LIBRARY 9 Michigan Static University This is to certify that the thesis entitled Interacting Carbon and Light Limits to Macrophyte-Growth presented by Sarah Kate Liehr has been accepted towards fulfillment of the requirements for M.S. degree in Fisheries 8: Wildlife Major professor Date July 28, 1978 0—7 639 INTERACTING CARBON AND LIGHT LIMITS TO MACROPHYTE GROWTH BY Sarah Kate Liehr A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Fisheries and Wildlife ABSTRACT INTERACTING CARBON AND LIGHT LIMITS TO MACROPHYTE GROWTH BV Sarah Kate Liehr This study was conducted to deve10p a laboratory tech- nique for measuring the growth kinetics of macroPhytes. Elodea canadensis and Ceratophyllum demersum were grown under a variety of light conditions in microcosms contain- ing defined medium. The pH was measured at regular inter- vals, and the amount of carbon fixed by the plants was calculated. Although there was a problem with algal inter- ference, it was possible to obtain usable data by this method. The data were used to calculate Monod growth equa- tions describing the interaction of limiting carbon and light levels on the growth of the plants. The plants were found to respond in a manner similar to the response of algae grown under similar conditions- The data indicate that elodea is able to grow at a faster rate than cerato- phyllum and that the macrophytes may be able to outcompete the green alga Chorella vulgaris at low CO2 levels. ACKNOWLEDGMENTS I would especially like to thank Darrell L. King for his guidance throughout this study, and for making my Master's program a valuable educational experience. I would also like to express my thanks to my parents, with a Special thanks to my mother for typing this thesis; to Clyde Anderson for his encouragement and assistance; to J.D. and Marie Davis for befriending a stranger; and to Judy and Pat and Fred and Chris and Tony and Don and Mabel for being my family. This study was supported by grant USDI A-909-MICH from the Office of Water Research and Technology. ii TABLE OF CONTENTS List of Tables List of Figures Introduction Methods Experimental Procedures Microcosms Light Alkalinity, pH Sampling Collection of Plants Initial Plant Carbon Content Data Calculation Procedures Carbon Calculations Growth Rate Results and Discussion Growth Rates as a Function of Carbon Threshold cozf Kinetic Equations of u as a Function of COZf Kinetic Equations of u as a Function of COZf and Light Utilization of Kinetic Growth Analyses iii Page No. vi 14 24 25 26 31 37 Comparison and Elodea Comparison with Algae Conclusions Appendix Bibiography of the Growth Rates of Ceratophyllum of the Growth Rates of Macrophytes iv 44 52 53 S4 LIST OF TABLES Page No. TABLE 1. Initial conditions for the ceratophyllum 17 and elodea microcosms. TABLE 2. Growth kinetic constants (average) with 27 95% confidence intervals. Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure LIST OF FIGURES Microcosm.with air lock used to study growth rates of C. demersum and E. canadensis. Lighting arrangement used in maCrophyte growth study. Typical curve of pH or Cfixed.With time for microcosm studies. Graphical representation of the 8/“ vs. S transformation of the growth rate formula. Typical curve of pH as a function of time for C. demersum microcosm under a light intensity of 3875 lux. Representative curves of pH as a function of time for C. demersum under four light intensities. Representative curves of pH as a function of time for E. canadensis under three light intensities. Representative curves of carbon fixed and free C02 as functions of time for C. demersum under four light intensities. Representative curves of carbon fixed and free C02 as functions of time for E. canadensis under three light intensities. Growth equations for C. demersum under four light intensities. xi Page No. 10 13 18 19 21 22 23 28 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. Growth equations for E. canadensis under three light intensities. Linear relationship of “m x with light for C. demersum and E. canadensis. Linear relationship of log Ks with log light for C. demersum and E. canadensis. Linear relationship of log C02q with log light for C. demersum. Relationship of Ks and CO2q with light. Typical curves of biomass and substrate concentration with time (a), and specific growth rate with time (b) from microcosm studies. Theoretical graph of the specific growth rates of two organisms competing for substrate S. Specific growth rates of C. demersum and E. canadensis under three light intensities. Three dimensional graphs of the specific growth rates of E. canadensis (a) and C. demersum (b). Comparison of the light relationships of “max for C. vulgaris. C. demersum and E. canadensis. Comparison of the light relationships of C02q for C. vulgaris and C. demersum. Comparison of the specific growth rates of C. demersum and C. vulgaris. 30 32 33 34 36 38 4O 42 4.3 46 47 49 INTRODUCTION As the population of man on earth increases, increased nutrient loading of our aquatic systems is inevitable. This nutrient loading stimulates plant production, resulting in extensive weed growth, a process often referred to as eutro- phication. Although abundant plant growth is usually con- sidered a nuisance, some aquatic plants are more desirable or useful than others in specific situations. water used for different purposes, such as human consumption, recrea- tion, land irrigation, have different criteria for determin- ing which plants are most desirable. For example, when aquatic plants are used in the treatment of wastewater, it is desirable to have a plant species that can grow in high pH conditions, can easily be harvested, and can be used for some purpose after it is harvested, such as for livestock feed. Therefore, it is desirable to be able to manage aquatic systems for the selection of the dominant plant species. This can be done by comparing growth kinetics of different plants. The purpose of this study was to develop a technique for the measurement of the growth kinetics of two macrophytes, Elodea canadensis and Ceratophyllum demersum, and to determine how their growth is affected by limiting levels of carbon and light. 