INVESTIGATIONS ON THE ROLE OF DISSOLVED ORGANIC MATTER IN DETERMINING ECOSYSTEM STRUCTURE AND FUNCTION: THE PLANKTON AND PHOTOHETEROTROPHY Dissertation for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY KELTON R. McKINLEY 1975 A‘— A... A.-.“ -_.. LI 31>: f": ’ when Stage: II Umenéty f"; c A__.. This is to certify that the thesis entitled Investigations on the Role of Dissolved Organic Matter in Determining Ecosystem Structure and Function: The Plankton and Photoheterotrophy presented by Kelton Ray McKinley has been accepted towards fulfillment of the requirements for Ph 0 D 0 degree in Botanx 94.44de Major profesg Date 22- WIT-,5 0-7639 fir? BINbENG BY I?” II HUAB & sans IBUUK amuspv ”IE-I I! 3.8.335?" ' ' ‘ ABSTRACT INVESTIGATIONS ON THE ROLE OF DISSOLVED ORGANIC MATTER IN DETERMINING ECOSYSTEM STRUCTURE AND FUNCTION: THE PLANKTON AND PHOTOHETEROTROPHY BY Kelton R. McKinley Within the broader context of the cycling of dis- solved organic materials, this study examines the occur- rence of the phenomenon of photoheterotrophy, the light— mediated assimilation of organic compounds at or near natural substrate concentrations, in the phytoplankton of lake systems. The pelagic zone of Lawrence Lake, an oligotrophic, dimictic, temperate, hard-water lake in southwestern Michigan, was selected as the study site. Extensive infor- mation is already available on Lawrence Lake, the result of intensive study for a number of years. The uptake of an organic compound, glucose, and photolithotrophic carbon fixation were monitored simultaneously. Light and dark bottle uptake of organic and inorganic carbon was measured throughout the annual period during three sampling periods throughout the daylight hours and at three depths within the water column. Kelton R. McKinley The study revealed that light bottle uptake of organic material was significantly greater than dark bottle uptake on the average, 9.2 ng m.3 hr.1 vs. 6.3 ugC In"3 hr“l (n=252). Annual averages attributable to photo- heterotrophic uptake and chemoheterotrophic uptake were 2.6 ugC m-3 hr-1 and 6.9 ugC 111-3 hr"1 (n=360) respectively. Photoheterotrophic activity represented 67.6% of chemo- heterotrophic activity on a comparative, annual basis for the daylight period (n=360). The patterns of chemoheterotrophic activity and photoheterotrophic activity were significantly related to the variables of months, depths, and time of day. Chemo- heterotrophic activity generally increased throughout the daylight period and with depth in the water column, with maximal values generally observed during the sunset- incubation series and the lO-meter series. Generally high and uniform activities with reSpect to depth were observed during periods of water circulation. Increasing activity at depth during the stratified summer period was also observed. Maximal values of photoheterotrophic activity were observed during spring circulation and during late summer stratification. Activity was generally greater at depth and during morning and midday incubation periods. There was an apparent shift during the daylight period in the area of maximal uptake from 2 and 6 meters in the morning to 6 and 10 meters as the day progressed. Thus it Kelton R. McKinley appears that chemoheterotrOphy and photoheterotrophy may be both temporally and spatially separated with respect to activity within the water column on a diurnal as well as seasonal basis. Heterotrophic uptake was compared to observed photolithotrophic fixation. Comparisons between the two techniques were difficult because of differing levels of precision. However, it is clear that photoheterotrophy may contribute significant additional carbon to photosyn- thetic organisms under conditions not favorable to inor— ganic fixation (e.g., at depth and under ice cover). The study revealed that dark bottle chemoheterotrophic esti- mates may lead to serious underestimates of organic cycling, since significant quantities of organic carbon were assimilated in the light. Photoheterotrophy represents a key feedback loop at a trophically significant level and may play an impor- tant determining role in phytoplankton succession and community structure over time. INVESTIGATIONS ON THE ROLE OF DISSOLVED ORGANIC MATTER IN DETERMINING ECOSYSTEM STRUCTURE AND FUNCTION: THE PLANKTON AND PHOTOHETEROTROPHY BY ) gs Kelton R. McKinley A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1975 C) Copyright by KELTON RAY MCKINLEY 1975 DEDICATED to the memory of the late and dear MARTHA PARRY Whose woods these are I think I know. His house is in the village, though; He will not see me stopping here To watch his woods fill up with snow. My little horse must think it queer To stop without a farmhouse near Between the woods and frozen lake The darkest evening of the year. He gives his harness bells a shake To ask if there is some mistake. The only other sound's the sweep Of easy wind and downy flake. The woods are lovely, dark, and deep, But I have promises to keep, And miles to go before I sleep, And miles to go before I sleep. Robert Frost Don Genaro glanced at me with piercing eyes and then turned his head to look into the distance, towards the south. "I will never reach Ixtlan," he said. His voice was firm but soft, almost a murmur. "Yet in my feelings . . . in my feelings sometimes I think I'm just one step from reaching it. Yet I never will. In my journey I don't even find the familiar landmarks I used to know. Nothing is any longer the same . . . ." "I left. And the birds stayed, singing." Carlos Castaneda Journey £9 Ixtlan ii ACKNOWLEDGMENTS The author would like to express his sincere appreciation to Dr. Robert G. Wetzel, W. K. Kellogg Bio- logical Station and Department of Botany and Plant Path- ology, Michigan State University for his unwavering support through the inevitable bad periods as well as the good periods in any graduate career. The breadth of his knowl- edge of aquatic systems and his dedication to Science are a continuing inspiration. Materials and technical assis- tance were always made readily available whenever neces— sary. His valuable criticism in the preparation of this manuscript is particularly noted. Appreciation is also extended to Dr. George H. Lauff, Director, W. K. Kellogg Biological Station, Michigan State University for financial assistance and personal interest during the course of my studies. Of particular importance in my graduate education have been the discussions and exchanges with other gradu- ate students. Sincere appreciation in this regard is expressed first to Dr. Judith S. Warner, and to G. Milton Ward and Amelia K. Ward. Appreciation is also due Dr. R. A. Rough and Gordon L. Godshalk. iii The careful and much needed assistance provided by Janet Strally and Jayashree Sonnad during the course of this investigation is gratefully acknowledged. Thanks are due Dr. Charles E. Cress, Department of_ Crop and Soil Sciences, Michigan State University and particularly Dr. John L. Gill, Department of Dairy Science, Michigan State University for advice and statistical con- sultation. I would also like to express my appreciation to the other members of my graduate committee, Drs. D. J. Hall, Department of Zoology, Michigan State University, Brian Moss, School of Environmental Sciences, University of East Anglia, England, and Dr. M. J. Klug, W. K. Kellogg Biological Station and Department of Microbiology and Public Health. Interactions with them, both during formal course offerings and in personal discussions, have been very valuable in the formulation of concepts. Use of the Michigan State University computer facilities was made possible through support, in part, from the National Science Foundation. These investigations were supported, in part, by the National Science Foundation Grant #GB-40172 to R. G. Wetzel and K. R. McKinley and the Energy Resources and Development Agency Contract E(ll-l)-1599, C00-lS99-95 to R. G. Wetzel. Also, support was provided by National Science Foundation Grants #GB-15665 and #GB-31018X to iv G. H. Lauff, et al. (Coherent Areas Program for Investi- gation of Freshwater Ecosystems). I would also like to express my love for my wife, Linda. We have come a very long way together. TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . viii LIST OF FIGURES. . . . . . . . . . . . . ix I O IIqTRODUCT ION O O O O C O O O O O I O l A. General Introduction and Historical Considerations. . . . . . . . l B. Dissolved Organic Matter - Distribution and Sources . . . . . . . . . . 4 C. Dissolved Organic Matter - Nature and Action I O O O O O O O O O 9 D. Photoheterotrophy. . . . . . . . . 15 II. PURPOSE OF THE INVESTIGATION . . . . . . 24 III. SITE FOR THE STUDY. . . . . . . . . . 26 IV. SAMPLING DESIGN. . . . . . . . . . . 35 V 0 METHODS C O O O O O I O O O O O O 3 7 VI. ASSUMPTIONS, CALCULATIONS, AND STATISTICAL ANALYSIS 0 O O O O O O O O O O O O 4 4 A. Organic Uptake. . . . . . . . . 46 B. Photoheterotrophic Uptake . . . . . . 47 C. ChemoheterotrOphic Uptake . . . . . . 48 D. Percent Comparison . . . . . . . . 49 E. Mean Values and Annual Means . . . . . 49 F. Inorganic Fixation . . . . . . . . 50 VII. RESULTS AND DISCUSSION . . . . . . . . 52 A. Heterotrophic Activity . . . . . . . 52 1. Bottles x Months x Times . . . . . 56 2. Bottles x Months x Depths. . . . . 61 3. Bottles x Times x Depths . . . . 65 4. Photoheterotrophy and Chemohetero- trophy . . . . . . . . . . . 68 B. Photosynthesis. . . . . . . . . . 76 vi VIII. IX. PERSPECTIVES AND INTEGRATION SUMMARY . . . APPENDICES Appendix A Organic Carbon Uptake Values Appendix B Inorganic Carbon Uptake Values. REFERENCES CITED vii Page 86 94 96 107 118 LI ST OF TABLES Table Page 1. Analysis of Variance table for photohetero- trOphic uptake of glucose. Three-way factorial split plot design. Significance levels are indicated by asterisks: (***) significant at the 0.1% level, or better; (**) significant at the 1% level; (*) significant at the 5% level; (n.s.) not significant at the 5% level, or better . . 54 2. Analysis of Variance table for inorganic carbon fixation estimated by the l4C-method. Three-way factorial split plot design. Significance levels are indicated by asterisks: (***) significant at the 0.1% level, or better . . . . . . . . . 80 viii LI ST OF FIGURES Figure Page 1. Morphometric map of Lawrence Lake, Barry County, Michigan. Contour lines in one meter intervals. Central sampling station indicated at point A . . . . . . . . 28 2. ISOpIeths of temperature distribution (°C) in Lawrence Lake, 1974 . . . . . . . . 3O 3. Isopleths of oxygen concentrations (mg 1.1) in Lawrence Lake, 1974 . . . . . . . . 33 4. Three-way interaction plots (BOTTLES x MONTHS X TIMES). Graphs represent Light and Dark responses in ugC m' hr'1 for all incubation periods across months as SR, sunrise ( ); MD, midday (----); and SS, sunset (°--°). Contrasts between Dark and Light bottles for each incubation period are also given. Sheffé S values for each series of graphs are indicated by vertical bars. The greater of the two, the difference for significance at the 1% level; the lesser, at the 5% level. Each point represents a mean of 12 samples. 58 5. Three-way interaction plots (BOTTLES x MONTHS X DEPTHS). Graphs represent Light and Dark responses in ugC m‘ hr‘l for all depths across months, as 2 meters ( ), 6 meters (----), and 10 meters (----). Contrasts between Dark and Light bottles for each depth interval are also given. Sheffé S values are indicated by vertical bars. Each point represents a mean of 12 samples . . . . 63 ix Figure Page 6. Three-way interaction plots (BOTTLES X TIMES X DEPTHS). Graphs represent Light and Dark responses in ugC m for all depths across incubation periods, as 2 meters ( ), 6 meters (----), and 10 meters (°°°'). Contrasts between Dark and Light bottles for each depth interval are also given. Sheffé S values are indicated by vertical bars. Each point represents a mean of 28 samples . . . . . . . . . 67 7. Estimated values for photoheterotroghic1 uptake of glucose during 1974 as ugC m Histograms represent the means of four replicate samples. Uptake values for each depth interval (2, 6, 10 meters) and each incubation period (SR, MD, 88) are indicated for each month. Bars denote plus or minus standard error (:SE) about the mean. Negative mean values are indicated by stippling . . . . . . . . . . . . 70 8. Estimated values for chemoheterotro ic u take of glucose during 1974 as ugC m‘ Histograms represent the means of four replicate samples. Uptake values for each depth interval (2, 6, 10 meters) and each incubation period (SR, MD, SS) are indicated for each month. Bars denote plus or minus standard error (:SE) about the mean . . . 73 9. Percent comparisons between photoheterotrophic and chemoheterotrophic uptake values during 1974. Histograms represent the means of four replicate samples. Percent comparisons at each depth interval (2, 6, 10 meters) and each incubation period (SR, MD, SS) are indicated for each month. Bars denote plus or minus standard error (+SE) about the mean. Negative mean values are Indicated by stippling . . . . . . . . . . . . 75 10. Annual mean values for chemoheterotrOphic uptake, photoheterotrophic uptake, and percent comparison values ((Photo/Chemo) *10 .0). HeterotrOphic uptake as ugC m‘ hr’ . Error bars give 99% confidence limits about the mean (n=360) . . . . . 78 Figure Page 11. Estimated values for inorganic carbon fixation during 1974 as mgC m'3 hr‘l. Histograms represent the means of four replicate samples. Uptake values for each depth interval (2, 6, 10 meters) and each incubation period (SR, MD, SS) are indicated. Bars denote plus or minus standard errors (:SE) about the mean . . . . . . . . 82 12. Idealized trophic scheme emphasizing the cycling of organic carbon. Dissolved organic carbon (DOC), dead particulate organic carbon (POC). Major pathways indicated by arrows. 