. . .HW. . . . “Nagy?” llquyuzllymluullmuswlW mun Michigan State University {HES This is to certify that the thesis entitled Factors Influencing Specific Growth Rates and Seasonal Abundance of Eutrophic Lake Phytoplankton presented by Gary F. Marx has been accepted towards fulfillment of the requirements for Ph.D. degree in Botarg and Plant Pathology QNIMU“ Major professor 0-7639 OVERDUE FINES ARE 25¢ PER DAY V PER ITEM 0 \ Return to book drop to remove w this checkout from your record. E; I" Q. “Ni. FACTORS INFLUENCING SPECIFIC GROWTH RATES AND SEASONAL ABUNDANCE OF EUTROPHIC LAKE PHYTOPLANKTON By Gary F. Marx A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1979 ABSTRACT FACTORS INFLUENCING SPECIFIC GROWTH RATES AND SEASONAL ABUNDANCE OF EUTROPHIC LAKE PHYTOPLANKTON By Gary F. Marx The seasonal succession of dominant phytoplankton populations and the daily concentrations of major nutrients in a central Michigan waste treatment basin were monitored over the 1977 growing season in order to study the factors controlling the growth of the various species. A short bloom.of a centric diatom, chlotella Sp., immediately after ice break was followed by a bloom of a small flagellated green alga, Chlamydomonas sp. These dominant species were followed by the coccoid green alga, Scenedesmus communis which produced the largest standing crop of the year. Two small blooms of Pandorina morum, a flagellated green alga, followed and a large growth of the nitrogen fixing blue- green alga Anabaengpsis elenkinii subsequently dominated from mid-July through late August. Three of the dominant species, §, communis, P. mgrgg, and A, elenkinii were isolated and cultured for growth kinetics studies related to carbon and nitrogen, both of which were suspected of being important causal factors in the observed species succession. Presumptive evidence indicates that S. communis declined because of an interaction of limiting quantities of nitrate and free C02. Early growth of S, communis was probably limited by low irradiance due to Gary F. Marx shading by earlier pOpulations. The upper limit of A. elenkinii growth was determined by low levels of free C02, while the week to week fluc- tuations were related to light attenuation (primarily due to self- shading). The onset of A. elenkinii growth seemed to be delayed by low temperature. Data suggested that two minor blooms of E, 22522 may have been due to mixotrophic growth, possibly using as a substrate dis- solved organic matter released from stationary or declining populations of the other two species. In this phosphorus-rich hypereutrophic lake, carbon and light attenuation due to large algal standing crops, as well as the availability of dissolved organic substances, seem to be the major factors controlling annual algal productivity. While a simple, kinetics based model may not be able to accurately predict the weekly specific growth rates of the various species, pre- dictions of approximate periods of growth may be quite accurate, making such a model of considerable value for practical use. This work is dedicated to Debby, who was always there to help smooth the rough edges. 11 ACKNOWLEDGMENTS I want to extend my appreciation to my committee members, Drs. P. G. Murphy, C. D. McNabb, R. G. Wetzel, and D. L. King for their critical evaluation of this manuscript and helpful suggestions. In particular, Dr. Murphy provided outstanding guidance throughout my doctoral program. I would like to thank him for his encouragement, and for always being an accessable source of helpful counsel. Also, many discussions with Dr. King helped greatly to clarify and refine my thoughts related to this project. iii TABLE OF CONTENTS Page LIST OF TABLES ooooooooooooooooooooooooo ooooooo oooooooooooooo .0000. V1 LIST OF FIGURES. ..... .0.000.......00.0.0.0.0000 ........ ..0..00..0. v11 INTRODUCTION......00..0....00.00.00000.0.....000...0.....000.0...0 1 METHODSANDMATERIALSooooooooooooooo ..... 0.....0... ....... .000..0. 6 The System.................................................. 6 Field Study......... ...... . ....... .......................... 9 Kinetics Experiments........................................ 10 Growth Medium............................................... 13 Ph.Measurements............................................. 14 Treatments.................................................. l4 Calculations................................................ 14 Organic Carbon Experiments.................................. 16 RESULTS..0.00.00000000000 ....... . ...... .....00..0. ........... 000.. 18 Phytoplankton............................................... 18 Secchi Disc Transparency.................................... 21 Temperature................................................. 21 Chemical Parameters......................................... 22 Laboratory Experiments...................................... 24 Scenedesmus........................................... 24 Anabaenopsis.......................................... 35 Pandorina............................................. 41 Heterotrophy Experiments.................................... 48 DISCUSSION...0.000.....000.0..0..00.....0.....0...000..0.0...0000. 53 General Discussion.......................................... 53 Laboratory Kinetic Data.................. ........ ........... 59 Predicted vs. Actual Trends................................. 65 Scenedesmus................................................. 68 Anabaenopsis................... ..... ........ ....... ......... 69 Pandorina............ ....... ..... ......... . ............ ..... 72 iv TABLE OF CONTENTS-cont inued Page CONCLUSION..0000000.0 0000000000000000 C 00000 0 0000000000 00.....00... 75 LITERATURE CITED ......... . ........ . ..... . ............ ............. 78 APPENDICES. .............. 0000.. ...... 000.0..00....000.....0...000. 83 LIST OF TABLES TABLE Page Kinetic values for S, communis growth related to free 002 and initial nitrate concentration obtained (a) by calculation using linear regression of transformed data and (b) from graphs fitted to data by visual approximation. (a) correlation coefficients indicate fit of linear equation to transformed data0.0000000000.00.0.0.00..000....000.00..00...0.0.0.00.0.00.00.27 Kinetic values for A, elenkinii growth related to free C02 and initial nitrate concentration obtained (a) by calculation using regression of transformed data and (b) from graphs fitted to data by visual approximation. (a) correlation coefficients indicate fit of linear equation to transformed data......0.00.0...0..0.0.00....000......00.....00000.0.0.0.0000.38 vi LIST OF FIGURES FIGURE Page 10. ll. 12. Diagram of MSU Water Quality Management Project indicating water flow patterns. Drawn to scale. X indicates sampling area. (MOdified from IWR, 1976)....00.....0......0.0.0000......08 (A) Light chamber, (B) light-dark chamber, and (C) experimental arrangement used in kinetics experiments...........12 1977 growing season time course measurements of (a) phyto- plankton populations, (b) Secchi disc measurements, (c) mean temperature, (d) depth-time temperature profile, (e) phosphate, (f) nitrate and ammonium nitrogen, and (g) free C02.............l9 Daily pH measurements of S. communis growth experiments at four initial nitrate concentrations in light and light-dark Chambers00.0....0.......00....0....00....00..0..00..00000..00...26 Specific growth rate (pg) of S. communis over free C02 levels at various initial nitrate concentrations.......................28 Relationship of Hg , nab: and us of S. communis at an initial nitrate concentration of 0.10 mg/l-N............................31 Regression equations and lines of K and Co values from light and LD chambers at different initiaI nitrate concentrations.....32 Comparison of field growth rates (pf) of S. communis to estimates (corrected for day length) calculated with data from light '(ug) and LD (nab) cmmersoooooococo-00.000.000.00...“ Daily pH measurements of A. elenkinii growth experiments at four initial nitrate concentrations in light and light-dark Chamber800...00....00.00.0.00..00.0000.00..00.00...0...0..000...37 Specific growth rate (pg) of A, elenkinii over free C02 levels at various initial nitrate concentrations................40 Relationship of mg, “ab: and p3 of A, elenkinii at an initial nitrate concentration of 0.01 mg7l—N....................43 Comparison of field growth rates (pf) of A. elenkinii to estimates (corrected for day length) calculated with data from light (mg) and LD (nab) chambers...........................45 vii LIST OF FIGURES--continued FIGURE Page 130 14. 15. 16. 17. 18. 19. 20. 21. Specific growth rate (u ) ofP P. morum over free COZ concen- tration at various initIal nitrate concentrations...............47 Relationship of pg, “ab, and ”s forP . morum at an initial nitrate concentration of 0. 