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"' .Q': A';L~m" 0 {Egg 32:71:. ervj)‘ ~ . 5" q?“ ' q'. 90‘ ' JX~£fii€z£ ‘:%§‘2:‘ L‘ . l; Lfi‘gq‘ r 1.‘ '15-&;:‘L\ “1""? WEN ‘. \r‘bnzfl ‘ ‘- "JUL. 1&5." ILA: may” . LEVI? G"'" ,3“ _._\ ". 3“}..‘35‘1 f‘ [V ‘I- l- , . v.--. u . {TIL n- .,.. IE3} I ““3435: .. I.,".' 1: ‘73:? "-"N w- \ I \ IL (. '. '. -. "" mfg. .: .. ‘ . C .x‘ {fly‘dr’ikwux ." '5' p.“ . ) “Mg.“ . .‘2’2 F" W.‘ III‘I“:I.H.'.\] b. I! . [‘11:]! W2“ “9%. 3f ”0.63, ‘0 ,. 'u C “1.7“:0‘ I. 3'g A PA R Y * Miuillgln Static University WHES'S This is to certify that the thesis entitled INTERACTIONS OF CARBON AND NITROGEN METABOLISM WITH CHANGING LIGHT INTENSITY IN NATURAL POPULATIONS AND CULTURES OF PLANKTONIC BLUE-GREEN ALGAE presented by Amelia Kay Ward has been accepted towards fulfillment of the requirements for PhoDo degree in Botany 21.14, Jug! Major professor Robert G . Wet ze 1 Date ’6 C'y‘l‘\\73 0-7639 CT 112001 01006 02 “Er; is I IIIII III III IIII III III IIIIIII IIIIII ' 2579 © Copyright by Amelia Kay Ward 1978 ii INTERACTIONS OF CARBON AND NITROGEN METABOLISM WITH CHANGING LIGHT INTENSITY IN NATURAL POPULATIONS AND CULTURES OF PLANKTONIC BLUE-GREEN ALGAE By Amelia Kay Ward 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 1978 ABSTRACT INTERACTION OF CARBON AND NITROGEN METABOLISM WITH CHANGING LIGHT INTENSITY IN NATURAL POPULATIONS AND CULTURES OF PLANKTONIC BLUE-GREEN ALGAE By Amelia Kay Ward This study dealt with the factors contributing to the occurrence of blue-green algae in the plankton of lakes. Blue—green algal popula- tions were examined in two different aquatic systems, moderately produc- tive Lawrence Lake and hypereutrophic Wintergreen Lake, with regard to inorganic nitrogen source, light intensity and regime, and species of blue-green algae present. Unicellular and colonial blue-green algae in Lawrence Lake occurred with depth under continuously low light condi- tions with NOB-N or NBA-N as a nitrogen source; whereas, filamentous nitrogen-fixing species in Wintergreen Lake occupied the upper water strata and were exposed to variable light intensities over a diurnal period, when combined, inorganic nitrogen sources were low. In order to understand the relationship between light and nitrogen source better among natural populations, representative species of blue- green algae, including isolates of Aphanizomenon flos-aquae, Microcystis aergginosa, and Anabaena flos—aquae, were grown in laboratory cultures under continuously high, variable, and continuously low light at intensi- ties similar to those in the lakes. 0f three inorganic nitrogen sources utilized (NH4-N, N0 -N, and NZ-N), NH -N always resulted in highest 3 4 Amelia Kay Ward growth rates with all light regimes. However, intermittent exposure to high and low light intensities over a diurnal period had several advan- tages for N -fixing cultures compared to those grown with NO -N or 2 3 NHa-N. Growth could not be maintained at continuously low light for Nz-N cultures, and cultures transferred to continuously low light from a higher light intensity decreased in photosynthetic rates within 4 to 8 hours. Cultures grown with NO3-N or NH4-N maintained a more constant photosynthetic rate over this period. Furthermore, periodic exposure to low light ameliorated inhibition of photosynthesis in cultures grown under high light conditions. Because nitrogen fixation saturated at lower light intensities than carbon fixation, nitrogen-fixing cultures showed a greater disparity in cellular carbon and nitrogen content than NO3—N or NH4-N grown cultures when exposed to continuously high versus continuously low light intensities. Hence, a more uniform cellular carbon and nitrogen content could be maintained with nitrogen-fixing cultures exposed to an array of light intensities daily. Regardless of nitrogen source, periodic exposure to high light intensities was neces- sary to keep cultures from becoming low—light adapted. The molecular weight of dissolved organic carbon released from axenic cultures was dependent on nitrogen source and light intensity. From 36 to 70 per cent of released dissolved organic carbon was in the molecular weight range of less than 500 Daltons. Natural populations of blue-green algae from Lawrence and Winter- green lakes appeared particularly well adapted to the habitats in which Amelia Kay Ward they occurred. Maximum growth rates for non-nitrogen-fixing populations in Lawrence Lake subjected to a continuously low light regime could be maintained with NBA-N as a nitrogen source. High light adapted nitrogen- fixing populations in Wintergreen Lake exposed to a gradient of light intensities could respond maximally to high light intensities when exposed, survive brief periods of low light intensities as well as main— tain a balanced C:N ratio. This work is dedicated to my parents, Ralph and Ruthelle Jones, who first encouraged me to ask questions. iii ACKNOWLEDGMENTS I extend appreciation to my committee members, Drs. R. G. Wetzel, M. J. Klug, P. G. Murphy, and C. P. Wolk for their critical evaluation of this manuscript and helpful suggestions. In particular, Dr. Wetzel has provided outstanding guidance throughout my graduate training. I would like to thank him.for his unwavering encouragement, professional counsel, and, occasionally, timely silence. Many people at the Kellogg Biological Station have contributed to the completion of this project, giving of themselves professionally and personally in countless ways. Among them are Kelton McKinley, John Molongoski, Gordon Godshalk, Wilson Cunningham, Joyce Dickerman, Jan Grace, Jim Grace, Polly Penhale, Art Stewart and Claudia Weaver. I thank Janet Strally, Jay Sonnad, and Pat Brown for cheerful and conscientious technical assistance. Werds seem inadequate in expressing my gratitude to my husband, Milton Ward, who, with typical unselfishness, has given advice and constant moral support during these past years. Financial support for portions of these studies was provided by the National Science Foundation (GB-40172, BMS-7S-20322, EMS-75—l9777, GB-28900X) and the Department of Energy, formerly U. S. Energy and Resource Development Agency (E-1101599, COO-1599-134). iv TABLE OF CONTENTS LIST OF TABLES oooooooooooooooo o oooooooooooooooooooo 0000000000000 Vii LIST OF FIGURES. ooooooo ooooooo00.00000000000000000.000.000.000... Viii H ImeCTION...0000 .......... .00... ..... 0... ....... 0.0.0.0....00 Occurrence................................................ Nitrogen Sources.......................................... Interaction of Light, Nitrogen Source, and Photosynthesis. Objectives................................................ 0‘wa co “TERIALSANDMETHODSooooOQQ 00000 0 00000000 .0......0.0.0.0....0.0 Field Procedures.............. ..... ....................... 8 General................................................ 8 Physical-Chemical...................................... ' 8 Biological............................................. 11 Laboratory Procedures..................................... 12 Culturing Techniques................................... 12 Cellular Carbon and Nitrogen........................... 14 14Carbon Fixation...................................... 17 Molecular Weight Fractionation of Released Dissolved Organic Carbon...................................... 17 OVERVIEWOF SNDY SITES.0000....0 ....... ..00000....0.000000..0.. 19 “SUIJTS.....0000.00 0000000000000000000000 .0000. 00000000 .00000.0. 21 Lawrence Lake......................... ...... ......... ..... 21 Occurrence of Blue-green Algae......................... 21 Sources of Nitrogen.................................... 21 Light Regime............. ...... ........................ 25 Wintergreen Lake..................................... ..... 27 Occurrence of Blue—green Algae..... ......... ........... 27 Sources of Nitrogen.............. .......... ............ 27 Light Regime........... .................. .... ..... ..... 33 TABLE OF CONTENTS--continued Page Growth Rates, Nitrogen Source, and Light Intensity ....... . 33 C:N Ratios, Nitrogen Source, and Light Intensity.... ...... 38 Changes in Rates of Assimilation of Carbon and Nitrogen with Changes in Light Intensity......... ..... .......... 38 Differences in Photosynthetic Response with Changing Light Regime........................................... 49 Molecular Weight Fractionation of Released Dissolved Organic Carbon......................................... 54 DISCUSSION...................................................... 57 Overview........................... ............. ... ....... 57 Nitrogen Source and Growth Rates.......................... 59 Light Regime and Nitrogen Source.......................... 62 Assimilation Rates of Carbon and Nitrogen with Changing Light Intensity........................................ 65 Adaptation to High and Low Light Intensities.............. 65 Molecular Weight Fractionation of Released Dissolved Organic Carbon......................................... 66 CONCLUSIONS..... ............................ ... ...... ........... 68 LITERATURE CITED ..... .......... ......... ........................ 70 vi LIST OF TABLES TABLE 1. 4. 