90 xi INTRODUCTION General Introduction and Historical Considerations Dissolved organic matter (DOM) has received con- siderable attention in recent years and much effort by many individuals has led to information concerning the sources, cycling, and measurement of DOM in natural waters. However, while we do know a great deal about DOM, we have yet to understand its roles as they relate to the orga- nisms of the freshwater community. There has been much speculation and investigation in an attempt to elucidate these functional roles. As early as 1885 various workers (Pearcey, 1885) reported mutually antagonistic relationships between various members of freshwater and marine communities. This resulted in a fairly extensive literature concerning the possible role of non-predatory relationships in the sea (e.g., Bigelow, 1931; Russell, 1936; Herdman, 1924, as cited by Lucas, 1947). Johnstone, Scott, and Chadwick (1924) were among the first to suggest that plankton com- munities somehow influence one another via a large scale group symbiosis, so that the plankton present in one area of the sea must depend, in part, on the type of plankton which preceded it in time. As noted by Lucas (1947), their suggestions seemed to be the direct result of earlier statements by Brandt (1898) and Nathansohn (1909). In 1931, Akehurst proposed his famous scheme of "starch and oil" groups in the phytoplankton, which is now only of historical interest. Working out an elaborate and detailed theory of the seasonal succession of algal types, he proposed that the phytoplankton comprised two distinct groups, which he distinguished on the basis of metabolic storage products (i.e., starch and oil). He further proposed that each population produced a toxin inhibitory to its own members, but at the same time stimulatory to members of the other metabolic group. Con- temporaries of Akehurst began emphasizing the importance of non-predatory interactions on both an ecological and an evolutionary scale. Hardy (1935) proposed his well known and often discussed theory of "animal exclusion." Allee (1931, 1934), in a view which included both community and evolutionary considerations, discussed the problems of mass physiology wherein the influence of aquatic organisms in conditioning the medium surrounding them by the addition of secretions and excretions also influenced the actual association of organisms. In this scheme "animal exclusion" appeared to be but an instance of a much more general class of non- predatory relationships dependent upon and related to the production and subsequent accumulation of external organic substances (Lucas, 1947). Perhaps the most vociferous proponent for non- predatory interactions was C. E. Lucas, who examined the phenomena of the influence of organism upon organism through the release of extracellular materials in a series of extensive reviews and provocative papers (1936, 1938, 1944, 1947, 1949, 1955, 1961). He coined the term "ectocrine substances," based in part upon the considerations of Huxley (1935) and as a direct analogy to the endocrine system and hormones, for that group of substances mediating ecological relationships by non-predatory means (Lucas, 1947). His examples of relationships mediated by "ecto- crine substances" were drawn from almost all areas of science ranging from the close association of many insects and plants and the proposed role of nectar and scent, to animal phermones, and to the simple observation that oxygen was at one time merely a metabolic by-product on which a large number of important interactions are now based. Lucas accurately observed that while an important part of the study of antibiotics and microbiology is specifically concerned with extracellular products and the interaction of organisms via those extracellular products, little attention is paid to the occurrence of those inter- actions in nature. As McIlwain (1944) and Waksman (1945) pointed out, microorganisms are in particularly intimate contact during their growth in common media and are found to exhibit mutual interactions to a high degree, both in the sense of symbiosis and antibiosis. There is no reason to suspect that this is not the case in nature. To the contrary, this is probably good evidence to support the claim that such interactions play an important role in the environment (Pan and Umbreit, 1972). Since that time a number of excellent, extensive reviews concerning the nature of dissolved organic matter and the roles which extracellular products are believed to play have been published (Fogg, 1962, 1966, 1971; Hellebust, 1974; Provasoli, 1958, 1963; Saunders, 1957). , No attempt will be made to review this literature concern- ing DOM and extracellular products. However, since some treatment of the subject is in order, only that material of particular significance, or of more recent publication will be discussed. Dissolved Organic Matter - Distribution and Sources Some of the first attempts at the quantification of dissolved organic matter in lakes were performed by Birge and Juday (1926) during their survey of Wisconsin lakes, 1911 to 1917. They found that the concentration of dissolved organic carbon (DOC) in 13 Wisconsin lakes ranged from 4.00 to 13.22 mg DOC 1.1 with a mean value of 6.23 mg DOC 1-l (n=28). In their work on two Wisconsin rivers the range was from 9.58 to 15.23 mg DOC 1-1. The average concentration in seawater is approximately 2 mg DOC 1'1 with a maximum of 20 mg 1-1 (Provasoli, 1963). There are many sources of DOM (Saunders, 1957; see also the review by Hellebust, 1974), but in the oceans the major source is undoubtedly due to the secretions or lysis of the plankton, particularly the phytoplankton (Provasoli, 1963). This is probably not true of most bodies of fresh- water, however. Thomas (1971) found the release of DOM by phyto- -l plankton to range from 0.11 mg C m-3 hr in the Continental Shelf waters to 1-2 mg C m-3 hr-1 in the estuarine waters. There was a general seaward trend of decreasing productivity and the quantity of DOM released, but an increasing per- centage release of fixed carbon as DOM in a seaward pro- gression. Values for percentages of photoassimilated carbon released as DOM ranged from < 7% in estuarine waters and < 11.6% in Continental Shelf waters to < 44% in the western-most Sargasso Sea. Extracellular release of dissolved organic materials approximated 1-20% of the total carbon fixed in the tropical coastal waters off India (Samuel, Shah, and Fogg, 1971). In general the quantity of excreted organic matter seems to be proportional to photosynthetic carbon fixation over a wide range, increasing markedly under conditions of light inhibition, low light, or near the end of a bloom condition (Fogg, Nalewajko, and Watt, 1965; Hellebust, 1965; Ignatiades and Fogg, 1973). In the near shore and estuarine areas a signifi- cant contribution to the DOM pool may be made by the macrophytic vegetation. Sieburth (1969; Sieburth and Jensen, 1968) demonstrated a release of carbon in organic form from 4.4 mg c 100g’1 hr"1 1 -l for Chondrus to 54.2 mg C 1009- hr for fruiting Ascophyllum. A carbon balance for Fugue during spring conditions indicates that approxi- mately 30% of the total carbon, or 40% of the net carbon fixed daily is exuded by the plant. EEEEé beds, which can exceed a density of 10009 C m-2 and fix approximately 16.5g C m—2 day-l, are capable of the release of extra— cellular organic material equivalent to 5-7g C m-2 day-l. Khailov and Burlakova (1969) in their study of DOM release from 18 species of macrophytes from the Barents Sea and Black Sea regions found similar rates of release. In the Barents Sea macrophytes release rates for different species ranged from 0.9 to 2.9 mg organic matter per gram 1 dry weight of plant per hour (mg g- hr-l) in March to 1.7 l to 9.8 mg g- hr.1 in June. The release rates for the species of the Black Sea area ranged from 0.5 to 1.6 mg g“1 hr.1 in slowly growing plants to 1.25 to 6.1 mg g-1 hr.1 in fast growing plants. They calculated the quantity of total DOM released on a yearly basis as a percentage of gross production to be 39% for brown algae, 38% for red algae, and 23% for green algae. With these estimates and the consideration that approximately 30% of gross production may be released as DOM through decomposition, the remainder being consumed by herbivores, they further estimated that as much as 70% of gross production may be released as DOM. The picture in freshwater is complex, but it has been studied in some detail. In Lawrence Lake, a small hard-water lake in southwestern Michigan, the concentration of the DOM pool varies from 1.5 to 9.6 mg C l-1 on a yearly basis with a mean of 5.6 mg C l"1 for all depths and sam- pling periods (Wetzel, et 31., 1972). A maximum quantity of DOC generally occurs in September and October prior to overturn. The in situ_measurement of the secretion of dis- solved organic compounds by phytoplankton has been followed for nearly five years (Miller, 1972; Wetzel, unpublished). The rates of algal release of extracellular products during photosynthesis in Lawrence Lake ranged from 0.0 to 22.5 mg C m-2 day-1 with a mean of 7.3 mg C m"2 day-1. The maximum observed rates never exceeded 3.8 mg C m—2 day—1 in the epilimnion. The annual mean percentage secretion of phytoplanktonic primary production was 5.7%. A higher percentage of secretion occurred at lower depths. Expressed as the mean percentage secretion of all dates and samples, 23.5% of the phyt0p1anktonic particulate production was secreted, an annual average determination which includes all depths. The release of dissolved organic matter by sub- mersed macrophytes has been studied extensively in axenic cultures (Wetzel, 1969a, 1969b; Allen, 1971b; Wetzel and Manny, 1972b; Hough and Wetzel, 1972, 1975). The rates of secretion of DOC by both submersed and floating-leaf macrophytes varied from 0.05 to over 100% of photosyn- thetically fixed carbon. The rate of release was dependent upon a number of environmental variables including light and ionic composition of the medium (Hough and Wetzel, 1972; Wetzel, 1969a, 1969b). In situ analysis of secretion rates by Najas flexilis in Lawrence Lake ranged from 1-3% of photosynthetically fixed carbon during the day-light period (Miller, 1972). Nearly a two-fold (2X) increase in percentage of secretion rates was found in the dark (Hough and Wetzel, 1972). A significant portion of the DOC entering Lawrence Lake is allochthonous, approximately 20.959 C mm2 year-l (Wetzel, et 31., 1972). However, these materials, largely terrestrial, humic compounds, are highly refractory bio- logically and as such not subject to rapid bacterial degradation (see also Wetzel and Manny, 1972a, 1972b; Wetzel and Otsuki, 1973). These studies coupled with work on the decom— position rates of DOM in both marine and freshwaters (Wetzel and Manny, 1972a; Ogura, 1972) and on the anaerobic and aerobic decomposition of algal cells (Otsuki and Hanya, 1972a, 1972b) have resulted in a fairly complete knowledge of the material transport of DOC (see especially Wetzel, 33 31., 1972, for freshwaters). Dissolved Organic Matter - Nature and Action Knowledge of the nature of the DOM and its mode of action is more limited. The qualitative composition of the DOM varies considerably in both time and space. Some attempts have been made to clarify that composition and an extensive literature has developed. Much of the work has been done with isolates of extracellular products from various algal cultures (e.g., Berland, 33 31., 1972; Myklestad and Haug, 1972; Hellebust, 1965; Otsuki and Hanya, 1972a, 1972b; Nalewajko and Lean, 1972; Kroes, 1971, 1972; Fogg and Watt, 1965; Sieburth, 1969; Sieburth and Jensen, 1968, 1969). Work has also been done in both salt and freshwaters (e.g., Birge and Juday, 1926; Carlucci and Bowes, 1972; Clark, Jackson and North, 1972; Ohwada and Taga, 1972). The composition of DOM has been approached by a number of different means, including examination and classification by various extractive chemical techniques (e.g., ether extract, chloroform soluble, steam volatile, yellow water soluble pigments), or by functional group (e.g., amino acids, peptides, proteins, carbohydrates, 10 lipids, fatty acids, organic acids, aldehydes, ketones). Some isolation and characterization of specific compounds have been performed (e.g., glycolate, mannitol, glycerol, proline, a number of vitamins, enzymes, some sexual sub- stances, and hormones). Often, particularly with vitamins, the concentrations of specific compounds were followed through time using bioassay techniques. The most compre- hensive, but dated, review of this entire subject was written by Vallentyne (1957) (see also Provasoli, 1963). Hellebust (1974) has written the most recent review of extracellular products. The functions which DOM is believed to perform in nature were summarized by Saunders (1957) under the following four topics: (1) as an energy source, or pro- viding essential, basic elements for the synthesis of cellular materials; (2) as accessory growth factors either essential to the growth of the organism, or stimulatory to the growth of the organism; included here could also be the various enzymes and sexual substances which, while often not directly linked with the growth of the organism, may be indirectly linked to that process and the propagation of the species; (3) as a toxic substance, including both auto- and heteroantibiosis; and (4) as an organic complex with various trace elements, chelation, which may produce either a beneficial or a detrimental effect depending upon the element and the nature of the chelatory binding. Much ll evidence for the above processes is offered both in Saunder's review (1957) and in the other general reviews mentioned earlier. More recent work has generally tended to support these proposed roles, for example: chelation (Kroes, 1972; Moebus, 1972; Wetzel, 1965, 1971); accessory growth factors (Provasoli, 1969; Carlucci and Bowes, 1970); and antibiosis (Moebus, 1972; Berland, 31 31., 1972; Fitzgerald, 1969; Kroes, 1972). An apparent contradiction is evident in studies concerning organic materials as an energy source. 