l o o «« A.‘ . II. a...- .\'-' .I-‘V""‘ A..Iv.-< " ‘. '1' u 0" n'f'flfi «4‘ 1—1) ~o h|"'-"' I,n-V‘ J-. I u. .. 0" 0"," t “.1." n.’ - 'v' .D l ..\ '. L I QAZCV it." ..v;‘u,\\a O c. «In . —_ V_ r“ 'VUDOOOVfl- ”CQ‘OO’QWIOFM.5...“..ognnu09”“ ‘— ADSORBED 0N CLAY PAR; TO A GREEN ALGA ‘ - Th -¢—-~ .a-g-c... 06.- ' IICL ”'MOW'— CONCOW 0.0"- AVAILABILITY OF PHOSPHORUS43L ~ p esis ”for the. Degree of M . 8'. MI e HIGAN; STATE UNIVERSITY LOUiS A. HELFRICH 972 -oto 0 . -r . I . ... 'a..~o' OH L-a. . _¢ n . - . o c 0 . Q o . o - . l .. . _: r , -. -o- 1. .. v . . . . . u l o . ‘ l v . _ . . . - - .’ . . J - . . . a . , . . .9 . ' a . . . - . ~ ‘ Q . . . _ . O o . - Q . . _ ! O t - I n - I . . . . y . . - . . . . . .. _ . D -‘ n . I C 0 g . _ s .. ~ 0 - _ I n _ _ . . ' - . . o . r . - — . . - . _ - - c . . . . . A o - .r_' - ‘ . . . . - . . . , v . ' a o A c I ' o -- . o . V . . v ‘ v .Q I v . i ' l ‘ . o . . ' I O . . . . . ‘ o - o ' n . ‘ o . o l . t + : o _ . n . C ‘ l I I 0 o . .n _ - - . a . - . . - u - . _ _ . . O- - . - - ' - ‘ 7 '.'- I - . ’ _ t ',4 ..,. . - - , ‘ a o n r .'. v. .‘ V. ~ .. ‘ . r n t O V '0 .. ‘ ° ' . ‘0 . . . o . In. .I . - o , 0" ‘ : o ‘ - .A . .‘ ‘ . K— - ' -o (- ‘1 ' . ‘ u A . - . - ‘, . '2' . -. ‘.- ._- .0 0- " .- . y _' .. . . -' .. « « ,,..,. a. . a _ ‘ o o . t ,. . .~,t-;\;,'-_. -. - 9 o ‘0‘ ’1‘ ‘.’-’ . .- - n .C‘ . gouac- ' " o ‘J '- . L -. '0 ‘. . , . < _. . - ' 0 O . :. ' ‘. U U .. 4 ' c . '. .. o . U Q .0 - _.‘ . 7‘ . .O ‘_. t ' t '. m'.‘ 'o' 1‘... -. a "71'.‘ - --i - _.-Q ‘ I. . ;¥ ‘A I-.. . 15¢ Q C .-1'c2 _\ - ”I L 'LTRARY L5! 4 Michigan State E.) University ' V v .z'x 5r amnuié nyfl ”1; .410“; & 80W kg - BOOK BINDERY INC. '- LIBRARY BINDERS t 3 i } gammy. 3mm ABSTRACT AVAILABILITY OF PHOSPHORUS-32, ADSORBED ON CLAY PARTICLES, TO A GREEN ALGA BY Louis A. Helfrich The exchange of radiophosphorus between clay particles, culture medium and phosphate-limited Pandorina £9535 cells was examined in order to determine the availability of phosphorus adsorbed on clay particles to algal cells. The cultures were maintained in three light regimes in an attempt to equate the uptake and release of 32F with the pH and dissolved oxygen concentrations associated with the fluctuating light conditions. Of the total amount of adsorbed 32 P present in all the cultures a mean uptake of approximately 30% was found in the cells after the second day. The greatest uptake of the tracer by the algal cells occurred in the cultures which received constant light and exhibited the highest dissolved oxygen levels as well as the highest pH values. 32 the greatest loss of adsorbed P by the clay was found in those cultures which received no light and Louis A. Helfrich exhibited the lowest dissolved oxygen levels and the lowest pH values. AVAILABILITY OF PHOSPHORUS-32, ADSORBED ON CLAY PARTICLES, TO A GREEN ALGA BY Louis A. Helfrich 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 1972 a»? A ACKNOWLEDGMENTS I extend my sincere appreciation to all the people who helped in the completion of this research. Especially to my graduate committee members, Dr. N. Kevern, Dr. B. Ellis, and Dr. C. McNabb for their invaluable advice. Appreciation is also expressed to Dr. R. Cole for his aid in preparation of the manuscript, and to Dr. W. Conley for his help in the statistical analysis of the data. Special thanks to my wife, Karen, for her encouragement and review of the manuscript. Financial support of this research was supplied by the Michigan State University Agricultural Experiment Station. ii TABLE OF CONTENTS Chapter Page INTRODUCTION . . . . . . . . . . . . . . 1 METHODS AND PROCEDURES. . . . . . . . . . . 4 Culture Conditions . . . . . . . . . . . 4 Measurement of Growth, pH and DO. . . . . . . 7 Clay Preparation . . . . . . . . . . . . 9 Radiological Techniques. . . . . . . . . . 10 Sampling Procedure . . . . . . . . . . . 11 Sample Fractionation. . . . . . . . . . . 12 RESULTS. . . . . O . . . . . . O . . . 18 32F Uptake by Cells and Loss From Clay. . . . . 18 Effects of Light and Time on 32P Uptake . . . . 21 Effects of Light and Time on 32F Release . . . . 28 Activity in Aqueous Phase . . . . . . . 33 Dry and Organic Weight of Algal Cells . . . . . 36 Effects of pH on 32F Movement. . . . . . . . 38 Effects of DO on 32P Movement. . . . . . . . 38 DISCUSSION. . . . O . 0 . . . . . . . . 43 REFERENCES CITED. . O O . O . . . . . . . 49 APPENDICES Appendix A. Equations for Correction of Counter Dead Time and Radioactive Decay . . . . . . 52 B. Mean Activity in P. morum for Each Light Regime During the Four Experimental Days With Standard Error and Coefficient of Variation . . . . . . . . . . . . 53 iii Chapter Page C. One-Way Analysis of Variance of the Effects of Light on the Uptake of 32P by Algal Cells for Each Experimental Day . . . . . 54 D. Mean Activity in Clay Particles for Each Light Regime During the Four Days With Standard Error and Coefficient of Variation . . . . . . . . . . . . 55 E. Two-Way Analysis of Variance of the Effects of Light and Time on the Loss of Adsorbed 32F From Clay Particles With Ducan's Multiple Range Test for Mean Separation . . 56 F. Mean Activity in Culture Medium for Each Light Regime During the Four Days With Standard Error and Coefficient of Variation . . . . . . . . . . . . 57 G. Correlation Matrix of Intercorrelations for Eleven Parameters Measured. . . . . . . 58 iv Table 1. 2. B-l. C-l. D-l. E-l. F-lo G-lo LIST OF TABLES Composition of Modified Chu-lO Medium . . Mean Dry and Organic Weight of g, morum in Each Light Regime for Four Days Wltfi Standard Error and Coefficient of Variation. . . . . . . . . . . Mean Activity in P. morum for Each Light Regime During the Four Experimental Days With Standard Error and Coefficient of Variation. . . . . . . . . . . One-Way Analysis of Variance of the Effects of Light on the Uptake of 32F by Algal Cells for Each Experimental Day . . . Mean Activity in Clay Particles for Each Light Regime During the Four Days With Standard Error and Coefficient of Variation. . . . . . . . . . . Two-Way Analysis of Variance of the Effects of Light and Time on the Loss of Adsorbed 32F From Clay Particles With Ducan's Multiple Range Test for Mean Separation. Mean Activity in Culture Medium for Each Light Regime During the Four Days With Standard Error and Coefficient of Variation. . . . . . '. . . . . Correlation Matrix of Intercorrelations for Eleven Parameters Measured . . . . . Page 36 53 S4 55 56 57 58 Figure 1. 