5. Growth rates (5) of Aphanizomenon flos—aquae and Microcystis aeruginosa grown with three nitrogen sources and under three light regimes: High (continuously 1500-1800 Lux), Variable (see Figure 3), and Low (continuously 150-200 Lux).......... Changes in C:N ratios throughout growth with Aphanizomenon flos-aquae and Microcystis aergginosa grown with three nitrogen sources and under three light light regimes: High (continuously 1500-1800 Lux), Variable (see Figure 3), and Low (continuously 150-200).................................. Changes in cellular carbon and nitrogen content in N -fixing cultures of Aphanizomenon flos-aquae and Anabaena flos-aguae exposed to continuous light of four different intensities (L - 2000 Lux; L - 1300 Lux; L - 800 Lux; L - 150 Lux). Changes in cellular carbon and nitrogen content in cultures of Microcystis aergginosa grown with N0 -N and NH -N and exposed to continuous light of two different inte sities (L1 - 2000 Lux; L - 150 Lux). T - initial time of expo- sure; T - time ter 10 hours of exposure with Aphanizome- £22.flos-aguae and Anabaena flos-aquae, and after 12 hours exposure with Microcystis aeruginosa........................ Molecular weight fractionation of dissolved organic carbon released from cultures of Microcystis aeruginosa grown with NO -N and NH -N and exposed to high (1500-1800 Lux) and low (1 0-200 Lux light intensities. 2 Total refers to the per- centage DEM of total dissolved organic 14carbon filtered through glass fiber filters. C.V. . coefficient of variance Molecular weight fractionation of dissolved organic carbon released from cultures of Anabaena flos-aguae (G-R) grown with Nz-N and exposed to high (1500-1800 Lux), medium (800 Lux) and low (150-200 Lux) light intensities, 1 Total refers to the percentage DPM of total dissolved organic 1“carbon filtered through glass fiber filters. C.V. - co- efficient of variance.. vii Page 35 39 43 55 56 LIST OF FIGURES FIGURE Page 1. Bathymetric map of Lawrence Lake, Barry County, Michigan... 9 2. Bathymetric map of Wintergreen Lake, Kalamazoo County, M1Chigan...0.0..000.....0............0.................00.0 10 3. Light regime for cultures grown under variable light intem1tie80.000.00.0............0.......0...0......000.... 15 4. Spectral composition of Vita-lite bulbs compared to three depths in Gull Lake: -—-Gull Lake at 0 (upper), 2.0 (middle), and 6.0 meters (lower); .... Vita-lite 1amps..... 16 5. Depth-time diagram of isopleths of temperature (0C) in Lawrence Lake, Michigan, l975.............................. 22 6. Depth-time dist ibuti n of i3 situ rates of primary produc- tion in mg C m7 day“ , Lawrence Lake, Michigan, 1975...... 23 7. Concentrations of NBA-N (Hg 1-1, :;S.D.) at four depths in Lawrence Lake, Michigan, 1975.............................. 24 8. Light penetration in Lawrence and Wintergreen lakes as percentage surface light intensity during maximum develop- ment of summer blue-green algal populations. Arrows delineate strata occupied by blue-green algae.............. 26 9. Saturation curves for photosynthesis for blue-green algal populations in Lawrence and Wintergreen Lakes Qt S.D.)..... 28 10. Depth-time diagram of isopleths of temperature (0C) in Wintergreen Lake, Michigan, 1975........................... 29 ll. Concentrations of NH -N (ug/l, 1 S.D.) at four depths in Wintergreen Lake, M1 higan’ 1975000....0.0000000000000000... 30 12. Concentrations of chloroph ll §_(mg/m3) in situ rates of primary production (mg C/mglhr, + S.D.), and rates of acetylene reduction (11M ethylene71/hr, i 5.0.) at 0.5 and 2.0 meters in Wintergreen Lake, 1975...................... 32 viii LIST OF FIGURES--continued FIGURE l3. 14. 15. 16. l7. l8. 19. 20. Changes in photosynthetic activity/unit biomass (DEM/mg C/hr) with growth for cultures of Aphanizomenon flos-aguae grown with N -N, NO3-N, and NH -N at continuously high, variable and continuously low Tight intensities (i;S.D.).. Changes in photosynthetic activity/unit biomass (DEM/mg C/hr) with growth for cultures of Microcystis aeruginosa grown with N0 -N and NH -N at continuously high, variable and continuougly low light intensities (1 S.D.)........... Increase in relative rates of carbon fixation (DEM/10 md/ hr, 1 S.D.) and N -fixation (11M ethylene/10 ml/hr : S.D.) 'with increasing lfght intensities in cultures of Aphanizomenon flos-aquae.................................. Photosynthetic carbon fixation/nitrogen fixation assimila- tion ratios at 0.5 and 2.0 meters in Wintergreen Lake. Values were calculated from data presented in Figure 12... Changes in C:N ratios of N -fixing cultures of Aphanizome- non flos-aquae and Anabaena flos-aguae exposed to continu- ous light of four different intensities Qt S.D.). Cultures were sampled during early log phase growth....... Changes in C:N ratios in cultures of Microcystis aeruginosa grown.with NO -N and NH -N and exposed to con- tinuous light of two different int nsities Qi'S.D.). Cultures were sampled during early log phase growth....... Changes in photosynthetic rates/unit biomass (DPM/mg C/hr, 1:8.D.) with NO -N and Nun-N grown cultures of Microcystis aeruginosa (upper two lines) and N -fixing cultures of ‘Aphanizomenon flos-aguae (third liae) and Anabaena flos- aguae (fourth line) when exposed to continuously low light (150 Lux). Cultures were sampled during early log phase grwth................0.....0..........0..0........0...... Changes in photosynthetic rates/unit biomass (DPM/mg C/hr, 1:8.D.) with N -fixing cultures of Aphanizomenon flos- aguae (upper) and Anabaena floseaguae (lower) when exposed to continuously high vs. intermittent light intensities (2000 and 800 Lux). Cultures were sampled during early log phase growth.......................................... Page 36 37 40 42 44 45 47 48 LIST OF FIGURES--continued FIGURE 21. 22. 23. 24. Saturation curves for photosynthesis for NO -N and NH -N grown cultures grown at continuously low light intensities (150-180 Lux) for a)_Aphanizomenon flos-aquae, b) Anabaena flos-aquae (A-52), and c) Anabaena flos-aquae (ArllB-s-q-a) (: S.D.). Cultures were sampled during early log phase growth .................................... Saturation curves for photosynthesis for N -N grown cul— tures of_Aphanizomenon flos-aquae (upper graph) grown under variable (top line) or continuously high light (lower line) and for N0 -N and NH —N grown cultures of Microcystis aeruginosa lower graph) grown under variable light intensities (i;S.D.). Cultures were sampled during early log phase growth.................................... Saturation curves for photosynthesis for N0 -N and NH -N grown cultures of Microgystis aergginosa grown under con- tinuously high light (upper graph; arrow indicates light intensity cultures grown under) and after 47 hours of exposure to continuously low light (lower graph; arrow indicates light intensity to which cultures were trans- ferred) Q: S.D.). Cultures were sampled during early log phase growth ..... ......... ...... ...... ....... ............. Saturation curves for photosynthesis for N0 -N and NH -N grown cultures of Microcystis aergginosa grown under con- tinuously low light (T ; arrow indicates light intensity under which cultures were grown); after 27 hours of expo- sure to a high light regime (T ; arrow indicates light intensity to which cultures were transferred), and after 47 hours of exposure to a high light regime (T ) Q: S.D.). Cultures were sampled during early log phase growth ....... Page 50 51 52 53 INTRODUCTION Certain members of the blue—green algae are unique among primary producers in that they are capable of assimilating elemental nitrogen under extracellularly aerobic conditions. This ability extends the range of ecologically important inorganic nitrogen sources to include N2-N as well as NOB—N and NHA-N. As prokaryotes, these organisms have many physiological characteristics more similar to bacteria than algae. However, in a majority of their natural habitats, they function as primary producers, incorporating inorganic carbon photosynthetically, which is then available to the rest of the system in the form of particu- late or dissolved organic carbon. From a functional standpoint, then, blue-green algae seem more closely aligned to other algal groups than bacteria. However, those bacterial characteristics which make blue- green algae unique among algal groups should give them a competitive advantage under certain conditions in their natural habitat. Among the characteristics which distinguish blue-green algae from other algal groups are the ability to fix atmospheric nitrogen and the possession of gas vacuoles, whereby some members can alter their position.within a light or nutrient gradient. Occurrence Planktonic blue-green algae occur in several different habitats. Frequently, these algae are associated with surface "blooms" in 1 Eli eutrophic lakewaters during periods when measurable inorganic nitrogen of the water strata are low (e.g., Duong, 1972; Ganf and Horne, 1975; Horne, 1970; Horne and Goldman, 1972; Horne et 31., 1972). Nitrogen- fixing species are usually dominant in these systems, but uptake of combined inorganic nitrogen, in the form of N0 -N and NH -N concomitant 3 4 with N -N has been reported among phytoplanktonic assemblages almost 2 exclusively composed of blue-green algae (Dugdale and Dugdale, 1965; Billaud, 1969). Populations of blue-green algae can also become success- fully established in deeper, more oligotrophic waters. Particularly ‘well-documented examples of this type include accounts of species of Oscillatoria