'Much of the work in all categories has been carried out in pure or axenic cultures with artificially high concentrations of organic substrates, concentrations which would virtually never be encountered in the environment. This has been particularly true concerning organic materials as energy sources. Therefore, while a number of species were shown in culture to be capable of either heterotrophic growth, or the utilization of organic substrates, it appeared that this potential could not be realized in nature. In a series of experiments, primarily the work of Wright and Hobbie (1965; Hobbie and Wright, 1965a, 1965b; Hobbie, 1969; Allen, 1969a, 1971b; Wetzel, 1967, 1968; Parsons and Strickland, 1962), it was demonstrated that the kinetics of uptake for planktonic algal species followed zero order principles (diffusion kinetics), while bacteria were able to actively transport organic materials across membranes 12 (first order kinetics) and simply out-compete the algae at natural substrate concentrations. It was further demon- strated (e.g., with the marine pennate diatom Cocconeis diminuta; Cooksey, 1972) that the uptake of organic sub— strates by algae was not energy dependent (i.e., again diffusion mechanisms were shown to be operative). This was particularly true for the green algae on which many of the studies were performed (N. B. Wright and Hobbie's classic work (1966) is based on work with a single species of Chlamydomonas sp.). However, it should be noted that the green algae are especially suited for culture work because of the relative ease of their prOpagation on defined, synthetic media. Those species which require more complex or exotic media (e.g., soil extracts, and other less clearly defined mixtures) simply cannot be as easily maintained. Many species, particularly in the ChrySOphyta, Cyanophyta and Pyrrhophyta, cannot be isolated and main- tained at all with the methods presently employed. Within this context it is important to note the heterotrophic utilization of organic compounds by cryptomonad species demonstrated by Wright in 1964. Work notably by Allen (1971a) and Saunders (1972), has indicated that the uptake or organic substrates by ‘various algal species is possible at near natural substrate concentration levels (see also Bennett and Hobbie, 1972). .Allen's (1971a) work (with some substantiation in a similar 13 approach by Remsen, Carpenter, and Schroeder, 1972; and more recently P. A. Wheeler, University of California, Irvine, personal communication) consisted of a size fractionation of a plankton sample after exposure to 14C- organic compounds by filtration through a series of nine membrane filters ranging in porosity from 14.0 pm to 0.22 pm. The reduction in the maximum velocity of active trans- port following the size fractionation demonstrated that organisms between 3 um and 8 um were responsible for the majority of the active uptake of glucose and acetate and that organisms of less than 1.2 pm (i.e., bacterial size- categories) were responsible for only a minor portion of the substrate uptake. An examination of the control sample revealed that the algal organisms were predominately micro- flagellates in the size range of 4 pm to 8 pm. Few bacteria were observed. Those algae which were apparently responsible for the active substrate uptake are those organisms which are often overlooked (see for example Horner and Alexander, 1972) and are generally not, or not easily, maintained in culture collections. While there is some confounding associated with Allen's technique (e.g., particles in the 3 pm to 8 pm range to which bacteria would be expected to be attached) some limited autoradiog- raphy by Allen supports his conclusions.1 1It should be pointed out that these statements con— cerning the importance of dissolved organic materials do not supplant the work concerning physical and abiotic 14 Within this broad context the particular subject to be addressed in this work will be the utilization of organic materials as energy sources. In later works, aspects of stimulation-inhibition interactions and a theoretical overview will be addressed (McKinley, in prep.; McKinley and Wetzel, in prep.). The interesting and provocative papers by Ingram 33 31. (1973a, 1973b) suggested that the key to under- standing the importance of algal heterotrophy might lie in the interplay concerning the presence or absence of light. chemical factors and the productivity of the phytoplankton. Much informative work has been done in the past and is cur— rently being performed (e.g., see Moss, 1972 and also the work on the importance of pH by Kroes (1971, 1972) and O'Brien and deNoyelles (1972), but see also the discussion by Proctor (1957)). However, after reviewing the subject Hutchinson (1967) concluded that, while there was good correlative evidence between the physical and abiotic chem- ical factors and the phytoplankton, those factors alone could not account for the observed algal associations, pro- ductivity, and variations through time. In order to more fully understand the total picture of algal associations and productivity, the abiotic material must be coupled with the elucidation of the role of dissolved organic substances. A major contributing factor to the paucity of insight into the functional interactions of DOM has been the failure both in the past and currently (e.g., see the discussion by Kroes, 1972) to recognize that the interactions mediated via DOM are generally likely to be subtle. The ecological impact of red tide for example, is rather spectacular, but very rare. However, examination of competition equations (see Hutchinson's discussion, 1967) reveals that for orga- nisms with as short a generation time as the plankton, subtle differences, of which these organic substances are certainly capable, can make substantial differences in com- petitive interactions and consequently in community struc- ture in relatively few generations. This may seem obvious, but the technology and the techniques of the necessary sen— sitivity to detect those differences in nature and on a species-specific basis, as these interactions are likely to be (Lucas, 1947; Pan and Umbreit, 1972), have been lacking (Wetzel and Allen, 1972). 15 Indeed, by examining a number of papers already cited it appeared that often the difference between those observing heterotrophy, or not observing heterotrophy, revolved around whether or not the tests were conducted in the light, or in the dark. The light mediated uptake or organic compounds at, or near natural substrate concentrations by photosynthetic organisms has been termed photoheterotrophy. Although this subject has received considerable work and a signifi— cant resultant literature has accumulated over the years, little work has been done concerning its potential eco- logical role. PhotoheterotroPhy In 1928 Bristol Roach noted that a strain of soil alga, Scenedesmus costulatus, was able to accumulate cell carbon at low light intensities by a combination of photo- lithotrophic and photoheterotrophic pathways. Since that time much discussion and experimentation has occurred con- cerning algal heterotrophy. Several recent and excellent reviews are now available on this topic (notably Droop, 1974; Neilson and Lewin, 1974; and earlier, Danforth, 1962). It has generally been conceded that true chemo- heterotrOphic utilization (i.e., utilization in the dark) of organic compounds is in large part dominated by bac— terial forms. This is particularly true, because of the 16 relatively rare occurrence of chemoheterotrophic algal forms, the often artificially high concentrations of organics necessary for sustained dark growth, and since the appearance of the papers by Wright and Hobbie (1965, 1966) and Hobbie and Wright (1965a, 1965b; see also Sloan and Strickland, 1966; Munro and Brock, 1968); the concepts having gained nearly universal acceptance. However, while it is now clear that chemohetero- trOphy probably represents a bacterial specilization, it is not clear that algal heterotrophy 235 §3_must be com- pletely ruled out. Although little direct work has been done concerning the photoheterotrophic assimilation of organics since Bristol Roach's work with Scenedesmus, it is now apparent that a number of algae are capable of utilizing organic compounds at, or near, natural sub— strate concentrations in the light. A number of algal types from a variety of different taxa have shown this ability (see Droop, 1974). A brief examination of the pertinent ecological literature also reveals that those persons observing "algal uptake" of organic compounds at natural substrate concentrations have generally run their experiments in the light (e.g., see Ingram 3£_31., 1973a, 1973b; Pintner and Provasoli, 1968; Sheath and Hellebust, 1974; Lylis and Trainor, 1973; Bunt, 1969; Eppley and MaciasR, 1963). 17 While evidence for photoheterotrophy has accumu- lated, little has been done with this information and photoassimilation has generally only been viewed in terms of a laboratory phenomenon. A brief review of what is known of the process is instructive. It should be pointed out that portions of this discussion are in large part based upon more extensively studied pathways in bacteria and higher plants. However, as Neilson and Lewin (1974) point out in their more extensive review, algal biochemical pathways have generally not been shown to be truly unique and in general follow closely those of higher plants and other organisms. A contrast may be made between those organisms which are capable of growth at the expense of organic com- pounds in the dark (i.e., chemoheterotrophs) and those organisms which are able to grow by utilizing organic com- pounds in the light (i.e., photoheterotrophs). There is not necessarily a good correlation between the two pro- cesses (Stanier, 1973). The majority of photosynthetic organisms capable of organic utilization must be considered to be facultative chemoheterotrophs, or facultative photo- heterotrophs, since CO2 generally remains the predominant source of cellular carbon. Chemoheterotrophic and photoheterotrophic assimi- lation and metabolism of organic compounds may be discussed in terms of the utilization of three compounds and their 18 respective families of related compounds: (1) glucose, (2) acetate, and (3) glycolate. These substances probably represent major, lower molecular weight compounds avail- able within the environment. Little work has been com- pleted on other compounds. Glucose is the best understood of these compounds. Only two pathways for the dissimilation of glucose appear to be operational in algae: (l) the Embden-Meyerhof— Parnas (EMP) pathway and the pentose-phosphate pathway (Neilson and Lewin, 1974). Under aerobic conditions the pyruvate generated by the EMP pathway enters the tricar- boxylic acid (TCA) cycle where it is oxidized to carbon dioxide. The second pathway, and for blue-green algae the major pathway, for glucose utilization is the pentose- phosphate pathway. Here the initial product is glucose-6- phosphate which is dehydrogenated and oxidatively decar- boxylated to carbon dioxide and ribose phosphate. Under aerobic conditions ribose phosphate may be further oxidized to C02. The regulation of glucose metabolism and photo- assimilation is not well understood in algae. It has been most extensively studied with blue-green algae. Pelroy 33 31. (1972) and others (see also Pearce and Carr, 1969) have shown that the synthesis of glucose-6-phosphate dehydrogenase was specifically inhibited by ribulose-1,5- diphosphate generated during carbon fixation in the light 19 via the Benson-Calvin cycle, thus suppressing the pentose- phosphate pathway. Under these conditions exogenous glu- cose is then assimilated almost entirely as polysaccharide (Neilson and Lewin, 1974). This suppression is reversed by the inhibition of photosystem II through use of DCMU (Stanier, 1973). Pelroy 33 31. (1972) suggest that some type of constituitive permease which mediates glucose uptake by Aphanocapsa would account for the relatively high substrate affinities observed. Ohki and Katoh (1975) have some evi— dence for the operation of a sodium pump in the transport of glucose by having observed accelerated organotrophic growth upon the addition of sodium chloride. Thus chemo- heterotrophic utilization in the dark is dependent upon the ATP generated through the pentose-phosphate pathway. However, where cyclic photophosphorylation is operational, adequate ATP may be generated for glucose transport. (See also Neilson and Lewin, 1974 and Tanner, Grfines, and Kandler, 1970, for a discussion of transport of hexose in green algae). A coupling with light, and of particular interest concerning the distribution of light availability at depth within lakes, is also shown by a shift in the absorption peaks of pigments. The absorption spectra of organo- trOphically cultured cells of Anabaena variabilis show a marked reduction in the red range 600-700 nm, but little 20 reduction in the blue range 400-500 nm (Ohki and Katoh, 1975). Greatest overall relative changes in pigment absorption were also noted when cultures of organotroph- ically grown Chlorella vulgaris were illuminated with mono- chromatic light at a wavelength of 450 nm (Karlander and Krauss, 1966). The effect of light was additionally revealed by Ohki and Katoh (1975), who observed increasing growth rates for both lithotrophically and organotrOphically grown cells with increasing light intensity to a maximum of 15 hours per doubling. The maximum values were attained at 2.2 mw cm.2 for organotrophic growth and at the higher intensity of 3.5 mw cm.2 for lithotrophic growth. Addi- tionally they observed low, but significant rates of organo- trophic growth under conditions of light limitation where no discernible lithotrophic growth could be observed. Much work has been completed with acetate, also. Acetate is generally oxidized through intermediates of the TCA cycle. In order to provide carbon skeletons for bio- synthesis, acetate must be cycled through the glyoxylate pathway. In Chlamygobotrys and Chlamydomonas cells are apparently dependent upon photosystem II for reducing power under anaerobic conditions. Droop (1974) interprets this to indicate an O2 dependence for the re-oxidation of NADH2 irrespective of the source of ATP generation (i.e., either through metabolic pathways or from cyclic 19 MC unf 21 photophosphorylation). He further points out the close association between oxidative and photosynthetic assimi- lation of acetate by noting that photoassimilation is associated with high activity of the glyoxylate cycle and that a reduction in the enzyme activity of the carbon reduction cycle has been observed during the photohetero- trOphic growth of several species. However, the photo- heterotrophic uptake of acetate by Chlorella pyrenoidosa, Euglena gracilis, and Anacystis nidulans is apparently different in that they are dependent upon non-cyclic photophosphorylation. In Chlamydobotrys stellata and Chlamydomonas mundana, species which apparently photoassimilate acetate directly utilizing energy derived from cyclic photophosphorylation, Wiessner (1969) has shown a shift in photosynthetic pigment spectra during photoheterotroPhic growth. This shift is related to an increase in the chlorophyll proteins associ- ated with photosystem I with a maxima near 695 nm and an apparent decrease in the 655 to 675 nm range associated with photosystem II. (See the discussion on the composi- tion of the two pigment systems by Govindjee and Braun, 1974.) Glycolate, while not generally a normal product of photosynthesis, may be formed in abundance under conditions unfavorable for inorganic carbon fixation and favorable for photorespiration (i.e., low C02, high 02, and light). 