxide availability will tend to increase their relative loss rate from the photic zone under stratified conditions (King and Hill, 1978; Repko, unpublished data) . Therefore, with increased phosphorus availability and decreased levels of light and carbon dioxide, an important premium is placed on being able to remain suspended in the photic zone. While reduction of the availability of light and carbon dioxide will likely decrease the physiological growth rates of gas vacuolate blue-green algae too, as shown in Figure 20, the overall potential for the formation of surface blooms of these algae is a function of both growth rate and buoyancy. For Anabaena flos-aquae, reductions of growth rate at reduced light and carbon dioxide levels were more than offset by increases in cell buoyancy within a zone of decreased light and carbon dioxide shown in Figure 21. It is becoming increasingly evident that as nutrient enrichment and eutrophication of lakes progresses, inorganic nitrogen levels commonly become reduced in addition to levels of light and carbon dioxide. In many eutrophic lakes, inorganic nitrogen concentrations decline to very low levels by mid to late summer (Keeney, 1973; King, 1979; Spencer, 1981). Nitrogen depletion in eutrophic lakes occurs as a result of several processes including: plant uptake, ammonia volatili- zation, and denitrification. All of these processes tend to 105 be enhanced by phosphorus enrichment. Addition of the commonly limiting nutrient, phosphorus, will normally increase photo- synthesis and plant uptake of inorganic nitrogen. Increased photosynthetic activity in eutrophic lakes resulting in carbon dioxide depletion will increase epilimnetic pH values which enhances the loss of ammonia through volatilization to the atmosphere (Weiler, 1979; Galloway, 1980; Murphy and Brownlee, 1981). Finally, increased production of organic matter and decreased oxygen availability common in the hypolimnion of eutrophic lakes will tend to enhance denitrification losses. Nitrogen limitation in eutrophic lakes is commonly asso- ciated with the presence of blooms of nitrogen-fixing blue- green algae (Dugdale and Dugdale, 1962; Vanderhoef 35 31., 1974; Fogg, 1975; Schindler, 1977) . This has led to the common conclusion that nitrogen fixing algae become dominant under these conditions because inorganic nitrogen levels declined to such low levels that these algae have superior growth rates compared to algae which cannot fix nitrogen. While the phy- siological growth rates of nitrogen fixing algae are certainly higher than non-nitrogen fixers when inorganic nitrogen is in short supply, the results presented in this study indicate that the buoyancy and potential for surface bloom formation of the nitrogen fixer Anabaena flos-aquae is greatly reduced when inorganic nitrogen is not available (Figure 21) . Anabaena flos-agae would appear to be much more likely to form a surface bloom if some ammonia—nitrogen was readily available. 106 The presence of inorganic nitrogen appears to be most important during the initial establishment of a surface bloom of Anabaena flos-aquae. If the blue-green algal bloom succeeds a preceeding bloom of green algae and diatoms, a ready source of inorganic nitrogen may be supplied at this time by stressed and declining populations of non-blue-green algae, which through leakage and decay, may release nitrogen to the water column. Significant releases of nitrogen have been reported following deterioration of algal populations (Brezonik, 1972; Galloway, 1980; Spencer, 1981). IHowever, once»a surface bloom of Anabaena flos-aquae is established, the bloom may persist in the absence of any inorganic nitrogen as long as the upper epilimnetic levels of light and carbon dioxide remain sufficiently reduced to maintain the algae in the buoyant zone shown in Figure 21b. Such reduced light and carbon dioxide conditions would often be expected in lakes containing dense surface blue-green algal blooms. 'The growth rate of the algal population may be reduced under these conditions; however, the blue-green algal bloom may continue to persist for the remainder of the summer, as is often observed in eutrophic lakes. In summary, phosphorus enrichment and eutrophication of lakes tends to create conditions favorable for the formation of surface blooms of blue-green algae. Increased phosphorus availability often stimulates spring and early summer blooms of diatoms, greens, and other algae which subsequently tend to reduce light penetration and carbon dioxide levels in the 107 water column. As the water temperature rises and the lake stratifies, depletion of light and carbon dioxide tend to increase the sinking and loss rates of non-blue—green algae while increasing the buoyancy of gas-vacuolate blue-green algae. If phosphorus remains available, blue-green algal growth will continue and a surface bloom may form, particularly under