Fixation of nitrogen by algae and associated organisms in semi- arid soils: identification and characterization of soil organisms Item Type Thesis-Reproduction (electronic); text Authors Cameron, R. E.(Roy E.) Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 27/09/2021 10:16:14 Link to Item http://hdl.handle.net/10150/191420 FIXATION OF NITROGEN BY ALGAE AND ASSOCIATED ORGANISMS IN SEMI..ARID SOILS: IDENTIFCATEON AND CHARACTERIZATION OF SOIL ORGANISMS by Roy Eugene Cameron A Thesis Submitted to the Faculty of the DEPARENT OF AGRICULT(JRAL CHEMISTRY AND SOILS In Partial Fulfillment of the Requirements 'or the Degree of MASTER OF SCIENCE In the Graduate College UNIVERSITY OF ARIZONA 19S8 STATET BY AUTHOR This thesis has been sunitted in partial fu.lfiThnent of re- quiruents for an advanced degree at the University of Arizona and is deposit in the University Library to be made available to borrow- ers under rules of the Library. Brief quotations from this thesis are allowable without specialpermission,provided that accurate acknowledgment of source is made. Requests for permission for extended quotatiQn from or reproduction of this manuscript in whole or in part may be granted 'by the headof the major department or the Dean ofthe Graduate Go].- ].ege when in their judgment the proposed use of thematerial is in the interests of scholarship.In ail other instances, howe-er, per- mission must be obtained from the author. (C SIGNED: APPROVAL BY THESIS DIRECTOR This thesis has been approved on the date shown below: W. H. FULLER Head,Department of Agricultural Chemistry and Soils 1929 Born July 16th, Denver, Colorado 19)47 Graduated from Lincoln High School, Tacoma, Washington 19)47-19)48 Attended the University of Washington, Seattle, Washington 195)4-1955 1950-1952U. S. Army Medical Laboratory Technician 19)48-1950 Attended the State College of Washington, Pullman, Washing- 1952-195)4 ton. B. S. in Agriculture 1953. B. S. in Bacterio1or and Public Health 195)4. 1955-1956 Research Laboratory Analyst, Hughes Aircraft, Thcson, Arizona 1956-1958Graduate Student in Soils, University of Arizona May 1955 M. S. University of Arizona Honor Societies: Sigma Alpha Omicron (Bact.), Pi Tan Iota (Pre-Med.), Beta Beta Beth, and Phi Lambda Upsilon, Sigma Xi (Assoc. Member) 1 ACKNOWLEDGMENT The author wishes to express his appreciation to the staffof the Department of Agricultural Chemistry and Soils, Universityof An-- zorn for their cooperation during the progress of this research. To Dr. Fuller, the research director, the authoris indebted for the basic idea of this research. His willing expenditure of time, stimulating suggestions, constructive criticism and over-allsupervision greatly contributed to the production of this research. Gratitude is also expressed to other departments andindividuals whose cooperation was necessary due to the nature of the thesistopic. Sincere thanks are here extended to a few of these individuals: Dr. W. S. Phillips, Head of the Botany Department,for invaluabletime spent with the author on algal identifications; Dr. K,F. Wertauan, Head of the Bacteriology Department, for the use of facilities;Dr. R. Kuyken- dali, Assistant Professor of Horticulture, for theuse of experimental chelates; and to Mr. J. E. Fletcher, USDA Soil ConservationService, for advice, suggestions, and invaluable information0 The author also wishes to express his indebtedness to the U. S. Atomic Energy Commission for funds which made this workpossible0 TABLE OF CONTENTS INTRODUCTION 1 LITERAJRE REVIEW 3 Laboratory Research on Nitrogen Fixation by Algae . 3 Importance of Soil Algae in Agriculture U Methodology 23 EXPERI1'UNTAL METHODS AND MATERIALS 32 Collection of Samples and Isolation for Purpose of Identification of Microorganisms 32 Nitrogen Fixation in Pure and Mixed Culture 39 Preliminary Procedure 39 Culture Solutions and Conditions for Nitrogen Fixation Io Apparatus for Nitrogen Fixation Li3 Nitrogen Fixation by Soil Crusts I8 Analyses for Nitrogen Fixation in Nutrient Solutions 50 RESULTS AND DISCJSSION 53 Isolation and Identification 53 Description of Blue-Green Algae Observed 65 Nitrogen Fixation with Pure and Mixed Cultures . 83 Organisms Not Demonstrating Nitrogen Fixation . 83 Organisms Demonstrating Nitrogen Fixation . 85 Chemical Analyses of Soil Crusts 96 Nitrogen Fixation by Soil Crusts 99 SUWIA.RY 103 BIELIOGRkPHY io6 iii LIST OF TABLES, FIGURES, AND PLATES Thbles Number Page Culture Media for Algae , 37 Total Micronutrient Solution for Algae. 38 Conditions for Nitrogen Fixation in Pure Culture .. 1414 14. Conditions for Nitrogen Fixation in Mixed Culture. 