22 Under these conditions glycolate may represent the major excretory product (Tolbert, 1974). Because of this fact, glycolate has received considerable attention. Glycolate has not been shown to support heterotrophic (i.e., dark) growth of any alga (Neilson and Lewin, 1974). It has been shown to be utilized by a number of species in the light (K. G. Sellner, Dalhousie University, Halifax, personal communication, Thalassiosira; see also Palmer and Star, 1971, Pandorina; Miller, Chang, and Colman, 1971, and Lex, Silvester, and Stewart, 1972, blue-green algae; Nalewajko, Chowdhuri, and Fogg, 1963, Chlorella; and others). Glyco- late is metabolized first by an oxidation to glyoxylate and then in blue-green algae to malate and for several types of green algae to glycine, serine, hydroxypyruvate and glycerate (Neilson and Lewin, 1974). Thus in summary, what is implicated is a system of active transport for glucose involving light generated ATP and cyclic photophosphorylation. The metabolism of glucose, but not necessarily the uptake, may be regulated by the products of photosystem II and the Calvin cycle. The system apparently operates at light intensities below that for inorganic carbon fixation and may involve pig- ments in the blue range, a range of wavelengths which most often dominates at depth within aquatic systems. Other compounds which may also fit this general pattern would 23 include fructose, galactose and a few dissacharides (Ohki and Katoh, 1975; Stanier, 1973). The metabolism and transport of acetate and glyco— late are less well understood. Certain species of algae, which may utilize acetate directly, follow a pattern similar to that given above. Shifts in pigment maxima are observed and energy derived from cyclic photophos- phorylation is used in conjunction with photoassimilation. Other species, some converting acetate to carbon dioxide before utilization, follow different patterns. PURPOSE OF THE INVESTIGATION Given this brief background concerning the phenomenon of photoheterotrophy, it is necessary that that phenomenon be placed within some frame of reference with regard to the cycling of carbon and the dynamics of lake systems. An attempt was made in this study to examine whether, or not photoheterotrophic utilization of organic compounds could represent a significant pathway in the cycling of carbon and second whether it has any potential for further study in the elucidation of the spatial and temporal patterns of plankton within lake systems. With this objective in mind, the conditions of incubation, the area selected for study and the methods employed were all dictated by the attempt to achieve sensitive, short term measures with as little change as possible from natural systems. Until the importance of photoheterotrophy was demonstrated within an ecological context, work on the elucidation of its role in species specific responses, or maximum potentials through use of metabolic inhibitors could not be justified. A number of those species previously discussed as having demonstrated the greatest potential for 24 25 photoheterotrophic utilization are those species generally associated with a substrate; either soil algae, epiphytic algae on macrophytes, or algae in association with the benthic sediments or other areas where one might expect to find naturally higher concentrations of organic compounds. One would probably expect that the greatest contribution to algal organic carbon metabolism within natural systems will always be predominately associated with those areas. However, the strongest test case would be made by measuring the response of phyt0planktonic species, those not in association with substrates or organic concentration gradients. Certainly an important case may be made for photoheterotrophic cycling of materials in general, if it is shown to be significant where one would least expect it to contribute strongly. I The site selected was therefore based upon ease of experimental manipulation and sampling, since the general mechanisms for photoheterotrophic utilization as proposed to date are basic cellular constituent pathways and probably do not represent any specialization, or radical departure from normal cellular metabolism. SITE FOR THE STUDY Lawrence Lake, a small hardwater lake in south- western Michigan (85° 21' W, 42° 27' N), was selected for the study site. Lawrence Lake has been described in some detail elsewhere (Wetzel 33 31., 1972; Rich, 1970; Allen, 1969b). All samples for this study and for concurrent studies to be referred to throughout this work were taken at the central depression (designated A in the accompanying morphometric map, Figure 1). The total surface area is 5.0 hectares; the maximum depth is 12.6 meters with a mean depth of 5.9 meters. The lake represents a typical temperate, dimictic lake. It experiences periods of temporary meromixis about every fourth year. The lake is strongly stratified through- out the summer period (Figure 2) with maximum temperatures in 1974 of 25°C and minimum temperatures under ice of < 1°C. Complete mixing occurred in 1974 following ice loss in March and continued until stratification began during April. Maximum thermal gradients were achieved during the period July-August at a depth interval between 4 and 8 meters. Disruption of stratification began in September 26 27 .d ucflom um coDMOAUCH :oHumum mcflHmEMm Hmuucmu .mam>umucfl uwumE mco ca nosed usoucoo .cmmAQOHz .wucsoo auumm .oxmq monou3mq mo mmE oeuuoeoan02|u.H ousmfim 28 mmmems. z_ m4<>mukz. mDOFZOu Gun 0% o8 oo. 0 on 8H.”H mmwok Enon _ MW - _ w _ MN mmmemz 249.122 52:8 53% .sm.m..z; Roma mxaj mozmm>><4 EQBN 0.5.0 29 0 gm vnma xmq oocouBmA cw Auov coausnfluumflc ousumuomfimu mo mcuoHQOmHnl.m musmam 30 Owe >OZ FOO omm OD< fia— 15., ZDfi ><_2 mad. $.32 mmm 2.3. ('w) H1d3CI 31 with surface water cooling and an increase in depth of mixing. Autumnal turnover began in November and continued until ice cover was established in December. The oxygen profile (Figure 3) is typical for a lake of moderate to low productivity with maxima under ice and at all depths during spring mixing and a metalimnetic summer maximum in July associated with high values of photosynthetic production. The range of 02 concentration 1 to < 1 mg 1'1. The lake, while was from > 13 mg 1- experiencing reduced oxygan levels at depth during summer stratification did not become axoxic during 1974, although the relatively small volume of water below the 12 meter interval has occasionally had no detectable oxygen during late summer stratification in other years. The pH and alkalinity are typical of hardwater lakes in the region. Because of the buffering capacity of the bicarbonate system little change in pH is observed over the annual period (i.e., a range of 8.0 to 8.2 for the epi- and metalimnetic waters). Only at depth just above the sediments and near the end of summer stratifi- cation do values approach a pH of < 7.6. Alkalinity values ranged generally from 4.2 to 4.4 meq l.1 for the epilimnion and metalimnion during the ice free period. Values increased with depth under the ice (i.e., to 4.8 meq 1-1) and during the summer strati- fication period, approaching 5.0 meq l-1 in late summer. 32 .whma .oxmq mocmuzmq GA A H Ia 05v mcoflumuucoocoo cmmwxo mo mcuoamomHll.m ousmflm Owo >02 #00 dwm 03.4 Vko— 42. 235 N >32 ~54 «.45. mm“. m 26% n o N— ('W) Hld3C] 34 The typical late summer phenomenon of epilimnetic decal- cification was observed in 1974, with a concomitant increase at depth (i.e., a minimum epilimnetic value of < 4.0 meq 1-1, see the discussion by White and Wetzel, 1975). A low value of 3.6 meq 1'.1 during winter directly under the ice was probably associated with ground water intrusion during a period of melting. SAMPLING DESIGN The design selected for the study was a three-way factorial split plot design of the following model: Y = u + ai + Bj + aBij + aByijk + E(ijk)1 + 6m + a6 + Yk + “Yik + Bij im + 86jm + Yékm + aBdijm + ayéikm + BYijm + “Bysijkm + R(ijk)1m + U(ijklm)n where: i = 1 .... a = 7 j = l .... b = 3 k = l .... c = 3 l = l .... s = 4 m = l .... d = 2 n = 504 All factors were fixed with the exception of replicate sampling, which was considered to be random. Monthly samples were taken at fixed intervals at sampling site A throughout 1974. Within the constraints of sampling and the design seven months were utilized for the statistical analysis. Three additional months, differing slightly in sampling procedure, are included in 35 36 annual estimates. No attempt was made to select either cloudy or cloudless days. For each month three incubations were completed during the daylight hours. These fixed sampling periods of approximately equal duration consisted of incubations which were begun at sunrise (SR), incubations which ended at sunset (SS), and midday incubations (MD), the midpoint of which was temporally at the midpoint of the daylight hours between sunrise and sunset. For each month and sampling period, three fixed depths within the water column were selected: 2, 6, and 10 meters respectively. For each month, sampling period and depth, four separate Van Dorn samples were taken. The contents were mixed and paired light and dark bottles filled simultane- ously by alternating between the two bottles during filling. Separate Van Dorn samples were taken in order to better represent the sampling heterogeneity at a single point in space within the water column. In previous experiments it was shown statistically that replicate samples from a single mixed Van Dorn were not significantly different (McKinley, unpublished). The design permits not only an assessment of main effects (i.e., Month, Time of Day, Depth, and Light/Dark Treatment), but also of any interaction terms resulting in non-parallel responses across these effects. (See the discussion in the section on Statistical Analysis.) METHODS The treatment consisted of a simple light/dark contrast, as in 14C-inorganic photosynthesis estimates, here with the addition of tracer quantities of a radio- active organic compound. This method was selected in preference to the use of metabolic inhibitors, because of a desire to maintain the populations in as natural a state as possible during the treatment period. Inhibitors of photosystem II would certainly force potential photo- heterotrophic organisms to utilize organic carbon. However, the contribution that photoassimilated organic carbon might make toward the total cycling of carbon in lake sys- tems would be difficult to assess, since it is undoubtedly the interplay between available light and inorganic carbon sources which determine any role photoheterotrophy might play. It was with this consideration in mind that a tritiated organic compound was selected in preference to l4C-organic compounds despite the greater difficulties in handling and counting. First to assure that the increase in light bottles over that in dark bottles is indeed due to organic fixation, one must minimize any potential 37 38 re-fixation by the algae of inorganic by-products of chemoheterotrophic utilization in the light. Since the utilization of the organic material added was acceptably low (i.e., in one case 8% of the glucose added, and in general less than 2% of the 4-5 ug glucose 1'l added), even if 100% of the material utilized was metabolized and released as 14C02, one would probably not expect great amounts of activity to be observed due to refixation. Nevertheless it was felt that a more reasonable course 14 would be to utilize 3H-glucose rather than C-glucose; the resultant 3H20 would thus be diluted by 106 rather than 102 as with C02. An additional factor considered was the desire to assess the importance of photoheterotrophy to algal nutri- tion as well as overall lake metabolism. Therefore, if 3H-organic compounds were utilized for studying chemo- heterotrophic and photoheterotrophic responses, 14C- bicarbonate could be utilized simultaneously to measure inorganic photosynthesis. This was accomplished following standard 13 3133 light bottle/dark bottle techniques (see Strickland and Parsons, 1972). Glucose was selected from a variety of compounds that could have been used, for a number of practical con- siderations. First, as discussed previously, there is a relatively small family of compounds which have been shown to be utilized photoheterotrophically and glucose is one 39 compound that has been fairly well studied. Second, much work has been completed using glucose and its utilization by both photoheterotrophic and chemoheterotrophic organisms is well established. One other compound which was con- sidered was glycolate. Glycolate has often been considered to be the major excretory product of the phytoplankton. However, it is no longer clear that this is universally the case (Tolbert, 1974). There are certainly wide variations concerning quantities released both in space and time and from species to species (see the discussion by Hellebust, 1974). It is important to note that earlier estimates of excreted