2. LIST OF FIGURES Photograph of Culture Chamber Designed for Light and Temperature Control . . . . . A Modified Flow Diagram for Water Fractionation . . . . . . . . . . Photograph of Sample Fractionation in a Bromoethanol Density Gradient Showing Medium Zone (A), Algal Cells (B), Bromoethanol (C), and Clay Zone (D) . . . . . . . . . Mean Percent of Total Activity for P. morum and Clay as a Function of Time, Showing the 95% Confidence Limits. . . . . . . . Mean Activity for g. morum Versus Activity in the Clay Over Four Days With Continuous Light . . O . . . . . . . . . . Mean Activity for g. morum Versus Activity in the Clay Over Four Days With a 12-Hour Light Regime. . . . . . . . . . . Mean Activity for g, morum Versus Activity in the Clay Over Four Days With No Light . . Mean Activity for 3° morum in Each Light Regime as a Function of Days After Intro- duction of 32P Adsorbed to Clay Particles . Mean Activity in Clay Sediments for Each Light Regime as a Function of Days After Introduction of 32F Adsorbed to Clay Particles. . . . . . . . . . . . vi Page 14 17 20 23 25 27 3O 32 Figure Page 10. Mean Activity in Aqueous Phase for Each Light Regime as a Function of Days After Intro- duction of 32F Adsorbed to Clay Particles . . 35 ll. Mean pH Values in Control Cultures for Each Light Regime During the Four Days. . . . . 40 12. Mean Dissolved Oxygen Concentration in the Control Cultures for Each Light Regime During the Four Days . . . .. . . . . . 42 vii INT RODUCT ION Recently, a great deal of concern has been ex- pressed about the rapid eutrophication of many bodies of water manifested by nuisance growths of aquatic vegetation. Although many nutrients can potentially contribute to such undesirable productivity, phosphorus has been established as the nutrient which is most often limiting to such growth (Mackenthun, 1968). Phosphorus found in surface waters may originate from a variety of sources that include; sewage effluents, synthetic detergents, industrial wastes, and drainage from agricultural lands. An initial concern in reducing the phosphate problem in lakes and streams is to identify the magnitude of phosphorus sources and the relative availa- bility of phosphorus as a plant nutrient from the various sources. Much emphasis has been placed on the removal of phosphorus from wastewaters and detergents to effectively control the phosphorus concentrations and reduce environ- mental degradation. While implementation of this approach and technology can substantially reduce phosphorus levels in some natural waters it may not reduce plant production if the contribution of phosphorus from land drainage is sufficiently available for plant growth. This research was conducted to determine if phosphate adsorbed on clay particles, as representative of typical drainage water sediments, is available to phosphate- limited Pandorina morum and to examine some of the parameters which influence the availability of this nutrient. More definite information about the quantities of adsorbed phosphorus available for plant growth and the mechanisms which control this availability may ultimately lead to effective control of eutrophication. In the terrestrial watershed virtually all of the soluble phosphorus is readily fixed by silts, clays and clay minerals and is often considered unavailable for plant use (Murphy, 1939; Low and Black, 1948; Hsu, 1960). Despite fixation on soils, phosphorus is often conveyed in appreciable quantities to natural waters adsorbed on suspended soil particles, particularly clay, as a result of land drainage and erosion. Johnson and Moldenhauer (1970) suggest that enormous quantities of suspended sediments, often carrying water pollutants such as plant nutrients, are delivered to streams and lakes as a result of erosion. Other investigators have concluded that land drainage is a significant source of phOSphates in streams which drain agricultural lands and that rural runoff may be a major factor in stream phosphate pollution (Engel- bracht and Morgan, 1961; Weidner, gt_gl., 1969). Kohnke and Bertrand (1959) indicate that typical surface runoff is high in clay particles and adsorbed phosphates. Suspended sediment may play a major role in controlling the dissolved phosphorus concentrations in natural waters by providing sorptive sites. Keup (1968) concluded that significant quantities of phosphorus may be transported in flowing waters as bed-loads or with floating materials. Although phosphorus definitely reaches aquatic environments via runoff the availability of adsorbed forms of this nutrient to aquatic plants remains obscure. Several investigators reason that the amount of phosphate in sorptive chemical exchanges between the sediments and water is large enough to be significant in biological processes (Pomeroy, gt_gl., 1965; Harter, 1968). Golterman, g£_gl. (1969) reported excellent growth of Scenedesmus obliquus was obtained with mud as a sole phosphorus source. This paper establishes that phosphorus adsorbed to clay particles is available to Pandorina morum cells. METHODS AND PROCEDURES Pure cultures of Pandorina morum (18) obtained from the culture collection of Indiana University were used to examine the availability of radioactive phosphorus (32F) adsorbed on clay particles. Culture Conditions All glassware used in experiments was washed in potassium dichromate-sulphuric acid solution and