occurring within the metalimnion during summer stratifica- tion (e.g., Wetzel, 1966; Baker eg‘gl., 1969; Saunders, 1972; Klemer, 1976). Other groups besides Oscillatoria, however, have also been reported in this type of lowblight environment (Eberly, 1959; Baker and Brook, 1971). Various hypotheses have been advanced to explain the success of blue—green algal populations in these physiologically divergent habitats. Several factors must be considered in relation to those populations which develop at great depth in less productive lakes as opposed to sur- face populations common to eutrophic conditions. First, the temperature- density characteristics of the metalimnion may be more suitable for maintenance of buoyancy than the warmer, less dense water of the epilimr nion in that populations may exist poised in the lower water strata of the photic zone (Fogg, 1969). Second, there is some evidence that the resulting low-light conditions found at this depth are more favorable to growth of blue-green algae than other groups of algae, for example green algae (Mur 25.31., 1977). Third, existence in this deeper zone is closer to accumulated minerals in the commonly reduced conditions of the hypolimnion during summer stratification (Wetzel, 1975; Klemer, 1976). Maintenance within the photic zone, although important in oligo- trophic lakes, becomes increasingly critical in very productive eutrophic systems, where light can be attenuated rapidly over a short distance because of biogenic turbidity. Synthesis and collapse of gas vascuoles, which are unique to certain prokaryotes (walsby, 1972), provide potential- ly powerful mechanisms for maintaining buoyancy within the photic zone (Dinsdale and Walsby, 1972). When attenuation of light becomes extr-e, photosynthetic maxima are increasingly displaced and compressed toward the surface of lakes (Wetzel, 1966; 1975). Hence, gas vacuoles provide a means by which rapid movement in response to changing light conditions could be attained over a relatively short distance. Nitrqgen Sources Superimposed on effects of light and nutrients in general, are the more specific effects of nitrogen source on the success of different types of blue-green algal populations. From an energetic standpoint, preference of nitrogen source should follow the order NH4-N3’NO3—ND’N2-N. However, studies with natural populations and cultures of blue-green algae indicate the effect of nitrogen source on growth is considerably more complex. Although blue-green algae grow well on NO3-N and NBA—N as -N (in N -N has been suggested as the well as N -fixing species), NO 2 2 3 "preferred" source in culture media (Fogg, 1973), as well as the better nitrogen source for growth (Wetzel, 1975, p. 198). Reservations con— cerning the use of Nz-N and NH -N probably stem from culture studies in 4 which combined nitrogen supported better growth than N -N (Kratz and 2 Myers, 1955) although this is not always the case (Allen and Arnon, 1955; Singh and Srivastava, 1968), and undesirable side-effects can result from use of NH -N in culture. For example, the use of NH -N in culture 4 4 can cause a drop in pH to inhibitory levels (Singh and Srivastava, 1968), cell lysis (Pintner and Provasoli, 1958), and toxicity at high pH (Stewart, 1964). Paradoxically, while N -N and NH -N can present prob- 2 4 lems as nitrogen sources in culture, natural populations of blue-green algae are frequently Nz-fixers or populations which occur in systems with potentially high turnover rates of NHa-N (as in eutrophic systems or in the metalimnion of less productive systems). With nonrnitrogen- fixing populations of Oscillatoria agardhii in Lake Deming, Minnesota, Klemer (1976) found that enrichment with NHa-N resulted in increased filament numbers; whereas, enrichment with NOB-N or a combination of N03-N and PO4-P did not. Klemer concluded that Oscillatoria ggardhii was likely restricted to metalimnetic water strata because of nutrient limitations in the epilimmion (specifically NH4-N) rather than an in- herent oligothermy. Thus, nitrogen source varies considerably among planktonic blue-green populations depending upon other environmental conditions. Interaction of Light, Nitrogen Source, and Photosynthesis The combination of light and nitrogen source could be determining factors in the success of blue-green algal populations in that a specific light regime may be required to maintain optimal growth on a given nitrogen source. All processes of inorganic nitrogen assimilation in primarily autotrophic organisms are dependent upon photosynthesis for carbon for the incorporation of end products of nitrogen reduction. In addition, nitrate reduction, nitrogen fixation, and to some extent, ammonia assimilation are light-stimulated events in blue-green algae (Fogg and Than-Tun, 1960; Hattori, 1962; Healey, 1977). Nitrogen fixa- tion appears specifically associated with Photosystem I (Lex and Stewart, 1973) and to a lesser extent on Photosystem 11, depending on the physio- logical state of the cell (Cox and Fay, 1969) and rates of photorespira- tion (Lex 95 e1., 1970). The association of nitrate reduction with the photosystems is less well-defined, with nitrite reductase apparently more closely associated with Photosystem II (Fujita and Hattori, 1963; wolk, 1973) than is nitrate reductase (Stevens and Van Baalan, 1973). Of potential importance to natural populations of blue-green algae are the different effects of light on nitrogen assimilation and photosynthetic carbon fixation. Cobb and Myers (1964) established dif- ferences in response to light intensity between nitrogen fixation and carbon fixation in Anabaena cylindrica; that is, nitrogen fixation rates tended to saturate at lower light intensities than carbon fixation rates. They hypothesized that Anabaena cylindrica possessed a mechanism whereby the cellular C:N ratio played a role in other cellular events, specifically heterocyst formation. Also, a positive correlation was reported by Kulasooriya egugl. (1972) between C:N ratio and heterocyst development in Anabaena cylindrica. Therefore, differences in assimila- tion rates in response to light between nitrogen fixation and carbon fixation affect the cellular C:N ratio, which in turn may affect other aspects of metabolism as specific as heterocyst development or more generally to "more balanced growth" (Peterson 25 31., 1977). Less is known of the relative rates of NHA-N and NOB-N assimilation in relation to photosynthetic carbon fixation and light intensity among representa- tives of natural populations of blue-green algae. In summary, planktonic blue-green algae are capable of utilizing a number of inorganic nitrogen sources. Their presence in surface waters of eutrophic lakes and at depths of low light intensities in more oligotrophic systems indicates a competitive advantage in these habitats, which differ considerably in nitrogen source, light intensity, and light regime. Therefore, among the many factors which contribute to the success of blue-green algae in lakes, the interactions of light, nitrogen source and photosynthesis offer particular potential. Objectives This research focused on the dynamics of two different types of blue-green algal populations in lakes of differing trophic status. The first part of the investigation was devoted to a study of various physical-chemical and biological factors of the lake systems. Of partic- ular interest were 1) source of nitrogen, 2) light regime, and 3) species of blue-green algae present. The second part of the investi- gation was directed at elucidating mechanisms operative in the establish- ment of blue-green populations by using unialgal or axenic laboratory cultures of representative species. Isolates of Microcystis aeruginosa, Aphanizomenon flos-aquae, and Anabaena flos-aquae were employed to examine growth rates, cellular carbon and nitrogen content, N -fixation 2 rates and carbon fixation rates under conditions designed to simulate those found _i_n_ 3:153 in the two main types of observed habitat. Although Rodhe (1948) recognized a "time of exposure" factor with regard to phytoplankton response to light and temperature, no experi- mental studies have incorporated, in a systematic manner, differences in light intensity 22g light regime as they occur in natural systems. Of emphasis in this study were the effects of continuous light of one intensity versus variability of light intensity in the context of the lake populations under investigation, and how these effects would be manifested in the parameters listed above. The presence of dissolved organic carbon and particularly dissolved organic nitrogen is intimately associated with natural populations of blue-green algae (e.g., Pearsall, 1932; Fogg, 1971) and is potentially of great importance to the detrital dynamics of aquatic ecosystems (Rich and wetzel, 1978). In certain strata of lakes where concentrations of combined, inorganic nitrogen sources are low, dissolved organic nitrogen released from the phytoplankton populations can represent a significant input of nitrogen to that portion of the system. Besides providing a substrate for the bacterial component of the community, these organic nitrogen compounds, upon mineralization, may provide an inorganic nitro- gen source in the form of NHh-N for the algal component. Of particular interest in this study were