glycolate by the Calkins colorimetric test with acidified 2,7-dihydroxynapthalene may have often given overestimates, since the color reaction is not specific and aldehydes, organic materials oxidizable to aldehydes and, of most interest, nitrate interfere with the assay (Tolbert, 1974). It is also important to note the discussion of light quality versus glycolate excretion by Ignatiades and Fogg (1973). The quality of light used in the majority of the work with cultures does not closely resemble the quality of light available at depth in aquatic and marine systems. Studies with Chlorella have shown higher uptake of aspartic acid when cultures were supplemented with blue light. On the other hand, glycolate excretion is apparently enhanced by red and white light, while no detectable amounts of 40 glycolate were observed under illumination by blue light (Becker, D6hler, and Egle, 1968). (See also the review by Voskresenskaya (1972) on the effects of blue light on carbon metabolism.) However, glycolate remains an impor- tant excretory product, especially under conditions favor- able for high rates of photorespiration (i.e., high con- centration of 02, low concentration of C02, and high pH (Tolbert, 1974)). Of critical importance to this experiment was the assessment of photoheterotrophic utilization in relation to chemoheterotrophy. In addition to some problems con- cerning the physical handling of glycolate, there is some confusion concerning its utilization by bacteria. R. T. Wright (Gordon College, Massachusetts, personal communi- cation) has shown high respiration values by bacteria incubated with glycolate, but virtually no growth, or cellular accumulation of radioactive label. He hypoth- esizes that glycolate may represent an energy source rather than a carbon source, and/or a co—factor in the metabolism of other organic carbon skeletons. By adding up to 300 mg 1-1 glucose he was able to show an increase in growth in the presence of glycolate. Until the role of glycolate metabolism in bacterial chemoheterotrophy is better understood, its utilization in the assessment of photoheterotrophic utilization versus chemoheterotrophic utilization must be held in question for mixed populations. 41 1) D-Glucose-2-3H (specific activity, 500mCi mmol- was selected for use because of the relatively stable metabolic carbon site for 3H attachment. The quantity of glucose added for these studies was 4-5 ug glucose liter-1. This concentration of glucose was achieved by dilution of the radioactive substrate without the addition of any non- radioactive carrier. The quantity of material was suf- ficient and not depleted significantly during the short incubation intervals, 2.4 to 3.4 hours. The quantity utilized is also well below those levels normally observed for diffusion mechanisms and is within the range of con- centrations reported for naturally occurring glucose (i.e., from undetectable levels to nearly 200 ug glucose liter-l in sea water (Vaccaro 33 31., 1968; Hicks and Carey, 1968)). The utilization of an organic compound to measure the "heterotrophic potential" of planktonic populations, in much the same manner that tracer quantities of 14C- bicarbonate are used to estimate photosynthetic activity, is not a new idea. Parsons and Strickland (1962) proposed its use and discussed the accompanying problems. It has since been used in that way by Paerl and Goldman (1972) and McKinley (1971, unpublished manuscript). 14C-bicarbonate (@ 4.6 or 5.1 One milliliter of uCi ml-l) was added simultaneous to the addition of one milliliter of 3H-glucose solution to each light/dark pair of bottles (125 m1 Pyrex glass—stoppered bottles). 42 Procedures for isotope utilization followed closely that of Strickland and Parsons (1972). 13.3133 incubations were generally less than 3.4 hours for organic uptake and 3.5 hours for inorganic fixation. Three-hour incubations preclude any conclusions concerning maximal instantaneous rates of fixation, since measures are averaged over a relatively long period of time. Therefore, as will be seen later, while the maximum instantaneous rates of inorganic carbon fixation would be expected prior to the midday maximum and the concomi- tant maximum of solar irradiance, the highest average sus- tained rates of fixation were found during the midday incubation periods. Samples were returned to the laboratory and 50 ml aliquants from each bottle were filtered through 0.22 pm Millipore filters (< 1/2 atm pressure). Filters were stored under dessication, until acid fumed to remove any residual Ca14CO3 which may have precipitated during the incubation period. Filters were then combusted in an oxygen atmosphere in a Packard Tri-Carb Oxidizer (Model 305).2 The combustion materials were thus isotopically 2Blanks were burned between each sample to reduce the possibility of cross contamination. "Carry-over" and "memory" on the collecting columns were carefully monitored for each oxidation series. 43 separated and collected as 3H20 and 14CO2 in scintillation vials.3 3Scintillation cocktails: a) 3H-cocktail 10 m1 Insta-gel (Packard Instrument Co.) 14 . C-cocktail 3 m1 Monoethanolamine (C02 trap) 9 m1 Absolute Methanol (Solvent) 7 m1 Scintillator consisting of 15 g PPO l g bis-MSB Scintillation grade Toluene to make 1 liter. b) ASSUMPTIONS, CALCULATIONS, AND STATISTICAL ANALYSIS The following assumptions were made in calculating the activity represented by the observed uptake of 3H- glucose: (1) That the isotOpic discrimination effect for 3H- glucose is 1.00. One can calculate on a random probability and weight basis that the discrimination against l4CO2 should be 1.045. Empirical observation has given support to the figure 1.06 which has received general acceptance. 3H20 on the other hand because of a proportionally smaller weight for the water molecule would be expected to have an associated factor of 1.11 or 1.10. For 3H-glucose, because of its relatively greater weight, one could calcu- late a figure of 1.01 (i.e., 182 g mole'l/lso g mole-1). Since this factor is generally unknown and close to 1.0 it was felt that 1.00 would give the least biased minimum estimate for glucose uptake. (2) Since the natural glucose concentration at the time of incubation was not determined it was assumed that the minimum conservative estimate would be represented by the following: 44 45 3Héglucose added = H-glucose available H-glucose measured H-glucose utilized The result of this assumption is two-fold. First the amount of organic uptake calculated by this method represents a minimum figure. Any naturally occurring glucose concurrent to observed utilization rates would be in addition to that injected. Therefore the glucose available would have been increased and the radioactive pool diluted prOportionately. In other words if there were 10 pg glucose liter.1 available naturally, the addition of 5 ug glucose liter.1 would raise the total figure to 15 pg and the estimate of the quantity utilized should have been increased by 3X. As the natural concentration approaches zero the proportional comparison between observed and projected approaches 1.00. Should values of naturally occurring glucose, or total similar competing organic compounds, approach 50 or 100 ug liter-1, the appropriate factors become 11X and 21X respectively, assuming these concen— trations are below saturation levels for uptake kinetics. Thus for values within the expected range of 10 to 20 ug glucose liter"l the estimates must be considered to be very conservative minimal estimates. The second result of this assumption is that the amount added is independent of concentration except on a 46 random strike probability basis. This will be true only within the additional constraints of the physiological tolerance limits for the organisms, that no substrate limitation occurs, and that the concentrations are within the expected range for the environment. The figure of concern for additions of the same relative magnitude then equals the specific activity of the material utilized (e.g., in this case SA = l mmol/500 mCi). Organic Uptake The calculation for converting the raw counts per minute (CPM) from the 3H-glucose uptake series was as follows: ugC m'3 hr'1 = (CPM*CON1*CON2*CON3*CON4*CON5*SA* TOPEF)/((A*ESR)+B)*BF*SS*TIME(J)* RE(K)*DF(K) where: CPM = raw counts per minute A = the slope for the calculated quench correction curve B = the intercept for the quench curve ESR = the External Standard Ratio (quench) BF = the bottle factor (corrects all bottles to 125 m1) SS = sample size (50 ml) 47 TIME(J) = the incubation time in hours RE(K) = the recovery efficiency for known standards with the oxidizer instrumentation DF(K) = the isotopic decay factor for 3H TOPEF = the isotopic discrimination effect (assumed to be 1.00) CONl = 1.0 (weighting function, not needed here) CON2 = 1000ml x 1000 l = 106 1 m3 con3 = luCi/(2.22 x lo6 dpm) SA = the specific activity (lumol/SOOuCi) CON4 = 6umol C x 12.001ug C x mg_C umol3H-giucose umol C 1000 pg C CONS = 1000 pg C/mg C This basic calculation was performed for every sample and the results from paired light bottles and dark bottles were used for further statistical analysis and estimation. Samples within the design for statistical analysis numbered 504; total n equaled 720. Photoheterotrophic Uptake Photoheterotrophic uptake was estimated as the dif- ference between light and dark bottle pairs. In order to arrive at the estimate it is necessary to assume the following:4 4This is much the same assumption used to estimate photolithotrOphic uptake of C02 (i.e., photosynthesis); calculated as the difference between photolithotrophic uptake less chemolithotrophic uptake. 48 Light bottle uptake = Photoheterotrophic uptake + Chemoheterotrophic uptake + Background Dark bottle uptake = Chemoheterotrophic uptake + Background Therefore light bottle less dark bottle yields an estimate of the proportion of the total heterotrophic uptake observed due to photoheterotrophic uptake (i.e., "PHOTO" in Appendices A and B). Chemoheterotrophic Uptake Chemoheterotrophic (i.e., "BACTERIAL") uptake was estimated by the following: -1 = CPMdark -3 ugC m hr (AfESR) + B - BKG(K) *(CON1*CON2* CON3*CON4*CON5*SA*TOPEF)/BF*SS* TIME(J)*RE(K) where: BKG(K) = background calculated for each oxidation series This calculation overestimates the contribution to total heterotrophic fixation by chemoheterotrophic orga- nisms, since the background used for the calculation repre- sents machine background (i.e., background associated with the oxidizer and scintillation counter). A proper control would have consisted of a sample "killed" at the time of 49 injection of the organic compound to account for any absorption and adsorption in the sample. With this in mind, any comparisons between chemo- heterotrophic uptake and photoheterotrophic uptake must be considered minimal estimates of photoheterotrophic potential. Any increase above this machine background would lower chemoheterotrophic estimates and proportionally increase the proportion due to photoassimilation in any comparison of the two. Percent Comparison The percent contribution of photoheterotrophic utilization with respect to chemoheterotrophic uptake was calculated as: PCTBC = (Photoheterotrophic uptake/ Chemoheterotrophic uptake)*100.0 Mean Values and Annual Means Mean values for all estimates were calculated from the four paired samples for each time, depth, and month (see Appendix A). Standard deviation and standard error were calculated to aid in graphing. Annual means were calculated from all sample esti- mates (n=360) for photoheterotrOphic uptake, chemohetero- trophic uptake, and percent bacterial uptake (i.e., (Photo/Chemo)*100.0). Standard deviation, standard error, coefficient of variation (CV), and 99% confidence intervals 50 about the mean were also calculated. An arcsine trans- formation was used for the percentile data, since the range of observed values was greater than the interval from 30 to 70%. Inorganic Fixation Calculations for inorganic carbon fixation followed a similar pattern: 3 l mgC m" hr- = CPM*CON2*CON3*ALK(JJJ)*PHFTR(JJJ)* CONl*CON5*TOPEF/RE(K)*((A*ESR)+B)* BF*SS*AA*TIME(J) where: TOPEF = the isotopic discrimination effect (1.06) CONl = dilution factor for alkalinity determination (20.0) CON2 = ml per bottle (125.0) AA = the activity per m1 added in uCi ALK(JJJ) = the alkalinity determination per depth and month (m1 0.02N H SO from titrations) 2 4 PHFTR(JJJ) = the pH factor for each depth and month Other factors remain the same as previously given. Photosynthetic (photolithotrophic) fixation was estimated as the difference between light bottles and dark 5See the table of values by Bachmann in Saunders, Trama, and Bachmann (1962) . 51 bottles. Chemolithotrophic production (dark bottle less background) was not calculated. Mean values for each of the differences of the four pairs were calculated along with standard deviation and standard error. These values were tabulated along with estimates of photoheterotrophic and chemoheterotrophic production from the same sample for comparison purposes (see Appendix B). Percent comparisons for mean values of photolithotrophic, photoheterotrophic, and chemohetero- trophic production were also calculated. The preceding calculations were carried out on a Hewlett-Packard HP 2100A computer. The statistical analysis was accomplished through cooperation with the Application Programming section of the Michigan State University Computer Labora- tory. A Control Data Computer System CDC 6500 was used for the analysis. RESULTS AND DISCUSSION HeterotrOphic Activity An examination of the analysis of variance table for the heterotrOphic uptake of 3H-glucose reveals a number of significant effects (Table 1). Most notable of the main effects is the "Bottle" effect, for which the treatment contrast light versus dark is highly significant. The exact probability that the difference observed is due to random chance alone is <0.0005. Thus there is very strong evidence of a difference between light and dark bottles. It is also clear that light activity is greater on the average than dark (i.e., 9.2 ugC m-3 hr“1 vs. 6.3 ugC m”3 hr-l). The treatment main effects consisted of Months (J,J,A, etc.), Time of Day (SR, MD, SS), Depth (2, 6, 10 m.), and Bottles (Light, Dark). Other than the main effect "Bottles," which represents a clear, controlled treatment, the other main effects represent fixed treatments which are confounded by a number of environmental changes. In order to correctly interpret the resulting differences it is necessary to remember the numbers and types of changes these treatments may represent. For example, changes observed over months may be due to species population changes, 52 53 .Houumn no .Ho>ma mm on» an DGMUAMHcmHm no: A.m.cv “Hm>oH mm see us nemoeeeemem 1.1 lem>me we we» as semoeeeemem Alec “sesame no .Hm>me we.o we» as uemoeeeemem 14.41 .mxmeemumm an emumoeeee mum mao>ma OUGHOHMHcmHm .cmwmmc uoam uflamm HMHHouomw >o3lmmusa .omoooam mo oxmums oanmouuoumumnouosm MOM manna mocwflum> mo mammamc¢II.H manna 54 mam am.eNNm Nmuoe me.m mad aN.GNoe aouam Nmaeflmmm maaa.a mN.N 4N oa.as mapuom x auama x meNe x auaoz maaa.N ea.aH e as.me mauuom x auama x mafia seme.N am.mN NH Nm.aae mauuom x auama x auaoz sem.a GH.oN Na GN.HNH meuuom x msee x auaoz «eme.e mH.eN N Hm.me mauuom x :uama ..