suc- cessively rinsed in double-distilled water. In order to prevent contamination from dust and to permit gas exchange, the flasks were stoppered with polyurethane foam plugs. Both the glassware and the culture medium were autoclaved for 30 minutes at 15 psi, and all transfers, innoculations and samples were taken with sterile serological pipetts in order to retard bacterial growth. Axenic cultures of stock a, morum were grown in 150 ml erlenmyer flasks placed within culture chambers designed for the regulation of photoperiod and temperature (Figure l). The conditions in the flasks were: solution volume, 100 ml; temperature, 2712 C; light intensity Figure 1. Photograph of the culture chamber designed for light and temperature control. provided by gro-lux fluorescent lights, 120 ft-c; agitation daily by hand, and initially equivalent nutrient concentrations. The culture medium, Chu-lO (Chu, 1942), was modified to double strength in all major nutrients to ensure adequate growth (Table l). A week prior to the tests these cells were transferred after centrifugation to a series of flasks containing the modified Chu-lO medium with all nutrients except phosphorus, insuring that the cells would become limited in phosphate during subsequent growth. In an attempt to examine the effects of light on the uptake of adsorbed 32 P by the algal cells a series of nine flasks was established according to a factorial design (3 cultures X 3 light regimes). Thus one-third of the cultures each received 24 hours of light per day, 12 hours of light per day, and the final third was maintained without light. Control cultures in each of the light regimes were used to monitor changes in pH and dissolved oxygen, while other control flasks without algal cells were used to demonstrate changes in the phosphorus equilibrium between the medium and the clay particles. Measurement of Growth, pH and DO With differing light regimes, corresponding changes in algal growth, pH, and dissolved oxygen concentrations of the cultures were examined to gain some insight on the TABLE l.--Composition of Modified Chu-lO Medium (Chu, 1942). Nutrients Concentration m M Ca(NO3)2 . 4H20 40.0 MgSO4 . 7H20 80.0 KZHPO4 10.0 NaHCO3 40.0 T_ra_c_e. NaETDA 3 . 0 0 FeCl3 . 6H20 0.08 MnCl2 . 4H20 0.06 ZnCl2 0.01 CoCl2 . 6H20 0.002 NaMoO4 . 2H20 0.01 Vitamins* 812 0.03 Biotin 0.0003 Thiamin . HCl 0.00003 *Modified by Dr. Brian Moss (personal communi- cation). release and uptake of 32 P. Algal growth (standing crop) was determined gravimetrically to the nearest milligram. The pH changes were monitored by meter to the nearest tenth of a unit. The sodium azide modification of the Winkler method was used for dissolved oxygen determination. Fromm (1958) developed a semi-micro procedure for measuring the, dissolved oxygen concentration. This method is rapid, accurate, and allows for determinations in a small volume of water. The MnSO4 solution, alkaline KI solution and phosphoric acid in the usual concentrations were con- veniently stored in rubber-stOppered serum bottles. Phosphoric acid was substituted for sulfuric acid in these micro-determinations as sulfuric acid may cause the liberation of iodine from the alkaline KI solution (Fox and Wingfield, 1938). Clay Preparation Kaolinite, a well known descriptive type clay mineral whose role in phosphate fixation in soils has been established, was used to represent typical suspended and settleable sediments in aquatic systems. A 5% suspension of kaolinite was homogenized in a blender and this suspension was slowly introduced into an ion exchange column containing a medium porosity Amberlite IR-120 resin. This exchange replaces Ca ions with H ions. The addition of 1N NaOH to the eluant maintained the pH above 8.5 as 10 well as replacing the H ions with Na ions. In order to obtain a relatively uniform particle size the Na-clay suspension was vigorously shaken and allowed to settle. Stokes's Law (Beaver, 1956) describes this settling as a function of the relationship between the radius of a particle and its rate of fall in a liquid. Reionization of the clay suspension replaced the Na ions with H ions, and by spontaneous decomposition of the clay surface the H-clay is transformed into an acidic Al-clay. Clay particles prepared in this manner readily adsorb phosphorus. The tracer, carrier-free 32 P as orthophosphate in 0.02 M HCl, was added to the clay suspension which was continually stirred for three days to insure that a large percentage of the tracer was adsorbed to the clay. Experiments were initiated by adding 5 ml of the radio- active clay, calculated to give about 8,000 cpm/ml in the cultures. Radiological Techniques Radiophosphorus, 32P, was chosen as the tracer because it provides accurate measurements of the phosphorus concentrations in each of the three major categories; culture medium, clay particles, and algal cells. This isotope emits a single high energy beta particle (1.712 mev), has excellent tracer qualities, and a satisfactory half-life (14.3 days). 11 Radioactivity was measured with an internal gas flow, end-window gieger detector equipped with a low background anti-concidence unit. To insure consistent counting geometry all samples were mounted in polished steel planchets providing a total area of 2.54 cmz. In order to facilitate uniform distribution of the sample in the planchets two drops of normal hexane were added during the drying process. Drying of the samples was accomplished using a hot plate at 105 C. The counting efficiency, determined daily, averaged about 38.25% for a simulated 32P standard of natural uranium. Counting times were determined to be significant at the 95% level (Overman and Clark, 1960). Background, sample geometry and back- scattering all remained relatively constant. Preliminary experiments demonstrated that self adsorption, in such small samples along with a high energy tracer, was negligible. All measurements were