145 Description of Samples and Genera of Algae Within Them. 514 Genera of Algae Found in Arizona Soils 58 Nitrogen Fixation in Aqueous Solution by Blue-Green Algae in Pure Culture 87 Nitrogen Fixation in Aqueous Solution by Blue-Green Algae in Mixed Culture 88 Nitrogen Fixation in Aqueous Solution by Blue-Green Algae in Mixed Culture 89 Summary of Nitrogen Fixation in Culture Solutions . 91 Chemical Analyses of Samples of Soil Crusts Containing Algae and/or Lichens 97 Nitrogen Fixation by Soil Crusts in Moist Chambers . 101 Figures Following Number Page 1. Nitrogen Fixation by Soil Crusts in Moist Chambers , . 101 Plates Following Number Page i, Typical Sample Collection Area 32 iv Following Page Number Close-up of Algal Crusts 32 Close-up ol' Conspicuously Raised LichenCrusts . 32 314 14. Incubation of Soil Crusts in Moist Chambers Experiment, 314 . Growth of Nostoc sp. in Nitrogen Fixation Experiment on Nitrogen Fixation with Lyngbya . 314 Diguetti . .. Nitrogen Fixation Urder Conditionsof Controlled Tem- perature, Light, and Use of FilteredAir . 143 Temperature Using 8 Nitrogen Fixation Conducted at Room Natural Light and Filtered Air . 143 Nitrogen Fixation by Soil Crusts 143 Photoinicrograph of Chroococcus rufescens 81 Photoinicrograph of Anabaena sp. 81 Photomicrograph of Aphanocapsa grevillei . 81 V INTRODUCTION Nitrogen is one of the elements essential for plant growth.It is also the least abundant major essential element in the soil. In aria and semi-arid regions of Southwestern United States there isvery lit- tie organic matter in the soil, and therefore very little nitrogen (1b9),since soil nitrogen usually is almost wholly in the organic form. Amounts of organic matter in virgin Arizona soils usually do not exceed 0.1 to 1.0 percent (103). Soils lose nitrogen by one or more processes. Since Arizona soils are low in nitrogen, constant replenishment is required tomain- tain maximum crop production. Nitrogen losses may be balanced by re- plenishuients through the addition of animal andgreen manures, crop residues, artificial fertilizers, or by fixationprocesses involving symbiotic or nonsymbiotic microorganisms. In temperate regions agricultural soils left fallow forsome years are found to gain in nitrogen and organic matter, even though no manures, crop residues, or artificial fertilizers are applied (200). This increase is believed to be brought about by fixation of atmospheric nitrogen by microorganisms. Nitrogen fixation is considered as an Ira- portant means for gaining soil nitrogen, and of economicimportance in the maintenance of soil fertility. Although nonsymbiotic fixation has been reported to be especially important in desert soils (lit8),it has received little attention compared with the vast amountof research on 1 symbiotic nitrogen fixation. To better evaluate the importanceof nonsymbiotic nitrogen fixation in soil fertility, itis essential to gain information about the characteristics of theorganisms involved, their habits, a.nd the factors influencing their vitalprocesses. The problem involved in this research considers the identificationof soil algae, particularly the blue-green algae, and thedetermination as to which of these algae are nitrogenfixers. A study of their habitsis necessary for cultur- ing purposes and to obtain quantitative-i nfonnation on nitrogen fixation by the organisms in question. The general method used to solve these problems consisted of the procurementof soil crusts and the isolation and identification of organisms withinthe crusts0 The organisms ob- tained were then cultured and nitrogehfixation determined in pure and mixed cultures, using available laboratoryfacilities and equinent1 LI1RATURE REVIEW Laboratory Research on Nitrogen Fixation by Algae Frank, in 1859 (89), was the first to receiveattention for in- vestigation of fixation of free atmosphericnitrogen by blue-green algae in soil cultures,He found that there were gains in soilnitro- gen but mistakenly proposed, on the basis of his impureculture experi- ments, that all algae possessed the abilityto fix atmospheric nitrogen, In1891Schloesing and Laurent (202) suggested that algaeprobably sup- plied carbohydrates to nitrogen-fixing bacteria,and in189k Kosso- witsch (133) concluded that algae did not fixnitrogen even though some of his flasks of mineral salt solutions gained in nitrogenfixed with the organism Nostoc present. Bouilhac (3), Bouilhac and Giustini- ani (32, 33) further investigated the fixationof nitrogen with associ- ations of algae and bacteria. They inoculated sand cultures of buck- wheat, mustard, corn and cress with impure culturesof blue-green