the qualitative differences among dissolved organic compounds released by blue-green algae under different condi- tions of light and nitrogen source. MATERIALS AND METHODS Field Procedures General: Sampling on Lawrence and Wintergreen lakes began in April, 1975, shortly after ice-off and continued through October, 1975, before autumnal circulation. Water samples were collected with an opaque Van Dorn water sampler from the central depression of each lake (Figures 1 and 2) every two weeks at four depths (2, 4, 6, and 10 meters in Lawrence; 0.5, 2.0, 3.0, and 4.0 meters in Wintergreen). Physical- chemical parameters which were monitored included temperature, light penetration, pH, alkalinity, and concentrations of N03-N and NH -N. 4 Ig_situ rates of photosynthesis and nitrogen fixation were also measured. Physical-Chemical: Temperature and Ligh». Temperature measurements were made with a Yellow Springs Instrument Tele—thermometer (Model 43TB). Light pene- tration as percentage of surface light was determined using an underwater photometer (Rich and Wetzel, 1969). ApH and Alkalinity. Measurements of pH were made utilizing either a Beckman Expandomatic (Model 76A) or Coleman (Model 38A) prmeter. Alkalinity in meq 1..1 was determined by titrating 50 milliliter water samples with 0.02 N H SO to pH 4.46 to 4.48 indicated by a mixture of 2 4 brom—cresol red and methyl orange (Amer. Publ. Health Assoc., 1976). .amwfinoaz 33550 than .deA mason—33 no use oauuoamnumm .H 933nm new»! I 32”.;- (580 rung hf ma w»! 23.5.: or 5:8 Exam 321.2. » R oum my?) wozmmzé... £0.00 0.1.. 10 WINTERGREEN LAKE KALAMAZOO COUNTY, MICHIGAN R.9W.,T.IN. Sec.8 ELEVATION 271m, AREA '5‘. h. ° so I00 no M! 15.3 CONTOUR INTERVALS IN METERS Figure 2. Bathymetric map of Wintergreen Lake, Kalamazoo County, Michigan . ll Nitrate and Ammonia. Water samples were filtered through pre- combusted (525 C for 45 minutes), glass-fiber filters (Reeve-Angel, 984H) and analyzed for NHA-N by the procedures of Harwood and Kuhn (1970), and for NO -N by the cadmium reduction technique (Wood SE 31., 3 1967). Biological: Primary Productivity. lg §1E2_rates of photosynthesis were obtained by incubation over an approximately four hour period with NaHll‘CO3 (Strickland, 1960). One milliliter of NaH14CO3 was added to a water sample in 125-ml stoppered glass Pyrex bottles and incubated at depths from which the samples had been collected. After incubation, aliquots of either 50 ml (Lawrence Lake) or 25 ml (Wintergreen Lake) were filtered from the bottles through Millipore HA filters (0.45 um pore size). Filters were desiccated, fumed with HCl (Wetzel, 1965), and analyzed by GeigereMfiller radioassay (Nuclear-Chicago D-47 of known counting efficiency). Results were reported as mg C/mg/hr. Nitrogen Fixation (Acetylene Reduction). The procedure for esti- mating igmgigg rates of nitrogen fixation followed closely those described previously by Ward and Wetzel (1975). However, the vials were .not flushed with a nitrogen-free gas prior to incubation in order to provide a more natural atmosphere above the samples and an extra set of controls for determining ethylene transformation (Flett eg 31., 1975) was included. The procedure for detecting ethylene transformation was as follows: a known amount of purified ethylene (Matheson Gas Products, Joliet, Illinois) was injected into killed (0.2 m1 2% HgClz) and 12 unkilled samples prior to incubation. Ethylene from these vials was measured concurrently with regular samples. A significant decrease in ethylene concentration in the head-space of unkilled samples compared to that in killed samples would indicate ethylene transformation. No sig- nificant decrease was noted, therefore ethylene transformation was assumed absent. Gas analyses were performed on a flame-ionization Varian Aerograph gas chromatograph (Model 600-D) equipped with a stain- less steel column (3 mm x 2 meters) packed with Porapak-N (80-100 mesh). Pigments. Concentrations of chlorophyll a_(corrected for pheo- pigments) were determined by filtering known volumes of lakewater through Millipore AA (0.8 pm pore size) filters. Pigments were extracted from the filters in 90% basic, aqueous acetone and absorption of the super- natant measured by a Hitachi-Perkin Elmer spectrophotometer (Model UV-VIS 139). Calculations were those of Parsons and Strickland (1963), Westlake (1969), and Wetzel and Westlake (1969). Phytoplankton. Algal samples were preserved with Lugol's solution (Ward and Whipple, 1959, p. 2000) and examined by the sedimentation technique in settling chambers with a Wild inverted microscope. Laboratory Procedures CulturinggTechniques: Representatives of blue—green species found in Lawrence and Wintergreen lakes were grown in cultures in the laboratory. Isolates of Microcystis aeruginosa Kfitz emend. Elenkin, clone NRC-l (SS-17), and Aphanizomenon flos-aquae (L.) Ralfs), isolate NRC-566, were used most l3 frequently in experimental work. Isolates of Anabaena flos—aquae (s-29-f-6), Anabaena flos-aquae (A—llB-s-q-a), and Anabaena flos-aquae (Ar52) were used periodically. These strains were isolated by Drs. Wayne Carmichael and Paul Gorham (Carmichael and Gorham, 1975) from the phytoplankton of hardwater lakes in Canada. In addition, a culture of Anabaena flos-aquae (G—R) was provided by Dr. G-Y Rhee, New York State Department of Health, isolated from Lake Erie. Samples from cultures of Microcystis aergginosa (SS-17) and Anabaena flos-aquae (G-R), peri- odically streaked on Plate-Count agar (Difco) and examined microscOpical- 1y (1000 X, phase contrast, Zeiss microscope) showed no bacterial growth. All other cultures were unialgal, but bacterized. Algae were maintained in batch culture on modified Moss medium (1972). The concentration of nutrients was based approximately on world averages for fresh waters and hence was less than concentrations of nutrients employed in other algal media (e.g., ASM-l; Carmichael and Gorham, 1975). The following modifications were made: NaSiO °9H20 and 3 the vitamin mixture were deleted; NaHCO3 concentrations were doubled (4.0 g/l); Tricine* (Sigma Chem. Co.) was included as a buffering agent (130 mg/l); and the pH before autoclaving was adjusted (0.1 N NaOH) to 7.3-7.5 to yield a final pH after autoclaving of 8.0 i 0.1. In prelimi- nary experiments no difference in algal growth was found between Ca(N03)2'4H20 and KNO3 as a NOB-N source. Thereafter, KNO3 was substi- tuted as a NO3-N source. Concentrations of NH4C1 and KNO3 were adjusted *(N-Tris(hydroxymethyl)methyl glycine). 14 to yield a final concentration of 5.0 mg N/l in experimental media. The medium was buffered rigorously; therefore, little fluctuation in pH occurred regardless of nitrogen source. A maximum increase of pH to 8.3-8.5 occurred in certain late log phase cultures. Stock and experimental cultures were grown in Sherer growth cham- bers at a temperature of 23.: 1.0 C. Light was supplied by Vita-lite fluorescent bulbs (Luxor) at an intensity of 150-200 Lux, variable (Figure 3), or 1500-1800 Lux (high) on a light regime of 17 hours light: 7 hours dark. Light intensities simulated those found lg §i£g_during mid-morning in July between a depth of 0.5 meters and 2.0 meters in Wintergreen Lake and 6.0 meters in August in Lawrence Lake. Surface light intensities were not used in order to avoid confounding effects of photoinhibition. The spectral composition of light from Vita-lite bulbs, determined utilizing a scanning spectroradiometer (Model SR, Instrumenta- tion Specialties Company, Inc.), was found to be similar in quality to that at 2.0 meters in Gull Lake (Kalamazoo County, Michigan), a hard- water lake similar in chemical composition to Lawrence and Wintergreen lakes (Figure 4). Cellular Carbon and Nitrogen: A known volume of algal culture was filtered onto 13-unn diameter pre-combusted (525 C for 45 minutes) glass fiber filters (Reeve-Angel 984H). Filters were rinsed with glass distilled water to remove dis- solved carbon and nitrogen contaminants left on the filter from the culture medium, wrapped in tin, and pelletized. Pellets were combusted in a Carlo-Erba elemental analyzer (Model 1104), interfaced with an 15 L MH M L L__..L_J_.l__1___l 0500 0800 1000 1200 1600 2200 = 200 LUX 800 LUX 1500 LUX L M H Figure 3. Light regime for cultures grown under variable light intensities. l6 p W/cm2 1.000 b .1 000 ~ .. 400 I- 400 450 500 550 000 650 700 750' WAVELENGTH (nm) Figure 4. Spectral composition of Vita-lite bulbs compared to three depths in Gull Lake: Gull Lake at 0 (upper), 2.0 (middle), and 6.0 meters (lower); ---- Vita-lite lamps. l7 automatic digital integrator (Columbia Model 081-208) and Monroe (Model 1305) digital print-out. Two blanks (glass fiber filter and tin) and three standards (Cicloesanone-Z,4-dinitrofenilidrazone, Carlo-Erba) were assayed with every 18 samples. Cellular carbon values were corrected for carbon contamination (l-SZ of sample values) in the calculations. Nitrogen contamination was undetectable. 