corrected for counter dead time and radioactive decay (Appendix A). SamplinggProcedure The experimental flasks, incubated under the same conditions as the control cultures, were removed from the chambers at the end of the dark period every day and thoroughly swirled. A 10 ml sample was taken from each culture with a pipette. From each of these samples a one milliliter aliquot was dried by evaporation and counted to determine the activity in the whole sample. The remaining 12 9 ml of the sample was fractionated for further isotope analysis. From each of the control cultures an 8 ml water sample was drawn into a syringe and used to determine dissolved oxygen concentrations. The flask was then swirled and the pH of the culture determined. Sample Fractionation Centrifugation and density gradient separation procedures were used to fractionate the water sample into three major categories: culture medium, clay particles and algal cells. These methods allow for the concentration and separation of the sample into homogeneous fractions in order to determine the quantative distribution of 32P in each category. Lamers (1966) developed a scheme for natural water fractionation which was modified for use in this study (Figure 2). The samples were initially separated into two fractions by centrifugation at 2500 rpm for 20 minutes. The supernatant culture medium and any colloidal clay was removed by cautious aspiration, separated by centrifugation at 9000 rpm for 20 minutes and prepared for radionuclide analysis. The remaining fraction of algal cells and clay particles was resuspended in isotope free medium and homogenized. This suspension was slowly introduced over 2-bromoethanol (density 1.77, viscosity 4.5 cp) and centrifuged at 2500 rpm for 20 minutes. Bromoethanol is a water soluble alcohol which forms a density gradient Figure 2. 13 A modified flow diagram for water fractionation (Lamers, 1966). Pellet (Algal Cells+Clay) Resuspension Homogenization Density Gradient Centrifugation 14 Initial Water Sample Aliquot for Standard Centrifugation Supernatant (Medium+Colloidal Clay) Centrifu ation Pellet (Clay) Supernatant (Medium) Assay Assay Pellet (Clay)- As ay -—-Supernatant (Algal Cells) A883! 15 through which the denser clay particles pass, but the gradient has a higher density than the algal cells, effectively fractionating the sample (Figure 3). Each fraction of the sample was removed by cautious aspiration and dried for analysis. Figure 3. 16 Photograph of sample fractionation in a bromoethanol density gradient showing medium zone (A), algal cells (B), bromoethanol (C), and clay zone (D). l7 RESULTS Radioactive phosphorus, adsorbed to clay particles, is available to phosphate-limited P, mgggm cells. Initially all of the phosphorus introduced to the cultures was adsorbed on clay particles. Thus, any phosphorus tracer evidenced in the algal cells must have been derived from that adsorbed on the clay. 32P The mean percentage of the total amount of present in the algal cells and clay particles for each day is shown in Figure 4. It appears that an equilibrium between the phosphorus in the algal cells, aqueous phase and the clay sediments was established, without regard to light regime, after the second day. The percentage of the tracer in the algal cells increased rapidly to a maximum of approximately 30% in two days and remained relatively constant until the end of the experiment. 32 P Uptake by Cells and Loss rom C ay The activity sorbed by algal cells in all three light regimes was highly correlated with a loss of activity 18 Figure 4. 19 Mean percent of total activity for g, morum and clay as a function of time, showing the 95% confidence limits. Mean Percent of Total Activity 1001 20 F. ~ -——-Algal cells \ \ - - - Clay particles \ \ \ \ ‘L \ \f _ - - ~ - i ----- .i 50' o , . ' fl 0 I 2 V 3 4 Days After Addition of P32 21 from the clay particles. As was expected, a negative correlation was shown describing the compensatory phenomenon of a loss of 32 P from the clay particles with a subsequent gain of the tracer by the algal cells. Those cultures maintained under constant light (complete darkness and constant light) showed the highest correlations with r values of -0.90 and -0.82 respectively. These values were significantly different from 0 at the 0.01 level. The uptake of 32 P by the cells in the lZ-hour light regime also was correlated with the loss of activity from the clay sediments having a correlation coefficient of -0.67, which was significant at the 0.05 level. The degree to which the loss of the isotope by the clay is correlated with a gain in activity by the cells is illustrated for each light regime (Figures 5, 6, 7). The shape of the ellipse is a function of the correlation between the two variables (loss by clay versus gain by algal cells), and the area of the ellipse is a function of the confidence coefficient (Sokal and Rohlf, 1969). Effects of Li ht and Time on P Uptake An attempt was made to follow the fate of adsorbed radiophosphorus in order to distinguish any differences in the concentration of the tracer in the cells as a function of light or time. The mean activity found in the algal cells for each of the three light regimes during the four Figure 5. 22 Mean activity for g, morum versus activity in the clay over four days with continuous light. 23 3% 4 d 2 I 25.6253 2.8 .82 5 33:2 Activity In Clay (CPM x103/ml) 24 Figure 6. Mean activity for g, morum versus activity in the clay over four days with a lZ-hour light regime. 25 1 1 2 ' :E\.o§:a2 2.8 .82 s 3.53 Activity In Clay (CPMX103/ml) Figure 7. 26 Mean activity for g, morum versus activity in the clay over four days wIth no light. I—_‘__ m-_ - 27 G. 1. 