1 iM5l...in...l3}fluV4.n|-1nfl1nflu 1”. IL. E" A ,. . .. . . T. METHODS Experimental Procedures The purpose of this study was to quantify the inter- active effect of carbon and light limits on growth responses of Elodea canadensis and Ceratophyllum.demersum~ Plants were collected from natural pOpulations and then were grown in the laboratory under artificial lights in microcosms containing a defined inorganic nutrient medium. Microcosms The experimental microcosms used in this study were similar to the microcosms used by Sievers (1971), Young (1972), and Klemovich (1973). They consisted of one-liter Erlenmeyer flasks with rubber stoppers in which two holes had been drilled. One hole contained a glass tube with an air lock to maintain atmospheric pressure within the micro- cosm-while minimizing recarbonation from the atmosphere. The other hole contained a rubber serum cap for the removal of samples (Figure 1). Samples were taken with a hypodermic syringe through the rubber serum.cap so that the medium was not exposed to the atmosphere. The growth medium (Kevern and Ball, 1965) contained all nutrients in excess except carbon, which was limited by the alkalinity of the medium (see Appendix). A running total of the volume removed for sampling was recorded. '3 )2 I L--. —-—..4 )——— \— f“ \\\l FigLre i. Mlcrocosm with air lock used To study grow’rh rates of C. demersum md E. ccmdensis. 9.1.92.2 Continuous light was provided by two 40-watt "Gro Lux" fluorescent lights mounted on a wooden frame (Figure 2). A range of light intensities was obtained by covering the lights with various combinations of black cheese cloth and fine mesh, black wire screen. Light intensity and energy units were measured with a weston footcandle meter Model 756 and a LI—COR Quantum Sensor, Model LI-l9ZS. Alkalinity,gp§g The initial carbonate-bicarbonate alkalinity was mea- sured by the titration method (Standard Methods, l97l). All pH measurements were obtained with a Corning Model 12 research pH meter with a general purpose glass semi- microelectrode. The pH meter was standardized against standard buffer solutions at each sample time, and the standardization was checked between sample measurements. Sampling Each sample, collected from the microcosm with a syringe, was injected into a 50 ml beaker which contained nitrogen gas, and was capped with a rubber stopper. The rubber stopper had one hole for injecting the sample and the nitrogen gas into the beaker, and a second hole for the pH electrode. The purpose of this method was to minimize recarbonation from the atmosphere during the time required to record an accurate pH measurement of the sample. side view mm view FigLre 2. Lighting arrmgemem used in mocrophyte growth study. Collection of Plants Elodea canadensis was collected from the second lake of the water Quality Management Project on the south portion of the Michigan State University campus at East Lansing. 9253: tophyllum demersum was collected from the concrete ponds in back of the Limnology Laboratory on Kalamazoo Street on the Michigan State University campus. The plants were collected and taken to the laboratory where they were allowed to ac- limate to room temperature. After they were sorted and put in beakers of clean medium, the plants were placed under ex- perimental light conditions to allow them to acclimate to light prior to the initiation of the experiment. Initial Plant Carbon Content A sample of plants collected for each experiment was weighed, dried at 105° C for 24 hours, and weighed again. These data were used to obtain a wet weight vs. dry weight curve. Initial carbon content of these dried plants was an- alyzed using a Perkin-Elmer Model.240 Elemental Analyzer. The plants actually used in the experiments were weighed for wet weight, and the carbon mass was extrapolated from the curves . Data Calculation Procedures In this study, plant growth was measured as a net up- take of inorganic carbon from the medium. As carbon dioxide is fixed by the plants and carbon is removed from the medium, the CO2 concentration is controlled by the following equilibrium reactions (King, 1970): -— + 2 : HCO3 + H ‘— H2C03 <— C02 + HOH (l) - - = Hco3 + OH —\¢_ co3 + HOH (2) These reactions can be added, resulting in — = 2HC03 + HOH .\__——\ co2 + co3 + 2HOH (3) As CO2 is removed by photosynthesis, the carbonate ion dom- inates the system. If carbonates do not precipitate from solution, as is the case with media dominated by monovalent cations, CO2 is further removed by the following reaction: co3‘ + 2 HOH —\—_—> co2 + HOH + 2 0H“ (4) As this reaction occurs, carbonate-bicarbonate alkalinity is converted to hydroxyl alkalinity, but the total alkalinity does not change (King, 1970). Carbon Calculations l Growth, or net uptake of inorganic carbon, was deter- mined by calculating the change in total inorganic carbon in the medium as a function of time. The amount of inor- ganic carbon in the system at any time can be calculated by the following equation (Sievers, 1971): E2 2C02 - a K1+H+K2 H + 2K 2 where: ECO2 = total inorganic carbon moles/l a = carbonate-bicarbonate alkalinity, corrected for hydroxyl ion concentration, eq/l H = hydrogen ion concentration, moles/l Kl = first dissociation constant of carbonic acid K2 = second dissociation constant