«NN.GN Hm.Na N Na.mNN mauuom x mane «esmN.N am.mm a mm.NmN mauuom x spec: sesam.eaa ea.meaa a ea.msoa mauuom aa.a awe mm.amNN a mouse maNm.o mo.a eN an.eea auama x msNe x auaoz eeeem.e em.Nm e me.ama nuama x msae .«eHm.NH aa.aa NH am.mmm auama x auaoz «.mmfl.e ea.NN NN no.emm maee x auaoz «eeaa.ae ma.aam N aa.ama auama seemm.mm Na.mmm N mm.ame msee «eahm.am vo.mvm m NN.mhom SDGOE UHumHumum .m mumsmuw Gmwz EOflmem meMDmvm MO 55m OUGMHHM> MO OUHSOm mo mmoumoc manna moccaum> mo mamaamcd 55 temperature changes, or organic loading and increasing con- centrations of organic materials (N.B. this would be especially true in late summer at depth). Changes of activity throughout the daylight period could be due to changes in light intensity and quality, or a periodism within the organisms themselves due to end product inhibition, or other biochemical feedback systems. Differ- ences over depth at nearly any time of the year could be related to a number of these parameters (e.g., light inten- sity and quality, temperature differences, pOpulation differences among the water strata, and organic loading). Realistically, all of these factors probably interact in such a way as to yield the changes observed. Since a number of three-way interactions are signi-3 ficant, or very nearly significant,6 it is necessary that these interactions be examined carefully in order to correctly interpret the differences observed. Understanding main effects alone is not sufficient for a prOper interpre- tation of differences observed. Careful selection of different comparisons may give insight in selecting those parameters most likely to be responsible for the changes observed (e.g., we may contrast differences with depth at turnover with those during a stratified period to 6Source of Variance Probability of F Statistic Month x Time x Bottle 0.047 Month x Depth x Bottle 0.005 Time x Depth x Bottle 0.09 56 approximate a comparison of light and pOpulations versus temperatures). Bottles x Months x Times Figure 4 represents the pattern of uptake observed in light and dark bottles over months and at times of day. Each point is the mean of 12 samples (i.e., replicates summed across depths). The relationship between light and dark uptake is presented in two ways to more clearly demonstrate the patterns observed. On each series of graphs, and those that follow, two distances are denoted by vertical bars (here 2.36 and 2.94, and 1.82 and 2.49). These distances represent the value calculated by the Sheffé S Method for 3 posteriori comparisons of means (here any two points) (Kirk, 1968). The larger of the two (e.g., 2.94 vs. 2.36) represents the minimum distance two points would be separated to be considered different at the 1% level. In other words, if the distance between two points of interest is greater than this value, they may be considered to be significant at the 1% level. The smaller of the values (here 2.36) represents the distance for the assignment of significance at the 5% level. While the Sheffé test is useful as a means of selecting those points of interest for comparison, as with all 3 posteriori tests multiple use at a low significance level will lead to some Type I errors, the rejection of a true null hypothesis. Thus if we are testing whether or not 57 .monEmm NH mo came a mucomoumou ucflom comm .Hm>mH mm on» an .Hommma may “Hm>ma NH on» um mocmoflmacmflm MOM mononMMHc may .03» may no umpmoum one .mumn Havauum> ma pouncapcw mum mammum mo moflumm comm MOM mosam> m Wmmmnm .:m>Nm Oman mum cofluom coeumnsocw comm How mwauuon unwed can xnmo cowsuon mummuucoo .A....Y Duncan .mm cam “AIIIIV SMUUNE .02 “AIIIV mmflucsm .mm mm mnucoe mmouom mcoflumm coauwnsocfl Ham MOM an“: IE om: CH noncommou xumc can unmfla pammmaama maameo .Amazea x amaze: x mmaeeomc muoaa aoaemmemuae mmzummaaeuu.e magmas pg C/m? hr _a N N ‘1" 9 9' 5 58 BOTTLES X MONTHS X TIMES pg C/msyhr 'JTJ'ATTS’OTNTDT MONTHS DARK n=l2 pg C/m7’hr 1138 1249 Sunrise LIGHT DARK dd ‘PQ‘P 59 two population means are the same, we reject the null hypothesis if the distance is greater than the Sheffé S value. If the value at the 5% level is applied numerous times to the same data set we would expect to reject a null hypothesis (i.e., declare the means to be different) which should have been accepted (i.e., the means are not differ- ent) one time in twenty on the average. Care must be exercised in this respect. By comparing dark bottles across all months some general observations may be made. The sunrise (SR) incu- bation represented the lowest activity across all months. The only exceptions to this statement are observed where mean values were not significantly different. Midday (MD) incubations usually yielded increased activity over that observed at sunrise and intermediate to that of sunset (SS). Late afternoon, pre—sunset (SS), incubations generally resulted in the greatest chemoheterotrophic activity throughout the daylight period. This finding agrees well with the diurnal "bacterial" activities observed by Saunders (see the discussion in Saunders, 1969). An exception to this rule was observed in the November and December incu— bations, where sunset values represent an intermediate range between midday values and sunrise values (midday and sunrise being significantly different, with midday representing the higher value, but with the sunset value not significantly different from either). In general across all months and times there was a pattern of increasing activity until 60 turnover and ice cover in December. This is also in agree- ment with the patterns of chemoheterotrOphic activity observed over an annual period by Hobbie (1969). Light bottle activity (i.e., chemoheterotrOphic dark bottle activity plus photoheterotrOphic activity) showed a different pattern. Here the greatest total activity was represented by the midday values across nearly all months, followed by sunset and then sunrise values. In general the greatest total activity across all months and times occurred during the late summer period from July to November. By examining the plots contrasting light and dark incubations at each time and across all months, the relative magnitude of chemoheterotrophic activity can be compared to total activity and to photoheterotrOphic activity, the distance between the two lines. The greatest difference between light and dark activity occurred during the midday incubation period. The maximum for the months within the design occurred during the months August, September, and October as previously discussed. In general the difference between light and dark bottles was greater throughout the year for sunrise incu- bations than for sunset incubations. The most marked decrease in photoheterotrophic activity occurred during the sunset incubation under ice. 61 Bottles x Months x Depths Figure 5 depicts the three-way interaction for BOTTLE X MONTHS X DEPTHS (n=12). Dark bottle activity across all months and depths again showed the general increase up to the time of autumnal circulation and then an apparent decrease under ice. Activity generally increased with depth across all months. IAn exception is the elevated 6 meter value during August, which corresponded to peak values for both total and photoheterotrOphic activity. The power of the 3 posteriori test in examining differences is demonstrated here very well. During June, early in the stratified period, values at 2 meters and 6 meters were not statistically different in activity, but both were different from that of 10 meters. As stratifi- cation progressed 6 meters values were not statistically different from those either at 2 or 10 meters, but values for 2 and 10 meters were different. By August with summer stratification fully established the 2, 6, and 10 meters samples represented different strata of water with statisti- cally different uptake rates. During September the peak uptake values at 6 meters decreased with concomitant increasing activity at 2 and 10 meters. During October, uptake rates for the 2 and 10 meter strata were again statistically different, but values at 6 meters were not different from those of either 2, or 10 meters depths. During turnover and under ice with isothermal conditions, 62 .mmHmEMm NH mo some n mucmmoumou “seem comm .mumn amoeuuo> an cmDmONccfi mum modam> m wmmozm .co>Hm Oman mum Hm>uoucw spawn comm HOm mmauuon unwed ccm xumc cmozuon mummuucou .A....c mumuoe oa can .Annuuc muouoe m .A V muouoa m on .mnucoe mmouom mnudmc Ham NOw any: mus cm: :H noncommon xumc can unqu ucomoumou madmao .lmmeama x mmezoz x mmaeeomc muoHa aoeuomumuaN mmsnmmuaasu.m mesmea 63 wrezoz o w . < . a - z . . - .. - a . 5.3 .n Song m .o. 0 W... N? .2 W 828:0- .ON .a.z.o.w < a. a. P b h xmad Eo.._/\/\.I .n «7: .m— 829:0 .ou. to.z.0.m.<.1.s. ¥m Sc coumoNUCN mum mosHm> m wmmocm .co>Nm Omam mum Hm>uoucfl cumoc como How moauuoc ucmfiq ccm cumc coozuoc mummuucoo .A....V muouoa ca ccm .AIIIIV muouoe m .A c muouofi N mm .mcoHHom coflumcsocw mmouom mcumoc cam How anuc mIE Um: ca noncommou xumo cam ucmflq ucomoumou mammao .Ammeama x mmzHe x mmaeeomc muoaa aoNuomumuaN mmzummuaeuu.a mesmea mOOEma 20:23:02. mm OE mm b b (F xmNm mumc Houum .Huuc maE um: mm mxmum: oecmouuououom .Ao.ooas AOEocU\ouocmvv mosam> comHHmmEoo ucoouom cam .oxmums oflcmouuonouocouocm .mxmums oflcmonuOHopocOEoco Mom mosam> cmoE Hmscccnu.oa ousmflm 78 lNBOBBd ow cm 00 om P mlmlaa Emoeod mZoa wa.o ocp um ucmoNMNcmflm Assay "mxmfluoumm ac cmumoflccw ohm mHo>oH oocmoNMNcmHm .cmflmoc uon ufiamm Hmfluouomm mmzloouca .cocuoEIo ac woumeflumo coaumxflm cocumo oflcmmuocfl MOM oacmu oocmflnm> no me New m5 mamuu.N maame 80 mom NN.Nma empoe Naa.a aaa em.NH poupm Hmamemmm eesea.aa ma.o eN mm.NN mauuom x chama x maee x apaoz .m.ea.ae em.m e am.ma mNuuom x auama x meNe mmeea.mm NN.m NH eN.aN meuuom x cpama x auaoz «eeee.NN Nm.N NN eN.om mapuom x msNe x chaos «eeea.emm Nm.mm N ma.eoa mapuom x cuama «emao.HmN HR.GN N Ns.mm mauuoa x msNe «camm.¢ma mm.NH m ma.vh mauuom x ch02 «m.aa.emeN me.mNN H Ne.mNN mappom mmo.o mma na.ma 4 Houum .e.mm.aa aa.o eN em.AN amama x mafia x auaoz ms.aa.am mm.v e mm.NH amama x meNe «eeaN.Ne Ne.m NH He.me apama x chaos mesee.mm am.N NH ma.em mace x cuaoz «seam.mNa mm.mm N Ne.aoa apdma meeeN.Nom Ra.mN N ee.am maee .«eee.mma am.ma a Hm.oa amaoz oeumflumum m ohmswm cmoz Eocooum moumoqm mo Eom oUGmNHm> m0 condom mo mooumoc macme oonNum> wo mflmwamcc 81 Figure ll.--Estimated values for inorganic carbon fixation during 1974 as mgC m‘3 hr'l. Histograms represent the means of four replicate samples. Uptake values for each depth interval (2, 6, 10 meters) and each incubation period (SR, MD, SS) are indicated. Bars denote plus or minus standard errors (:SE) about the mean. mg C/m3/ hr 82 MONTHS JAN FEB MAR 2m N 7 504 6m 5;). 10m 25. 0.0 + __ JUN JUL AUG 50. 2m 50. 6m ‘ I 59. 'IOm SEP DEC ”N3... W 5 M m 5.0 50. 10m 25. 0.0 fl —-— _ SR'MD'ss"'SR'M0'ss' 'SR‘MD'ss' 'SR'MD‘ss‘ INCUBATION PERIODS 83 metalimnetic peak in activity during August was again seen. High epilimnetic values were also found during March and October and under ice in January. Generally the contri- butions to overall fixation were dominated by epilimnetic values; an exception during the metalimnetic peak in August has been noted. The contribution by 10 meter populations was usually minimal, particularly so under ice cover. As have been reported by others on a number of occasions, greatest activity was generally associated with morning and midday incubation periods. A comparison was made between those values obtained for chemoheterotrophic uptake, photoheterotrophic uptake, and photosynthesis. On an individual basis the numbers were highly variable ranging from zero (and slightly negative values) to greater than 250% for photoheterotrophic uptake (photoheterotrophic uptake/photosynthesis) and greater than 1000% for chemoheterotrophic uptake (chemohetero- tr0phic uptake/photosynthesis). Because the values were so variable and, since the precision associated with each of the measures is quite different, these comparisons may be useful in only a very general way. According to Strickland and Parsons (1972) the precision associated with the radioactive carbon method at the 1.5 mg C m—3 hr.l level is approximated by 0.15/no'5 mg C m-3 hr.l for a 7-hour incubation and 5 pCi of activity added, where n is equal to the number of determinations. For this work n equals 4, therefore the correct value should 84 5 -1 lie within the range : 0.15/(4)0' , or 3 75 pg C 111-.3 hr . This agrees well with the estimates in this study. With mean values for photoheterotrophic uptake and chemoheterotrophic uptake of 2.6 and 6.9 pg C m—3 hr—l reSpectively, the only times where one would expect to quantify significant contributions to the carbon pool would be those times where photosynthetic values were near, or below the sensitivity of the l4C—method. This is not to say that these values do not contribute significantly to total carbon metabolism and fixation. Additionally, while it is instructive and useful to compare the two types of heterotrOphic uptake for a single substrate, the natural concentration of which is