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Examining the Nutritional Requirements of Acidophilic Archaea Eastern Illinois University The Keep Masters Theses Student Theses & Publications 2014 Examining the Nutritional Requirements of Acidophilic Archaea "Ferroplasma acidarmanus" strain fer1 Yudong Qu Eastern Illinois University This research is a product of the graduate program in Biological Sciences at Eastern Illinois University. Find out more about the program. Recommended Citation Qu, Yudong, "Examining the Nutritional Requirements of Acidophilic Archaea "Ferroplasma acidarmanus" strain fer1" (2014). Masters Theses. 1367. https://thekeep.eiu.edu/theses/1367 This is brought to you for free and open access by the Student Theses & Publications at The Keep. It has been accepted for inclusion in Masters Theses by an authorized administrator of The Keep. For more information, please contact [email protected]. Examining the Nutritional Requirements of Acidophilic Archaea "Ferroplasma acidarmanus" strain fer1 (TITLE) BY Yudong Qu THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master in Biological Sciences IN THE GRADUATE SCHOOL, EASTERN ILLINOIS UNIVERSITY CHARLESTON, ILLINOIS 2014 YEAR I HEREBY RECOMMEND THAT THIS THESIS BE ACCEPTED AS FULFILLING THIS PART OF THE GRADUATE DEGREE CITED ABOVE THESIS COMMITTEE MEMBER DATE THESIS COMMITTEE MEMBER DATE THESIS COMMITTEE MEMBER DATE The Graduate School~ EA'iTER.N ILLINOIS UNIVERSITY .. Thesis Maintenance and Reproduction Certificate FOR: Graduate Candidates Completing Theses in Partial Fulfillment of the Degree Graduate Faculty Advisors Directing the Theses RE: Preservation, Reproduction, and Distribution of Thesis Research Preserving, reproducing, and distributing thesis research is an important part of Booth Library's responsibility to provide access to scholarship. In order to further this goal, Booth Library makes all graduate theses completed as part of a degree program at Eastern Illinois University available for personal study, research, and other not-for-profit educational purposes. Under 17 U.S.C. § 108, the library may reproduce and distribute a copy without infringing on copyright; however, professional courtesy dictates that permission be requested from the author before doing so. Your signatures affirm the following: D The graduate candidate is the author of this thesis. D The graduate candidate retains the copyright and intellectual property rights associated with the original research, creative activity, and intellectual or artistic content of the thesis. D The graduate candidate certifies her/his compliance with federal copyright law (Title 17 of the U. S. Code) and her/his right to authorize reproduction and distribution of all copyrighted materials included in this thesis. D The graduate candidate in consultation with the faculty advisor grants Booth Library the non­ exclusive, perpetual right to make copies of the thesis freely and publicly available without restriction, by means of any current or successive technology, including by not limited to photocopying, microfilm, digitization, or internet. D The graduate candidate acknowledges that by depositing her/his thesis with Booth Library, her/his work is available for viewing by the public and may be borrowed through the library's circulation and interlibrary loan departments, or accessed electronically. D The graduate candidate waives the confidentiality provisions of the Family Educational Rights and Privacy Act (FERPA) (20 U.S. C.§ 1232g; 34 CFR Part 99) with respect to the contents of the thesis and with respect to information concerning authorship of the thesis, including name and status as a student at Eastern Illinois University. I have conferred with my graduate faculty advisor. My signature below indicates that I have read and agree with the above statements, and hereby give my permission to allow Booth Library to reproduce and distribute my thesis. My adviser's signature indicates concurrence to reproduce and distribute the thesis. "/\ J ( Jh\ o\0rcJ 5 Ll.RJ\M LS Graduate Degree Program < Please submit in duplicate. Examining the Nutritional Requirements of Acidophilic Archaea "Ferroplasma acidarmanus" strain ferl Master's Thesis Research, Spring 2012 - Fall 2014 YudongQu Department of Biological Sciences Eastern Illinois University Charleston, IL 61920 Acknowledgement I would like to take this opportunity to gratefully acknowledge the help and advising of Dr. Kai F. Hung, as my graduate research supervisor in the Department of Biological Sciences at Eastern Illinois University, for his untiring support, patience and encouragement, and for helping disseminate my research in scientific conferences throughout the research project. I am also grateful to Dr. Steven L. Daniel and Dr. Scott J. Meiners at the Department of Biological Sciences at Eastern