of carbonic acid. - To calculate total inorganic carbon by this formula, it is necessary to know the initial carbonate-bicarbonate alka- linity, a; the temperature, to calculate K1 and K2; and the pH at given time intervals. As the pH rises with plant carbon fixation, the alkalinity has to be corrected by sub- tracting the increasing hydroxyl ion concentration. An increase in plant biomass, or carbon fixed (C ) can be fixed calculated by measuring pH and calculating the total inor- ganic carbon (ZCOZ) at the specified time intervals and applying the following equation (Young, 1972): C = AZCO2 = 2C02(initial) - 2C02(final) (6) fixed The relationship of pH with time and of C with time fixed can be represented by similar curves with the general shape shown in Figure 3. Growth Rate Plant biomass can be described at any time t by the equation M = Moeut (7) M = biomass at time t 3 ll initial biomass (t=0) specific growth rate. 1: II The specific growth rate (u), or the instantaneous rate of change of biomass per unit biomass, does not have to be con- sidered as an intrinsic constant value. In this study, u was considered to be a variable, and was calculated as follows (Young and King, 1973): AM/At (Mtz - Mtll/(tz -t1) (8) 1.1 At = m = (Mt2 + Mtl)/2 where: “At = spicific growth rate during time increment, hr“ M = biomass increment (inorganic carbon fixed), moles-C/l 10 Figure 3. time Typical CUTVC oi‘ pH or Time For microcosm sTudies. Cm wiTh 11 tl,t2 = boundary parameters of time increment, hr"l At = t2-tl = time increment, hr'l m = average standing crop biomass during the increment, moles C/l. The specific growth rate can be related to some limiting nutrient concentration by Monod's application of the Michaelis-Menten enzyme kinetic equation to whole organisms, S =umax Ks + S (9) i where: u = growth rate, hr-1 = maximum growth rate, hr‘l max = substrate concentration 8 K = substrate concentration at 8n 5 max The limiting nutrient in this study is carbon. Assuming plants can only use free carbon dioxide (C02F)’ the growth rate can be written as a function of COZf‘ The CO2f concen- tration can be calculated by the following equation derived by Harvey (1957) and Park (1969): 2 _ H cozf‘ a K1(H + 21'; (1°) where: * I O CO2f = H2C03 (aq) including C02 (9), moles/l a = carbonate-bicarbonate alkalinity corrected for hydroxyl ion concentration, eq/l H = hydrogen ion concentration, moles/1 12 73 ll 1 first dissociation constant for carbonic acid N M II second dissociation constant for carbonic acid. Equation 9 is usually used to describe relationships between specific growth rate and a limiting nutrient in situations where an assumption is made that growth will continue until the substrate has been completely removed. This assumption cannot be made with regard to aquatic plants and CO2f concentration, because there is a certain minimwm CO2f concentration required by plants for growth to be sus- tained (Klemovich, 1973). This threshold concentration (Sq ) can be accounted for by modifying Equation 9 as follows: S - Sq u = u - ~ - max (KS Sqi + (S Sq) (11) where: S0. = the minimum.substrate concentration required to sustain growth. The constant Sq is obtained from the data as the sub- strate concentration where u goes to zero. The other con- stants, pmax and Ks were calculated using the 8/“ vs. S transformation of Equation 9 shown in Equation 12 and in Figure 4. 8/11 = KS/umax + (l/umax) S (12) This transformation was chosen because it gives more accu- rate estimates of the constants than the commonly used double-reciprocal transformation (Dowd and Riggs, 1964). S/u F lane 4. 13 Sime'l/“mx Graphical representation oi? the S/u vs. S transformation 0? lbs growth rate formula. RESULTS AND DISCUSSION This study involved the application of a microcosm method previously used to study algal growth kinetics to the study of macrophyte growth kinetics. Since the method had not been used for this purpose before, preliminary experi- ments were necessary to determine if the method could be ap- plied to macrophytes. The preliminary experiments showed that the plants did grow within the microcosms, but that diatoms and green and bluegreen algae naturally associated with the macrophytes also fixed carbon from the medium. This algal interference made it impossible to quantify the carbon fixed by the macrophytes alone. Therefore, to be able to use plants from natural populations, it was neces- sary to develop some techniques to eliminate or at least minimize algal interference. Scanning electron photomicrographs of elodea indicated that significantly fewer algae were present on the tips than on the older parts of the plants (L. Koivuniemi, personal communication). To minimize the initial algal pOpulation introduced into the microcosms, only the 2-4 cm tips of elodea and the 6—10 cm tips of ceratophyllum were used in this study. Copper sulfate was added to the medium at a concentra— tion of 15 umoles Cu/l to slow algal growth. This concen- tration was selected after preliminary studies indicated 14 15 that it did slow algal growth while not perceptibly changing the initial growth rate of the plants. This concentration of 15 umoles Cu/l was not high enough, however, to complete- ly stop algal growth, but it did postpone any noticeable growth of the algae. 