6 Activity In Clay (CPMxlo‘le) 31f q 1 2 I C csaopxsaov 2.8 .82 a. £52 28 days was determined (Figure 8). The standard error and coefficient of variation of the cellular activity for each photoperiod is shown in Appendix B. An almost linear increase in cell activity was shown in all three light regimes as the experiment proceeded. Those cultures which received constant light exhibited the greatest uptake of the tracer, reaching a maximum concentration of approxi— mately 2900 cpm on the third day. In order to further investigate the effects of light on the activity sorbed by algal cells a one-way analysis of variance was conducted for each of the four days (Appendix C). Ducan's multiple-range test was used for comparing treatment means. The activity in the cells was significantly different (P<.05) for all light con- ditions on each day except the third when no significant difference in algal activity was determined between the cultures in the 12-hour light regime and those which received no light. Effects of Light and Time on 32F Release The mean activity of the clay particles for each light regime during the four days is shown in Figure 9. The standard error and coefficient of variation of the activity in the clay for each light regime during the four days is given in Appendix D. A rapid decrease in 32 the concentration of adsorbed P was shown during the 29 Figure 8. Mean activity for g, morum in each light regime as a function of days after introduction of 32P adsorbed to clay particles. Activity In Algal Cells (CPM X103/ml) 3O 31 2+ ——2eiv light regime ’ ° - - - 12hr light regime I / _._ om light regime J Days After P32 Introduction 31 Figure 9. Mean activity in clay sediments for each light regime as a function of days after introduction of 32P adsorbed to clay particles. Activity In Clay (CPMX103/ml) 61 32 ————-24 hr light regime — - 12hr light regime -- - - 0 hr light regime Days After P32 Introduction 33 first two days. The greatest loss of phosphorus from clay sediments occurred in the cultures kept in constant darkness. A gain in activity of the clay particles on the fourth day in the 24-hour light regime occurred simultaneously with a loss of 32 P by the algal cells in the same light conditions. The effects of light and time on the release of 32P from the clay particles were considered simultaneously by a two-way analysis of variance (Appendix E) and the treatment means were compared with Ducan's multiple range test. A significant difference (P<.001) in the release of the tracer by the clay was shown among light regimes and days. Activity in Aqueous Phase With the introduction of relatively high concen- trations of adsorbed 32P, the amount of tracer in the aqueous phase increased (Figure 10). The standard error and coefficient of variation for the activity in the water is given in Appendix F. The increase in the levels of the isotope in the culture medium occurred in all three light regimes and continued for the four days. The greatest amount of activity was released to the aqueous phase by the clay particles in the cultures maintained without light. No significant difference in the amount of activity found in the aqueous phase occurred between the cultures receiving 24 hours and 12 hours of light. In 34 Figure 10. Mean activity in aqueous phase for each light regime as a function of days after intro- duction of 32P adsorbed to clay particles. 35 300 W ------- 24 hr lighf regime _ — - I2 hr light regime —— -- 0 hr light regime / 7: Control ' E '/ \ a -/ e ‘ ./ o. g 20 / g / a. . a -/ 8 / . / 3 -/° q / ........ '1’ s lOO'l / I’w”’ 1'? / firsa — - was r A .2 ' g .//‘ r ./ .I oo 1 5 5 7; Days After P32 introduction Ii”- 36 32P found in the comparing the average concentrations of culture medium with the activity assayed in the cells, it is apparent that E, mgggm can concentrate radiophosphorus at least 40 times that amount found in the water. Three control flasks, without algal cells, were maintained in each light regime in order to monitor the physical equilibrium of the tracer between clay particles and the aqueous phase. The mean activity in the medium after the first day was 75 cpm/ml in all light regimes, and this value remained relatively constant throughout the experiment. 0 A a Ce 8 The dry weight of the algal cells in all light regimes remained relatively constant during the four experimental days. The mean dry and organic weight of g, mgggm in each light regime for the four days with standard error and coefficient of variation is shown in Table 2. No significant growth was evident in any of the light regimes during the four days; this was probably a function of the large algal samples necessary for analysis. The amount of organic matter was distributed in almost direct proportion to the dry weight. The organic matter was highly correlated with the dry weight (Appendix G) as would be expected. 37 TABLE 2.--Mean Dry and Organic Weight of g. morum in Each Light Regime for Four Days With Standard Error and Coefficient of Variation. Day Photoperiod Dry WeightiSE S(lOO%) (hrs) (ug) X I 24 228 1 16.62 12.62 12 210 1 16.84 13.86 0 245 : 12.68 8.95 II 24 219 1; 15.84 12.54 12 248 _'l_-_ 20.23 14.13 0 240 3; 11.67 8.40 III 24 181 _+_ 5.69 5.44 12 213 1 17.32 14.06 0 237 ;_i-_ 14.99 10.94 IV 24 185 i 4.84 4.54 12 219 -_I-_ 17.44 13.78 0 183 1 12.73 12.07 Day Photoperiod Organic Weight:SE S(100%) (hrs) (ug) x I 24 204 1 16.80 14.26 12 184 L: 15.57 14.65 0 226 3; 12.01 9.20 II 24 201 3; 15.39 13.26 12 170 :i_-_ 20.42 20.75 0 217 i 9.60 7.64 III 24 158 i 5.24 5.73 12 189 1 16.07 14.65 0 181 _+_-_ 33.85 32.44 IV 24 161 1 7.64 8.21 12 204 1 19.55 16.62 0 161 1 13.92 15.00 38 32F Movement Effects ofng on The pH values for all three light regimes increased during the four experimental days (Figure 11). Initially all the cultures were slightly acidic caused by the intro- duction of the acidic clay particles. The greatest increase in pH was shown by those cultures in the 24-hour light regime. The cultures maintained in the other light regimes showed a slight increase in pH during the four days. The pH values were significantly correlated (P<.01) with the uptake of the tracer by the algal cells for all light regimes during the experiment. When the uptake of 32 P was maximum the pH in all light regimes ranged between 7.3 and 7.9. Effects of DO on 32F Movement The concentration of dissolved oxygen in the algal cultures increased in all light regimes during this experiment (Figure 12). As was expected, the highest levels of dissolved oxygen were found in the cultures which received constant light, reaching a maximum concen- tration of 10.2 mg/liter on the fourth day. The oxygen levels found in the cultures maintained in the dark are lower but seem to increase proportionally with the cultures in the 12-hour light regime. The greatest release of 32P occurred in the dark cultures which also evidenced the lowest oxygen concentrations. Figure 11. 