01 mg/l-N............................SO Response of populations of P. morum in culture to the ad- dition or non-addition of acetate at high and low nitrate concentration8.0.00.....0....0000.........0.000.00.00.0.....0..0Sl Plot of various levels of specific growth rate (u ) for S. communis showing relationship to various combinations of free C02 and initial nitrate concentrations.....................S6 Co values (minimum 002 required for growth) for S. communis from light (pg) and LD (nab) chambers showing relationship to initial nitrate concentration................................S7 Specific growth rate (u ) 0f.§3 communis (S), A, elenkinii (A), and S, morum (P) compared over free C02 concentration at four initial nitrate concentrations..................................60 (a) “max values for S. communis (S) and A, elenkinii (A) showing relationship to initial nitrate concentration. (b) Co values for S. communis (S) and A. elenkinii (A) showing relationship to initial nitrate concentration...................63 Points representing combinations of mean 002 and nitrate concentrations of weeks during which growth of S. communis (S. c. ), é;- elenkinii (A. e. ), md P. morum (P. m.) occurred Two points connected indicate weeks- when two species both increased.......................................................67 Comparison of field growth rate (uf) of A, elenkinii with Secchi transparency showing similar patterns of variation.......70 viii Introduction Many factors that may control the seasonal rise and fall of phytoplankton populations have been studied. A review paper by Lund (1965) concludes that the general seasonal succession of species can be related to the interaction of light and temperature, but indicates that other factors are also important. Lund considers the influence of nutrients, particularly phosphorus, nitrogen, silicates and carbon (un- der enriched conditions) potentially important. Also discussed is the importance of a number of micronutrients, including magnesium, potassium, iron, manganese, cobalt, molybdenum, zinc, sodium, calcium and chlorine. Other potentially influential factors include extracellular organic substances released by algae and higher plants, and the effects of grazing and parasitism. More recent studies have provided additional evidence that phyto- plankton seasonality may be primarily a result of nutrient dynamics. Studies of algal distributions in the Great Lakes (Schelske and Stoermer, 1971, 1972) indicate that the phytoplankton are primarily phosphorus limited. The increasing phosphorus inputs associated with domestic pollution increases the growth of the dominant diatom populations caus- ing the depletion of the silica reserves in the water. As silica levels decrease, the diatoms (with a high silica requirement) are gradually replaced by green and blue-green algae, which require little silica. Intense growth in highly enriched lakes can result in reduction of nitrate to undetectable levels. Only the nitrogen fixing blue-green algae have the capability to grow well photosynthetically under these conditions and often form dense surface blooms after the nitrate-re- quiring species have declined (Mess, 1972; Fogg et al. 1973; Schindler, 1977). Moss (1972, 1973a, 1973b, 1973c) investigated the distributions-of a large number of algal species which he divided into an oligotrOphic group and a eutrophic group by their tolerances to varying levels of different nutrients and pH.' The eutrophic group grew well above a pH of 8.85 while those in the oligotrophic group did not. Moss suggested that the oligotrophic species were limited at high pH by lack of suf- ficient free COZ. King (1972) provided evidence implicating free C02 limitation in an observed seasonal periodicity in a sewage lagoon. It was shown that the green algae (more oligotrophic) were limited at a much higher level of free CO2 than were some blue-green algae, so that as the dominant green algae grew rapidly early in the season, free CO2 was decreased to self-limiting levels and blue-green algae rapidly attained dominance. ‘ A number of studies have used measurements of species specific population growth and uptake kinetics values as a means of obtaining more specific information of the growth responses of individual species to varying levels of limiting factors (Caperon and Smith, 1978; Goldman and McCarthy, 1978; Goldman, 1974, 1977; Eppley and Thomas, 1969; Droop, 1974; Tilman and Kilham, 1976; MacIsaac and Dugdale, 1969). The specific growth rate is assumed to follow the Michaelis-Menten or Mbnod (1949) expression for enzyme kinetics: u = “max S/KS + S (1) where u is the specific growth rate, “max is the maximum growth rate, S is the concentration of the single limiting nutrient, and K8 is the half saturation value, that is, the concentration of S at which n - kumax. Dugdale (1967) suggested that the various algal species may have different values of “max and Ks for the different potential limiting factors and these may be the basis for their differential distributions. For example, it would be advantageous for a species which occurred in nutrient deficient areas to have a high efficiency at extracting the limiting nutrient from very low ambient levels (low Ks)’ wheras in enriched areas a higher Ks would suffice, and a high growth rate (high “max) would be necessary to compete with other fast growing species which would be favored by the abundance of nutrients. Evidence for this proposition was produced in a series of studies by Tilman in which most outcomes of competition between two species of planktonic diatoms with experimentally determined uptake efficiencies could be predicted using this information (Titman, 1976) when concen- trations of limiting nutrients were experimentalIy varied. Using this information, 70 percent of the variance in the distribution of the two species of diatoms along a natural phosphate-silicate gradient in Lake Michigan could be explained (Tilman, 1977). Using data from the literature, King (1970) determined efficiency values for free CO2 uptake for a number of common planktonic species and, using these data, was able to explain the seasonal distribution of various green and blue-green algae. Other studies have determined these rate values for different species and used this information to simulate or explain species distribution with various degrees of success (Eppley et a1. 1969; Droop, 1968; Kilham, S., 1975: Kilham, P., 1971; Lehman et al. 1975a, 1975b). The majority of these studies have focused on a single limiting factor as the sole agent controlling specific growth rates. Droop (1974) has shown that under steady state conditions only one nutrient can be limiting at any one time. There is no evidence, however, to show conclusively that population growth in the natural environment responds to the rapidly changing conditions there in the same fashion as to steady state conditions in the laboratory. In addition, several studies have shown a strong interaction between light and various come monly limiting nutrients (King and King, 1974; Davis, 1976; Senft, 1978) demonstrating that some limiting factors may in fact interact to control growth in natural systems. An important aspect of phytoplankton ecology not often considered in these studies is the specific sinking rate from the euphotic zone of the different species populations in relation to stress conditions. Eppley et a1. (1967) observed an inverse relationship between growth rate and sinking rate for several species. Eppley et a1. (1967) and Smayda (1974) provide evidence that sinking rate can be directly re- lated to physiological stress, such as that due to nutrient deficiency. While increased sinking rate may increase uptake rates of limiting nutrients and thus increase specific growth rate (Titman and Kilham, 1976), such increases do not compensate for the increased loss rates caused by the accelerated rate of sinking (Canelli and Pubs, 1976). Studies in fully light containers (e.g. continuous cultures) provide estimates of the maximum physiologically attainable uptake efficiencies of the test species since, even when the cells have become stressed and sink to the bottom, they continue to photosynthesize until their physiological limits are reached. King and Hill (1978) have shown that the inclusion of sinking can substantially increase the Ks values and the lower tolerance limits obtained in kinetics studies, providing a ,more accurate estimate of the ecological efficiency, that is, the uptake efficiency attainable under natural conditions. 