1 Another strategy used in the attempt to minimize the effect of the algae was to start with a large plant biomass. A larger initial biomass results in a faster macrophyte carbon fixation rate. This strategy was used with the hope that the carbon would be removed from the medium by the macrophytes rapidly, yielding a free carbon dioxide concen- tration which limited further macrophyte activity before the algae had a chance to become established. None of these techniques were entirely successful at eliminating algal growth. The preliminary studies indica- ted, however, that data could be obtained if all three methods were used simultaneously. Therefore, the study was conducted using all three methods to minimize algal interference. The combined technique for elimination of algal inter- ference worked fairly well at the high light intensities, especially for ceratophyllum. Under light intensities of 3875, 2585, and 1290 lux, ceratophyllum removed the carbon fast enough to lower the COzf concentration to the point where growth stOpped before the algae started to grow. Under a light intensity of 690 lux, ceratophyllum did not grow fast.enough to allow determination of the point in time 16 when the macrophyte stopped growing. It was not possible to distinguish this point for elodea under many of the light conditions used, even though elodea generally grew faster than ceratophyllum. Elodea appears to have had larger pOpulations of algae associated with it. The preliminary experiments also were used to determine the range of light conditions for the study. Plants under light intensities less than 540 lux did not grow well, so only light intensities over 540 lux were used. Alkalinities between 3.4 and 4.0 meq/l were used in all experiments. The bottles were kept at room temperature, and remained at a fairly constant temperature of 25° C. Initial conditions for the experiments are given in Table l. The pH measurements were taken at 12 hour intervals until the pH stOpped rising, usually between 10 and 16 days. A typical curve of pH as a function of time is shown in Figure 5. A smooth curve was drawn through the data points to eliminate deviations due to incomplete mixing and slight temperature variations. As can be seen in Figure 5, the pH rose sharply, then reached a plateau before starting to rise again. This plateau was interpreted as the point where macrophyte growth stopped. The subsequent rise in pH was due to algal growth. Representative pH curves for ceratophyllum under four light conditions are shown in Figure 6. The curves indicate that the plants were able to continue growing to higher pH values under higher light intensities. This figure also TABLE 1. Ceratophyllum Elodea Light (lux) 3875 3875 3885 3875 3875 3885 2585 2585 2585 2585 1290 1290 1290 1290 690 690 690 690 3875 3875 3875 3875 3875 3875 3875 2585 2585 1290 1290 690 690 17 Initial conditions for the ceratophyllum and elodea microcosms. 3.759 3.759 3.981 3.981 3.835 3.835 3.981 3.981 3.835 3.835 3.981 3.981 3.835 3.835 3.981 3.981 3.835 3.835 3.483 3.483 3.474 3.474 3.474 3.759 3.759 3.759 3.759 3.981 3.981 3.981 3.981 Initial Organic Alkalinity Carbon (u einstein m"2 sec‘l) (meq/l) (mmoles 17.457 19.666 17.816 18.752 13.514 13.785 19.656 18.819 14.169 14.216 21.774 22.332 15.145 15.506 25.841 28.663 16.762 17.144 17.521 35.310 2.579 4.941 6.782 8.509 12.111 11.191 12.518 13.500 12.072 14.588 16.615 C/l) 18 1 I a 43 93 144 192 240 J. time (hairs) F igLre 5. Typical CU‘VC of pH as 0 chtion 04‘ time For a C. demasu;n_ microcosm mder a lig'tt intensity 04‘ 3875 lux. 19 10.50- -- 1025-- 3875 lux 10.004 2585 km 9.75.. 1290 (LIX W 950 690 lux 9.254 9.00-- 8.75 a as as 144 1552 240 Figure 6.. Representatiive ctrves 01" pH as a (motion 04‘ time For C. demersun mder 900“ light intensities. 20 shows that higher light intensities result in steeper pH curves, and thus the rate at which the pH rises increases with increasing light intensities. Similar pH curves are given in Figure 7 for elodea with three light intensities. There was too much algal interference at the lowest light intensity (690 lux) to obtain usable data. These curves demonstrate that elodea responds to various light intensities in a manner similar to ceratophyllum. The two highest light intensities allowed growth to continue to almost the same pH value. The curves illustrate, however, that there was a difference in the rate at which that pH was attained. The pH data were used to calculate the amount of carbon fixed by Equations 5 and 6. The results are shown in the top half of the graphs in Figures 8 and 9 for ceratophyllum and elodea respectively. Even though the initial biomass is not taken into consideration, the curves demonstrate that as light intensity increases, the amount of carbon that can be fixed by the plants and the rate at which that carbon is fixed also increases. Concentrations of C02f were calculated using Equation 10. The lower half of the graphs in Figures 8 and 9 show the mirror-image relationship of CO2f to carbon fixed. These graphs indicate that higher light intensity not only allows the plants to fix more carbon, but also allows the plants to grow to lower CO2f concentrations. .. .n-IJ 21 3875M! 10.504 1290 lux F igire 7. Representative CU'VGS 04‘ pH as a i‘mction oi‘ time l‘or E. canadensis mder three light intensities. 22 3875M 2585 lux 0.8 1- 0.7 ~- 85" 1290 lux 0.54 0-4" 690 lux Cm (mules/ll 0.24- 0.9-i- 590 IUX 1290 ile log 00, amiss/ll time curs) F inn 8. Representative oLrves 0+“ ccrbon i‘ixed mdl‘ree coaasi‘mctimsoitimel‘or C. demeram Lnder l‘ar lld'li' intensities. 23 .- 3875lux 1" 2585M: 0.8» 1290 iux Cmm (moles/0 ii 1290M loo co. annulus/o $ 2585i!“ 3875M l I r 1 0 4e 95 144 192 250 the (hairs) F laws 9. Representative was 04' ocrbon i-‘ixed md i‘ree 002 as l-‘Lnotions 0i lime tor E. ocmdersls mder ttree llmt intensities. 