unknown, comparisons between organic and inorganic carbon pools are more difficult and prone to err. Any percentage must be considered minimal for both photo- and chemoheterotrophic contributions, since (1) the natural, organic substrate concentration and thus the dilution is unknown, and (2) the pool of competing, or readily utilizable compounds which also would increase estimates of total heterotrophy are equally unknown. These precautions in mind concerning minimal values, the following generalizations were made. The percent con- tribution of photoheterotrOphy to overall carbon fixation in epilimnetic waters is probably always minimal. This is generally true for metalimnetic waters as well, but values greater than 1% are encountered during early morning 85 incubations and during conditions of low light (e.g., ice cover, cloudy conditions, etc.).' Ingeneral, based on mean values (n=4), the greatest photoheterotrophic contribution to toal fixation occurs at depth, 10 meters, and during the sunrise incubation period. The midday values at depth contributed greater absolute amounts, but proportionally lesser amounts as compared to photolithotrophic fixation. Sunset values were intermediate to these. The highest values occurred under ice during January and December (i.e., >8%). Higher values were also encountered during late summer. Bacterial values, chemoheterotrophic uptake, followed a similar pattern. Highest relative values were observed under ice, 20-50%. These values may be an artifact of the method, however, since the mean value for photo- lithotrophic fixation is less than 75 ug C m-3 hr.1 in both cases. At depth heterotrophic activity probably ranges from 0 to 10% of photosynthetically fixed carbon, as minimally estimated here, with photoheterotrophic activity generally 2-5% based on means and chemoheterotrophy <10%. PERSPECTIVES AND INTEGRATION Additional work is unquestionably needed. However, this study has clearly demonstrated the direction that work should take and has given considerable insight into the workings of an important feedback pathway in the regulation and cycling of organic carbon in lake systems between the phytOplankton and the dissolved organic carbon pool. Regardless of the agent of uptake, it has been demonstrated that measures of heterotrOphic potential in aquatic systems may lead to serious underestimates depending upon whether, or not these incubations are carried out in the light, or in the dark. Within the constraints of the statistical design (n=252) light bottle uptake averaged 9.2 ug C m-3 hr-l, while dark bottle uptake averaged 6.3 pg C m—3 hr-l; light bottle, or total, represented 146% of dark bottle estimates. Averaged across the annual period, the combined estimate for photoheterotrophic uptake plus chemoheterotroPhic uptake yielded a similar figure, 138%, as an estimate of total heterotrophic fixation versus chemoheterotroPhic potential. 86 87 While these values themselves point out the impor- tance of consideration of this heterotrophic pathway, it must be recalled, that depending upon time of day, depth, and month this error may be many times greater. Thus until better information becomes available we must consider this to be a major pathway in the cycling of certain specific organic compounds within lake systems. Within the overall scheme of cycling of materials in lake systems it is unimportant whether, or not the agent involved belongs to algal groups within the plankton, or to the bacteria. Stanier (1973) points out that the dominant nutritional mode among non-sulfur purple bacteria is photo- heterotrophic uptake of organic materials. However, the wealth of evidence concerning algal uptake already dis- cussed, coupled with limited microautoradiographic studies with l4C-glucose in association with this investigation, point to algal species as being those organisms primarily responsible for the observed, sustained annual uptake. More important than the annual uptake values and the implications for organic cycling based upon a single organic compound during the daylight period is the impor- tance of this link in the cycling of materials at a signi- ficant point in the trOphic scheme of organization. Since Lindeman's (1942) provocative paper, ecologists have attempted to place in prOper perspective those pathways responsible for the major flux rates in ecological systems. This work in large part has now been accomplished and it is 88 clear that organic carbon (i.e., detritus) plays a central role in the structuring and the functioning of a majority of systems studied in some detail (see the discussion by Wetzel §£_al., 1972; Saunders, 1969; Jordan and Likens, 1975; and Hobbie et al., 1972). It is equally clear that we are generally lacking in any understanding of how and why those rates function as they are observed. Key to this understanding is the eluci- dation of a number of feedback loops within that system. Within this framework those organisms, which influence the pool of dissolved organic carbon and are themselves in some manner directly affected by the composition of that pool, are extremely important insofar as their position within the ecosystem and their influence upon system structure. Those organisms, which occupy these important seats within the system, are without doubt those organisms wherein a majority of carbon cycling occurs and are confined to the lower trOphic levels. Figure 12 depicts such an idealized trophic relationship. Photoheterotrophy thus represents not only an important pathway in the cycling of organic materials, but meets the criterion listed above concerning those 100ps by which the biogenic drivers in the system may also be regulated. Chemoheterotrophic assimilation certainly Operates as the major mechanism of organic utilization, when one considers the non-daylight hours where photoheterotrOphy is 89 Figure 12.—-Idealized trophic scheme emphasizing the cycling of organic carbon. Dissolved organic carbon (DOC), dead particulate organic carbon (POC). Major pathways indicated by arrows. 90 co2 .3 - x 3 Photosynthesis o? Photoheterotrophy Phytoplankton Zooplankton and m 91 inOperative, and the fact that a much greater variety of organic compounds are probably readily utilizable by chemoheterotrophic pathways. However, the really important questions concerning photoheterotroPhic growth have yet to be addressed. If algal species are indeed involved, as the evidence indicates they are, which species possess the ability to photoassimilate organic materials? All species are certainly not equally capable of organic uptake. If the observed uptake is not to be considered a generalized constitutive phenomenon, then it becomes important to ask which species are responsible for the majority of the uptake observed at various times throughout the year. How the structure of the phytOplankton community is influenced over time by the ability of certain organisms to utilize organic substrates is also an important question. Are those species which supplement carbon uptake able to replace other species over time because of this advantage; how then do photoheterotrOphic capabilities influence phytOplanktonic succession rates? Photoheterotrophic uptake of organic compounds may also represent a key to the understanding of the existence of populations at depth and under ice, con- ditions not favorable in the extreme to photolithotrOphic fixation (see the discussion by Rodhe, 1955; Bernard, 1963). Of much interest would be work coupling the release of extracellular products, either during the course of normal cellular metabolism or photorespiratory pathways, to the potential for assimilation. These "wasteful" processes 92 may not be nearly so costly metabolically, if a measurable proportion of "lost“ organic compounds could be successfully recovered at a time when photosynthetic fixation of in- organic carbon is no longer optimal. PhotoheterotrOphy may represent a partial explanation for the apparent lack of selection for a more complete retention of photosynthetic by-products. Of particular importance on an evolutionary scale would be the Species relationships between those capable of photoassimilation and those reSponsible for the majority of extracellular products found in aquatic systems (whether, or not these processes are concurrent within the same species). Patterns of excreted organic matter by the plankton with relation to photoassimilation would be instructive. Of importance would be the quantification and qualification of the compounds in the organic carbon pool. A clearer idea of the competing, diluting pool would be gained for a more complete comparative assessment of heterotrOphic processes. Lastly, an important point discussed early in this work concerns the relative importance of photoheterotrOphy in the pelagial zone versus its importance in the littoral zone. Certainly strong evidence has been presented for the photoheterotrophic pathway in lake systems. However, as discussed earlier those species most often reported to possess photoheterotrOphic potentials were those associated with natural zones of concentrations of organic materials. 93 Certainly epiphytic algae and those species associated with sediments would be expected to benefit measurably should photoheterotrophic ability be possessed by many members of those associations. Therefore it is probably within the littoral zone and not the pelagial zone where one would find the greatest quantitative contributions to algal nutrition. Unquestionably the biochemical and physiological mechanisms of organisms at lower trophic levels are inti- mately tied to the structure of ecosystems. How organic and inorganic carbon pools, photoheterotrophic, photo- lithotrophic, and photorespiratory pathways, and organic and inorganic nitrogen pools collectively interact to influence metabolism on a diurnal basis and species compo— sition on an annual basis is yet to be addressed. Cer- tainly this interplay will prove to be important. Its elucidation will depend upon insight into questions of broad ecological importance. SUMMARY Ample evidence has been presented in this study concerning the importance in nature of the phenomenon of photoheterotrophy. As compared with chemoheterotrOphic activity for glucose during the daylight period, photo- heterotrophic activity equaled 67.6% on a comparative basis in a hard-water lake in southwestern Michigan. Consequently, studies of heterotrophic uptake utilizing dark techniques may seriously underestimate total activity. The pattern of photoheterotrOphic activity as com- pared to chemoheterotrophic activity demonstrated that the two heterotrophic processes are separated in space and time on a daily as well as a seasonal basis. PhotoheterotrOphic activity generally was skewed toward the morning and mid- day, with predominating activity shifting to increasing depths in the water column as the day progressed. Maximal values were observed during the spring and late summer. Chemoheterotrophic activity generally increased throughout the daylight period and with depth within the water column. During isothermal lake conditions uniform chemoheterotrophic activity with respect to depth was observed. 94 95 Heterotrophic fixation as compared to photolitho- trophic carbon fixation indicates that photoheterotrophy may contribute significant amounts of carbon to photo- synthetic organisms under conditions not favorable to inorganic carbon fixation (e.g., the low irradiance at depth and under ice cover). The importance of photoheterotrophy in phytoplank- tonic species succession, the potential importance in the littoral zone both epiphytically and in association with the sediments, and to the overall cycling of organic carbon and the structure observed in lake systems is discussed. APPENDICES APPENDIX A ORGANIC CARBON UPTAKE VALUES APPENDIX A ORGANIC CARBON UPTAKE VALUES Appendix A is a tabular presentation of calculated values (UQC m-3 hr-l) for organic carbon uptake in Lawrence Lake during 1974. The data are arranged as four light bottle/dark bottle pairs for each Time, Depth, and Month of sampling. The difference between light and dark estimates (Photoheterotrophy) is found in column "PHOTO UPTAKE." Chemoheterotrophic uptake is presented in column "BACTERIAL UPTAKE." PhotoheterotrOphic estimates divided by chemo- heterotrophic values and multiplied by 100 are placed in column "PERCENT OF BACTERIAL UPTAKE." Means, standard deviations, and standard errors are indicated. 96 97 000.0 «00 0.0.0. «00 000.0 1% 0000.0 5000.0! 0050.0! .000.00 500.00 :00 050.00 «00 000.00 ax ..00.0! 0500.0.! 0000.0! 0005.55. 000.0 :00 000.0. n00 00..0—I .0 0050.50! .000.! 0000.0! 0000.00! 0M35 —I no _ page: ”mm—Glam a HUsu vb mg: ON 0 IA IDS sugar—bu...— 100 005.00 «00 000.50 «00 000.50— ux 00—5.—0— 0005.00 0500.0— 000—.—0~ 000.00 .00 —0—.00— «00 _00.00— «x 0.5—.000 00__.50— 0050.000 —_00.0l 000.00 “mm 0-0.00_ Mam 500.00— IX 0000.500 ~500.50_ 0000.0_— 0500.00 HMdFLD Ad—flmhvdm LO #000000 550.— n00 00_.0 n00 500.0 ax 0050.0 050—.0 0000.0— ~050.0 000. .00 005. n00 500.0 ax _000.0 0000.0 0000.0 0000.0 00—. "00 ~00. n00 000.— ix 0—00.— 0000.— 0050.- 50—0.0 00\0l\000 Ad—flflhUdn 050.00 «00 000._ «mm 050._ "um 000.00 «cm 000.0 «00 _0_.0 mam 500.00 ax 0n0.5 1X 050.0 IX 0000.00 0—00.0 0—00.0 0005.0 5050.0— 0000.00 0555.5 0—05.0 ~000.5 50.0.q— 00—0.00_ 000_.0 00—0.0 00—0.0 000~.0~ 5000.0 5-00.0 0005. 0000.0 000~.0 :0— 000.00 «mm 500. umm 000.— «00 005.00 n00 0—0. «am 00_.0 now —00.00 ax 000.0 1% 000.0 3% 0000.50— —00~.0 0005.0 0000.0 0000.0 0550.0— 0000.0 ~00—.— 00—0.0 0000.5 0—00.50 0000.0 0000.— ~000.0 ~00_.0 0500.00 0000.0 0000.0 000—.0 000—.0 00 000.0 "mm 000. «00 ~0—. «00 000.0— n00 050. «00 000. u00 000.0 ax 005.0 ax 50—. ax 5000.5! 0500.0 —050.I _000.0 0000.0 ~000.5 0000.0 0000. 0000.0 0550.0 00—0.0~ 0000.0 0000. ~000.0 0005.0 0005.0— 0000.0 0000. 000—.0 0000.0 :0 02(500 ¢=\0t\000 00\0t\000 00\02\000 ¢0\0fl\000 A<~00PU<0 mudhmb mkdhmb Hidhmb 02450: 00 E020.— i—SE<0 09.00.— mg g0 mar—L00 Flu—‘— KO~P<0DOB~ FHQKDQ 005.~ “Hm —00.0_ "mm 0—0. "mm 005. «00 000.0 «00 005.00 n00 000._ n00 000.— "00 .50.0 ax 000. ax 000.0 u 0—0.I ax 000—.0 0000.0 0000.0— 0500.00I 050_.0 _000.~I 0000.0 0000.— 0050.0 0050.5 0000.0-1 ~0——.5 ~005.l 0000.5 0—00.