'This study utilized batch cultures to determine kinetic efficiency values of three species of phytoplanktonic algae to determine if a nutrient based model could provide an explanation for their occurrence in a seasonal sequence of populations that occurred in a waste treatment basin during the 1977 growing season. The application of the Mbnod model was modified to allow an examination of possible simultaneous limitation by two limiting nutreints and the effects of specific sinking rates on algal growth to determine the importance of these fac- tors in influencing the observed periodicity. The effects of algal heterotrophy and variable water transparency were also examined in an attempt to assess the possible influence of these factors on the ability of the nutrient based model to predict population dynamics. Methods and Materials ThegSystem The study was conducted on Lake 3 of the Michigan State University Water Quality Management Project located at T4N, R1, 2W, Secs. 1, 6, 31,. and 36, Ingham County, Michigan on the MSU campus. The project is designed to improve the water quality of secondary sewage effluent from the East Lansing sewage treatment plant. It consists of four man-made lakes with a total surface area of 16 ha. and a mean depth of 1.8 meters connected in sequence by gravity flow lines (Figure 1). Effluent is pumped 7.25 kilometers from the treatment plant to the site where it is discharged into Lake 1. The water then moves by gravity flow, the rate of flow dependent on the discharge rate, through Lakes 2, 3, and 4 and is finally discharged into the local watershed (Institute of water Research, 1976). Levels of inorganic phosphate and nitrate decrease substantially through the system due to the intense growth of phytoplankton and aquatic macrOphytes, with a decrease in total phosphorus from ca. 0.75 to 0.07 mg/l and nitrate-N from ca. 5.0 to 0.2 mg/l in the final dis- charge from the initial effluent (Institute of water Research, 1976). Lake 3 was chosen for this study as it was the only one of the four which was phytoplankton dominated throughout the majority of the growing season, the others being primarily macrophyte dominated. This algal dominance reduced the need to consider competition from macrOphytes as Figure 1. Diagram of MSU Water Quality Management Project indicating water flow patterns. Drawn to scale. X indicates sampling area. (Modified from IWR, 1976). .H munwfim ll a major factor influencing algal growth. Although macrophytes occurred in substantial amounts at one point in the summer, they were subsequent- ly harvested and greatly reduced. Field Study water samples were collected weekly from a small area (Figure 1) of Lake 3 of the project from March 29 through October 30, 1977. Three samples were taken on each date at a depth of 0.5 meters using a non- metallic Kemmerer sampler. Measurements of the temperature at depth intervals of 0.3 meters and Secchi disc transparency were also made during this period. Samples were transported to the laboratory in a darkened container and immediately fixed with a solution of iodine, potassium iodide, and glacial acetic acid (Prescott, 1970). After standing for one hour, measured amounts of the samples (depending on algal density) were filtered onto 0.45 pm pore size Millipore membrane filters which were allowed to air dry. The filters were mounted on large (2" x 3") microslides using Type "A" immersion oil, and covered with a large coverglass for storage until counting (Amer. Public Health Assoc., 1976). Cell densities for each species in the samples were determined by cell counts (McNabb, 1960; Amer. Public Health Assoc., 1976). Twenty fields at high power (950K) were counted, the mean number per field multiplied by a factor to obtain total cells per filter and divided by the volume of sample filtered to obtain cells per milliliter for each species in the samples. Cell volume per ml was calculated by determin- ing a mean volume per cell by first measuring cell dimensions of 100 cells of each species, calculating their cell volumes and determining a mean value. Cells per ml was then multiplied by cell volume per cell 10 to obtain cell volume per ml (Amer. Public Health Assoc., 1976). Daily measurements of various water chemistry parameters (Appendis 1) were provided by the MSU Institute of Water Research. Kinetics Experiments Three of the species that dominated the phytoplankton of Lake 3 for various periods during the 1977 growing season Scengdesmus communis Hegewald (formerly S. quadricauda), Anabaenopsis elenkinii Miller, and Pandorina morum (Muell.)Bory , were isolated and grown in unialgal cultures in the laboratory. By examination and multiple regression studies of the field data it was determined that nitrate and free CO2 were the most likely factors limiting to the growth of the dominant species. The kinetics experiments were designed to test the effects of varying nitrate and free C02 concentrations on the growth of these dom- inant species. The experiments were conducted using batch cultures in specially de- signed culture chambers (King and Hill, 1978), using the methods of Young and King (1973). Each treatment consisted of paired containers, a light chamber and a light-dark (LD) chamber. The light chamber (Fig- ure 2a) consisted of a 1000 m1 Erlenmeyer flask sealed with a rubber stopper with two holes. Into one hole was fitted a serum cap through which samples could be extracted; the other accomodated an air lock to minimize gas exchange with the air to reduce recarbonation of the medium and to maintain atmospheric pressure in the chambers. In this chamber, the maximum.physiological growth efficiency of the species was measured. The LD chamber was constructed from three 500 ml Erlenmeyer flasks fused as shown in Figure 2b. The bottom section of the container was painted black and covered with aluminum foil to create a darkened zone, 11 Figure 2. (A) Light chamber, (B) light-dark chamber, and (C) experimental arrangement used in kinetics experiments. 12 Cl nul uni l SERLM CAP VALVES \ L.___l I “7 5 CLEAR GLASS 5 CHAMBER ' (A) Figure 2 . 13 into which sinking cells would fall and be prevented from photosyn— thesizing. This chamber enabled the measurement of kinetic values in a situation more similar to field conditions than was possible in the light chambers. ‘ Illumination of the chambers was by two horizontal 35-watt cool white flourescent light tubes, which provided a constant intensity of 3200 lux. The cultures were lighted from the side to minimize light penetration into the dark portion of the LD chambers. Light chambers were raised on platforms to compensate for the greater height of the LD chambers (Figure 2c). The lights were oriented so that all eight champ bers could be simultaneously illuminated. Light intensity was measured using a Heston 756 footcandle meter. Growth Medium The growth medium used (Appendix 2) was modified from Bold's basic medium (James, 1974) to include bicarbonate for a carbon source and to be dominated by monovalent cations to reduce the possibility of carbon- ate precipitation during photosynthesis. All nutrients were assumed to be well in excess of need except for nitrate and CO2 which varied ac- cording to treatment. The medium was autoclaved at 121°C, 1.05 kg/cmz, for 15 minutes, cooled overnight and bubbled with air for four hours to return it to atmospheric equilibrium. Alkalinity was then measured using the potentiometric method (Amer. Public Health Assoc., 1976) and adjusted to 2.00 meq/l with autoclaved NaHCO to simplify calculations. 3 The algae used to seed the cultures were from cultures of the three species isolated from Lake 3 and maintained in the laboratory in iden- tical medium and under similar light conditions to those used in the 14 experimental cultures. Prior to each experiment, the algae were subcul- tured in fresh, nitrate free medium and allowed to grow until the pH of the medium had increased 1-2 units (3-5 days). These cultures were then well mixed and, for each treatment, equal amounts were concentrated by centrifugation, decanted, and resuspended in fresh medium. This pro- tcedure was performed twice for each treatment. In this manner, equal amounts of inoculum were added to each experimental chamber. pgiMeasurements The progress of growth in the cultures was monitored through daily measurement of pH. The pH was measured to the nearest 0.02 unit using a Horizon Model 5996 pH meter calibrated frequently with standard buffer solutions. Samples for measurement were removed from the chambers with a hypodermic needle inserted through the serum cap in the top of each chamber. The samples were then deposited, slowly to minimize recarbon- ation, into small containers into which the probe was lowered. Treatments The population growth rates of the three species were measured in relation to varying concentrations of free C02 and nitrate. The treat- ments consisted