24 The C curves were next incremented by time to ob- fixed tain the change in carbon biomass per time increment. Aver- age carbon biomass was calculated by adding the total Cfixed to the initial carbon biomass and averaging over the time increment. These data were then used to calculate the specific growth rates by Equation 8. Growth Rates as a Function of Carbon There is some question as to what form of carbon aqua- tic plants, such as ceratophyllum and elodea, use as their actual carbon source. There have been reports in the liter- ature (e.g. Raven, 1968, 1970: Steemann Nielson, 1947, 1960) of aquatic plants and algae directly taking up bicarbonate ions as a carbon source at high pH. These studies, however, do not offer definitive proof that it is the bicarbonate ions and not the equilibrium CD that is actually taken up 2f by the plants. There have also been reports that algae respond to the total inorganic carbon concentration. Goldman, et a1 (1974), using chemostat studies, concluded that the green algae Scenedesmus quadricauda and Selenastrum capricornutum respondeinetically to the total inorganic carbon concen- tration in the water, even though only one form of carbon may be assimilated. King and Novak (1974) used Goldman, et al's data to compare KS values calculated by using total in- organic carbon, bicarbonate ion, and equilibrium CO2 as the 25 substrate concentrations. The conclusion of this recalcula- tion was that it is more likely that the algae were respond- ing to C02f concentration than to total carbon or bicarbonate ion concentrations. There is also direct evidence from field data that ceratophyllum uses COZf as its carbon source. Craig (1978) constructed isopleths of percents of “max with alkalinity vs. pH, using COZf and HCO3- concentrations as the sub- strates in calculating the percentages of “max‘ When HC03- was used as the substrate, the isopleth representing a u of zero eliminated some of the alkalinity - pH range where ceratoPhyllum is known to occur. When cozf was used as the substrate, none of the known range of ceratophyllum was eliminated. Thus, Craig concluded that ceratophyllum responded only to the C02f concentration. The examples cited support the theory that aquatic plants use only COZf as their carbon source. Since there is no conclusive evidence that aquatic plants do respond to other inorganic carbon concentrations, COZf was used as the carbon substrate in this study. Threshold C02f Since aquatic plants do not continue to grow until the COZf concentration reaches zero, it is necessary in the cal- culation of growth rates as a function of CO2f to know how low the CO2f concentration can drop before the plants are no 26 longer able to fix carbon. This threshold COZf concentra- tion-(COZq) was obtained from the data as the COZf con- centration that resulted in a u of zero. Due to algal interference, however, it was not possible to define the exact threshold concentration in all microcosms. The cera- tophyllum microcosms were relatively free of algae except at the lowest light intensity (690 lux). The elodea micro- cosms, however, had significant algal interference in the low CO2f ranges at all light intensities. In the microcosms where algal interference occurred at low CO2f concentra- tions, it was impossible to get reliable estimates of the CO 2q Kinetic Equations of u as a Function of COzf To describe u as a function of C025, it is necessary to know pmax and Ks as well as the COZq’ as described in Equa- tion 11. The constants u and K w a d ' max 3 ere c lculate in Equation 12. Table 2 is a table of these kinetic constants as the average of all experiments at a given light intensi- ty. It is obvious from the 95% confidence intervals that algal interference was particularly significant for elodea and for both plants at the lower light intensities. The values listed in Table 2 were used as the values of the parameters in Equation 11, resulting in equations for the growth rate of ceratOphyllum and elodea. Figure 10 is a graph of the growth rate equations for ceratophyllum for 27 TABLE 2. Growth kinetic constants (average) with 95% confidence intervals. Light (lux) ”max (hr-l) KS (umoles COZf/l) C02q(umoles COZf/l) Ceratophylygg 3875 .001012 1 .000256 .50999 t .08623 .26451 t .08276 2585 .000773 f .000094 .79134 t .19636 ,.47757 t .13547 1290 .000267 f .000124 2.0497 t 1.8799 1.6999 1 1.7350 690 .000196 ---- * 5.2919 --- * --- * --- * 11.9.42; 3875 .003538 f .008843 .35229 t .23504 --- * 2585 .002218 --- * .26353 ---- * -—-- * 1290 .000543 --- * .83101 --- * --- * * - not available 28 .moEmcoE. #8: 58 been chateau .o to... manage £265 .3 choc $8.95 .8 10¢ i mmoood .. . 8.80.0 29 four light intensities. These curves illustrate the drama- tic effect of light intensity both on the rate and the ex- tent, or minimum COzf, to which the plants will grow. The poor estimate of the CO2q from the 690 lux data is responsi- ble for flattening the growth rate curve for that light intensity. It is still possible, however, to compare the maximum growth rate at this light intensity with the maximum growth rates at the other light intensities in this figure. There is clearly a relationship between light and “max such that “max decreases as the light intensity decreases. It can also be seen in the top three curves that the C02q in- creases with decreasing light intensity. In other words, the plants require higher C02f concentration to sustain growth as the available light decreases. The growth rate curves for elodea are shown in Figure 11. Again, poor estimates of the CO2q were responsi- ble for flattening the lower ends of the growth curves. The H values show the same trend as ceratOphyllum of max decreasing with decreasing light. This figure indicates that there is a possibility that CO2q increases with de- creasing light for elodea as it does for cerat0phyllum, but the data here are not adequate for drawing a definitive conclusion. 