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Ann Arbor, MI48106 STABILITY IN CLOSED ECOLOGICAL SYSTEMS: AN EXAMINATION OF MATERIAL AND ENERGETIC PARAMETERS A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN MICROBIOLOGY DECEMBER 1991 BY Jonathon Shaffer Dissertation Committee: Francoise Robert, Chairwoman Agnes Fok Roger Fujioka David Karl Bruce McConnell © copyright 1991 by Jonathon A. Shaffer iii ACKNOWLEDGMENT "All men must escape at times from the deadly rhythm of their private thoughts. It is part of the process of life among thinking beings." - R. Chandler. This dissertation is dedicated to the memory of Clair Edwin Folsome - the father of closed system ecology and my mentor. When searching a book on baby names, Ivone noticed that the name Clair Edwin translates as an "illuminating friend" - he was that exactly. Clair showed by example that science was not a burden but a privilege, an enjoyment and an exercise of spirit. His offering ~o the world of "little planets in bottles" was part paradigm and part whimsy. I hope in my dissertation the element of wonder didn't get too lost in numbers. I thank my colleague Andrew Brittain for being incorruptible, and for the intelligence to make this research exciting. It would otherwise have been a very dark laboratory after Clair's death. I also thank Don Obenhuber for getting me interested in CES in the first place. I thank Susanna Murata and Charles Buck for their decency as scientists and human beings and simply for their friendship. I thank Edward Siwak for his wise insights into iv the human condition and for a million laughs. I thank Ron Kennedy and John Spencer for showing me the ropes. And I thank Debra Brittain, Cindy Fukunaga, Ira Fujisaki, and Bettye Shines for being with me. You all mean a lot. I thank my dissertation committee for a job well done. I especially thank my chairwoman Francoise Robert for unselfishly rising to the occasion after Clair's death. This dissertation owes much to her. My mother, father, brother, sister, wife and children I wouldn't know where to begin - perhaps there is something in this work that Louie, AUdrey and Jondi-Dondi can use some day to make the big CES a better place to live. v ABSTRACT Closed ecological systems (CES) are gas-sealed microcosms. One-liter CES have been shown to maintain life in a consistent manner for over 25 years (Folsome and Hanson, 1986). Operationally: stability was defined as a constant total biomass (live plus dead organic material) for a period exceeding one turnover of all carbon pools. CES differing solely in total concentrations of carbon (30-150 roM), nitrogen (4-13 roM) and phosphorus (0.3-0.9 roM), or in a fundamental aspect of configuration (i.e., gas-open versus true materially-closed microcosms), were compared to determine: 1) which systems attain a stable state; 2) how biomass levels varied relative to material composition; and 3) whether a given stable biomass concentration was related to some unique set drawn from other parameter-defined relations. Twenty-seven of a total of 28 CES prototypes achieved stability in about 60 days. Stable biomass-C levels (typically 10-40 roM) could be described solely as a linear function of the total C concentration. Limiting N concentrations depressed expected biomass levels, whereas limiting P conditions caused an increase in biomass. vi Limitations in Nand P coincided to those predicted by the Redfield biomass C:N:P ratios (106:16:1). Various parameters measured in stable CES, such as detrital-C, live-C, carbon production, respiration, chlorophyll-a concentration, viable counts, and heterotrophic substrate range, were differentially affected by total C, N, and/or P concentrations. Furthermore, for a given radiant energy flux and comparative physical conditions, data showed no unique level of stable biomass, nor was a given biomass level