calaneather conditions when vertical mixing is reduced. While nitrogen depletion gives a physiological growth advantage to nitrogen fixing blue-greens, establishment of surface blooms of these algae in addition to non-nitrogen fixing blue-green algae is enhanced by the presence of. inorganic nitrogen. Once a surface bloom of blue-green algae is established, the bloom itself may create conditions of reduced light and carbon dioxide favorable for persistence of nitrogen fixing blue-greens such as Anabaena flos—aquae even in the face of severe inorganic nitrogen depletion. From the data and discussion presented here, it appears that development of a surface bloom of buoyant blue-green algae requires a preceeding bloom of non-blue-green algae to create favorable conditions of reduced light and carbon dioxide. While phosphorus enrichment of lakes normally stimulates the formation of the requisite preceeding algal blooms, other factors may interfere with the development of these preceeding blooms such as zooplankton grazing or depletion of other nutrients or micronutrients. 108 [j NON'BLUEGRE EN V 71 BLUEGREEN J> o l OJ 0 I N W PHYTOPLANKTON (mm3/fl) a 8 l I POND l POND 2 POND 3 POND4 Figure 27. Seasonal means of the density of nonblue-green and blue-green phytoplankton in Ponds 2 and 3 with sparse cladoceran zooplankton, and Ponds 1 and 4 with abundant cladoceran zooplankton. 109 Additional evidence to support the dependence of surface blue-green blooms on preceeding algal blooms is provided in a recent field study conducted in four hypereutrophic ponds (Spencer, 1981). In this study, the development of spring and early summer blooms of non-blue-green algae was prevented by elevated cladoceran zooplankton grazing pressures in two of the ponds. In contrast, sparse cladoceran populations in the other two ponds allowed establishment of dense populations of diatoms, green algae, and flagellates in spring and early summer. Mean seasonal phytoplankton standing crops in the four ponds are shown in Figure 27. In Ponds 2 and 3, with sparse cladoceran densities, light penetration was severely reduced in the water column with Secchi disc measurements often falling to less than 0.5 meters. Early season blooms of non-blue- green algae were replaced in mid-summer by dense blue-green algal blooms which persisted until late summer. The dominant blue—green algae were Anabaenopsis Elenkinii and Microcystis sp., both of which contain gas vacuoles. In Ponds l and 4 with dense cladoceran populations, light penetration was quite high and light intensities greater than 100 pE/mz/sec were frequently measured at the pond bottom at a depth of 1.8 meters. Blue-green algae were rarely encountered in these ponds, with the exception of a single small bloom in Pond 4 (Figure 27). This bloom was immediately preceeded by a bloom of diatoms and flagellates which reduced light penetration in Pond 4 for a short period of time. 110 Other researchers have also reported the reduction or disappearance of blue-green algal blooms in eutrophic lakes following increases in zooplankton populations and associated increases in light penetration (Schindler and Comita, 1972; Shapiro, 1979; Lynch and Shapiro, 1981). Increases in zooplankton densities in these studies occurred following reduction of pflanktivorous fish populations through winter kills or other means. The preceeding discussion and models concerning the role of light and nutrients in regulation of blue-green algal population in lakes may be used in evaluating the appearance and competitive advantage of various types of common bloom forming blue-green algae. There are a number of different types of gas vacuolate blue-green algae, each of which may gain a competitive advantage over the other types under certain conditions. One major distinction among the gas vacuolate blue-green algae is whether or not they are capable of nitrogen fixation. This ability'is primarily restricted to those algae which contain heterocysts, such as Anabaena, Aphanizomenon, and Anabaenopsis. When the availability of inorganic nitrogen is low and blue-green blooms are present, they are often dominated by nitrogen fixing species as mentioned previously. Nitrogen fixing blue-greens should have a competative advantage under these conditions since they can supplement their nitrogen needs through fixation of nitrogen gas. However, nitrogen fixation is quite energy demanding and may be disadvantageous 111 when inorganic nitrogen is available in abundance. Common examples of non-nitrogen fixing blue-greens which may dominate when inorganic nitrogen is readily available include Microcystis and Oscillatoria. A second major distinction between common gas vacuolate blue-green algal types lies in the growth form of the algae. Common bloom forming blue-green algae usually are found in either of two forms: large colonies of cells, or individual cells or filaments. Large colonies