39 Mean pH values in control cultures for each light regime during the four days. .—_1_—i__- _‘ ‘— —‘ _._. pH 40 IO! —-—24 hr light regime - - -l 2 hr light regime --- - 0 hr light regime Days After P32 Introduction Figure 12. 41 Mean dissolved oxygen concentration in the control cultures for each light regime during the four days. Dissolved Oxygen (mg/liter) HT 42 6'1 -----24 hr light regime _ - 12 hr light regime -— - 0 hr light regime —— Saturation Days After P32 Introduction DISCUSSION The results presented in this paper serve to emphasize the potential role of clay particles in supplying phosphorus necessary for algal nutrition. That phosphate- limited 2, mgggm can obtain this nutrient adsorbed to clay particles has been demonstrated. Originally the only 32P adsorbed to source of activity in the cultures was as clay. The relatively high concentrations of radio- phosphorus found in the algal cells demonstrates a movement of the isotope from the clay to the cells. Of the total 32P adsorbed to the clay particles approximately 30% was assayed in the algal cells, without regard to light regime, after two days. Rigler (1956) showed a similar uptake of 32 P from lake water by plankton, stating that the percentage of activity in plankton at the surface (0-2m) reached a maximum of 50% while in deeper water (8-9m) a maximum uptake of 30% was reached in three days. The 32 P adsorbed to clay particles in this laboratory study was nearly as available to algal cells as free radio- phosphorus found in the aqueous phase in a natural lake system. 43 44 Phosphate-limited g, mgggm_has the ability to rapidly sorb large amounts of radiophosphorus far in excess of that found in the water and in much greater quantities than can be explained by a simple physical equilibrium. Phosphates are strikingly concentrated by Euglena: 100,000X, Volvox: 140,000x, Pandorina: 285,000x, and Spirogyra: 850,000x (Round, 1970). Because many microorganisms have evolved in environments which have very low nutrient concentrations, it may be character- istic for these algae to have mechanisms for the uptake of nutrients from low levels. It may also be possible for some algae to alter the chemical composition of their immediate environment through metabolic activity, affecting the availability of adsorbed phosphates. A relatively stable equilibrium developed in the cultures after the second day through compensatory changes in the activity of each phase. Initially the clay particles rich in adsorbed 32 P released this isotope to the medium and the cells. Kurtz (1945) suggests that as adsorbed forms of phosphate increase in amount their solubility in water increases rapidly. The rapid loss in cellular activity in the 24-hour light regime on the fourth day was correlated with a simultaneous gain in activity by the clay, evidence that the system can reverse itself to maintain equilibrium. Bigger and Corey (1969) noted a similar exchange and showed that subsoil particles low in 45 phosphorus may absorb this nutrient from over-laying water, reducing the concentration in Solution. The movement of 32 P from the clay and its availa- bility to algal cells appears to have been largely governed by pH and chemical equilibria reactions. In general, high phosphate adsorption by clays is favored by a low pH. Stumm and Morgan (1970) state that maximum adsorption of orthophosphates on kaolinite clay occurs at a pH near 3. Ohle (1937) noted that maximum adsorption by sediments takes place at pH 5.9 and there is a decrease above and below this value. MacPherson, gt_al. (1958) found a pH above 6.5 created a greater increase of phosphorus in the water. The predominant dissolved orthophosphate species over pH range 5-9 are H P04 and 2 HPO4, both of which are available to algae cells. The relationships between total phosphate in solution and pH was determined using the solubility product principle. The dissociation product constants of the two chemical species Al(OH)3 and AlPO4 - ZHZO (Variscite) were 33.8 and 22.4 respectively. The ionic strength of the solution, determined from the major nutrients in the 4 Chu-lO medium, was 22.949x1o‘ M. The phosphate activity coefficients of 0.949 for H2PO4 and 0.811 for HPO4 were determined by the Debye-Huckel equation (Klotz, 1958). From these values a minimum phosphorus solubility of about 0.349 mg/l was calculated for pH 6.2. At pH 7.0, 7.4, 46 and 8.0 the phosphorus solubility rapidly increased to approximately 3.12, 14.5, and 169.0 mg/l respectively. A similar trend of the effect of pH on soluble phosphorus concentrations was shown by Lindsay and Moreno (1960). At pH values approaching 8 and higher the tendency for precipitation of phosphorus becomes enhanced and may be related to the formation of CaHOP 32 4. The distribution of P among clay particles, algal cells and in solution was correlated with pH and appears to follow the predicted trend calculated for phosphorus solubility as a function of pH in the cultures. As the pH increased from the initial pH 6.2 the activity in solution, as well as the activity in the cells increased reaching maximum levels at pH 7.4-7.8. It should