of four initial nitrate concentrations (10.0, 1.0, 0.1, and 0.01 mg/l-N) with free CO2 being allowed to vary over the course of the experiment. Instantaneous growth rates were calculated daily as CO2 declined. In this manner, an examination of the effects of the inter— action of these two factors on the growth of these species was accomplished. Calculations The calculation of specific growth rates followed the methods of King and King (1974) and King and Hill (1978), the kinetic variables 15 (“max’ Ks) being a function of free C02. Total carbon (£002) and free CO2 were calculated using carbonate-bicarbonate equilibrium equations (King and Novak, 1974) in conjunction with measurements of pH, alkalin- ity, and temperature. Carbon fixation for photosynthesis was assumed to draw exclusively from the carbonate-bicarbonate alkalinity and all carbon removed from the alkalinity was assumed to be due to fixation by the algae. Total carbon and free CO2 were calculated using the daily pH meas- urements, alkalinity (due to the sealed container) and temperature were constant. Algal growth was assumed to follow a first order growth equation: Mt = M.o eugt (2) where Mt is the mass at time t, M6 is the initial mass, and "g is the specific growth rate. ug was calculated by dividing the change in 2C02 per day by the average biomass (carbon fixed) over the period of measurement. The specific growth rate was assumed to follow the Michaelis-Menten or Monod model, modified to include a minimum required concentration or threshold level of free C02: “8 .. “max {cozl - co (Kc-Co) + (Tcozl-Co) where “max is the maximum specific growth rate, {C02} is the concentra- tion of free 002, Kb is the concentration of CO2 at which “g - 8 "max’ and Co is the minimum concentration of C02 required for growth. Kc and "max were calculated using a linear regression of a ug/{COZ} vs. pg l6 transformation of the specific growth rate equation (Dowd and Riggs, 1965). Co was the concentration of free CO at which growth ceases 2 in culture. While in the light chambers, sinking cells fell to the bottom and continued to photosynthesize, in the LD chambers sinking cells were re-. moved from the growing biomass of the population. Because of this re- moval, the accrual of active biomass was slower in these than in the light chambers and the rate of this accrual is calculated somewhat differently. Since both chambers were incubated under identical con- ditions, it was assumed that p8 in the lighted zone was the same in both chambers at the same free CO2 levels. To calculate accrued biomass (ab), the change in total carbon per day was divided by the pg that occurred at that particular CO2 concentration in the light chamber. The rate of biomass accrual (pab) was calculated then as before by dividing the change in accrued biomass per day by the mean accrued biomass for that period. This value may be positive or negative depending on whether there was an increase or decrease in ab over that period. The specific growth rate (us) represents the total growth rate when effects from sinking are prevented in the light chamber. The biomass accrual rate (pab) represents the specific growth rate minus the specific sinking rate (us). Therefore ps can be calculated by sub- tracting pab from us. Qgganic Carbon Experiments The field data provided evidence to suggest that the two growth periods of _I_’_. m were in response to increased amounts of organic matter which stimulated its growth through either heterotrophy or mixotrophy. Two experiments were designed to test this possibility. 17 In the first, the response of g, 92532 to additions of acetate (a simple organic substrate) at high and low nitrate levels was measured. Only light chambers were used in this experiment, with culture medium and illumination as before. The four treatments were as follows: high nitrate (10 mg/l-N) with acetate (10 mg/l); high nitrate without acetate; low nitrate (0.01 mg/l-N) with acetate; and low nitrate with- out acetate. A replicate of each of these treatments was maintained in complete darkness. Cultures were inoculated as before and pH was measured daily. To account for the possibility of heterotrophic growth which would not predictably affect pH, cell counts were made approx- imately every other day using a Sedgewick-Rafter counting cell (Amer. Public Health Assoc., 1976). Values for cell volume per ml were cal- culated as before. In the second experiment, the response of S, 39593 to growth in medium filtered from growing cultures of S, communis and A, elenkinii was compared to sterile medium of similar initial pH. The purpose of this experiment was to test the possibility that the organic substrate which stimulated the growth of S, 99522 in the lake was that released from the populations of the other species during their log growth phases. One culture of S, communis and one of A, elenkinii, inoculated and cultured as before were allowed to grow until the pH of the nitrate free medium reached 9.0. The cultures were then filtered through Reeve Angel 984 H glass fiber filters (0.5 pm porosity)to remove the algae, and bubbled with CO2 gas until the pH equalled that in the sterile medium (approximately 7.5). All three were inoculated with 2, ggggg_as before, with daily pH measurements and cell counts every two days. Results Phytgplankton Populations of five algal species dominated Lake 3 for various periods during the 1977 growing season (Figure 3a). The dominant phytoplanktonic alga at the beginning of the sampling period (3/29) was Cyclotella sp., a centric diatom, which peaked in early April at a cell volume of 238 mm3/l. Cyclotella declined rapidly and was immediately replaced by a volvocalean green alga, Chlamydompnas sp., which reached its maximum development the next week at 205 mm3/l. Soon after the subsequent rapid decline of Chlamydomonas from dominance, a large bloom of Scenedesmus communis, a common chlorococcalean alga, occurred and reached its peak in early May with a cell volume of 297 mm3/l, the larg- est of the season. This population then decreased precipitously to 27 m3/1 by 24 May. A volvocalean green alga, Pandgrina morum, reached its highest value that week at 116 mm3/1, its major growth period occurring while S. communis was still dominant. ‘2, 22523 exhibited a second peak of 113 mm3/l in early July during a small bloom of Anabaenqpsis elenkinii which later dominated the phytoplankton. This pattern of growth, with major increases occurring during periods of reduced light due to large pop- ulations of other species, suggested that S, mgggm_growth may have been augmented by heterotrophic uptake of organic carbon. A nitrogen fixing blue green alga, Anabgengpsgg elenkinii (Nostocales) 18 19 Figure 3. 1977 growing season time course measurements of (a) phyto- plankton populations, (b) Secchi disc measurements, (c) mean temperature, (d) dept-time temperature profile, (e) phosphate, (f) nitrate and ammonium nitrogen, and (g) free C02. 20 l PuosmArstpo; -,--- ummnog) _ W-OAMMONIUMml-tzl Figure 3. 21 showed a small peak of 15 mm3/1 at the end of June, then increased through early July to plateau at 123 mm3/1, then increased to a maximum of 181 mm3/l in late August. By late September, A, elenkinii had declined to undetectable levels. Secchi Disc Transparency Measurements of Secchi disc transparency (Figure 3b) correlated well with measurements of total phytoplankton cell volume per liter (r - 0.77). The transparency was very low early in the season when populations of gyclotglla, Chlamydomonag, Sggnedesmus, and Pandorina were abundant, varying from 0.3 to 0.7 meters during this period. The transparency increased greatly with the decline of these populations towards the end of May, to 1.6 meters. With the onset of the second S. m bloom and subsequent A. elenkinii growth, the measurement _ decreased the depth of transparency to about half of that which occurred during the earlier blooms. This decrease in transparency may have resulted from the blue-green algae being highly concentrated at the surface due to the presence of gas vacoules in the cells allowing flotation. Temperature Mean water temperature increased gradually from a low of 6°C in early April to a maximum of 29°C in early July (Figure 3c). The shal— low nature of the lake caused lake temperature to reflect changes in air temperature causing the observed weekly fluctuations throughout the season. Depth profiles of temperature (Figure 3d) show the absence of stratification for the major part of the growing season. Only two periods of slight stratification occurred, one lasting two weeks in mid- April when a maximum difference of 5°C between surface and bottom water 22 occurred, and the other in late May, lasting for three weeks, with a maximum.difference of 