indicative of some unique parameter-defined system configuration. Gas-open systems were similar in most attributes to closed systems except for about a 2- to 6-fold relative increase in detritus, fluctuating biomass levels, and longer time periods necessary for stabilization. These data suggest that laboratory-scale material closure may facilitate ecosystem stability. Overall, results suggest that CES prioritize the establishment of some characteristic bioelemental pool structure over that of energy flux or efficiency of energy utilization. Results also indicate that it should be possible to predictably scale-·up small CES as regards technological applications. vii TABLE OF CONTENTS ACKNOWLEDGMENT.. ..•• . .iv ABSTRACT .•.. .••• .. vi LIST OF TABLES.. •.•• .. xi LIST OF FIGURES •....•.••.•....•••.•. xaa a LIST OF ABBREVIATIONS. • • ••••....•. xiv KEY TO CARBON-NORMALIZED POOLS AND TURNOVERS .•..•. .xv CHAPTER I. BACKGROUND 1.1 Summary of Closed Ecological System Research .1 1.2 Scope of this Dissertation ••....•.•. .4 CHAPTER II. INTRODUCTION 2.1 In Vitro Microecosystems. ....••....5 2.2 Gas-open Microcosms in Ecologic Research •••••.•6 2.3 Closed System Biology .•...•..•.... .10 2.4 Terminology .•...••..•...••••• •. .12 CHAPTER III. LITERATURE ~EVIEW 1: CLOSURE AS A UNIQUE RESEARCH TOOL 3.1 Biology and Nonequilibrium Thermodynamic (NET) Theory ..••.•......•14 3.1.1 Problems in Applying NET Theory to Biology. ••.....•.•• 15 3.2 Bioregenerative Space Life Support •.•••••. 18 3.2.1 The Engineering Approach to Life Support . 20 3.2.2 The Holistic Approach to Life-Support ....23 CHAPTER IV. LITERATURE REVIEW 2: ECOLOGICAL CONCEPTS IMPORTANT TO CLOSED AND OPEN MICROCOSMS 4.1 Per~istence and Stability....•..•..•.•• 30 4.2 Persistence in sediment~Type Microecosystems... 31 4.2.1 Persistence in Nutrient-Amended Microecosystems.•..•.......•.. 31 4.2.2 Persis'tence in Nutrient-Poor Open Microcosms. •..••.. .33 4.2.3 Factors Influencing Persistence in Microecosystems. ...•..... .37 viii 4.3 The Concept of stability in Ecology. 38 4.3.1 stability in Microecosystems . .. 43 4.3.2 stability as Balanced Growth in (Open) Microcosms ...... .• .. 46 4.3.3 stability as Balanced Growth in CES: Importance of Closure ...........•50 4.4 Total Biomass Concentration as the Indicator of Microecosystem stability. ..•..... .... 52 CHAPTER V. DISSERTATION GOALS AND OBJECTIVES 5.1 Introduction.•....•.........•.... 54 5.2 Open Versus Closed Microecosystems 54 5.3 Stability and Material Potential in CES..•....•55 CHAPTER VI. MATERIALS AND METHODS 6.1 Closed System Media. .. 57 6.2 Total Biomass Carbon. .. •. ..... .. 60 6.3 Respiration. ••••. .. •.... 62 6.4 Live Biomass Carbon .... 65 6.5 Carbon-Specific DNA Synthesis Rate. .. •. .66 6.6 Chlorophyll-a. ..•....•....... •. .69 6.7 Heterotrophic Viable Counts and CFU Morphotypes ..•70 6.8 Metabolic Processes. .•.•...• .•. .71 6.9 Epifluorescence Acridine Orange Microscopy ••.•. 76 6.10 Heterotrophic Substrate Range•...•....... 78 6.11 Parameter Relations. ..•...... •. .. 78 CHAPTER VII. EFFECTS OF BIOLOGICAL DIVERSITY ON STABILITY IN CES 7.1 Preliminary Gnotobiotic Experiments... 0 ••••• 81 7.2 Effects of Inoculum Concentration .. •... 84 7.3 Experimental Design. ..•.. .. • ....•..•87 7.4 Results ................... G ••• 88 7.5 Discussion. .... 108 7.6 Conclusion. ................... 110 CHAPTER VIII. ASPECTS OF STABILITY AND MATERIAL BUDGETS IN OPEN VERSUS CLOSED MICROCOSMS 8.1 Introcluction. ..... .. ........•... .111 8.2 Experimental Design. .. 113 8.3 Results. .•.... .. •........... 114 8.4 Discussion. .... .. 127 8.5 Conclusion...................•..137 ix CHAPTER IX. RESPONSE OF STABLE CLOSED SYSTEMS TO OPENING 9.1 Introduction. • • ••••••••••••••139 9.2 Experimental Design •••••••••••••••• 139 9.3 Results. , Cl ••••••••••••••••••••141 9.4 Discussion. ••••••••••• • .148 9.5 Discussion Open