30 ‘ .mo:_m8..c_ L8: 85.: .85 $8898 .m. to» £52.38 £380 5.8mm .3 Poor. .586 :83 .ivood 31 Kinetic Equations of u as a Function of CO2f and Light The above discussion clearly shows that the parameters of the growth equation are related to the available light intensity. These relationships can be expressed as mathe— matical equations. King and King (1974) found the relation- ship of "max with light for some green algae to be linear, or: “max = a + bL where: L = light intensity If macrophytes respond in a similar manner, then they 'should also show a linear relationship. This theory was tested for "max vs. light by using linear regression to calculate a and b as shown in Figure 12, resulting in the following equations: u (ceratophyllum) = -3.43 x 10‘5 + 2.78 x 10‘7 L hr‘l max r=.897 (14) u (elodea) = -8.74 x 10‘4 + 1.15 x 10"6 L hr‘l max r=.927 (15) where: L = light intensity in lux The correlation coefficients of .897 and .927 indicate that the equations gave a fairly good fit to the data. The parameter KS and cozq for ceratophyllum are also functions of light and can be expressed as a linear rela- tionship on a log-log scale as shown in Figures 13 and 14. 0.0041 0.003- 0.002- um at") 0.001- 32 5X .0111 gig x r =- (1%? r=0.897 L l 4 L I r I 1000 2000 3000 4000 Ilmt (iux) Figire 12. Linear relationship 04‘ with light For C. demersun m0 . ocmdensis. 33 x Economies I 0.71 0-5? 0.5" '- 0.4a- 03 U 0.1: log 00, (umoies/l) e a l? ,8 i l l i 3 l 2.8 3.0 3.2 3.4 3.5 log lid‘li 0100 F igwe 13. Linear relationship at log KS with log light For C. demersum m0 E. cmadensis. 34 I 0.81 0.51 0.4-1 0.2- U 0.0- -02-~ log 00., (“moles/b -O.6-+ T -0.8- 1P as 3.0 5.2 5.4 3.0 log I m (qu) 4 F iglre 14. Linear relationship oi‘ log C029 with log limt i‘or C. demersum 35 Again using linear regression, the following equations were obtained: 4 -l.25 Ks(ceratophyllum) = 1.45 x 10 L umoles COzf/l r=-.901 (l6) 2 -.76 Ks(elodea) = 1.63 x 10 L umoles COZf/l =-.790 (l7) 5 -l.57 C02q(ceratophyllmm) = 1.07 x 10. L umoles COzf/l r=-.878 (18) where: L = light intensity in lux These equations can be seen graphed on a straight scale in Figure 15. The relatively low correlation coefficient for Ks(elodea) again indicates difficulty with algal inter- ference. Equations 14, 16, and 18 can now be substituted for the parameters in Equation 11 to obtain an expression of the growth rate of ceratophyllum as a function of light for the light conditions of this study. The resulting equation is: S ' f3(L) hr-l ‘1 = f1““ (ngL) - f3(L)r+ Y§- {3an ‘19) where: f1(L) = (ceratophyllum) Equation 14 umax f2(L) = Ks(ceratophyllum) Equation 16 f3(L) = COZq (ceratophyllum) Equation 18 S = C0 in umoles/l 2f 36 — - — Ks (firemadensis) ——-—- K, (C. demersum - —- COS, QM) 003. (umoies/ll Flare 15. Relationship oi‘ KS and 0020 with light. 37 It is not possible to write such an equation for elodea since no Cqu data are available. Utilization of Kinetic Growth Analyses Analyses of kinetic growth rates, such as that attemp- ted in this study, allows us to compare the relative ability of different species of organisms to compete with each other. Organisms in nature must compete for resources which are limited, and a prime means of competition is their growth rate. The set of conditions that physiologically allows an organism to grow is often referred to as that organism's niche. The niche that an organism occupies is defined, or bounded, by the threshold concentrations of limiting resources. As seen in this study, these threshold concentrations are not determined by single limiting fac- tors, but by the interaction of limiting factors. The growth rate is not constant under all conditions where the organism is physiologically able to exist, but approaches zero as the substrate concentration approaches the boundar- ies of the organismis niche. This can be seen graphically by the typical curves shown in Figure 16 of biomass and sub- strate concentration with time, obtained from microcosm studies. The top curve, of biomass with time, is a typical logistic growth curve. Initially, the biomass increases ex- ponentially, but as the substrate concentration decreases, so does the rate at which the biomass increases. Finally at time t, when the substrate concentration has been reduced to (O) bimiass abstrate (b) F lgLre 16. Typical mas o? biomass cnd substrate concentration with time (a), 010 specil‘ic growth rate with time (b) i‘rom microcosm studies. 39 the threshold concentration, growth stops and the biomass no longer increases. The bottom curve of Figure 16 shows graphically this effect of the substrate concentration on the specific growth rate- When growth staps, the population has reached its maxi- mum biomass. This limit of the logistic growth curve is commonly described as a carrying capacity. The carrying capacity approach to the logistic growth curve is purely empirical and offers no explanation of the environmental factors that impose this limit. For that reason, it is of very limited use in designing management strategies. The kinetic approach, however, directly relates the growth rate of the organism to the limited substrate concentrations, and therefore offers management application. The niche of an organism only defines the conditions under which the organism has the physiological ability to grow. The ability to grow under certain conditions, how- ever, is not sufficient to determine how competitive that organism will be when challenged by others. The ability to compete depends on the rate at which an organism grows rela- tive to the rate of growth of competing organisms. By using this kinetic approach, it is possible to compare growth rates under defined conditions, and thus compare the rela— tive competitive abilities of the organisms. This compari- son can be made graphically by drawing the growth rate curves on the same graph, as in Figure l7.