may consist of numerous individual cells packed together in a common mucilage such as Microcystis, or tightly'packed bundles of individual filaments such as Aphanizomenon which may form visible green "flakes". Common blue-greens that normally form individual filaments include Oscillatoria, Anabaena, and in some cases Aphanizomenon. In general, the growth rate of individual cells or filaments is higher than for large colonies due to their greater surface/volume ratios promoting more effective nutrient uptake (Smith _e_t a_l., 1981; Holm e_t_ a_l., 1983; Ganf and Schache, 1983). Several possible advantages have been suggested for large colonies. Walsby gt 31. (1983) suggests that large colonies of gas vacuolate blue-green algae have faster vertical migration rates than individual cells or filaments of similar density because of reduced friction associated with a larger particle size according to Stoke's Law. A second suggested advantage of large colonies is resistance to zooplankton grazing (Holm _e_t a_l. , 1983) . Although I 3 112 grazing and growth of zooplankton on blue-green algae often is considered negligible (Arnold, 1967; Porter, 1973), Holm t al. (1983) have shown that large colonies of Aphanizomenon are grazed much less effectively than single Aphanizomenon filaments. Reduced vulnerability to grazing is suggested as an explanation to their field observations of the presence of large Aphanizomenon flakes in clear lakes in the presence of abundant Cladocera. In the absence of abundant Cladocera, singleuAphanizomenon filaments were more commonly observed due to their superior growth rates in the absence of significant grazing pressures (Smith gt gt., 1981; Holm gt 21., 1983). The data and models presented in this paper suggest another potential advantage for the formation of large colonies of gas vacuolate blue-green algae. Algal cells in the interior of large colonies are exposed to reduced light and carbon dioxide as compared with exterior cells, individual filaments, or single cells. With such reductions in light and carbon dioxide, interior cells should become buoyant at higher external light and carbon dioxide levels than individual cells or filaments. Increased interior cell buoyancy' may result in positive buoyancy for the entire colony under light and carbon dioxide levels where individual cells or filaments are non-buoyant. This mechanism provides an alternative explanation to the interaction between Cladoceran density and Aphanizomenon growth form described by Holm gt gt. (1983). In those lakes with elevated Cladoceran densities, light penetration was 113 increased by removal of phytoplankton by zooplankton grazing. The appearance of Aphanizomenon flakes under these conditions may be because large colonies represent the only growth form which was buoyant under the high light intensities found in these lakes. A similar hypothesis concerning the advantage of large colony size was advanced recently by Paerl (1983) who suggested that large Microcystis colonies may become more buoyant because of reduced availability of carbon dioxide to interior cells. Management of Blue-Green Algal Blooms Regardless of the type of gas vacuolate blue-green algae, the appearance of blue-green algal blooms in the surface waters of lakes represents a serious degradation of water and lake quality. While this is generally true in areas where recreation and water quality are the primary lake management concerns, it should be noted that maintenance of blue-green algal blooms may be desirable in some areas of the world such as parts of China, Japan, and Africa. In these areas, blue-green algae are used as a food source for humans, fish, and/or livestock (Watanabe, 1970; Moriarty and Moriarty, 1973; F099 gt gt., 1973; Pirie, 1975). Blue-green algae may also be considered desirable since in some cases they may increase the production of rice and other crops through the supply of nitrogen through nitrogen fixation (Fogg gt gl., 1973). Although my research has been directed at predicting conditions under which blue- green algal blooms may form and how these algal blooms may be prevented, this same information may potentially be turned 114 around and be useful in attempts to enhance the production of blue—green algae where this is desirable. A number of different management alternatives are presently in use now, or have been proposed, aimed at the elimination, reduction, or prevention of the formation of blue- green algal blooms. Management techniques can be classified into two basic categories: chemical control or biological control. Chemical Control The appearance of blue-green algal blooms in lakes has been correlated repeatedly with increased nutrient inputs, particularly phOSphorus. From the interacting mechanisms presented here, it is clear that reduction of phosphorus availability represents the most effective means of reducing or eliminating the appearance of surface blue-green algal blooms in lakes. Such phosphorus reduction would reduce total algal production and increase the availability of light and carbon dioxide, all of which would reduce the buoyancy and growth of blue-green algae. The best known example of successful rehabilitation of a eutrophic lake plagued by blue-green algal blooms through reduction of phosphorus inputs is Lake Washington (Edmondson, 1972). In many cases, however, significant reduction of phosphorus inputs are not feasible or are too costly to engineer. In these cases, alternative management strategies often are considered. 