1liCarbon Fixation: Rates of photosynthesis were measured by incubating 50-ml aliquots from experimental cultures with 0.25 ml of Na14CO3 (specific activity, 5.12 uCi/ml) for 30-60 minutes. Smaller amounts (5-10 ml) were filtered onto Millipore HA (0.45 um pore size) filters for radioassay. Molecular Weight Fractionation of Released Dissolved Organic Carbon: Dissolved organic carbon released from blue-green algal cultures was obtained by incubating cultures with NaHlACO3 of high specific activ- ity (approximately 100 uCi added to 300 ml of culture) under various light intensities. After approximately 4 hours of incubation, cultures were filtered through Reeve-Angel glass fiber filters (984H). The re- sulting filtrate was the "Total" fraction and aliquots (25 ml, 2 deter- minations) from this fraction were passed through Amicon membranes of the following designation and nominal molecular weight cut-off: PM 30== 30,000 Daltons; PM 10 = 10,000 Daltons; UM 2 = 1000 Daltons; and UM 05== 500 Daltons. These fractions were acidified to pH 3 with H3P04, purged for 10 minutes with CO2 to remove residual inorganic carbon, and then 18 subjected to 1yophilization and scintillation radioassay by the tech- niques of McKinley e5 31. (1976). Results were reported as the percent- age disintegrations per minute (DPM) of the "Total" fraction. OVERVIEW OF STUDY SITES Lawrence and Wintergreen lakes are small, hardwater lakes located in southwestern.Michigan. However, whereas Lawrence Lake is considered oligo— to mesotrophic, Wintergreen Lake is hypereutrophic. These lakes have been and continue to be the subject of intensive limnological research and considerable information on the physical-chemical and bio- logical interactions within these systems has been accrued from previous studies (e.g., Wetzel egflgl., 1972; Manny, 1973). In summary, Lawrence Lake, located in a small depression of land and bounded on several sides by emergent macrophytes, has a rather small surface area to volume ratio (maximum depth = 12.6 m., surface area - 4.9 ha.). Suppression of primary productivity within the openrwater portion of the basin is directly related to the chemistry of the lake- water, which is strongly alkaline and carbonate-rich. Nutrients, such as phosphorus, and trace metals tend to precipitate out, complex with CaC03, or are otherwise made physiologically unavailable to the phyto- plankton (Wetzel, 1972; Wetzel and Manny, 1978). Because of the inter- actions of basin morphometry and chemical characteristics of the water as well as aspects of the watershed in general, Lawrence Lake retains a considerable capacity to withstand rapid change. The system is chemical— ly well-buffered and this capacity is reflected in various biological parameters of the system as well. For example, although extensive l9 20 seasonal variations exist among many parameters (e.g., primary produc- tion, oxygen concentration and pH), there are not marked day to day or, in some case, week to week fluctuations (cf. review by Wetzel, 1975). Although of similar geological background to Lawrence Lake, Wintergreen Lake has a much larger surface area to volume ratio (maximum depth - 6.3 m., surface area - 15.8 ha). The higher percentage of shallow water has encouraged greater macrophyte production over the years in relation to Lawrence Lake and has been one factor contributing to the hypereutrophy of the lake (Manny £5 91., 1978). However, the establishment of the W. K. Kellogg Bird Sanctuary at this site in the recent past has greatly accelerated this condition. The organic input derived from 4600 kg dry weight of waterfowl feces annually (Manny _e_t_ _a__1., 1975) has eroded the buffering capacity of the system still inherent in Lawrence Lake, by means of a complex of interacting factors (cf. wetzel and Allen, 1970). As a result, Wintergreen Lake exhibits frequent and violent oscillations in annual biological and chemical properties. RESULTS Lawrence Lake Occurrence of Blue-green Algae: Lawrence Lake exhibited temperature characteristics typical of a dimictic, north temperate lake (Figure 5). During summer stratification, the depth of the epilimnion extended to about 6 meters by August. Typically, the blue-green populations occurred within or just above the metalimnion and were made up predominantly of colonial and unicellular forms such as Microcystis spp., Gomphosphaeria spp., Coelosphaerium spp., Chroococcus spp., and Aphanocapsa spp. Blue-green algae are often associated with eutrophic systems; however, even in moderately produc- tive Lawrence Lake, productivity rates associated with the blue-green algal populations in summer were higher than at any other time of the year (Figure 6). This late summer metalimnetic blue-green algal associ- ation has developed similarly each year for 11 years of continuous analysis (Wetzel, unpublished data). Sources of Nitrogen: Concentrations of NHa-N in Lawrence Lake were consistently low in the upper water strata (Figure 7), ranging from 25 to 100 ug NHb-N/l, however in the hypolimnion, NH4 of summer stratification progressed. At the time of the strong —N accumulated, increasing as the period 21 DEPTH (m.) . I) 22 I .. uI 3 ‘\ / ‘2 i 1 1 JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC' 1975 c’ Figure 5. Depth-time diagram of isopleths of temperature ( C) in Lawrence Lake, Michigan, 1975. DEPTH (m.) 23 L 1 NOV DEC Figure 6. Depth-time distribution of in situ rates of primary production in mg C m?3 day :1, Lawrence Lake, Michigan, 1975 . 24 I I I T T ' 2m 50. '1 o e : : : 4 4m 50- ‘ o : 4 . . v 4 L 6m g so: ‘ E . O i % 1 i i 1 '9' 400- 1001‘ I . z 300- I V a 7 I 150'- " too- ‘ SOI- " l l l L l l Figure 7. Concentrations of NH -N (pg 1-1, : S.D.) at four depths in Lawrence Lake, Michigan, 1975. 25 development of blue-green algal populations in August, a reservoir of NH4-N was available in the hypolimnion. A significant decrease in NHa-N concentrations at 6 meters indicated utilization of that nitrogen source at that depth, presumably by blue-green algae. Because of the close physical association of the bacterial and the algal components, it is not possible to distinguish the relative importance of each to the uptake of NHa-N. However, the utilization of NHa-N by bacteria, e.g., nitrifying bacteria, would not preclude concomitant utilization by the blue-green algae or vice versa. The problem could only be resolved directly by microautoradiography, assuming a suitable radioactive nitro- gen source. The occurrence of the blue—green populations was correlated with low levels of NH -N in the epilimnion (2 and 4 meters), a depletion l. of NBA-N at 6 meters and accumulation of NHA-N at depth. Concentrations of N0 -N were measured periodically and were much higher than NH -N con~ 3 4 centrations, in the range of 2-5 mg NO3-N/1 (cf. Wetzel, 1975, p. 204). Light Regime: Blue-green populations occurring in Lawrence Lake were exposed to a continuously lowhlight regime. Figure 8 illustrates a representative light profile taken during mid-August with the arrows indicating the depths occupied by blue-green algae. Light in this water stratum had been attenuated to between 9-11% surface light intensity, corresponding to approximately 180-240 Lux. Measurements of diurnal productivity rates during August indicate little, if any, movement above this stratum daily (McKinley and wetzel, 1978). Samples from populations exposed to light intensities varying from 150 to 2400 Lux were low-light adapted in 26 7, SURFACE LIGHT INTENSITY 0.1 1.0 10 100 if I f ..F 1 1 0 1 WINTERGREEN LAKE i; <. 35 d? 2.0m. 1 L fJ—__L___I 0 9 I5 2I 27 JULY 1975 Figure 16. Photosynthetic carbon fixation/nitrogen fixation assimilation ratios at 0.5 and 2.0 meters in Wintergreen Lake. Values were calculated from data presented in Figure 12. 43 Table 3. Changes in cellular carbon and nitrogen content in N -fixing cultures of Aphanizomenon flossequae and Anabaena flos-aquae exposed to continuous light of four different intensities (L I 2000 Lux;L .1300 Lux;L = 800 Lux;L - 150 Lux). Changes in cellular carbon and nitrogen contenf in cultures of Microcystis aergginosa grown N03 -N and NH -N and exposed to continuous light of two different intensifies (L1 - 2000 Lux; - 150 Lux). T - initial time of exposure; T - time after 18 hours of exposure with Aphanizomenon flos-aquae and Anabaena flos-aquae; and after 12 hours exposure with Microcystis aeruginosa. SE 1': i 1? Species Tl'mg C/l T]_mg N/l T2 mg C/l T2 mg N/l n-4 n-4 n-4 n-4 Aphanizomenon L1 2.10 L1 0.37 L1 3.02 Ll 0.45 flos-aguae L2 2.29 L2 0.42 L2 3.25 L2 0.51 (NZ-N) L3 2.14 L3 0.38 L3 2.62 L3 0.46 L4 2.18 La 0.39 L4 2.06 L4 0.38 Anabaena L1 4.58 L1 0.96 L1 6.34 L1 0.97 flos-aguae L2 4.57 L2 0.95 L2 5.95 L2 0.96 (NZ-N) L3 4.47 L3 0.91 L3 5.15 L3 0.93 L4 4.67 L4 0.95 L4 4.86 L4 0.96 Microcystis L1 0.78 Ll 0.15 L1 1.50 L1 0.22 aergginosa L4 0.46 L4 0.08 L4 0.78 La 0.14 (NO3-N) Microcystis L1 1.02 L1 0.21 L1 1.94 L1 0.37 aeruginosa La 1.03 L4 0.20 L4 1.28 L2 0.27 (NHa-N) C: N RATIO C:N RATIO Figure 17. 44 Apmzm'e' '°” 2000 Lux flos-aquae 6.5 r- .. I300 Lux I OJJr ‘ 800 Lux 5’5 I I 150 Lux ‘ f L l l 1 l l a; 6.5 _ A I na 2000 Lux . flos-aquae 1300 Lux 6.0 p d l 1 5.5 __ j 800 Lux _‘ I, I I 150 Lux 5.0 b j « ,L 1 L l L 1 1 j J 0 4 8 12 HOURS Changes in C:N ratios of N -fixing cultures of Aphanizomenon flos-aggae and Anabaena flos—aquae exposed to continuous light of four different intensities (1 S.D.). Cultures were sampled during early log phase growth. 6.5 6.0 $2 '5 5.5 .2. C) 5.0 6.0 92 ’2 (I E C) Figure 18. 45 ' ' ' ' ' 2000 t MIcrocystIs aerugInosa Lux NO3-N q 150 Luxa ’- d : ‘5 e #7 I i i’ I " MicrocystIs aeruginosa ‘ M4'N 2000 Lux 150 Luxfi: Changes in C:N ratios in cultures of Microcystis aeruginosa grown with NO -N and NH -N and exposed to continuous light of two different intensities (1 S.D.). Cultures were sampled during early log phase growth. 