- Figure 17 is a theoretical graph of the growth rates of two organisms, A 40 u (W1) 8ch) 3.00 3x 3 (mg/0 FIgLre 17. Theoretical g'cph 04' the specii‘ic growth rates 04‘ two organisms competing For abstrate S. 41 and B, competing for substrate S. As seen in this figure, when the substrate concentration is greater than Sx' organ- ism A has a larger growth rate, and thus would be dominant over organism B. When the substrate concentration is less than Sx, organism B has the competitive advantage and would be expected to dominate. When the substrate concentration equals Sx’ neither organism has a competitive advantage over the other. This theoretical example illustrates how easily the relative competitive abilities of organisms can be compared by this method. Comparison of the Growth Rates of Ceratophyllum and Elodea The conditions of this study might represent a nutrient enriched, hyper-eutrophic situation in which carbon and light are the limiting factors to the growth of plants. In such a situation, plants compete with their n values within the boundaries defined by their cozq. The growth rates of ceratOphyllum and elodea are compared in Figure 18 for 3875, 2585, and 1290 lux. The lower ends of the elodea curves are dashed to indicate best estimates of Cqu values. The sig- nificance of these curves is that they indicate that elodea is a better competitor for carbon, i.e., has a higher growth rate, at all light levels used in this study. This rela- tionship is dramatically illustrated by the three-dimension- al graphs in Figure 19. This implies that elodea should be able to out-compete ceratophyllum in carbon limited situa- tions, at least when the light conditions are within the 0.0044“- 3875 lux E. omadensis 0.003" E- 0.002» :1 / C. da'neram‘ 0.001-- / / / : /’ , + e E. candasis 01'“) \ \ 0.001-- / C. deme'sun :1 , # / / / 0.002-L 1290 lux 1 0.01.1 _E_.:cmadensis /-- ’- "Tdemersun l I ' 1 / 1 —1.'0 0.0 i 1.0 log 00., (uncles/I) F IgLre 18. Specific g'owth rates 01° C. demerSLm cnd .ocnadensis mder trree limt intensities. s we 6,. E 3 ch .31 0 new... born 05 . “.0 00 an... 1 m“. m em... 88 .u 8 . km A .m H ab 0 E any) 44 range used in this study. However, ceratophyllum does exist in some nutrient enriched, highly eutrophic situations where it is not excluded by the growth of elodea. It is possible that elodea has never been introduced into these lakes, but if it has been introduced, the existence of ceratophyllum: indicates that the limit to plant growth in these situations is more complex than a relatively simple carbon-light inter- action. Data are not now available to determine growth rates of macrophytes relative to phosphorous, nitrogen, or other nutrients and their interaction with limiting carbon and light. Comparison of the Growth Rates of Macrophytes with Algae This type of kinetic analysis also can be used to describe competition between macrophytes and algae. In a study done by Hill (1977), microcosm techniques similar to the ones used in this study were used to obtain kinetic data for the growth of the green alga Chlorella vulgaris. When used for algae, the type of microcosm used here provides data on the physiological ability of the algae to fix car- bon, since algae that sink to the bottom of the microcosm continue photosynthesis. This is not very realistic when compared to a lake, in which algae that sink are often re- moved from the photic zone. Therefore, in addition to the "light" microcosms used here, Hill used bottles in which the lower half of the microcosm was shaded. This technique provides a situation in which algae that sink to the bottom 45 of the microcosm are not able to continue photosynthesis. In this way he was able to get data for the physiological growth abilities of the algae from the "light" microcosms and data on the effective growth rate from the shaded micro- cosms, which take into consideration the sink character- istics of algae. Data from Hill's "light" microcosms were used to cal- culate equations for "max for chlorella as a function of light (D. L. King, personal communication). Figure 20 shows the linear relationship of “max for chlorella with light. The “max light functions for ceratophyllum and elodea are included on this figure. The lines indicate that chlorella has a much higher maximum growth rate than either elodea or ceratophyllum. The line for chlorella, however, represents its physiological ability to fix carbon and does not con- sider the fact that algae sink. Hill's data also indicate that chlorella is physio- logically able to grow at much lower CO2f concentrations than ceratophyllum. Again, this is not a true indication of chlorella's actual ability to compete. Figure 21 shows recalculations of Hill's data (D. L. King, personal commu- nication) for the physiological Cqu and the cozq from the shaded microcosms compared to the COquor ceratophyllum obtained in this study. The curves indicate that if the algae did not sink, they would be able to grow to much lower C02f concentrations than ceratophyllum. However, when sink properties of algae are taken into consideration, 46 mitosis .m. 3.5m: .o Ebola .o .5... 3: .6 85.0.6.2 to. 8.. 15 8.60:8 .8 .13.... 8? L a mEbBSoU 25:6: 8... . ea . shill) .. .«od (t—J-i) M" - .36 47 55898.0 .096 2.8.3 .o 8.1 88 t 8.88.5.8 16.. as .5 8.880 .8 8.3.... as 8.. 8.9 . 88 . as 8.2 8. II D a-O'F c-OI r0" v-Ot tOI 001 301 m888§ts=s§84881 .o 888823.36m280 8» 88.. .80 .8888?