Environmental Disturbance of Oligotrophic Bacteria and Effects on Water Quality in Deep Karstic Pools
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Environmental Disturbance of Oligotrophic Bacteria and Effects on Water Quality in Deep Karstic Pools by Andrea J. Hunter By Andrea Hunter Water Resources Program University of New Mexico Albuquerque, New Mexico 87131-0001 www.unm.edu/~wrp/ Publication No. WRP-11 April 2004 Note: This report is the Professional Project report of Andrea J. Hunter, submitted in partial fulfillment of the requirements for the Master of Water Resources degree at the University of New Mexico (May 2001). The project was supervised and approved by the following committee: Dr. Clifford Dahm, Department of Biology, UNM (Chair); Dr. Michael E. Campana, Water Resources Program and Department of Earth and Planetary Sciences, UNM; and Dr. Diana Northup, Department of Biology, UNM. ACKNOWLEDGMENTS I would like to acknowledge and thank Dr. Penny Boston for giving me the opportunity to continue her microbial work with biofilms and coliform monitoring in Lechuguilla Cave. Her added knowledge of lab procedures and cave protocols has helped me tremendously. To Dr. Cliff Dahm, my committee chair, who has spent numerous hours with me reviewing methods and results. His classroom encouragement, numerous protocol advice and professional style will stay with me throughout my career. I also thank Diana Northup, committee member, for her continuing guidance and support during the past two years. Her incredible resourcefulness extending from biological concepts to 'life as a graduate student formalities' has saved me from many sticky situations. Thank you to Michael Campana, Director of the Water Resources Program and committee member, for financial support, classroom encouragement and the opportunity to participate in the Masters of Water Resources Program. Thank you to the following cave guides and/or field help: Bob Bellospirito, Carlsbad National Park Service (Stan Allison, Dale Pate and Jason Richards), Laura Dong, Andy DuFrane, Dave Hamer, Robert Monczka, Mark Stover and Jim and Val- Hildreth Werker. I would like to give photo credits to Bob Bellospirito, Kenneth Ingham, Mark Stover and Val-Hildreth Werker. Lab help has been very much appreciated from Penny Boston, Diana Northup, John Craig, David Faguy, Mike Spilde and Jake Turin. Thank you to the following people and organizations who have helped fund this project: Dr. Michael Hunter D.O., Cave Research Foundation, National Speleological Society, Waste Management Education and Research Consortium, and the Charles H. and Anne Morrow Lindbergh Foundation. Additionally, I would also like to acknowledge the following funding sources from The University of New Mexico: Office of Graduate Studies-Research Project Travel and Student Research Allocations Committee Grants, 3% scholarship tuition waiver, and The Water Resources Program. Finally I would like to acknowledge my parents, Dr. Michael and Laura Hunter, and sister, Jessica, whose financial and emotional support has helped a great deal. ENVIRONMENTAL DISTURBANCE OF OLIGOTROPHIC BACTERIA AND EFFECTS ON WATER QUALITY IN DEEP KARSTIC POOLS by Andrea Jo Hunter B.S., Environmental Science/Biology, Northern Arizona University, 1998 M.W.R, Master of Water Resources, University of New Mexico, 2001 ABSTRACT Active intervention of humans in pristine water resource environments has caused both surface and ground waters to degrade in quality and natural purification capacity (Geldreich 1996). Groundwater is often considered to be less impacted than that of surface water due to the absence of climatic changes and storm water migrations (Geldreich 1996). Shallow karstic limestone areas (less than 30 m deep) have a greater chance of surface contamination since surface water runoff can rapidly percolate through areas lacking a protective bedrock layer. A deeper (485 m), cavernous environment such as Lechuguilla Cave, however, remains isolated from most percolation contamination making it an ideal, nearly virginal ecosystem for answering water quality and microbiological questions (Turin and Plummer 2000). When humans intrude on these pristine ecosystems, however, the resident microbial communities are jeopardized. Human-associated bacteria, prevalent on hands and fecal material, increase the chances for coliform contamination. Despite numerous conservation attempts made by Carlsbad Caverns National Park, researchers, mapping, exploration and restoration teams, some Lechuguilla cave waters have become contaminated with coliforms within the past several years. Research conducted during this study found coliforms residing between 0.4 and 27 CFU’s (colony forming units) / 100 mls in six different water locations. This study also addressed the persistence of these coliforms and their potential