Approaches to assessing microbial communities in soil, two examples: Biosurfactant production and phenanthrene degradation Item Type text; Dissertation-Reproduction (electronic) Authors Bodour, Adria 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 01/10/2021 06:55:39 Link to Item http://hdl.handle.net/10150/280136 INFORMATION TO USERS This manuscript has been reproduced from the microfiinn master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. 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ProQuest Infomnation and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 APPROACHES TO ASSESSING MICROBIAL COMMUNITIES IN SOIL, TWO EXAMPLES; BIOSURFACTANT PRODUCTION AND PHENANTHRENE DEGRADATION by Adria Angele Bodour A Thesis Submitted to the Faculty of the DEPARTMENT OF SOIL, WATER AND ENVIRONMENTAL SCIENCE In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 2002 UMI Number: 3061020 UMI UMI Microform 3061020 Copyright 2002 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Leaming Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 THE UNIVERSITY OF ARIZONA ® GRADUATE COLLEGE As members of the Final Examination Committee, we certify that we have read the dissertation prepared by Adria Angele Bodour entitled Approaches to Assessing Microbial Communities in Soil, Two Examples: Biosurfactant Production and Phenanthrene Degradation. and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy M^Maier Date Mark L. Brusseau Date •»- -/f Leland S. Pierson_ Date C :2 Charles P. Gerba DatV UO'^vO (XrS) fayne L'. Nicholson Date Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. .. Ks^j^ration Director Raina M. Maier Dace J STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the department or Dean of the Graduate College when in his or her judgement the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED: py^k^. DEDICATION To my husband, who supported me through this endeavor. 5 TABLE OF CONTENTS LIST OF ILLUSTRATIONS 7 LIST OF TABLES 8 ABSTRACT 9 CHAPTER 1. INTRODUCTION 12 EXPLANATION OF THE PROBLEM AND ITS CONTEXT 12 DEFINITION OF BIOSURF ACT ANTS AND BIOEMULSIFIERS 12 Biosurfactants 12 Bioemulsifiers 15 TYPES OF BIOSURFACTANTS 15 Glycolipids 16 Rhamnolrpids 16 Trehalose Lipids 21 Sophoro lipids 25 Mannosylerythritol Lipids 28 Other Glycolipids 30 Phospholipids, Neutral Lipids and Acidic Lipids 31 Lipoproteins 33 Surfactin, Fengycin, and Iturin 33 Lichenysin 39 Other Lipoproteins 40 Polymeric Biosurfactants 40 Emulsan 42 Other Polymeric Biosurfactants 43 SCREENING FOR SURFACTANT AND EMULSIFICATION ACTIVITIES 46 Screening for Surface or Interfacial Tension Reduction 46 Drop-Collapse Test 46 Tilted Glass Slide 47 Hemolysis of Red Blood Cells 47 Axisymmetric Drop Shape Analysis by Profile 48 Colorimetric Assay 49 6 TABLE OF CONTENTS Screening for Emulsification Activity 49 Emulsion Index 49 Turbidity Assay 50 INDUSTRIAL AND BIOTECHNOLOGY APPLICATIONS 50 Bioremediation 51 Biodegradation of Organics 51 Biodegradation of Organics in Metal-organic Co-contaminated Systems 54 Biosurfactants as a Flushing Agent 54 Rhamnolipid as a Flushing Agent - Metals 56 Industrial Processes 59 Medicine 61 Production of Fine Chemicals 62 Biological Control 64 SUMMARY 65 EXPLANATION OF DISSERTATION FORMAT 66 CHAPTER 2. PRESENT STUDY 67 REFERENCES 69 APPENDIX A: DISTRIBUTION OF BIOSURFACTANT-PRODUCING 92 MICROORGANISMS IN PRISTINE AND CONTAMINATED ARID SOUTHWESTERN SOILS APPENDIX B: PURIFICATION AND CHARACTERIZATION OF A NOVEL ... 117 BIOSURFACTANT FROM Flavobacierium sp. 36 APPENDIX C; TEMPORAL CHANGES IN AN INDIGENOUS 138 PHENANTHRENE-DEGRADING SOIL COMMUNITY DURING LONG-TERM EXPOSURE TO PHENANTHRENE 7 LIST OF ILLUSTRATIONS Figure Page L The types of aggregates formed by biosurfactants 14 2. The six major types of rhamnolipid produced by Pseudomonas aeruginosa. ... 17 3. The rhamnolipid biosynthetic pathway 20 4. Quorum sensing regulation of rhamnolipid (Rl) synthesis 22 5. Structure of trehalose lipids produced by species of Rhodococcus 24 6. Structure of sophorose lipids produced by Candida sp 26 7. Structure of mannosylerythritol lipids produced by Ca/iJjVsfa and L'5//7flgo 29 8. Structure of spiculisporic acid produced by Penicillium 32 9. Structure of the lipopeptides 34 10. The surfactin biosynthetic pathway 37 11. Regulation of surfactin synthesis 38 8 LIST OF TABLES Table Page L Lipoproteins produced by genera other than Bacillus 41 2. Polymeric biosurfactants from representative niicroorganisms 44 3. Stability constants for various organic ligands with cadmium and lead 58 9 ABSTRACT This dissertation is concerned with studying aspects o fthe ecology o fmicroorganisms from a functional perspective using different microbial populations in soils. In the first study, an investigation was done on the distribution of biosurfactant producing microorganisms. In the second study, temporal changes were observed in an indigenous phenanthrene degrading community following a long-term pubc cf phenanthrene. Bio surfactants are a unique class of compounds that have been shown to have a variety of potential applications in remediation of organic- and metal-contaminated sites, in enhanced transport of bacteria, in enhanced oil recovery, as cosmetic additives, and in biological control. The first part of the dissertation describes the first attempt at evaluating the distribution ofbiosurfactant-producing microorganisms in the environment. The goal of this study was to determine how commonly surfactant-producing organisms occur in pristine and contaminated sites by screening a series of 20 contaminated (metals and/or hydrocarbons) and uncontaminated soils. Results showed that biosurfactant-producing organisms are found in most soils with 45 of the 1305 isolates obtained during this study positive for biosurfactant production. These positive biosurfactants were grouped using repetitive extragenic palindromic (REP) PCR which yielded 16 unique isolates that were identified using 16S rDNA PCR. According to the 16S rDNA database search results there were only 11 unique isolates. However, isolates that had identical 16S rDNA database search results were different according to REP results and their surface tension analysis. This suggests that use of the present 16S rDNA database as an analysis tool may miss functional diversity in 10 populations analysis. The second part of the dissertation focuses on purifying and characterizing a novel biosurfactant discovered during the ecology study of biosurfactants. Culture supernatant of Flavobacterium sp. 36 grown in nitrogen-limiting mineral salts medium containing 2% glucose as the sole source ofcarbon and energy reduced surface tension to 40.7 mN/m After partial purification of the surfactant, it reduced surface tension to 26.0 mN/m indicating strong surface activity. The flavosurfactant was also a strong and stable emulsifier and enhanced the apparent solubility of hexadecane in pure water (0.003 ± 0.0002 mg/L) by more than 4 orders of magnitude. The flavosurfactant conditional stability constant for complexation with cadmium was measured to be 3.61 which is comparable to organic ligands such as oxalic acid and acetic acid. Mass spectroscopy indicates a mixture of 12 compounds that have molecular masses ranging from 626 to 682 g/mol. Nuclear magnetic resonance results indicates that the structure has a sugar, a fatty acid tail and one