46 In contrast to results with NZ-N and NO3-N, Microcystis grown on NHa-N displayed much less of a disparity between carbon and nitrogen assimilation at high and low light intensities as reflected in C:N ratios (Figure 18). Hence, the carbon and nitrogen content remained more uniform with changes in light intensity. The decrease in C:N ratio at low light with NHa-N grown cultures, unlike the N2 was the result of a greater increase in cellular nitrogen relative to -fixing cultures, cellular carbon at that light intensity (Table 3). Photosynthetic rates/unit cell carbon differed with nitrogen source at continuously low light (Figure 19). Cultures of Microcystis aergginosa grown with NBA-N resulted in higher photosynthetic rates than those grown with NOB-N. Further, rates of carbon fixation did not de- crease over an 8 hour period with these nitrogen sources. However, Nz-fixing cultures of Aphanizomenon flos-aguae and Anabaena flos-aquae transferred from higher light intensities to low light showed an immedi- ate decrease in photosynthetic rates, undoubtedly a factor in the decrease in cellular carbon over the same period. When exposed to continuously high light, however, carbon fixation rates of Nz-fixing cultures of Aphanizomenon flos-aquae and Anabaena flos-aguae decreased over an 8 hour period (Figure 20). The decrease in photosynthetic rates was more pronounced with the denser cultures of Anabaena flos-aquae (4-5 mg cell carbon/l) compared to the cultures of Aphanizomenon flos-aqUae (2—3 mg cell carbon/1). The decrease in photo- synthetic rates at continuously high light was ameliorated to some extent if cultures were exposed intermittently to a lower light intensity 47 I I 1 I NO3'N E 40000" n a ' »-- ‘ e I e CL f C) 2CLOCK)- ‘ N2‘N ‘50 Lux 1 l, 1 1 0 4 8 HOURS Figure 19. Changes in photosynthetic rates/unit biomass (DPM/mg C/hr, :_S.D.) with NO -N and NH -N grown cultures of Microcystis aeruginosa (upper two lines) and N -fixing cultures of Aphanizomenon flos-aggae (thirg line) and Anabaena flos—aquae (fourth line) when exposed to continuously low light (150 Lux). Cultures were sampled during early log phase growth. 48 1.000.000 'I .E D 3’ \ -I 12 O. 0 200.000 4 0 1,000,000 7 .E \ ‘é \ .. 12 O. 0 200,000 I Figure 20. Changes in photosynthetic rates/unit biomass (DPM/mg C/hr, :_S.D.) with N -fixing cultures of Aphanizomenon flos-aquae (upper) and Anabaena flos- aguae (lower) when exposed to continuously high vs. intermittent light intensities (2000 and 800 Lux). Cultures were sampled during early log phase growth. 49 (Figure 20), as would theoretically be the case in circulating epilimne- tic water. This alternation was particularly effective with the denser cultures of Anabaena flos-aquae. Differences in Photosynthetic Response with Changing Light Regime Photosynthesis of these species of blue-green algae became satur- ated at a lower light intensity (800-1000 Lux) when grown at continuously low light than those grown at variable or continuously high light (Figures 21, 22, and 23). Cultures of_Aphanizomenon flos-aquae and Microcygtis aeruginosa, grown under the variable light regime saturated at intensities similar to those of cultures grown under continuously high light (cf. Figure 22 with Figures 23 and 24). Cultures of Microgystis aeruginosa, once adapted to a high or low light regime, would not re-adapt rapidly whether grown with N03-N or NBA-N (Figures 23 and 24). In Figure 23 (upper portion), cultures of Microcystis were grown under continuously high light prior to assays of photosynthetic response to different light intensities (arrow indicates light intensity at which cultures were grown). Cultures were then trans- ferred to a continuously low light regime (lower portion of Figure 23; arrow indicates the light intensity to which cultures were transferred). Re-examination of photosynthetic response after 47 hours (including two dark cycles of 14 hours) indicated a similar saturation curve, although fixation rates/unit cell carbon had increased markedly. Similarly, cultures grown at continuously low light (T1, Figure 24) and then DPM/mg C/hr Figure 21. 50 500.000 250.000 500.000 250.000 up up Saturation curves for photosynthesis for NO -N and NH -N grown cultures grown at continuously low light intensities (150-180 Lux) for a) Aphanizome- non flos-aquae, b) Anabaena flos-aguae (A952), and c) Anabaena flos-aggae (A-ll3-s-q—a) (i S.D.). Cultures were sampled during early log phase growth. 51 300,000.. I ....... I N03—N 11’, DPM/mg C/hr Figure 22. Saturation curves for photosynthesis for N -N grown cultures of Aphanizomenon flos-aqgae (upper graph) grown under variable (top line) or continuously high light (lower line) and for N0 -N and NH -N grown cultures of Microcystis aeru inosa (lower graph) grown under variable light intensities (: S.D.). Cultures were sampled during early log phase growth. 52 900.000 300.000 HIGH -> LOW 1.200.000 DPM/mg C/hr 600.000 Figure 23. Saturation curves for photosynthesis for N0 -N and NH -N grown cultures of Microcystis aeggginosa grown under continuously high light (upper graph; arrow indicates light intensity cultures grown under) and after 47 hours of exposure to continuously low light (lower graph; arrow indicates light in- tensity to which cultures were transferred) (18.0.). Cultures were sampled during early log phase growth. LOW+ HIGH ...................... I ........... 1 sssssssssss {000.099. J 1.200.000 __ I ........ T2 E 600.000 '- \ O C» o E \ E 1.200.000 D Figure 24. 53 Saturation curves for photosynthesis for N0 -N and NH -N grown cultures of Microcystis aeggginosa grown unéer continuously low light (T ; arrow indicates light intensity under which culEures were grown); after 27 hours of exposure to a high light regime (T2; arrow indicates light intensity to which cul- tures were transferred), and after 47 hours of exposure to a high light regime (T ) (i S.D.). Cultures were sampled during early log phase growth. 54 transferred to continuously high light showed little modification in saturation curves when examined after 27 hours (corresponding to T on 2 the graph and including one 7 hour dark period) and 47 hours (corre- sponding to T on the graph and including two 7 hour dark periods). 3 However, there was a significant decrease in photosynthetic rate/unit cell carbon. Molecular Weight Fractionation of Released Dissolved Organic Carbon From 36 to 70% of released dissolved organic carbon from axenic cultures of Microcystis aeruginosa and Anabaena flos-aguae (G-R) were filterable through Amicon UM 05 membranes and, hence, in the molecular weight range of less than 500 Daltons (Tables 4 and 5). Filtrates from NO3-N-grown cultures exposed to high light intensities were of lower molecular weight than those grown at low light. In addition, NHh-N- grown cultures produced a larger percentage of lower molecular weight compounds than NO -N-grown cultures. Nitrogen-fixing cultures of 3 Anabaena flos-aquae (G-R), in contrast, tended to produce a larger percentage of lower molecular weight compounds when exposed to low-light intensities. 55 Table 4. Molecular weight fractionation of dissolved organic carbon released from cultures of Microcystis aeruginosa grown with N0 -N and NBA-N and exposed to high (1500-1800 Lux) and low (1 0-200 Lux) light intensities. Z Total efers to the per- centage DEM of total dissolved organic carbon filtered through glass fiber filters. C.V. 8 coefficient of variance. Nitrogen Light Source Intensity Fraction 2 Total (C.V.) N03-N High PM 30 72.5 (27.1) PM 10 70.5 (27.7) UM 2 54.9 (17.6) UN 05 47.9 (22.8) NHa-N High PM 30 87.5 ( 1.8) PM 10 84.4 ( 1.2) UM 2 73.3 ( 0.4) UM 05 69.6 ( - ) N03-N Low PM 30 87.1 (13.2) PM 10 91.1 (11.4) UM 2 54.0 (14.9) UM 05 41.9 ( 5.0) NRA-N Low PM 30 96.7 ( 4.2) PM 10 90.3 ( 3.5) UK 2 55.1 (10.3) UM OS 46.9 (27.1) 56 Table 5. Molecular weight fractionation of dissolved organic carbon released from cultures of Anabaena flos~aquae (G-R) grown with N -N and exposed to high (1500-1800 Lux), medium (800 Lux) and 16w (150—200 Lux) light intensities. Z Total refers to the percentage DPM of total dissolved organic 1"carbon filtered through glass fiber filters. C.V. - coefficient of variance. Nitrogen Light Source Intensity Fraction 2 Total (C.V.) NZ-N High PM 30 52.5 ( 4.6) PM 10 33.3 ( 5.6) UM 2 38.3 (14.9) UM 05 35.8 ( 2.2) Nz-N Medium PM 30 53.4 ( 4.5) PM 10 55.7 ( 5.4) UM 2 38.7 ( 6.1) UM 05 37.1 ( 6.5) Nz-N Low PM 30 70.7 ( 2.9) PM 10 76.3 ( 7.6) UM 2 52.7 (10.1) UM 05 48.9 ( 0.3) DISCUSSION Overview Highest growth rates and photosynthetic rates/unit biomass re- sulted with all isolates tested when grown with NHA-N in batch culture at either continuously high, variable, or continuously low light intensi- ties. Growth rates with NO3-N and N2 not surprising, then, that blue-green algae are frequently found in -N were significantly less. It is close proximity to relatively high concentrations of NHa-N as with the metalimnetic populations in Lawrence Lake or where turnover rates of NH‘-N would be rapid as in eutrophic systems such as Wintergreen Lake. Nitrate could serve as an alternative nitrogen source to Nfln-N in the metalimnion of Lawrence Lake, since adequate growth rates were