synergism with natural microbial biofilm populations, morphologically similar to Hyphomicrobium sp., whose growth is enhanced by VWR Tygon water siphoning hoses. Alternative fungus resistant hoses not supporting biofilm growth were also researched finding Silicon Masterflex and Teflon as the best replacements for the current VWR Tygon hoses. When groundwater environments, such as Lechuguilla Cave, become contaminated the process of restoring water purity is very slow (Geldreich 1996). Pools within Lechuguilla are a necessary source of drinking water for cavers and are critical in addressing both microbiological and water quality issues. Most importantly, the natural waterborne microbial populations could be adapting to and relying on our human inputs. The potential for a shift in resident microbial communities is a real threat when any pristine environment is invaded. Implementing further cave management recommendations such as sterile procedures at water sources, removal of the current VWR Tygon hoses and installation of a more inert thermoplastic will discourage nonnative, human-associated microbial growth and minimize further impact at these delicate cave pools. TABLE OF CONTENTS LIST OF FIGURES x LIST OF TABLES xii INTRODUCTION 1 Impacts Caused by Human Visitation: Contamination of Pool Sources 1 Problems to be Addressed 5 Lechuguilla Cave Geology and Speleogenesis 6 Biofilms 8 Dimorphic Prosthecate Bacteria 9 Coliforms 11 Hose Materials 13 Natural Rubber Latex 14 VWR Brand Tygon 14 Norton Tygon 15 Silicone Masterflex 15 Nalgene Premium tubing 16 Teflon-FEP tubing 16 THE WORKING HYPOTHESIS 18 OBJECTIVES 18 METHODS 19 Sterile Techniques 19 Describing Dominant Microbial Strain 19 Establishing Coliform Presence and Quantity 21 Identification and Enumeration 23 Calculations 23 Testing Coliform Growth with/without Biofilm 25 Identifying Carbon/Nutrient Sources 27 Determining Biofilm Growth on Tubings 28 RESULTS 29 Describing Dominant Microbial Strains 29 Establishing Coliform Presence and Quantity 32 Testing Coliform Growth with/without Biofilm 35 Identifying Carbon/Nutrient Sources 36 Determining Biofilm Growth on Tubings 37 DISCUSSION 38 Describing Dominant Microbial Strain 38 Establishing Coliform Presence and Quantity 38 Testing Coliform Growth with/without Biofilm 41 Identifying Carbon/Nutrient Sources 42 Determining Biofilm Growth on Tubings 43 CONCLUSIONS 45 MANAGEMENT RECOMMENDATIONS 46 SUGGESTIONS FOR FUTURE STUDY 49 APPENDICES 50 Appendix 1-Pictures of Study Sites in Lechuguilla Cave, Carlsbad 50 Caverns National Park, NewMexico. Figure A-Deep Secrets Pool collection site (located in the western branch. Figure B-Red Lake collection site (located in the western branch). Figure C-Lake Louise study area (located in the western branch). Figure D-Big Sky Pool sample collection (located in the southwest branch). Figure E-Oasis Pool (located in the western branch). Figure F-Snow White Passage drinking source (courtesy of Robert Monczka; located near E-F Junction). Figure G-Pearlsian Gulf study area (located in the southwest branch). Appendix 2-Protocols for Sample Collection in Lechuguilla Cave. 53 Coliform Quantification Protocol Coliform Positive/Negative Test Water Collection Tubing Experiment Directions for Biofilm Collection Slide Collection Appendix 3-Protocol for Acridine Orange/INT Slide Staining Procedure. 57 Appendix 4-Hyphomicrobium Medium Protocol. 59 Appendix 5-Protocol for Coliform/Biofilm Experiment. 60 Coliform/Hyphomicrobium Inoculations Preparation of Glucose Solutions Test for Coliforms Serial Dilution Appendix 6-Protocol for Total Coliform Media Preparation. 62 Appendix 7-Data from E. coli growth in the presence 64 of Hyphomicrobium culture and biofilm. Appendix 8-Directions to the Spectrophotometer for 65 Hyphomicrobium Readings. Appendix 9-Research and Collecting Permit Required for Entrance to 66 Lechuguilla Cave. Appendix 10-Investigator Contact Information 68 Appendix 11-Additional Tubing Details and Contact Information 69 LIST OF REFERENCES 70 i LIST OF FIGURES Figure 1-Location of Lechuguilla Cave (modified from Northup et al. 1992). 1 Figure 2-Plan view map of Lechuguilla Cave and study sites 3 (Modified from Northup et al. 1992). Figure 3-Red Lake study area has been closed due to coliform 5 contamination in the water source. Figure 4- Entrance Pit into Lechuguilla Cave (Andi Hunter). 6 Figure 5-“Model of hydrogen sulfide reaction with dissolved oxygen 7 near the water table, Carlsbad Cavern” (Hill 1987). Figure 6-Biofilms from siphoning hoses transferred to a cavers water bottle. 8 Figure 7-Electron micrograph of a thin section of a single 10 Hyphomicrobium cell (Madigan et al.1999). Figure 8-Sterile techniques applied while collecting microbial samples 19 at Lechuguilla Pools (Andi Hunter). Figure 9-Sterile slides hung at the interface. 20 Figure 10-The