115 The most common alternative to phosphorus control is another chemical control method and involves the use of algicides, such as various forms of copper sulfate. While such treatments are generally effective in killing the algae, there are a number of problems with the use of algicides. Chemicals must be added repeatedly during the growing season since algae (n: other plants often become re-established following treatment. Repeated chemical treatments are expensive and may result in accumulation of undesirable levels of toxins in the lake. Another possible side effect of chemical control of plant growth is severe oxygen depletion following bacterial degradation of large quantities of dead plant material. A recent paper by Smith (1983) suggests another method of chemical control of blue-green algal blooms. Smith gathered lake data from a variety of sources and found a correlation between low nitrogen/phosphorus ratios and dominance of lakes by blue-green algae. He suggests that the addition of inorganic nitrogen to such lakes thereby increasing the nitrogen/ phosphorus ratio may result in a reduction of blue-green algal dominance. The addition of nitrogen for this purpose also have been proposed by others (Flett gt gt., 1980; Barica gt gt., 1980; Murphy and Brownlee, 1981). However, my research indicates that the addition of inorganic nitrogen to lakes with low nitrogen availability' may’ greatly increase the potential for the formation of Anabaena flos-aguae blooms. 116 Similar conclusions may be drawn from studies of Oscillatoria where addition of inorganic nitrogen increased the buoyancy of this alga as well (Walsby and Klemer, 1974; Konopka gt gt., 1978). Further cautions to the addition of inorganic nitrogen as a management alternative come from observations of eutrophic surface waters receiving high inputs of inorganic nitrogen which are dominated by dense blooms of non-nitrogen fixing blue-green algae (Zevenboom and Mur, 1980; Paerl, 1983; Reed, 1969). Biological Control A number of researchers have suggested the potential control of blue-green algal blooms through the introduction of viruses to attack and destroy' blue-green. algal. cells (Safferman and Morris, 1964; Goryushin, 1967; Walsby, 1970). The success of such procedures have only been demonstrated on a small scale, and appear limited by the specificity of each virus which may attack only a single species or strain of blue- green algae. The culture of large quantities of viruses also may be difficult and expensive. Furthermore, resistant strains of blue-green algae may develop following treatment. Another biologically mediated technique for potential control of nuisance blue-green algal blooms involves biomanipu- 1ation of fish populations (Shapiro gt gt., 1975). Shapiro suggests that by increasing the density of large piscivorous fish, or by totally eliminating all fish, zooplankton densities and grazing pressures will be increased and nuisance phyto- plankton blooms may in turn be reduced or eliminated in 117 eutrophic lakes. This has been demonstrated among others by Schindler and Comita (1972), Helfrich (1976), Shapiro (1979), Lynch and Shapiro (1981), and Spencer (1981). The data and discussion presented from my research suggests that the dis- appearance of blue-green algal blooms following biomanipula- tion may result indirectly from increased zooplankton grazing activity through removal of grazable non blue-green phytoplank- ton thereby increasing the availability of light and/or carbon dioxide causing reductions in blue-green algal buoyancy. There are a number of problems with biomanipulation as a management method. While undesirable phytoplankton popula- tions may be reduced with this technique, increased light availability may subsequently lead to undersirable increases in growth of submerged macrophytes and periphyton (Hurlbert gt gt., 1972; Spencer, 1981). Another potential complication is the occasional appearance of large colonial blue-green algae such as Aphanizomenon under the resulting high light conditions as observed by Lynch and Shapiro (1981) and Holm e_t _a_]_._. (1983) . Finally, the required management of the fish populations may be neither feasible to maintain nor desirable to all lake users. In summary, the inevitable consequence of continued