12°C. The absence of continuous stratification reflects the almost continual mixing of the entire water column of Lake 3. Chemical Parameters Thirteen parameters in Lake 3 were monitored daily by the Institute of water Research at MSU. Orthophosphate, nitrate nitrogen, and free CO2 were examined in detail as these are most often the nutrients implicated in studies of nutrient limitation of photosynthesis in eutro- phic fresh waters. Micronutrients were assumed to be in abundance as these lakes are supplied with secondary effluent from treated domestic wastewater. Orthophosphate (P023) underwent considerable seasonal variation but at no time decreased to levels limiting to algal growth (Figure 3e). In early spring, the phosphate concentration was approximately 1 mg/l but declined rapidly during the Cyclotella, Chlamydomonas, and Scenedesmus blooms to a low of 49 pg/l in late May, a level in the range of the minimal concentrations necessary for optimal growth in most algal species including‘S. communis (Hutchinson, 1957; Wetzel, 1975). The levels subsequently increased to a high of 1.6 mg/l in mid-August and declined somewhat to the end of the sampling period. Due to the ade- quate levels of orthophosphate in the lake throughout the growing sea- son it was decided that, under these conditions of heavy artificial enrichment, phosphorus was not likely to be an important factor influ- encing the periodicity of algal populations observed, and was not considered further in laboratory experiments. Nitrate nitrogen fluctuated considerably less than phosphate 23 (Figure 3f), declining from an early high concentration of 6.0 mg/l to undetectable levels by mid-May. Nitrate was not detected again until the end of July when a small concentration (0.1 mg/l-N) appeared for two weeks. The early decline coincided with the growth of the three early species suggesting that the decline was due to uptake by these popula- tions. Ammonia nitrogen was very low throughout the sampling period except for two peaks of about 1 mg/l which occurred in late July and early August. Since there seemed to be insufficient nitrogen as nitrate or ammonium to allow the large population growth of A, elenkinii, it was assumed that elemental nitrogen, N2, was used as a nitrogen source by the heterocystous blue green alga, species of which have been shown to have this ability (Fogg, 1974; Watanabe, 1951). The ammonium was presumably either taken up by the algae or was released into the air as ammonia (NH3) which occurs to an increasing extent when pH increases past the pK (9.2) for the dissociation of NH: into ammonia gas (Insti- tute of water Research, 1976) a level that was surpassed during most of this period. Free CO2 exhibited fluctuations within a narrowly restricted range, occurring outside the range of 0.1 to 1.3 pM/l on only two occasions (Figure 3g). For the most part, the trends seemed to be explicable on the basis of phyt0plankton growth and decomposition in the lake. The decline early in the season corresponds, as with nitrogen and phosphorus, to the large increases of Cyclotella sp., Chlamydomonas sp., and S. communis as well as S, Egggg_during that period. The increase in early June is likely due to the decomposition of those previous blooms, but the cause of the subsequent rapid decline of CO2 is unclear, but may have been due to uptake by macrophytes. The decline of 002 in late 24 July corresponded with the first major growth phase of A, elenkinii, while the late August decline corresponded somewhat less precisely to the second blue-green growth period. On the basis of these considerations, it was determined that nitrate and free CO , singly or in combination, were most likely the two most 2 important nutrient factors influencing the population dynamics of the three dominant species which were studied in the laboratory. The studies of population growth kinetics were based on these apparent correlations. LaboratorygExperiments Scenedesmus Daily measurements of pH were taken from each of the four culture treatments in both light and LD chambers and plotted against time. The extent to which S. communis could increase the pH diminished with decreasing initial nitrate concentration (Figure 4). The maximum pH values were lower for each treatment in the LD chamber than in the light, except for the 10 mg/l—N LD treatment, where attached growth on the chamber walls apparently caused a pH increase equalling that in the light chamber (see below). Kinetic values for ”max’ Kc, and Co were calculated (Table 1) using specific growth rate (us) vs. free CO2 concentration data and curves were calculated and plotted for the light, chambers (Figure 5). It is evident that pmax (the maximum growth rate) decreases ca. 20 percent over the range of decreasing initial nitrate concentrations. Co values (the minimum CO2 concentration at which growth can occur) increased by almost two orders of magnitude, from 0.0015 to 0.104 pM/l, as did the K.c values (half saturation value) increasing from 0.033 pM/l at an initial nitrate concentration of 10 mg/l-N to 0.795 pM/l at 0.01 mg/l-N. 25 Figure 4. Daily pH measurements of S, communis growth experiments at four initial nitrate concentrations in light and light-dark chambers. 26 5.5%.... .c ouswfim 2 . as” m con—Eosu stow-Em: 828358 . I I ' ~55. CO ? .J 692- 2 ,‘Uullul . 1111’ .1 1L .m canop—o in: D d ‘ d I 1‘ mDEmmOmZmUm . .6 :d H .O— . .O 0.64. .0 4rd .— I «0.6— "It: Table l. (a) Initial-N (mall-N) 10.0 1.0 0.10 0.01 (b) 10.0 1.0 0.10 0.01 Kinetic values for S, communis growth related to free C02 and initial nitrate concentration obtained (a) by calculation using linear regression of transformed data and (b) from graphs fitted to data by visual approximation. (a) correlation coefficients indicate fit of linear equation to transformed data. Light Chamber “max (day-1) 1.19 1.11 1.08 0.96 K c (HM/1) 0.0329 0.0404 0.4082 0.7950 Light-dark Chamber 0.036 0.050 2.30 5.90 C o (UM/l) 0.0015 0.0018 0.0369 0.1048 0.0015 0.011 1.010 3.00 0.75 0.76 0.70 0.65 28 .m:0fiumuuaoonoo mumuufic Hmfiuwaa macaum> um mHo>mH moo comm um>o mannaaou .1 mac A may mama nuaouw oawaooam .m ounwam 22$: N0o we: . o.— e. . x no . Se . . . . . . .\A\u O , 0 3:? o no a .o .a AU 8.1 U- J: an... an . we...” . . .. Jamal a a 6.9.. o . is: . 323328... 29 When data from the LD chambers (pab) are plotted for N031 = 0.10 mg/l-N (Figure 6)(the mean lake concentration the week when S, communis began to decline) it is clear that the inclusion of sinking as a factor further increases the values of Co (from 0.035 to 1.01 pM/l) and Kc (from 0.408 to 2.30 pM/l). The concentration of free CO2 in the lake during the time when S, communis first began to decline was 0.15 pM/l, a value which fell between the Co values of the pgand pab curves, provided evidence that the experimentally determined values may be extremes within which one could expect the field values to fall, when carbon and nitrate are the controlling factors. This relationship was tested for the remainder of the growth period by determining upper and lower estimates of specific growth rate in the field using the mean weekly concentrations of nitrate and free CO2 in Lake 3 in the following equation: {002} - C01 (4) (Kci-C01)+({C02}-Coi) pg = umaXi where umax’ Kci’ and Coi are the values of these variables that would be expected at the mean nitrate concentration of the 1th week. Kc and Co values (in units of erCOzf/l) from both light and LD chambers were plotted against initial nitrate concentration, linear regression equa- tions were calculated (Figure 7), and values were calculated from these equations for incorporation in equation (4). As previously mentioned, the Co value for the 10 mg/l-N LD treatment is probably inaccurate due to the attached growth on the walls of the chamber. However, the elim- ination of that point does not significantly change the prediction equation or the calculated Co values used in equation (4). Values for 30 Figure 6. Relationship of p , pa , and p of S, communis at an initial nitrate concentragion of 0.10 mg/l. 31 .o ounwfim :0.7 52:18 ”my: on. no . 56 .. m .2. . . m. an .X. qud.nu nu [hum :0: Ba 28...... u a I . 0 Es. 8:qu $353 :30: O m . . E5. :56 n m: a .0.— _\mEO—.on Z .33.: . mDEmmn—mZmUm 32 1.0 i' A .200 .100 000 100 T W U15 003,40 Figure 7. Regression equations and lines of Kc and Co values from light and LD chambers at different initial nitrate concentrations. 