be noted that lake sediments and most mineral-bearing waters are known to lie generally within a pH range 6-9, while agri- cultural soils are characteristically more acidic, pH<6. Significantly, the algal cells by altering the pH, at least partly determine the availability of adsorbed 32P. The greatest uptake of radiophosphorus occurred in the 24-hour light regime where photosynthetic activity was maximized and the pH reached 7.8. This condition was predicted, on the basis of pH and equilibria reactions, to provide the greatest availability of phosphorus. Numerous investigators have substantiated that the removal of oxygen from natural systems will allow the 47 release of inorganic phosphorus from bottom sediments (Mortimer, 1941-42; Hutchinson, 1957). A similar trend was described in this study, as the greatest release of 32F from the clay occurred in the cultures maintained in the dark and 12-hour light regimes. These cultures evidenced the lowest concentrations of dissolved oxygen, although oxygen was present at all times. Fitzgerald (1970) determined that phosphorus-limited Selenastrum and Cladophora did not respond by growth when exposed to as much as 2 mg of phosphorus as lake muds under aerobic conditions. Golterman, g5_31. (1969) reported Scenedesmus obliquus was able to increase tenfold or more with phosphorus from lake muds obtained from aerobic zones. This study indicates that adsorbed phosphates are available to P, mgggm although increases in growth rate were not evidenced. It is likely that the differences reported may have been due to testing procedures, characteristics of the mud samples, or requirements of the individual algae studied. In view of the vast reservoirs of phosphates absorbed to the sediments in natural waters, the pro- portion of phosphorus shown available to algal cells in this study is biologically significant. Phosphorus availability was largely controlled by pH and equilibria reactions and to a lesser degree by oxygen concentrations; while in natural waters with a substantial fraction of 48 iron present oxygen concentrations may play a major role in determining phosphorus availability. Increased emphasis and further research is needed in examining the role of adsorbed nutrients in the eutrophication of aquatic systems. General removal of soluble phosphorus at waste-water treatment plants is not the total solution if adsorbed phosphates from other sources are readily available to aquatic plants. Con- tinued effort should be made to understand the modes of nutrient losses and the effects of chemical gradients, pH, and other parameters on the availability of adsorbed forms of phosphorus. REFERENCES CITED REFERENCES CITED Baver, L. D. 1956. The mechanical composition of soils. pp. 48-80., In Soil Physics. John Wiley 5 Sons, Inc., New YOFE. 489p. Biggar, J. W. and R. B. Corey. 1969. Agricultural drainage and eutrophication.' pp. 414-445. In. Eutrophication: causes, consequences, correctives. Nat. Acad. Sci., Washington, D.C. Chase, G. D. and J. L. Rabinowitz. 1970. Principles of radioisotope methodalogy. Burgess Publishing Co., Minneapolis, Minn. 663p. Chu, S. P. 1942. The influence of the mineral composition of the medium on the growth of planktonic algae. ' J. Ecol. 30:284-325. Engelbrecht, R. S. and T. T. Morgan. 1961. Land drainage as a source of phosphorus in Illinois surface waters. pp. 74-79. In Algae and metropolitan wastes. Public Health Serv. Publ. SEC TR W61-3. Fitzgerald, G. P. 1970. Aerobic lake muds for the removal of phosphorus from lake waters. Limnol. Oceanogr. 15(4):550-555. Fox, H. M. and C. A. Wingfield. 1938. A portable apparatus for the determination of oxygen dissolved in a small volume of water. Jour. Exper. Biol. 15:437-445. ' Fromm, P. O. 1958. A method for measuring the oxygen consumption of fish. Prog. Fish. Cult. 22:137-139. Golterman, H. L., C. C. Bakels, and J. Jakobs-Mogelin. 1969. Availability of mud phosphates for the growth of algae. Verh. Internat. Verein. Limnol. 17:467-479. 49 50 Harter, R. D. 1968. Adsorption of phosphorus by lake sediment. Soil Sci. Soc. Amer. Proc. 32:514-518. Hsu, P. H. 1965. Fixation of phosphate by aluminum and iron in acidic soils. Soil Sci. 99:398-402. Hutchinson, G. E. 1957. A treatise on limnology. Vol. I. John Wiley & Sons, Inc., New York. 1015p. Johnson, H. P. and W. C. Moldenhauer. 1970. Pollution by sediment: sources and the detachment and transport processes. pp. 3-20. In Agricultural practices and water quality. Iowa State Univ. Press. 415p. Keup, L. E. 1968. Phosphorus in flowing waters. Water Res. 2:373-386. Klotz, I. M. 1958. Chemical thermodynamics. Prentice- Hall, Inc., New Jersey. 369p. Kohnke, H. and A. R. Bertrand. 1959. Soil Conservation. McGraw-Hill Pub. Co., New York. 92p. Kurtz, L. T. 1945. Adsorption and release of phosphate ions by soils and clays. Unpub. Ph.D. thesis, Univ. of Ill. Lib., Urbana, Illinois. Lammers, W. T. 1966. Natural water fractionation: theory and practice. Verh. Internat. Verin. Limnol. 16:, 452—458. Lindsay, W. L. and E. C. Moreno. 1960. Phosphate phase equilibria in soils. Soil Sci. Soc. Amer. Proc. 24:177-182. Low, P. F. and C. A. Black. 1948. Phosphate-induced decomposition of kaolinite. Soil Sci. Soc. Amer. Proc. 12:180-184. Mackenthun, K. M. 1968. The phosphorus problem. Jour. Amer. Water Works Ass. 60:1047-1054. MacPherson, L. B., N. R. Sinclair, and F. R. Hayes. 1958. The effect of pH on the partition of inorganic phosphate between water and oxidized mud or its ash. Limnol. Oceanogr. 3:318-326. Mortimer, C. H. 1941-42. The exchange of dissolved substances between mud and water in lakes. J. Ecology. 29:280-329. 51 Murphy, H. F. 1939. The role of kaolinite in phosphate fixation. Hilgardia. 12:341-382. Ohle, W. 1953. Phosphorus as the initial factor in the development of eutrophic waters. pp. 77-78. In nitrogen and phosphorus in water. Public HealEH Serv. Pub. 11. Cincinnati, Ohio. Overman, R. T. and H. M. Clark. 