maintained at continuously low light with all isolates tested in culture. The absence of large assemblages of blue-green algae in the NO -N rich, but 3 NHa-N poor upper strata in Lawrence Lake is less easily explained, but could be related to a competitive disadvantage under conditions of higher light with N0 -N as the main nitrogen source as compared to other algal 3 groups. In addition, other nutrients, besides NHb-N, could be depleted in the upper strata of the lake by late summer. For example, alkaline phosphatase activity is very high in these upper strata (Wetzel, unpub- lished data), indicating a phosphorus demand. In that context, one could predict that if the epilimnion became more nutrient rich at that 57 58 time of year in Lawrence Lake, a substantially larger volume of the lake would be occupied by blue-green algae. With the depletion of both N0 -N and NH -N in the photic zone of 3 4 Wintergreen Lake, nitrogen-fixing species have a competitive advantage over other algal groups. The proximity of these populations to the sur- face could be very important in maintaining growth, since periodic exposure to high light intensities was necessary for some N -fixing iso- 2 lates. The narrow nature of the epilimnion as well as the large surface area in Wintergreen Lake (conducive to rapid mixing), are ideal for maintenance of the N -fixing populations within an array of light inten- 2 sities daily. Presence of gas vacuoles in this circumstance may serve more to maintain populations within the general upper strata, thereby preventing sinking into the hypolimnion, than aligning cells to a specif- ic light gradient. During calmer periods, however, populations may become more appressed to the surface and, hence, be exposed to continu- ously high light intensities. Rapid turnover rates of NHfi-N in the epilimnion of Wintergreen Lake, associated with the decomposition and mineralization of dissolved and particulate matter of high protein content, could provide the nitro- gen source for the non-nitrogen fixing species such as Microcystis aeruginosa, which occur concomitantly (but in fewer numbers) with the nitrogen-fixing forms. Examination of Km values for NHfi-N for Microcystis aeruginosa and other species offers a potentially fruitful area of future research in this regard. in". . . .IIIIivuhu Bfi_ S9 Nitrogen Source and Growth Rates The above hypothetical overview of events deserves closer scrutiny in the context of other studies. Kapp gt El- (1975), in comparing 60 organic and inorganic nitrogen sources, found that NHACl supported high- est growth rates with égmenellum_guadruplicatum, a marine blue-green alga, under conditions of pH and NHh-N concentrations similar to this study (pH 8.2, 9.6 mg N/l). Therefore, toxicity or poor growth reported previously may, in some cases, be an artifact of culture conditions in which NHn-N was supplied at artificially high concentrations (Winkenbach .gg.gl., 1972), the medium was not sufficiently buffered, or when high cellular yields produced harmful side-effects under culture conditions. Although use of laboratory cultures is frequently subject to criticism for engendering artificial conditions not applicable to natural popula- tions, it is relevant in this study that NHn-N was supplied at concentra- tions lower than in many culture studies at 5.0 mg NHa-N/l. Also, pH of the medium was rigorously buffered to simulate the well-buffered proper- ties of the lakewater in which natural populations occurred. Further- more, conditions of growth resulted in cell yields during log phase growth within range of that found in some lakewaters (1.0 to 4.0 mg cellular carbon/l in cultures compared to 5.0 to 6.0 mg particulate carbon/l in Wintergreen Lake during periods of blue-green predomdnance). Conditions within the euphotic zone in lakes during summer stratifica- tion resemble aspects of continuous cultures and batch cultures. For example, Healey and Hendzel (1976) found that certain cellular constit- uents in natural populations of Aphanizomenon flos-aquae fluctuated in .&,'Z'. .»_-n 60 a manner more similar to batch cultures than continuous cultures. However, short-term culture experiments were performed during early log phase growth in this study, when nutrients would not be limiting. For example, cellular nitrogen content of cells (mg N/l) during early log phase growth was always well below inorganic nitrogen concentrations (mg N/l) available in the growth medium. The phenomenon of better growth with NH -N as a nitrogen source is I. not universal among algal groups, although generally NHh-N will be utilized preferentially to N0 -N (Morris, 1974). Moss (1973) found that 3 only 4 out of 13 freshwater species of non-blue-green algae grew better (doublings/day) with NHa-N than N03-N. Two of the four, isolates of Chlamydomonas reinhardii and Eaglena'gracilis (eutrophic forms), grew only with Nun-N and not at all with NOB-N. Moss' study makes a particu- larly useful comparison to this one since similar growth media were employed. Therefore, blue-green algae as well as eutrophic forms of non- blue-green algae seem particularly well adapted to growth with NHa-N. Variations in growth rate in response to different nitrogen sources have also been reported with marine phytOplankton (Antia g£_gl., 1975). Urea and ammonium generally resulted in best growth in 25 isolates tested. An additional reason for slower growth rates with N03-N could be related to apparent blue-light inhibition of induction of nitrate reduc- tase (Stevens and Van Baalan, 1974). The action spectrum for nitrite production (i.e., nitrate reductase) indicated a peak at 680 nm (Stevens and Van Baalan, 1973). However, induction of nitrite production was inhibited in the range of 430-480 nm, although nitrite production would 61 continue at a high rate if induction had occurred previously in cells exposed to white light. Reports of a decrease in nitrate reductase activity with depth in ocean systems (e.g., Eppley st 31,, 1970) could be related to the selective attenuation of red light and consequent predominance of blue light with depth. Predominant light with depth in hardwater Gull Lake, however, was in the green range (540—560 nm), as was that supplied by Vita-lite bulbs in culture experiments (Figure 4). The spectral composition of Vita-lite bulbs resembled most closely that at 2.0 meters in Gull Lake with some selective absorption of red light occurring relative to surface light. Further absorption of light in the red and blue range would occur in deeper water. Therefore, the differ- ences in growth rates between N0 -N and NH -N grown cultures in this 3 4 study may represent minimum differences in that further attenuation of red light could result in inhibition of induction of nitrate reductase in deeper strata, where populations would not be exposed periodically to light of similar spectral composition to surface light. Lower growth rates with N -N or NH -N may be 2 3 4 somewhat dependent on the isolate. Gentile and Maloney (1969) reported -N as opposed to NO maximum growth with a nitrogen fixing isolate of Aphanizomenon flos- .ggggg; however, Healey and Hendzel (1976) found an apparent nitrogen deficiency among some natural populations of Aphanizomenon flos-aguae, which they attributed to insufficient phosphorus supply (Stewart gt 21°: 1970; Stewart and Alexander, 1971). Raising concentrations of phosphorus, iron, EDTA, molybdenum, and including vitamins (Moss, 1972) did not enhance growth of N -fixing isolates in this study. Further, whereas 2 62 cultures of aphanizomenon flos-aquae tended to have higher C:N ratios than that of Microcystis aeruginosa, there was some overlap of range between the two species. Also, C:N ratios of Anabaena flos-aquae and Aphanizomenon flos-aquae were lower than that reported necessary to induce heterocyst formation in Anabaena_gylindrica, that is, approxi- mately 8:1 (Kulisooriya, 1972). Therefore, although growth rates were lower with Nz-N, C:N ratios did not reflect a nitrogen deficiency. Light Regime and Nitrogen Source The effect of lower growth with N -N as a nitrogen source was most 2 striking at continuously low light, where isolates of Aphanizomenon flos-aquae could not maintain growth. Some N2-fixing isolates of Anabaena flos-aguae, however, would grow at this light intensity, but only very slowly. The lower limit of growth at continuously low light could be an important factor in determining which blue-green algae domi- nate within a system. That is, it is generally believed that both epilimnetic and metalimnetic blue-green populations develop at their respective depths in a system (Brook 55 51., 1971; Reynolds and Rogers, 1976) as opposed to developing within the surficial strata and "settling" down through the water column. Hence, given an equal inoculum of several varieties or species of blue-green algae among depth strata in a lake, the interactions of light intensity (directly related to depth of the epilimnion), nitrogen source, and onset of thermal stratification could determine which species or sub-species predominate. For example, in Wintergreen Lake the shallow nature of the epilimnion as well as the 63 lake as a whole would guarantee rather rapid exposure to higher light intensities. The distance between the nutrient-rich, but poorly lighted hypolimnetic water, and the higher light regime of