phosphorus enrichment of most lakes is eventual dominance of the phytoplankton by nuisance blue-green algal blooms. The continuing inability to design and carry' out successful management strategies for controlling blue-green algal blooms has led to desperate extremes such as suggesting the detonation 118 of explosives in afflicted lakes to collapse gas vacuoles in the blue—green algae, causing them to sink (Walsby, 1968, 1970) . A number of management strategies have been presented; however, most are aimed only at treating the symptoms of the problem. Despite important interactions between carbon, light, and nitrogen in regulating blue-green algal blooms, effective reduction of the availability of phosphorus represents the only management strategy which treats the ultimate cause of surface blue—green algal blooms. CONCLUS IONS The buoyancy and growth of Anabaena flos-aguae was regu- lated by a three-way interaction between the availability of light, carbon dioxide, and ammonia-nitrogen. At elevated light intensities, Anabaena flos-aguae was non-buoyant regardless of the availability of carbon dioxide and nitrogen. At low light intensities, the availability of carbon dioxide and ammonia—nitrogen were important in buoyancy regulation of Anabaena flos-aquae» These two parameters had Opposite effects on buoyancy: decreased carbon dioxide levels generally increased algal buoyancy, while decreased ammonia-nitrogen availability decreased buoyancy. An index to the potential formation of surface blooms of Anabaena flos-aquae was provided by multiplying the specific algal growth rate by the percent positively buoyant cells yielding a maximum potential bloom doubling time of 5.4 days in the presence of NH4-N and 22 days without NH4-N, 119 120 Regulation of algal buoyancy, in response to changes in the availability of light, carbon dioxide, and ammonia- nitrogen, appeared to be mediated by interactions between cell turgor pressures which controlled the collapse of gas vacuoles; and cellular C/N ratios which controlled the synthesis rate of gas vacuoles. Increased phosphorus availability in lakes, that leads to increased algal production and decreased availability of light and carbon dioxide, creates conditions favorable for the formation of blue-green algal blooms by increasing the loss rates of non-blue—green algae while increasing the buoyancy of gas vacuolate blue-green algae. While eutrophic lakes often become nitrogen limited, the presence of available inorganic nitrogen will greatly increase the potential for the establishment of surface blue-green algal blooms. The inevitable consequence of continued phosphorus enrichment of most lakes is eventual dominance of the phytoplankton by surface blue-green algal blooms. APPEND ICES 121 APPENDI X I Composition of Algal Nutrient Medium Nutrient Concentration NaHCO3 168.00 mg/l CaCLz . 2H20 20.00 mg/l FeC13 . 6H20 6.66 mg/l MgSO4 . 7H20 40.00 mg/l KH2P04 30.3 mg/l NH4C1 Variable EDTA . 2H20 11.07 mg/l Thiamin . HCl 0.10 mg/l Biotin 0.5 ug/l B 2 0.5 ug/l Microelement solution 1 ml/l Composition of Microelement Solution Nutrient Concentration H3303 2.86 g/l MnC12 . 4H20 1.81 g/l CuSO4 . SHZO 0.08 g/l ZnSO4 . 7H 0 0.22 g/l Na M004 . gaze 0.24 g/l C0012 . 6320 0.40 g/l |22 mmH.o «Mazda uo>oz so“: mmINHIN cm mHH.o mamza< uo>oz agony“: moINHIN om ovo.c mac.o mm.v suds mmIHIN cm mmvo.oum mpo.aum mvc.o mamzfla 00>»: agony“: mmINHIN on wvc.c mqo.c cfl.cv usonuaz moIvIOH aw moH.aum mmH.oum m~..0um ov.~.ouw mmfiLWm ch.o ov°.c pm.v~A can: masouum SH mm~.c mam.o mc.wmA and: maIAIw ca vac.c qqc.o mm.~mA so“: mmINHIN cu S«H.oum oo~.°nm nm.Hum mNH.oum qu.oum mn.¢um vmc.c vmc.c mm.q usoauaz «oIescH ca who.o msc.o po.m agony“: mouumum cu mqm.o uwv.o Np.u uaoauaz moIHIe cu mno.a omo.o HH.~ agony“: moINHIN ea mNH.o m~.o m.mn spas maumuuu m mom.o m.o o.m agony“: mouuuum m m.om uo>uz H.Ho no“: mmIHIm u n.mq msosaa uu>oz guano“: maIHIG N laxmuaoea. AH\uoHoe:. .H\muaoea. zuvmz mama oomxna\ua .vmv umodm Ou swoon uncut ou :«mom acoaquomxm huqucoucu oaoamouna muons Noo mums: Noo Osman moo ucdomxnoum acuomeONQ .ousmmImOHu ncomnocd uom .oH gunman. onofiz aocmhosm on» ausuumcoo 0» comb anon .N Nuccammc LI ST OF REFERENCES LIST OF REFERENCES Allison, F.E., S.R. Hoover, and H.J. Morris. 1937. Physiological studies with the nitrogen fixing algal Nostoc muscorum. Bot. Gaz. 2§:433-463. Allison, E.M. and A.E. Walsby. 1981. The role of potassium in the control of turgor pressure in a gas-vacuolate blue- green alga. gt Exp; Bot. 33:241-249. Arnold, D.E. 1971. Injection, assimilation, survival, and reproduction by Daphnia pulex fed seven Species of blue-green algae. Limnol. Oceanogr. t§:906-920. Barica, J., H. Kling, and J. Gibson. 1980. Experimental manipulation of algal bloom composition by nitrogen addition. Can. gt Fish. Aquat. Sci. glzll75-ll83. Booker , M.J. and A.E. Walsby. 1979. 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