1960. Radioisotope Techniques. McGraw-Hill Pub. Co., New York. 476p. Pomeroy, L. R., E. E. Smith, and C. M. Grant. 1965. The exchange of phosphate between estuarine water and sediments. Limnol. Oceanogr. 10:167-172. Rigler, F. H. 1956. A tracer study of the phosphorus cycle in lake water. Ecology. 37:550-562. Round, F. E. 1970. The biology of the algae. Edward Arnold Ltd., London. 269p. Sokal, R. F. and F. J. Rohlf. 1969. Biometry. W. H. Freeman Co., San Francisco. 776p. Stumm, W. and J. J. Morgan. 1970. Aquatic chemistry: An introduction emphasizing chemical equilibria in natural waters. John Wiley 5 Sons, Inc., New York. 583p. Weidner, R. B., A. G. Christianson, S. R. Weibel, and G. G. Robeck. 1969. Rural runoff as a factor in stream pollution. J. WAter Pollut. Control Fed. 41:377-384. APPENDICES APPENDIX A EQUATIONS FOR CORRECTION OF COUNTER DEAD TIME AND RADIOACTIVE DECAY APPENDIX A EQUATIONS FOR CORRECTION OF COUNTER DEAD TIME AND RADIOACTIVE DECAY From the counted dead time (T = 1.5928x10-6cpm”1 ) a true count rate was calculated using the following equation from Chase (1959): n N = l - nT where N = true count rate, n = observed count rate, and T = counter dead time. Because 32F has a half-life of 14.3 days, radioactive decay represented the greatest correction factor. Time of counting was recorded for each sample and all samples were corrected to the time the tracer was added to the cultures by the following formula: f3 = {0.693% A 0 where A0 = initial activity, At = activity remaining after t, t = elapsed time, and e = base of natural logrithms. 52 APPENDIX B MEAN ACTIVITY IN E, morum FOR EACH LIGHT REGIME DURING THE FOUR EXPERIMENTAL DAYS WITH STANDARD ERROR AND COEFFICIENT OF VARIATION TABLE APPENDIX B B-l.--Mean Activity in P. morum for Each Light Regime During the-Four Experimental Days With Standard Error and Coefficient of Variation. Day Photoperiod Activity SE S(100%) (hrs) (cpm) x "' I 24 1849 44.91 4.20 12 1673 45.54 4.71 0 1309 30.12 3.98 II 24 2567 44.68 3.01 12 2364 31.44 2.30 0 2215 25.96 2.03 III 24 2937 67.11 3.95 12 2555 47.03 3.18 0 2401 50.53 3.64 IV 24 2224 36.11 2.81 12 2809 49.73 3.06 0 2504 30.28 2.09 53 APPENDIX C ONE-WAY ANALYSIS OF VARIANCE OF THE EFFECTS 32 OF LIGHT ON THE UPTAKE OF P BY ALGAL CELLS FOR EACH EXPERIMENTAL DAY APPENDIX C TABLE C-l.--One-Way Analysis of Variance of the Effects of Light on the Uptake of 32P by Algal Cells For Each Experimental Day. Day Source of Variation df SS MS .01 I Among Photoperiods 456279 228139 45* Error 29982 4997 Total II Among Photoperiods 187612 93806 25* Error 21952 3658 Total 111 Among Photoperiods 456936 228468 24* Error 55614 9269 Total IV Among Photoperiods 525978 262989 20* Error 78298 13049 Total 54 APPENDIX D MEAN ACTIVITY IN CLAY PARTICLES FOR EACH LIGHT REGIME DURING THE FOUR DAYS WITH STANDARD ERROR AND COEFFICIENT OF VARIATION APPENDIX D TABLE D-l.--Mean Activity in Clay Particles for Each Light Regime During the Four Experimental Days With the Standard Error and Coefficient of Variation. Day Photoperiod Activity SE S(100%) (hrSI (Cpm) x I 24 5934 65.35 1.90 12 5926 117.19 3.42 0 5785 83.56 2.50 II 24 5417 152.15 4.86 12 5436 178.70 5.69 0 5124 65.85 2.22 III 24 5223 152.11 5.04 12 5398 171.19 5.49 0 5091 100.38 3.41 IV 24 5728 68.88 2.08 12 5349 129.41 4.18 0 5063 89.72 3.06 55 APPENDIX E TWO-WAY ANALYSIS OF VARIANCE OF THE EFFECTS OF LIGHT AND TIME ON THE LOSS OF ADSORBED 32F FROM CLAY PARTICLES WITH DUCAN'S MULTIPLE RANGE TEST FOR MEAN SEPARATION APPENDIX E TABLE E-l.--Two-Way Analysis of Variance of the Effects of Light and Time on the Loss of Adsorbed 32F From Clay Particles With Ducan's Multiple Range Test for Mean Separation. Source of Variation df SS MS F.001 Between Days 3 2266324 755441 17.0* Between Photoperiods 2 665525 332763 7.5* Interaction 6 369683 61614 1.3 Error 24 1058385 Total 36 5933 5926 5785 5728 5436 5416 5398 5349 5223 5124 5091 6063 S6 APPENDIX F MEAN ACTIVITY IN CULTURE MEDIUM FOR EACH LIGHT REGIME DURING THE FOUR DAYS WITH STANDARD ERROR AND COEFFICIENT OF VARIATION APPENDIX F TABLE F-l.--Mean Activity in Culture Medium for Each Light Regime During the Four Days With Standard Error and Coefficient of Variation. Day Photoperiod Activity SE S(100%) (hrs) (cpm) x I 24 71.66 3.38 8.17 12 73.33 9.82 23.19 0 120.66 3.28 4.71 11 24 73.00 3.51 8.33 12 72.00 7.57 18.21 0 134.0 8.72 11.26 III 24 104.00 2.08 3.46 12 94.33 11.78 21.62 0 202.00 7.00 6.00 IV 24 117.33 5.04 7.44 12 107.66 7.33 11.79 0 276.00 5.51 3.45 57 APPENDIX G CORRELATION MATRIX OF INTERCORRELATIONS FOR ELEVEN PARAMETERS MEASURED Amav.0 Hm>ma H0.v Ammm.0 Hm>oa m0.0 coflumHmuuoo ucmowmacmwm u e woman unmflms man u 0 cmmhxo 00>Howmwo u Ha when u m mm a CH mCOAHomouonm u 0 made usmfles owsmmuo u 0 made sw hua>fiuo¢ u m moan unmwo3 mun u m mamam CH huabfluom u N woman unmflw3 owcmmuo u h ESHCOE cw >uw>fluod u H "wuoz 000.H emmm.0 0mm.0l 00N.0i emam.0l emmm.0l e00m.0 ema>.0 hNH.0i e0m0.0 m00.0 Ha emmm.0 000.H 0hH.0l 00H.0i emom.0i «mom.0l «000.0 emum.0 mm0.0l tomv.0 mm0.0 0H mmm.0i 05H.0l 000.H >v0.0 hem.0l ~N0.0l 000.0 mmm.0l mmH.0i «00.0 Hem.0 m 00N.0l 0va.0l «500.0 000.H 000.0l 000.0l 0m0.0 mm~.0l HmH.0l 000.0 00m.0 m emam.0l 00m.0i b¢0.0l 000.0l 000.H emm0.0 «www.0l «Hmm.0i 000.0 «vbm.0l hma.0l h «mom.0i tm0m.0i «No.0l 000.0l «mmm.0 000.H «www.0l www.0i mHH.0 NvN.0l maa.0l 0 emmm.0 e¢00.0 000.0 0m0.0 «050.0l «Nav.0l 000.H 000.0 emam.0l «mmb.0 «mam.0 m «mah.0 emum.0 mmm.0i 0m~.0l «Hmm.0i 5mm.0i 000.0 000.H «m0m.0 th.0 ema0.0i v nma.0l mm0.0i mmH.0i Hma.0l 000.0 0HH.0 emam.0l tm0m.0 000.a «H00.0l «www.ci m e0m0.0 conv.0 «00.0 000.0 «enm.0l Nvm.0i «mmb.0 hmm.0 ed00.0l 000.H v0a.0 m m00.0 mm0.0 Hem.0 00m.0 hmH.0i mHH.0i cmam.0 emm0.0i emmm.0l 00H.0 000.H H Ha 0H m m h o m v m N a .ooHSmmoz mumumemumm cw>mHm Mom mcoflumamunooumucH mo xwuumz coaumauuuouli.al0 mqmce xHDzmmmAw 58 HIHHIHIIIHIIIH 1202 ”HI”! 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