the surficial waters is relatively short. This relationship may facilitate the pre- dominance of Aphanizomenon flos-aquae in this lake during most years. Once established, populations of blue-green algae in Wintergreen Lake are exposed to an array of light intensities diurnally. The effect of variable light intensity diurnally in cultures of Aphanizomenon flos- lagggg was a lowered growth rate compared to those exposed to continuously high light. However, rates of photosynthesis/unit cell carbon measured between 1000-1200 hours at high light intensities for cultures grown under a variable light regime (cf. Figure 3) were greater than cultures grown with continuously high intensities during the same time period during the day. In addition, although 9 out of 17 hours of light sup- plied daily to cultures grown under a variable light regime were of low intensity, periodic exposure to high or medium intensities resulted in positive growth among N -fixing cultures not able to grow under continu- 2 ously low light. Therefore, with natural populations, mixing within the upper water strata could result in enhanced photosynthetic response during periods of exposure to high light intensity as well as a carbon and energy supply during brief periods of exposure to low intensities. This general hypothesis is supported by the photosynthetic response of Nz-fixing cultures of éphanizomenon flea-aquae exposed for several hours to continuously high, variable, or continuously low light. 64 An increase in the rates of photosynthesis/unit cell carbon during log phase growth was a characteristic of_Aphanizomenon flos-aquae, but not of Microcystis aeruginosa. Rates of photosynthesis/unit cell carbon were highly variable during growth for Microcystis grown under continu- ously high or variable light intensities and uniform for cultures grown under continuously low light. These data agree with those of Fallon (personal communication) and Konopka and Brock (in press) for changes in photosynthetic efficiencies of natural populations of Aphanizomenon flos-aquae and Microcystis agruginosa_during bloom conditions in Lake Mendota. Although intriguing, the consequences to success or sequence of blue-green populations are, for the most part, unexplained. As in Lake Deming (Brook £5 21., 1971), the metalimnetic blue- green populations in Lawrence Lake are active photosynthesizers rather than senescent populations settling out of the epilimnion. Among other factors, the higher concentrations of inorganic nitrogen as well as the greater depth of the epilimnion contribute to inhibit the development of nitrogen-fixing populations. The development of colonial and uni- cellular forms in Lawrence Lake as opposed to non-nitrogen-fixing fila- mentous forms is more problematic, but could be related to differences in the concentration and availability of nutrients in the hypolimnion, with higher concentrations supporting the filamentous forms such as Oscillatoria. In that case colonial and unicellular forms would reflect a less productive system, as is the case of Lawrence Lake. 65 Assimilation Rates of Carbon and Nitrogen with Changing_Light Intensity Aside from growth rates and photosynthetic responses, a further consideration with respect to light regime and nitrogen source is the difference between assimilation rates of carbon and nitrogen with chang- ing light intensities. This difference would be most applicable with the N -fixing populations of Wintergreen Lake, since cultures of 2 _Aphanizomenon flos-aquae and Anabaena flos-aguae grown with NZ-N showed a greater discrepancy between carbon and nitrogen content with changing light intensity than did cultures grown with either NO3-N or NHa-N. Vertical migration through a gradient of light intensities daily may result in more balanced growth for N —fixing populations (Peterson 2 .E£.El°» 1977), although the term "balanced" is not physiologically explicit. However, in this study, cultures of blue-green algae had a generally lower C:N ratio during the most rapid period of growth as was also the case with natural populations of_Aphanizomenon flos-aguae (Healey and Hendzel, 1977). Adaptation to High and Low Light Intensities All species grown in culture became low light adapted when grown at continuously low light and exhibited saturation curves similar to those of natural populations in Lawrence Lake. Cultures grown under variable or continuously high light regime exhibited saturation curves similar to the high-light adapted populations in Wintergreen Lake. 66 The saturation curves for low— and high-light adapted cultures appeared to resemble the "Cyclotella" adaptive type described by Jorgensen (1969) and Steemann-Nielsen and Jdrgensen (1968) in that initial slopes of the curves were alike, but the light-saturated rate for high-light adapted cells was higher than that for low light adapted ones. Adaptation in the "Cyclotella" type was, then, primarily via enzymatic processes. However, unlike the "Cyclotella" type, cultures of Microgystis aeruginosa did not adjust rapidly (up to 47 hours) to other light regimes, once adapted to either high or low light. Because of this effect, the blue- green algal populations in Lawrence Lake would not be expected to respond in the same manner as high-light adapted cells, even if they were exposed to light intensities encountered in the upper strata in the lake. Similarly, periodic exposure to high light may enable populations in Wintergreen Lake to remain high-light adapted and, hence, able to respond to higher light intensities with considerably higher rates of photosynthesis than low-light adapted populations. Moleculgr Weight Fractionation of Released Dissolved Organic Carbon Fractionation by Amicon ultrafiltration of dissolved organic carbon released by cultures of Anabaena flos-aguae (G-R) and Microcystis aeruginosa produced intriguing results in that light intensity as well as nitrogen source affected the molecular weight of the released com- pounds- Besides providing a substrate for aquatic bacterial populations, there is increasing evidence that released organic substances from 4m 2 .- I‘kU-F'I‘I'". . a l 67 blue-green algal populations affect the succession of algal populations as well (Heating, 1977, 1978). Further qualification of the nature of these substances offers another relevant and potentially fruitful area for future investigation. CONCLUSIONS Populations of blue-green algae in Lawrence and Wintergreen lakes are particularly well-adapted to the light and nitrogen conditions which occur in these two divergent habitats. Presumptive evidence is pre- sented that metalimnetic populations of blue-green algae in Lawrence Lake utilize NHé-N as a nitrogen source, although NO3-N is also avail- able in abundant supply. In this manner, these populations are able to maintain maximum growth rates under conditions of continuously low light. Among other factors, the morphometry of the lake basin and the general trophic status of the entire Lawrence Lake system interact to restrict populations to this stratum, hence suppressing potential productivity of these populations. Since productivity rates associated with the metalimr netic populations already contribute significantly to the annual phyto- planktonic productivity, enhancement of growth of the planktonic blue- green algae by increased rates of decomposition in the hypolimnion or enrichment of the epilimnion could markedly increase the annual primary’ productivity of the entire open water portion of the system. In highly productive Wintergreen Lake, on the other hand, high- light adapted nitrogen-fixing populations can be maintained in the surficial waters. Because of the shallow nature of the epilimnion, phytoplankton are well-mixed in the upper strata and, hence, are exposed to an array of light intensities during growth. The consequences of 68 gm 69 intermittent exposure to high and low light intensities are advantageous in maintaining a more uniform carbon and nitrogen content within the cell, which is more important to N —fixing populations exposed to dif- 2 ferent light intensities than to NO -N or NH -N utilizing populations. 3 4 In addition, in spite of rapid attenuation of light with depth, popula- tions can remain high-light adapted, hence responding maximally to higher light intensities when intermittently exposed. In this context, the pOpulations could be viewed as opportunistic, which undoubtedly is a competitive advantage in a habitat subject to rapid change such as Wintergreen Lake. The nature of the dissolved organic compounds released by phyto- planktonic populations is of particular interest, since these compounds are a direct link between the primary producers and the bacterial- detrital component and, hence, the ecosystem as a whole. All evidence from these culture studies indicate a substantial portion of the dis- solved organic compounds released, with all nitrogen sources and at all light intensities, is of low molecular weight and probably readily utilizable by bacteria. This finding was particularly true of organic carbon released from cultures grown with NH -N and that from N -fixing 4 2 cultures exposed to low light intensities. LITERATURE CITED LITERATURE CITED Allen, M. B. and D. I. Arnon. 1955. Studies on nitrogen-fixing blue- green algae. 1. 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