33 u were obtained from similar plots. max The actual growth rate of S. communis in Lake 3 was calculated as follows (Fogg, 1975): (1n N ) - (1n N ) = n+1 n . (5) p f1 7 days where pf1 is the specific growth rate of S. communis in Lake 3 during week 1, Nn is its cell volume on the sampling day previous to week i, and Nn+1 is the cell volume on the following sampling date. pg (physio- logical efficiency), and pab (ecological efficiency), were corrected for day length, and with pf were then plotted over the period of growth (Figure 8). The period from the initial S. communis appearance until its major decline at the end of May was included. During weeks 1-3 of this period, while S. communis was a minor component of the algal community, its measured field growth rate fell below the predicted range. In the following four weeks, during which it was the dominant species, the field values fell between the estimates calculated from laboratory data. Similar plots were constructed holding values of Khand Co constant to determine if the field variations in free CO2 alone could better account for the changes in pf. When the kinetic values obtained at NOS—N - 0.01 mg/l, pf was similar to the 118 curve but actually exceeded it for the portion of the growth period when the nitrate concentrations in the field were well above 0.01 mg/l. These results demonstrate the better fit attainable using both factors. A problem with this analysis is the likelihood that the values of Kc and Co calculated using the methods described may be artificially 34 D “g .6» ; _ A ”f ' ' 0 "ab .4" T3,. . . a I 3 .2-- ‘ :1 . - 0} " Scenedesmus -.2.- \ l '7 I 1 I 3 6 weeks q- I q- 4 Figure 8. Comparison of field growth rates (Hf) of S. communis to estimates (corrected for day length) calculated with data from light (pg) and LD (pab) chambers. 35 high. This error occurred because nitrate concentration was not kept constant in the chambers, and decreased over time due to uptake by the algae. Since the values of KC and Co come from data obtained near the end of the culture period when nitrate was considerably lower than initial levels, it is likely that the algae would grow more poorly, producing values that were higher (less efficient) than if nitrate maintained its initial concentration. These higher values would produce low values of p8 and “ab in the above analysis, possibly causing less agreement with field values. However, it is unlikely that a more accurate determination of the K.c and Co values would substantially change the trends exhibited by the p8 and pab curves and it is the agree- ment of pf with these trends, rather than whether or not it falls between the two curves, that is most significant. Anabaenopsis Initial nitrate concentration had little effect on the rate or extent of pH increase of A, elenkinii in the light chambers (Figure 9). When kinetic values of “max’ Kc and Co were calculated (Table 2) it was notable that the lowest “max value, 0.65 day -1, was obtained at the highest initial nitrate concentration, 10 mg/l-N, indicating possible growth inhibition by high levels of nitrate (Figure 10). In the LD chambers, a substantial decrease in the highest pH attained was found at the two lowest nitrate concentrations, 0.10 and 0.01 mg/l-N, and the values attained in the LD chambers were substantially less than those in the light chambers. When pab was calculated from the LD chamber data with N031. 0.01 mg/l-N (the approximate concentration of nitrate during the period of A, elenkinii growth) the Co and K.c values were over two orders of magnitude 36 Figure 9. Daily pH measurements of A, elenkinii growth experiments at four initial nitrate concentrations in light and light-dark chambers. 37 ZI._\0<< cos—cop? Em: .,m_maozm_23~8 we: 8 .o..o _\ss_o.o . 2 see .m_mm02m4 applicability of the kinetic data, they were applied to the chemical measurements from the field to determine the population changes the model would predict under the prevailing conditions in Lake 3 in 1977. Since kinetic data were obtained for only three species, predictions were make assuming only those three were in the system. To determine how the growth characteristics of the three species would compare under varying combinations of carbon and nitrogen concen-. trations, Hg curves for the three species at each initial nitrogen con- centration were poltted together (Figures 18 a-d). It is clear from these figures that at all nitrate and carbon dioxide concentrations con- sidered, "g for g, morum is substantially lower than that for one or both of the other two species. This result suggests that there is no combination of CO and nitrate concentrations within the ranges used 2 under which E. morum would have a greater Hg, and therefore a competitive advantage, over the other two species. The model predicts, then, that this species would at no time be able to compete with the other species, and so would not become dominant at any time. Since 2, morum clearly did dominate in two instances during the season, the implicit assumption 6O .mcowumuuooocoo oumuuaa Hafiufica know an coaumuucoocoo Nov moum um>o pmuoafioo Amv asuos .m can .A4v «wcwxcmam .< .Amv macsaaou .m «0 Awnv mumu :u3ouw afiwaomam .wH muowam =3... «8qu 32... N8 3.: o.— 8 no .3. o.— q._ 8 56 i .w a .1 m. u. 2-3586 .2 $2... «3 we: .23 «8 3.: 2 a s .3 e s a .3 «a 61 in the model of strictly autotrophic growth must be questioned, since in the model 2, mgggm_cannot compete on this basis with the other spe- cies. This disparity suggested some form of heterotrophic augmentation of growth, discussed in more detail in a subsequent section, or the involvement of other nutrients not investigated. According to the model then, only S, communis and g. elenkinii should be competitive under the conditions of CO2 and nitrate found in the lake and imposed in the laboratory. If figures 18 a-d are examined, it can be seen that at the two higher initial nitrate concentrations (10 and 1.0 mg/l—N), S. communis has the higher pg over all levels of CO2 and therefore would be expected to dominate over 5. elenkinii at those nitrate levels regardless of available free C02. However, at the two lower nitrate levels (0.1 and 0.01 mg/l-N) it can be seen that while S. communis has the greater pg at higher CO2 levels, the curves for the two species cross so that at low nitrate levels S. communis would dominate while CO remained high (greater than 0.7 to 1.9 pM/l, depending on the 2 nitrate-nitrogen concentration), but when CO2 drOpped below the cross- over point, 5. elenkinii would be expected to have a greater pg and to attain dominance. To demonstrate this relationship in a different manner, the physio- logical maximum growth rates (pmax) for the two species calculated at the four initial nitrate levels were plotted (Figure 19a). This figure clearly shows that with unlimited amounts of available C02 ,‘S. communis has a greater growth rate regardless of initial nitrate concentration and thus would be expected to dominate over the blue-green when CO2 is present in abundance. The advantage of 5, elenkinii becomes apparent when growth threshold values (Co) are plotted for the two species (Figure 62 Figure 19. (a) “max values for S, communis (S) and A. elenkinii (A) showing relationship to initial nitrate concentration. (b) Co values for S. communis (S) and A. elenkinii (A) showing relationship to initial nitrate concentration. 1.2~- : S '> im" 3‘ s .8 A 1 v 6.01 oil 110 {o Noéi'N (mg/l) g» .1-- K.) 5 E 3.01] a? A .001" 6.01 oil to f0 Nogi-N (mg/l) Figure 19 . 64 19b). While S, communis has the advantage at nitrate levels greater than 0.56 mg/l-N due to its ability to extract 002 to somewhat lower levels than the blue-green in that nitrogen range, A, elenkinii has a clear advantage over the green algae when nitrate nitrogen drops below the crossover point. At the lowest nitrate level, the blue-green algae can extract C02 to levels more than an order of magnitude lower than can the green, allowing it to grow well under conditions in which S, communis cannot survive. To summarize, the laboratory kinetic data for S, communis and A. elenkinii indicate that when nitrate is present in abundance, S, communis will dominate over all levels of C02. When nitrate decreases to levels below the 0.1 to 1.0 mg/l-N range, the green algae will be expected to dominate when C02 is abundant, but when it drops to levels below the 1.0 pM/l range, the blue-green algae should attain dominance. When these data were applied to the chemical trends in the field, the model predicted a clear pattern of growth of the two species. The early high nitrate and moderate C02 concentrations indicated rapid growth of S. communis for the first five weeks and a decline thereafter since the mean NOS-C02 point for week 6 fell below the physiological Co for the species. After the projected decline of the green algae, nitrate became undetectable in the water and CO2 fluctuated at low to moderate levels (0.1 to 1.0 pM/l). Under these conditions, an immediate rapid increase of A, elenkinii to high levels was predicted. Since nitrate remained low for the remainder of the growing season and 002 maintained its fluctuations between ca. 0.1 and 1.0 pM/l, the blue—green algae were expected to maintain dominance throughout that period. A short period of increased ammonium to ca. 1.0 mg/l-N in late July and early August 65 was predicted to temporarily give a competitive edge t°.