Illinois University, for being members of my thesis committee and for providing feedback to the research. I want to thank Dr. Steven L. Daniel for sharing the reagents and lab materials of his laboratories. I also want to thank Dr. Edward Treadwell from the Chemistry Department for sharing reagents. I also acknowledge the assistance from Caitlin M. Greene, who helped carried out experiments as an undergraduate researcher. My research was greatly supported by the Graduate Assistant Award by the Department of Biological Sciences, Eastern Illinois University. I am thankful to the Graduate School at Eastern Illinois University for awarding me the Williams Travel Award for attending scientific meetings including Illinois State Academy of Science (ISAS) and American Society for Microbiology (ASM). Last but not the least, I wish to thank my family, my parents Xiumin Zhu, Minhao Qu and my fiancee Ruohan Zhang for supporting me spiritually. 2 Contents Page no. Abstract 4 Introduction 6 Methods and Materials 17 Composition and making of ferl 's media and it's grow condition 17 Growth observation test 18 Limiting factors for fer 1 growth 19 Defined medium test 22 Results 24 Growth observation test 24 Limiting factors for ferl growth 29 Defined medium test 42 Discussion 47 Growth observation test 47 Limiting factors for ferl growth 49 Defined medium test 53 Future Direction 63 References 64 3 Abstract Ferroplasma acidarmanus strain ferl (ferl) is an archaeal microaerophilic acidophile (pH 0 to pH 3) with a chemolithotrophic metabolism. As an efficient consumer of sulfate, ferl plays an important role in the biogeochemical process of pyrite dissolution at acidic site. Confirming the genomic prediction of metabolic gene functions in this organism requires functional assays, which cannot be carried out until a defined medium is obtained. The objective of the current study was to replace the 0.1 % (w/v) yeast extract component in the growth medium, mfer, with defined components for carbon, nitrogen, and vitamin needs. In order to replace the 0.1 % (w/v) yeast extract in mfer, possible sources for carbon (glucose, short-chain carbon compounds, organic acid mixture), nitrogen (alanine, ammonia, nitrate, Casamino Acids), and vitamins ( l 0 vitamins) were tested in different combinations for their ability to support growth. Various growth conditions were also tested. Serial passages ( 11100 dilution) were carried out to eliminate carry-over effects. Growth was monitored by optical density (590 nm), visual inspection, and most probable number estimation, as appropriate. Multiple independent trials with at least 2 repeats of each trial were conducted for statistical analyses. Results showed that optical density reading is a relatively reliable method to quantify the growth of fer 1 in comparison with most probable number estimate (R2=0.904). A solid mfer medium was developed and shown to be able to support the growth of ferl, although no individual colony was observed. 48 well-plate was unsuitable for growth quantification but able to identify positive growth. Yeast extract was unable to support growth at either high (0.5% w/v) or low (0.01 % w/v) concentrations. Glucose had no effect on ferl 's growth with concentration of 0.1 % w/v or 0.01 % w/v. Increasing FeS04 concentration from 20 g/L to l 00 g/L inhibited growth of ferl. Base medium concentration significantly (p=0.046) increased the growth of ferl in a medium 4 with lower concentration of yeast extract and excessive accessible energy. No defined medium that support visible growth was found, mainly due to the unique carbon usage of fer 1. Elucidation of a defined medium to support growth of fer 1 continues. Discovering a defined medium will enable research into the metabolic capacities offerl, which will enhance our understanding of how extremophiles exist at the boundaries of life and raise the accuracy of bioinformatic predictions. 5 Introduction The current classification of biological organisms lists Archaea, Bacteria and Eukarya as the three domains of life, where Archaea has been incorporated as the third domain by Woese and Fox in 1977 (I) (Figure I). Archaea are unicellular, prokaryotic microorganisms. Although they share some bacterial (energy generation and gene organization) and eukaryotic (DNA and RNA polymerases) traits, Archaeal organisms are distinguished by their reliance on ether lipids in their cell membranes, unique rRNA structure, and many other unique characteristics (2-4). Most microbes found in extreme habitats, such as hot springs, salt lakes, hydrothermal vents, acid mine drainage (5), are archaea. Current phylogenetic classification separates the Archaea domain into four major phyla: Crenarchaeota, Euryarchaeota, Nanoarchaeota, and Korarchaeota. Most of the cultivated archaeal
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