§9msn§§d$881 we «OI a/salmmn ‘00 48 ceratophyllum can grow to lower C02f concentrations than chlorella for some light intensities. A growth rate equation also was calculated from Hill's data for both the physiological growth rate and the "effec- tive" growth rate of chlorella under a light intensity of 3875 lux. Figure 22 shows these curves compared to the growth rate curve for ceratophyllum.under 3875 lux. Again, the physiological growth rate of chlorella far exceeds that of ceratophyllum but when sink characteristics of the chlorella are taken into consideration, ceratophyllum has a higher competitive ability under some conditions. Chlor- ella has a relatively high specific growth rate, but it tends to sink out of the photic zone, especially when sub- jected to stress conditions (Hill, 1977). Even though ceratophyllum has a relatively low specific growth rate, its ability to maintain its biomass in the photic zone gives it a competitive advantage over chlorella. Figure 22 can be used to illustrate how growth kinetics can be used as a management tool. For example, if chlorella and ceratophyllum were the only competing plant species in a waste stabilization lagoon where carbon and light were the limiting factors, the dominant species could be chosen by adjusting the detention time of the water flowing through the lagoon. Longer detention times allow the plants to fix more carbon and thus create lower CO2f concentrations. From Figure 22, chlorella and ceratophyllum are equally compet- itive when the CO2f concentration is approximately 49 us -ui‘0r C. vulgg‘1_s° inllg'it microcosms 11,. - u (or C. vulggs in shaded microcosm um -u¥a'C.demersmlnlthtmia-ooosms 01“) 0M1“ fl o.®"' fl" 0015-- 0.010-- 0.0%“ "an A 1.0" 10" 10" 10' 1.0I 10' 00, uncles/D Flare 22. Cormmson o? the speoil‘lc g'owth rates 04‘ .demersLm md C. wigs. 50 l umole/l. If the detention time is adjusted so that plant photosynthesis could lower the COzf concentration to a value greater than 1 umole/l, cthrella would dominate. If the detention time was increased so that the plants could lower the C02f concentration to less than 1 umole/l, then ceratophyllum would dominate. This type of simple model should be used with caution, since it does not take all factors into consideration, such as shading effects from the plant species that becomes domi- nant first. Models of this sort also cannot always be applied directly to the field, as indicated by field data from Craig's study (1978) of ceratOphyllum in an enriched system. Craig reported a ”max of .067 day-1, or approxi- mately .0048 hour-l, which is considerably higher than the "max of .0010 hour.l found in this study at 3875 lux. This could easily be a result of higher light intensity in the field situation. He also reported a CO2q of 1.3 moles COZf/l, which is much higher than the value obtained in this study of .265 umoles COzf/l at 3875 lux. Craig's pH data were not collected directly in the plant mat, and thus the CO value represents an average for the water column. This 291 average would be expected to be higher than the C025 concen- tration present in the plant mat. It is obvious that more data are necessary to determine if laboratory studies of this kind can be applied directly to field situations. 51 This study was an attempt to develop a technique for designing a small model of plant growth that could have management application. At the same time, it points to some of the downfalls of the large universal eutrophication models currently being attempted. These models often are composed of unjustified simplifications, such as constant algal sink rates, no threshold values for limiting nutri- ents, single limiting factors, empirically derived rate‘ constants and simple ballpark estimates (e.g. Bierman, et a1, 1973; Bloomfield, et al, 1973). It is obvious from this study and studies done with algae (e.g. Hill, 1977; King and King, 1974) that plant growth cannot be accurately represented in such a simplified manner but that environ- mental factors interact to affect growth characteristics of plants. This study points out that u and C0zq are not max constants, but vary as a function of light. It is very likely that these parameters also vary as a function of other limiting nutrients, creating a very complex set of interactions in nature. Until we learn more about these interactions, attempts at constructing universal management models are futile. 1) 2) 3) 4) 5) CONCLUSIONS This microcosm method of studying growth kinetics can be used with macrophytes, provided the plants used are free of algae. Ceratophyllum.demersum and Elodea canadensis follow Monod growth kinetics in relation to carbon if all other growth requirements are met. Carbon and light availability interact to control the growth kinetics of Ceratophyllum demersum and Elodea canadensis in the same manner previously observed for the green alga Chlorella vulgaris. Elodea canadensis is a better competitor for carbon than Ceratophyllum.demersum at all light intensities used in this study. There are insufficient data at this time to determine whether or not laboratory data collected in this manner are directly applicable to field situations. 52 APPENDIX Composition of Inorganic Nutrient Medium Nutrient Concentration NaHCO3 varies KNO3 114.0 mg/liter CaC12 43.3 mg/liter FeCl3 4.0 mg/liter MgSO4-7HZO 40.0 mg/liter EDTA 2.0 mg/liter K2H1>04 8 . 0 mg/liter Microelement Solution 1.0 ml/liter Composition of Microelement Solution Nutrient Concentration H3BO3 2.86 g/liter MnC12°4H20 1.81 g/liter ZnSO4°7H20 0.22 g/liter (NH4)6Mo7024 0.18 g/liter CuSO4 0.05 g/liter Co(NO3)2°6H20 0.49 g/liter 53 BIBLIOGRAPHY Bierman, V. J., Jr., F. H. Verhoff, T. L. Poulson and M. W. Tenney. 1973. Multi-nutrient dynamic models of algal growth and species competition in eutrophic lakes. In Modeling the Eutrophication Process, proceedings-afa workshOp held at Utah State Univer- sity, Logan, Utah. E. J. Middlebrooks, D. H. Falken- borg, and T. E. Maloney, ed. 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