§- communis (since ammonium can be used as an alternate nitrogen source) but be- cause the pulse of nitrogen was relatively short and the large standing crOp of the blue-green created a poor light climate because of intense shading, the_S. communis was expected to be small. .As previously men- tioned, S, mgrgm_was not expected to appear under the assumptions of the model. Predicted vs. Actual Trends When these predicted population trends were compared with those which occurred in the field, substantial agreement was found. Growth of S. communis was restricted to the first five weeks of the growing season and one week in early August as predicted, and a major and long lived bloom of A, elenkinii occurred after the demise of S. communis, as predicted. A further demonstration of the agreement found between the predicted and actual growth trends is shown in Figure 20. Points representing combinations of mean CO2 and nitrate concentrations of weeks during which growth of the various species occurred were plotted to illustrate that each species grew under chemical conditions consistent with the findings of the kinetics studies. The points indicating S, communis growth are clustered in the region of high nitrate and low to moderate CO concentrations, A, elenkinii growth points are primarily below 1.0 2 mg/l-N and within the same CO range, and the g. morum points occur 2 where both nitrate and C0 are low (where none of the species would be 2 expected to grow). The figure illustrates a clear segregation of S, communis and A, elenkinii over nitrate concentration, indicating, as the kinetic data suggest, that nitrogen is the most important factor in 66 Figure 20. Points representing combinations of mean C02 and nitrate concentrations of weeks during which growth of S. communis (S. c. ), A. elenkinii (A. e. ), and P. morum (P. _m.) occurred. Two points connected indicate weeks —when two species both increased. 67 'l O 0 Cl '- C D E r 9 :1 I ~—d ll. ’ cs“ - ' Uo.1-- ‘ ‘ l-S c A'P. m. O-A.e. A A .01 i i = = t 0.1 1.0 10 NO§;N (mg/I) Figure 20. 68 determining when one or the other will attain dominance in this eutrophic lake situation. the segregation of these points into predictable com- binations of conditions further illustrates the agreement of the field growth of these species to the predictions (except for S, 22322). How- ever, the predictions did not precisely mirror the rates and times of growth found in the field. Scenedesmus While S, communis did undergo its major growth episode during the first five weeks as predicted, its rate of growth during the first three weeks was lower than predicted (Figure 8). Examination of the field data suggests two factors not included in the model that may have been responsible for this lower than predicted rate. During these first weeks the large populations of the early dominants (gyglotella sp. and Chlamydomonas sp.) inhibited light penetration as indicated by Secchi transparencies of only 0.3 to 0.5 meters, presumably causing a decrease in the growth rate that would have been attainable with those nutrient conditions had light been at saturating intensities. When the SAAAmy- domonas sp. bloom collapsed, the intensity of light increased to the point where it was presumably no longer limiting, and the growth rate increased to a rate determined by the ambient concentrations of free C0 and nitrate, as assumed in the model. The low temperature in the 2 lake, which did not reach 150 C until the fourth week of measurement, may also have contributed to the low growth rate recorded in the first three weeks. This close agreement between the predicted and actual growth of .S. communis strongly supports the contention that its growth was largely limited by interacting limits of nitrate and carbon dioxide. If pg is 69 calculated for the sixth week (when S, communis began to delcine), an increase in either free CO2 or nitrate caused the population to increase in both cases, further indicating an interactive limit. Anabaenopsis A, elenkinii underwent a rapid period of growth after the decline of S, communis and dominated for an extended period from July through September as predicted. However the onset of growth did not occur until a considerable period of time (ca. 4 weeks) had elapsed after the major decline of the green algae. The cause for this delay is uncertain but may be attributable to the low temperatures occurring in the lake. The temperature decreased following the decline of S, communis and growth of A. elenkinii began during the first week that the temperature exceeded 200 C after that decline. This growth continued as the temperature increased to a maximum of 290 C and ceased when it again decreased to o C. This evidence is circumstantial, but in general, blue-green algae 20 are known to have temperature optima in the 30-350 C range (Fogg et a1., 1973) suggesting that the temperature may have been an important factor in delaying the onset of A. elenkinii growth. That the growth of the blue-green was in agreement with the predic- tions of the model is further demonstrated in Figure 12, where pf falls between calculated values of p3 and pab on all but 3 of 12 dates. Exam- ination of the field data suggests light limitation may have been responsible for the unpredicted major fluctuation of pf in the first 5 weeks of growth. When growth rate of A, elenkinii (pf) is compared with Secchi disc transparency over the eleven week period of its growth, a close corres- pondence is found (Figure 21). The decrease in pf during the second and 70 .4-r Anabaenopsis .2-- “f I; O" .5 .2« Amps! Limoges; 1.2“ ,. Secchi m ‘3‘. transparency I": o E '4‘. C)" Figure 21. Comparison of field growth rate (pf) of A, elenkinii with Secchi transparency showing similar patterns of variation. 71 third weeks of growth correspond to a dip in transparency coinciding with the second bloom of 2, $9332, When that population declined with a concommitant increase in light penetration, pf for the blue—green increased dramatically. The two weeks of subsequent A, elenkinii growth decreased the transparency to its lowest levels of the summer, and pf decreased to correspondingly low levels and remained there, presumably because transparency did not improve. These correlations suggested that a limiting factor of the growth of the blue-green algae during its major bloom was a lack of light caused by shading from.£,'§gggm_or by self-shading. Since light de- creases exponentially as a function of cell concentration and depth (Fogg, 1975), the growth habit 0f.é° elenkinii of becoming concentrated near the surface due to the formation of gas vacuoles would tend to exacerbate the latter condition. Jewson (1976, 1977) and Jones (1977a, 1977b) domonstrated the influence of various light related phenomena including total daily irradiance as related to day length, and light attenuation caused by self—shading by dense populations. Jones provided evidence of limitation of photosynthesis because of intense self-shading by a blue-green bloom. The near zero growth rate of A. elenkinii meas- ured during weeks 6—11 of its growth was probably the net result of growth by those cells with sufficient light for net photosynthesis, and the sinking and death of cells with insufficient light. That the sink- ing of cells may be an important factor in keeping pf low (nearer to pab then us) during this period is indicated by the high sinking rate (p3) of the species under conditions of low C02 and nitrate (Figure 11). The source of carbon for this "stationary growth" was most likely diffusion of C0 into the water from the atmosphere. Schindler (1971) 2 72 and Emerson (1975) have shown that sufficient C02 can diffuse into the water to support large phytoplankton populations. The CO2 measured probably represents this source as well as respiratory CO circulated 2 up from the bottom and from decomposing algal cells. Evidence implicating a particular factor in the final decline 0f.é- elenkinii in September is inadequate. As mentioned earlier, the decline coincides with a dr0p in temperature to below 200 C. The concentration of such a large population at the water surface may have resulted in the accumulation of a self-produced autotoxin to levels fatal to the blue-green (Fogg, 1975). Concentration of blue-greens at the surface often results in high light intensity photoinhibition which can cause the demise of such a population. This may occur when photosynthesizing cells become over-vacuolated because of light limitation during a period of some turbulence, and are, in effect, trapped at the surface when becalmed (Fogg et a1. 