— 0000.0~ 0000.~— 0000.0! 0000.0 _00—.0 0000.0 0000.0 0000.0 000—.0— 0500.00 0000.0 0050._ 00—0.0 l0— 000.~ «00 005.0 umw 000. "mm 000. mum 00—.0 '00 0-0.0_ new 000. new 000. "00 000.0 ax 000.0— n0 005.0 ax 000. «X 5000.5 0000.0 0000.0 000_.00 0000.0 0000.— 0005.0 5000.0 0000.0 0000.5 0000.0 0000.0 0000. 0__0.0 0——0.0 _000.0 5000.5 000_.0I 0—00.0 0500.I 0050.0 0000.I 0000.0 0000.0 0—00.00 00—0.0 0550. 5000.0 E0 500. "00 000.0 umm 00‘. umm 000. «mm 05~.~ u 00_.5_ "am 500. "am 000. new 050.0 ux 000.0— «X 000.0 ux 500. ax 0000.0 50—0._ ~000.0 00.0.1 ~000.0 5000.! —005.0 0000.0 0505.~ 0000.0 0000.0- 0005.0 0050. 5000.0 0005.. ~000.— 0000.0 0000.00 00—0.0 0000.— 0000.0 —005. 0000.0 ~000.0 0000.0— 0000.0 0000. 0500.0 :0 00\0=\000 00\0E\00= 00\0t\000 m¥<00~I lO—P<0DUI— 00—0200 0 00<0 05 E0 00 0 IL .50 5003035800.— 0000.0 0500.0 0050.0 0000.0 5500.0 0000.0 00—0.0 000_.0 0005.0 000_.0 5000.0 0000.0 00\0I\000 HMdeD l0 HAP500 M0<0 045500 500—4 101 0NN.ON mum 009.00 unm $00 . on: sun “00¢.u0u 000D.00 enn0.—b— 00h0.h0— 00h.vo uflm ——0.0N— tam flab.00~ ax vafl.hflu 0N00.b~0 0000.0 nonh.'0_ th.h «Hm ”$0.0— mam 0h§.bfl xx hO—O.Q_ N'00.h0 0—h0.0— 000h.fl0 HM<fiaul l0—P10: n: :0 ~ P0000h000P0009000 nm0h.N— 0000.0 0000.0 00N0.0 NNV0.0 h000.0 h00~.0 0000.0 #000.» #000.h 0.00.0 0000.0 00\0=\UOD HMdPLD =0— :0 IN 00—.0. «mm 000. ”mm 000._ "um 0N0.0fl unw hb0.— new 0N0.“ «am 105 999.9- "x 99:... n 99:..- »x 99~...9- a9mm... ..99.9- «.99... .99u.9 .9u9.9u 9uou.o 9999.“ 9u99.9 «999... 9.~o.9«- 9999.". 9999.9: 9.99.«. 9nou.o 9999.“ 9999.9. 9.9u. «has... 999m... :9. 999.9« "mm 999. "mm «99.. "mm 9a..99 "am 999.. "am 999.: «:9 9N9.99 "x 999.9 "x 9N9.9 "x 999u.~- 9999.9 «9.9.- 99.9.9 9999.9 «999.99 9999.: 999..9 9999.9 “999.“. 9o.u.99. 999~.9 9999.9 9999.9 9999.~. 9999.99 o.«9.9 99.9.9 9999.9 9.99.". :9 999.9 «mm 9N9.. .mm 999. .59 999.9 ":9 999.“ .99 u.... ":9 «99.9. "x 999.9 u 999.. .5 9.99.9. 9999... 9999.. 9u99... 9~99.9. 9.99.9" 9999.9. ..99.9 9999.9. 9999.9. 9999.9. 9999.9. :99... 9999.9. acne... 9999.9 «999.9 99.9. 9999.9 999..9 an 52...: 559.58: 55:58: 55:58: 559.58: .<.¢mbo<9 mx<9.: 5599.: mx<00~l 0 flo00 h“ 00~P<0bolu Hm—flzbm __0m :00 Flhcflbaflflhfllbbcflm 106 000.00 n00 .80.08 «00 000.80 «X 0000.00! 000..00. 000..8. 0.00.0! 008.00 .00 000..0 «00 000.00 fix 0000.8! 0000.00 0800.0! .080.00 000.00 «00 800.00. .00 000.00 1% 080..0 0000..0. 0080.000 8..0..0 A<.0HPU<0 80 8200000 "00 n00 "mm «00 000.0. n00 00... «00 000.. "00 080.00 «00 080.0 900 080.0 n00 000.00! :0 0.0.0 10 08..0! «X 0080.00! 0000.0 8.00.0! 8080.0. 008..0 0.00.0! 0008.0 .0.0.! 0000.0 .000.0 0000.80! 0800.0 0000.0! 0800.0 0000.0 .800.0! 000..0 .000.! 0080.0 0..0.0 :0. 80—.00 I00 0.... «00 000.. «00 000.00 .00 000.0 :00 .00.0 .00 808.0! 1% 080.0 1% 000.! 1% 0000.! 0800.0 0800.! 0000.0 0800.0 0088.80 0080.0 0.00.0 8000.0 0000.0 008..00! 00.0.0 0000.0! 0000.0 0000.0 0008..0! 0088.0 8000..! 0000.0 0000.0 I 0 008.0. umm 00.. «mm 0.0. «00 000.00 u00 000. 000 000.. new 800.00 nx 0.0.0 u 000.0 u 8008.0 0080.0 0000. .000.0 800..0 00.0.00 0000.0 0000.0 ..0..0 0080.8 0000..0 0.00.0 0800.0 0000.0 0.00.0. 0000.80 0008.0 0800.0 0000.0 .000.8 £0 uldkmb 00\02\UOD 00\0E\00= 00\0E\UOD 00\0=\UOD 44.:HFU<0 03488: 0&(80: m¥0<=K<0 00 8000.8 00.0.0 00.0.0 .0...0 8.00.0 8880.0 0080.0 .000.0 0000.0 8000.0 0008.0 00.0.0 00\0I\UQD 00\0l\000 HHdFLD 048800 H040 048800 800.4 :0.F<0DUI. 00.0000 0.00 .50 8002:002000 0000.0 0.00.0 0000.0 0000.0 00. 0000.0 0000.0 0.00.0 0000.0 :0 0000.8 0000.0 0000.0 0000.0. 00 E\B\§ Hudhmb APPENDIX B INORGANIC CARBON UPTAKE VALUES APPENDIX B INORGANIC CARBON UPTAKE VALUES Appendix B is a tabular presentation of calculated 3 hr-l) for inorganic carbon uptake in values (mgC m- Lawrence Lake during 1974. The data are arranged as four light bottle/dark bottle pairs for each Time, Depth, and Month of sampling. The difference between light and dark estimates (Photolithotrophy) is found in column "C14 UPTAKE." PhotoheterotrOphic and chemoheterotrophic esti- mates (ugC m’3 hr-l) from the same light bottle/dark bottle pairs are placed in columns "PHOTO UPTAKE" and "BACTERIAL UPTAKE" respectively for comparative purposes. Means, standard deviations, and standard errors are indicated. 107 108 000. .00 .00. .00 000.8 .0 0000.8 000..8 0008.0 880.. .00 00..0 .00 000.0 .0 0008.0 .008.8 0000.0 0080.0 008. .00 000.. .00 000.0 .0 00.0.0. 0000.0 0000.8 8008.8 004800 44.008030 00\00\ODD 000. .00 000. .00 000. .00 00... .00 8.0.. .00 000. .00 000.0 .0 000.! JR 880. .0 0000.0 0000.. 0080. 0000. 0000. 00.0.0 00.0..! 0000. 0000. .000. 0000.0 0000.. 0000. 0800. 0800. 0008.0 0000..! .00.. 0000. 0080. :0. 800.. .00 000.. .00 000. .00 0.0.0 .00 080.0 .00 080. .00 000.0 .0 000.. .0 800. .0 8008.. 0000.0 0808. 8080. 8000.. 0.00.8 0000..! 0000. 0000. 00.0. 0.80.0 0800.! 0000. 0000. 0000. 800..0 0000.. 0000. 00.0. 0000. :0 000.0 .00 000.. .00 080.. .00 000.0 .00 00..0 .00 000.0 .00 800.0 .0 000.. .0. 0.0.0 .0 00.0.0 0000.0 0000.0 0000. 8000.0 0000... 0000.0 0.00.0 0.00. 8000.0 0000.8 0080.! .8.0.0 8000. 0800.0 8000.! 0000. 00.0. 0000. .080. :0 004800 004800 ¢0\0:\UOI 00\00\002 00\0I\ODI 44.008040 98000 004800 0048LD 004800 00\mu.\§ 00\mu.\08 0.0 008800 0.04.. 0.8.800 800.... 00.840000. 800000 000. .00 .00. .00 080.. .00 080.. .00 0.0. .00 080.. .00 .00. .00 800.0 .00 00..0 .00 000. .00 .00. .0. 000. .0 000.8 . 000.. .0. 8.0. .0. 00.0. 0000.! 0000. 0080. 0.00.0 000... 0800. 0000. 0000.! 0000. .000. 08.0. 0008.0 00.0..! 0000. 00.0. 0.00.! 8.00.! 0000. 0000. 0000.0 .000.0 0000. 0600. .00..“ 0080. 0800. 00.0. 0000.0 008..! 0..0. 0.00. 00. . 000.. .00 800. .00 .00. .00 000. .00 0.0. .00 000.0 .00 0... .00 .00.. .00 008. .00 .00. .00 000. .0 000. .0 000.0 .0 000.. .X 000. .x 0000.! 8080. 0000. 0000. 0000.0 00.0.0 0000. 0.00. 0000.! .000. 8000. 0008. 8.80.8 0000.. 800.. 0000. 8000.! 0000. 0000. 8000. 0000.0 0000. .080. 8000. 00...0 .000. 00.0. 0008. 0000.0 0000. 0.00. 0000. I0 008. .00 008. .00 080. .00 000. .00 000. .00 000.. .00 ..0.. .00 00... .00 00... .00 .88. .00 000..! .0 000.0 .0 800.0 .x 000.. .0 080.. .0 0000.0! 0000. 0800. 0000. 0000.0 .0.0.. 0000.. 8.00. 0000.! 080..0 0000. 0000.0 0000.0 0000. 0080.. .000. 0000.! 0000.0 0000. 0000.0 0000.0 8088. 0000.. 0000. 0080.0! 8000.0 0000. 008..0 00 0000.0 0000.0 8080.! .080. 004880 ¢0\nl\UBl. ¢0\0l\000 ¢0\0l\ool. 004800 000800 00\00\000 00\00\000 08000 004800 004800 004800 04.008040 08000 004800 0048LD E; 0.0 0.0.88 004.. 0.5.00... EAR... 00\.u.\§ 0.0 00.840000. 0400.0 . 0040 08 8040040 00 00.840000. 00.0000 0 00 000 80800800080008000 0000. 08.0. 0000. 00. 00 .008.. 0000.. .088.. 0000. 00\00\Ufll 109 008. .00 000.. .00 0.0.8 .0 .000.0 0000.8 0008.0 00.0.8 0.0. 000.. .00.0 .0 0000.8 08.0.0 0000.0 .0.0.0 000. .00 000. .00 000.0. .0 0000.0. ..00.0. 8000.0 000..0 .00 004880 00\0I\000 000.. .00 000.0 .00 080. .0 .000.0 .000.0! 0080.! 0808.0 000.. .00 00..0 .00 0.0.. .0 00.0. 0000.! .000.0 .00..! 088. .00 000.. .00 0.0.! .N 0000..! 0000.! 0800. 0.00.. 0H4880 08000 00\0l\000 000. .00 800. .00 800. .x 8000. 08.0. 000.. 0000. 0000. 0000. .000. 0800. .0.. .00 .00. .00 00... .0 8000.. 0000. 0800.. 0000. 008... 00.0. .000. 0000. 000. .00 .00. .00 000.0 IX 000..0 0.00. 0080.0 0800. 8000.0 0.00. 8000.0 .000. 00\00\OQI 00\0l\000 004800 004800 0.0 048800 0040 0 0040 000. .80. 08..0 0000. 0000. 0.00. 0000. 0088 08... 800.0 000.0 0000. 0000. 0800. 0080. NOON 000. 800.. 0.0.0 0080.8 .0.0.0 0000.0 0000.0 .00 .00 .0 .00 .00 .0 004800 04.008040 00\0fl\000 00\0fl\000 00.840000. 800000 00.840000. 8400.: 000. 00... 8.8.! 0000. 0000.! 0.00.! 0000.0! 000.. 000.0 00.. 0008.0 0000.. 0000.! 0000.0! 000.. 000.0 000.. 0080.0 00.0.0 0008.0! 0000.. .00 .00 .0 .00 .00 .0 .00 .00 .x 0M48LD 98000 0800. .000. 0080. 0008. 0000.. 0000.. 0000.. 0.00.. 0800.0 0000.0 0000.0 00.8.0 00\00\000 004880 008800800.0 0.0. 000. .00. 0000. 0.80. 0000. 000.. 00.. 080. 080. 0000. 08.0. .008. 088.. 800. 08.. .80.. 0000. 0000. 0008. 0000. .00 .00 .x .00 .00 .X .00 .00 viii—d .000. 0000. 0000. 0000. 0080. 0000. 0.00. 0000. .000. 0000. 0000. 8.00. 00\00\UQE 00\0l\002 024800 0.0 00 .88.. 000.0 800.8 0.00. 0000. 0000. 8000. 00... 000.0 000.0 0000. .000. 800.. .800. 000. 000.. 0.0.0 0000.0 .8 0000.0 .0 0080 8800 004800 .00 .00 .X 080- .00 .00 .x 0H48LD 44.008040 00\0l\000 00\0I\000 80... 000.0 080. 8000. 000.. 0000. 080.. 000.. 800.0 000.0 0080. 000.. .00.. 0000. 00... 000.0 000.0 0080. 0000. 0800. 0000. .080. 0000. 0000. 0.00. 0080. 0.00. 000.. 00.0. 0880. 0.00. 8000. 0000. "g 0 . 0 0 00020.9. mu<8Lb .00 .00 .X .! 0 .00 .00 .x ."08 .00 .00 .0 08000 000. .00 00.. .00 ..0.! .0 .000. 00.0. 0000. 0000. 000..! .000. 0000. 0000. 000. .00 000. .00 000. .N .000. 8800. 0008. 8800. 0000. 8000. 0080. 00.0. 880. .00 00.. .00 000.. .0 0008.. .000. 00...0 .000. 0008.. 0000. 0000.. 8000. 024800 00\0l\000 00\0I\UGI 004800 004880 0.0 008800 0040 08 80400008 00 008800 0040 048800 800.0 :0. 00 00 00.840000. 00.0000 0 0m :00 80000800080098000 0000. 8000. 0000. 0000. 000.. 00... .000. afl-~~ 0000.0 0000.0 .000.0 0080.0 00\0l\000 004800 008800 800.0 00. 00 110 0N0 . ham ”a! . new ONO . "mm ~o«.. new baa. .am nae. “am poo.a. 1x can. 1: can. ax abouao. va.... «can. «one. noun. oa.«.u. oaoa.u ovo.. coon. «no». coun.a. an»... gnaw. ”coo. anon. nono.o. nuao.u oao.. an.n. not». no. nae. new coo. .um one. .am boa.. .am aaa.. .nm e... «an so..o. .x on».. .x on... ax nova... oon0.au oeoo. when. ouov.. .oon.a. noob.- can... once. moon.. aoaa.o. «a... o¢ao. cone. enoa.. oo.o.a. .«o... our... nooe. nave.. :0 ea..« .um coa.. .mm m... .um oo«.o .nu ou..o ”no oo.. .am can... In uno.a 1x 9.... A: «auo.o. ¢aoe.n anuu.o cave. oooo.v an...» new... .«oe.v cane. ooav.o aapn.o. aona.. aao«.o cone. auvu.o oo.a.o. ooa«.. coco.» oeao. oo.... nu mu400.l 000.. :00 000.. .00 .00. «mm 000.0 «00 .0..0 mam 000. .00 0.0.... «X 00.0.0 IN 00. . ax 0.00.0 0.00.0 0000. 0000. .000. 0hhh.h 0.00.0 00.0. 0000. 000k. 000..0 00.0.0 0000. 0000. 0000. h.00.0 000k. 0h0.. 00.0. 0000. 20. N00. .00 000.. :00 .00. n00 0&0. lam 00..0 nan 000. n00 000.0 1% 000.0 «x 000. ix .00..0 000h.0 N000. .000. 0000. 0000.0 .00... 00h0. .000. 5000. 0000.0 0000.. 0000. 0000. 0000. 0000.0 0000.0 0000. 0000. .080. :0 000. :00 .0.. .00 000. :00 080. «00 000. u00 00.. u00 000.0 n... 80. . La 00.. n... 0000.0 .000.! bh.0.. 0000. 0000.. 0000.0 0000. 0000.. 0000. 0000.. 0000.0 0000. 0000. 05.0. 0000.. 0000.0 0000. 0000.. 0000. .000.. 20 HMdFmD mxdkmb fll\0fl\00£ 00\02\UQE 00\0=\OQI 44.00FU<0 09000 aldhmb 0M¢h00 HMdeD 00\E\Ug 00\0:\§ 0.0 E an E E0... 00.P<0000. Pflmlbm 00.. «mm 0.0. «00 00b. «00 00.. «00 . .0 . «00 000 . . «00 000 . . «00 000 . .50 000 . nun 000 . 0 «X 0. 0 . o nun 000 . new 0000. 000k. .000.. 0h0..0 .000..I .0.0.I 00.0. .000. 000.. 0000. 00.0. .0...h .000 I 0000. .000. 0000. N000. 0000. 5000.. 0000.0 .00..! 0000. 0000. .000. 0000. 00.0. 0000.. 0000.0 0000.. 0h0.. 0000. 0000. 00. IO. 00. . n00 000 . 5.0 000 . :00 00. . £00 000. «00 000. «00 000. '00 0.0. «00 000.0 1K 000.0 ax 000. .0 000. IN .00..0 0000. 0h00.0 0000.0 0000.. hh00. 0000. 0000. h000.0 08.0. 0..0.0 0000.0 0000. 0000. 0050. 08.0.. h00..0 .000. 00.0.0 0.00.0 0h00.l 0.00. 0000. 0000. 0000.0 0000. 000..0 00.0.0 0050. 0000. 0000. 0000. 00 I0 000. :00 00.. n00 000. "um 00.. «00 000. p00 N00. .00 000. n00 0h0. ”00 0...0 IN 000.0 "N 000. «X .00.. AN 00.0.0 .000. 0000.0 .000.0 h000.l 0500.. 0005. 0000.0 0..0.0 0000. h000.0 000h.0 00h0. 0000.. 00.0. 0.0—.0 .000.0 0000. 0000.0 00.0.0 0000.. 0000.. 0000. 0000.0 00.0.0 0000. 00...0 00 0000.0 0000. 0000.. 0080. 00.0.0 I0 00\0I\Uul 00\0=\00£ 00\0I\UQI HMdPLD HM999 99 9 99 999 99.99999999999999 113 000. uflm NON. lam NNO. nww hoo._ ham ONO.— :90 0'0. new 090.8 1% —N0.0 AN non. ax ohflN.o NOON.N NehN. to-n. ONOh. vho@.0 0080.» DONG. boot. ouch. OOOG.O cowh.N N0§0. GOO». auto. 0090.n ”NO—.n ~96”. o_o¢. ovhfl. IO— nmh.— uflm h_a.§ [Hm ”DN. mum Nov.” .90 Ono.o mam NOD. cam 000.0— I” 'ah.0 1x 00¢.— 3% 0959.0 CN'N. ”ON—.n «Nbo. 0N0h._ whao.—~ QaON.O— Q09N.N ”who. _a_o.N Nouo.o oooN.h— NDOO.N Obvo. thh.N 0.0—.Ou O—hb.l OONO.N vhbo. Nflao.N 20 who. sum OON.— "Hm Of_. new no—.— nan DOO.N mam OON. mam ~09.” IX O~D.N 1X onQ.N 1% _WOD.O o—O¢.— Nhoo.N ONNO. Qoao.m DONO.N ova—.0 ONNN.0 000a. nhOo.o Doe—.0 0000.~ GQ¢O.N ONoc. 9000.9 thO.N OONO. ooaa.N 0000. nhQO.N EN HIdFmD flM03 00 lO—FCflDUB— 00_0IDD :00 050 Egg.— 117 000.~ n00 0hfl.0 '00 NDO.N Ax 000D.N ~v~0.0 v0b¢.0 0~NN.- 000. umm D00.~ n00 DD~.N 1x h080.0 0000.0 hfl0fl.0 0000.0 0D0.~ n00 N0b.“ uam 0~0.D 1x 0NN0.0 0NON.O ~000.“ 0000.0 mfldhmb 0<~0HPU<0 ¢H\mfl\000 v~0.~ n00 080.0 .00 050. 1% hD00.~I Dt~0.' 0NDQ.~ 000~.~I Q0~.~ num 8N0." '00 000.~ ax 0000.I 0Dh§.v 000~.I ~00h.~ 0DO.~ «mm 000.“ mam 00~.0 1X N00~. 0NDN.v 00N0.0 ND00.~ HMdFmD 0P000 fll\nl\UOD 00~.~ umm 000.~ sum -0. "mm 050.0 «00 N50.“ «00 ~N0. n00 0~D.0 n” Dh~.fll 3%. D00. ax 00D0.0 N~00.Dl 00—0.I #090. 0000. 00flh.f ~D~N.I 0000. 0Db¢. 0NOD. 0NNN.0 0DN0.NI 0D00.I N000. 0000. «0D~.D ~0Dfl.l Nb~0. 000v. 0hbv. 20~ 0-.~ umm 00fl.~ n00 0N0. n00 000.N :00 ~ND.N «00 0'0. n00 0h0.D u D00.I mi 0- . IN 0h00.0 0NN0.I 0000. 00~v. NDOO. N0h0.0 0~§N.N 0§-. know. 00DD. VN~0.0 0000.0I 0N0~. N~h0. ~00D. 0Dbh.v h000.~l ND00. ¢~0v. NOND. l0 00w. uum D~0. umm «00. ~00 000. mam 000.~ :00 D00. .00 «~N.D Ix D00.“ 1% 000. u «OND.D 0000. h-h. 000v. 00h~.~ 0000.D 0DNN.N 0~00. N000. 000~.~ 0~0D.D «000.0 0D00. N000. 0Nfl~.~ «005.0 0NON.N 0005. N000. ~hhfl.~ EN HHdeD mldeD 00\nfl\oel 00\n:\oufl 00\n=\ofll A<~0HPU<0 09000 Hudhmb mudfimb mudbmb §\E\§ E\B\§ Q~U E8 “'20 04.5.00 :01— Iéuhdmbulu Pflmzbm -0. "mm 500. «mm 00D. num 000. «00 0N0. n00 0h0.~ «00 h0~.~ n00 0D0. n00 0N0. 1x 0~N.v ax ~v~.~ ax 0~0. 1x ONDO. 00N0. 0800. 0000.0 N000.I 0000.! ~N~0. ~000. 0000. 00D0.0 0~bh.~ 0000.! ~000.l D000. 000'. «000.0 ~0h0.~ 00N0. 0580. 0000. ~000.N fhflh.~ I I0~ «00. «Mm 000. «00 ~Nv. :00 ~00. 1H0 000. n00 N~0. «00 ~00. 000 ~00. u00 0+0. "X 0~N.v ax 0D~.~ u 0D0. fix ~00N. D~00. .0~00. hh0fl.¢ 00Nh.~ h0~0. 0000. 00~h. h~00.0 00N~.N N00~. 0000. 00DD. h000.0 0D00. DO0N. 0~00. 0800. ¢0N0.v 0000. N0~0 I I0 0*~. mum bf0. «mm 009. n00 000. uflw ~00. «00 000.~ 000 000. "00 0N~. n00 N00.~ 1N ~0~.0 "x 000.0 nfi Dub. 1% 0&0~.~ 000'. D0~D.~ ~0V0.D 000~.fl N~N¢.~ D0~'. h0'0.~ 0000.8 ~00O.N N00v.~ ~Dflv. 0~00.~ 00ND.N 0010.0 0N0h.~ 00~¢. DO0N.N EN b000.0 "$00.0 00\d§\00£ 00\n!\00£ 00\nl\ool mudhhb mlflhhb H¥