1973). Pandorina Predictions from the kinetic data indicated no growth of g, mgggm because under all conditions where it had the capability of autotrophic growth, S. communis or A, elenkinii had a greater p8 and thus were ex- pected to dominate S, mgggm_under all such conditions. However, this species did produce substantial populations on two occasions, both at times when nitrate and free 002 conditions were such that the kinetic data would predict no growth. These results suggested that a different factor was involved in the stimulation of this growth, possibly heterotrophic utilization of dis- solved organic material present in the water column. Palmer and Starr (1971) have shown that mixotrophic growth of E, 73 IEEEEE in axenic culture is stimulated by a variety of organic compounds including a number of metabolic intermediates and amino acids, as well as other compounds, including acetate. Approximately a third of the strains they studied were able to grow heterotrophically in the dark using acetate as a substrate. Another third were only stimulated by acetate in the light. Similar experiments for the strain isolated from Lake 3 showed stimulation of growth by acetate in the light but no growth in the dark. These results supported the possibility of photohetero- trophy (light enhanced heterotrophic growth) or mixotrophy (organic substrate stimulation of growth in the light over and above the auto- trophic growth possible at that light intensity (Palmer and Starr; 1971)). A large increase in pH during acetate stimulated growth in culture indicated autotrophic uptake of C02, providing preliminary evidence for for mixotrOphy as the mode of nutrition of g, mgggm_under these condi- tions. Since these cultures were not axenic and 10 mg/l of acetate is orders of magnitude greater than concentrations typically found in the field, further experiments are necessary to provide more convincing evidence for this. Possible sources of substrates for this growth are organic compounds released from dead cell autolysis, excreted from decomposing material . circulated up from the bottom. Experiments conducted here to determine if excreted organic matter from log phase cultures of S. communis and A, elenkinii would stimulate growth of g, mgggm_gave negative results. Sharpe (1977) has shown that there is little evidence to suggest that natural populations of phytoplankton release significant amounts of organic matter during their active growth phase. The growth of g, morum did occur primarily, however, during the 74 decline of earlier populations. This result suggests that the number of stationary phase and dying cells sinking through the water column would be great at that time. These would provide ample substrate for growth stimulated by organic matter as excretion from stationary phase cells is well documented (Sharpe, 1977). The large number of dead cells that could be expected under these conditions (Jassby and Goldman, 1974) would undergo decomposition during sinking and release much of their cell contents to the water column (Otsuki and Hanya, 1972). Recircu- lation of organic matter from the bottom sediments is also a possibility due to the frequent turnover of Lake 3 during the period of study. Based on the limited experimental evidence obtained and the above considerations, it seems possible that growth of S, mgggm_in Lake 3 was associated with mixotrophic nutrition. Further experimentation is necessary to reach a more definite conclusion. Conclusions A number of studies have shown that phytoplankton population fluctuations are influenced primarily by the dynamics of inorganic nutrients. This study provides further supportive evidence for that as well as evidence suggesting that other factors may at times be of over- riding importance. The occurrence of S, communis and A, elenkinii during specific periods of the growing season was shown to be largely due to the interaction of limiting levels of free CO and nitrogen, while 2 limited evidence suggested 2, mgggm_may have utilized organic matter to stimulate its autotrophic growth, allowing it to grow under conditions that would otherwise be unfavorable. Further evidence was presented to suggest that while inorganic nutrients are of primary importance in regulating phytOplankton popu- lation growth, temperature and light also play critical roles. Differ- ent groups of algae have different ranges of temperature tolerance and this may influence growth patterns within the constraints set by nutrient considerations. In this case, low temperatures may have been responsible for delaying the onset of A, elenkinii growth which could have occurred up to a month earlier had nutrients been the only con- trolling factors. The attenuation of light by dense phytoplankton blooms was implicated as a factor controlling growth rates of two of the populations. Evidence suggested that both S. communis and A, elenkinii were light limited during a portion of their growth due to this shading. 75 76 The results of the study do not provide evidence to enable a clear evaluation of the role of variable sinking rate in seasonal succession. Data from the LD chambers provide strong evidence that under stagnant conditions, sinking rate can significantly affect the ability of a species to survive under nutrient deficient conditions. However,-due to the frequent turbulence of Lake 3, the effects of sinking are dif— ficult to evaluate. While in only one case.did the field growth rates (pg) for S, communis and A, elenkinii exceed the pg values calculated, it is not clear that the difference between the two is due entirely to sinking. It would be expected, however, that under more quiescent conditions the effects of sinking could be substantial. Under these conditions, kinetics values determined from continuous cultures, which fail to provide a darkened zone, would not be expected to accurately estimate field values. This study suggests that while depletion of nitrate and ammonia may cause a shift in species populations from green to nitrogen-fixing blue-green algae, large standing crops can still develop, suggesting that nitrogen is not a significant factor limiting the annual productiv- ity of this system. Since phosphorus was always likely present in quantities adequate for optimum growth, it seems probable that light and carbon are the most important factors limiting the annual phyto- planktonic productivity of this system. Thes work has shown that while a simple, kinetics based model may not satisfactorily reproduce the week to week specific growth rates of the dominant species of planktonic algae, predictions of approximate periods of growth can be quite accurate. As more kinetic data become available on the more common dominant algal species of freshwater systems, 77 such a model has potential to predict species changes that would result from perturbations of existing nutrient conditions of lakes. Such a capability could be very useful in the analysis of the biological effects of various water resources related projects. More precise predictions of actual growth rates require the inclu- sion of more factors in a more complex polynomial model such as that proposed by Droop (1973). Such a model would likely provide more precise predictive capabilities and increase our knowledge of the pre- cise factors that influence algal growth and seasonal abundance patterns. 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Production in cultural solution of some amino acids by the atmospheric nitrogen-fixing blue-green algae. Archs Biochem. Biophys. 34: 50-55. Wetzel, R. G. 1975 Limnology. Philadelphia. W. B. Saunders Co. 743 pp. Young, T. C. and D. L. King. 1973. A rapid method of quantifying algal carbon uptake kinetics. Limnol. Oceanogr. 18: 978-981. APPENDICES Appendix 1. Water chemistry parameters measured by MSU Institute of water Research on Lake 3 using grab samples over 1977 growing season. Parameter Total Alkalinity Hardness Chloride Dissolved Oxygen Ammonium Nitrogen Nitrate Nitrogen Nitrite Nitrogen Total Kjeldahl Nitrogen pH Total Phosphorus Orthophosphorus Specific Conductivity Temperature Units mg/l CaCO3 mg/l CaCO3 mg/l C1 mg/l mg/l-N mg/l-N mg/l-N mg/l-N pH mg/l-P mg/l-P pmhos/cm °c 84 Appendix 2. Composition of culture medium used in algal growth kinetics experiments. Compound Mg804'7H20 KZHPO4 CaCl2 NaCl NaI-ICO3 NaNO3 EDTA KOH FeSO4-7H20 HZBO3 ZnSO4 MoO4 CoCl2 MnCl2 CuSO4-5H20 Concentration 50 mg/l 100 mg/l 25 mg/l 25 mg/l 100 mg/l variable 50 mg/l 31 mg/l 5 mg/l 11.4 mg/l 8.8 mg/l 0.71 mg/l 0.49 mg/l 1.44 mg/l 1.57 mg/l \ .l :7: : WM '- ICHIGAN s \WIHNIW 3129 TRTE UNIV. will} I Hi» 06290 LIBRQRIES u WI