Northumbria Research Link Citation: Lim, Jesmine (2014) Characterisation of the Prokaryotic community of Lake Suigetsu, Japan: towards a novel palaeoenvironment research biomarker. Doctoral thesis, Northumbria University. This version was downloaded from Northumbria Research Link: http://nrl.northumbria.ac.uk/id/eprint/27272/ Northumbria University has developed Northumbria Research Link (NRL) to enable users to access the University’s research output. Copyright © and moral rights for items on NRL are retained by the individual author(s) and/or other copyright owners. Single copies of full items can be reproduced, displayed or performed, and given to third parties in any format or medium for personal research or study, educational, or not-for-profit purposes without prior permission or charge, provided the authors, title and full bibliographic details are given, as well as a hyperlink and/or URL to the original metadata page. The content must not be changed in any way. Full items must not be sold commercially in any format or medium without formal permission of the copyright holder. The full policy is available online: http://nrl.northumbria.ac.uk/policies.html Characterisation of the Prokaryotic community of Lake Suigetsu, Japan: towards a novel palaeoenvironment research biomarker Jesmine Lim PhD 2014 Characterisation of the Prokaryotic community of Lake Suigetsu, Japan: towards a novel palaeoenvironment research biomarker Jesmine Lim Thesis submitted in partial fulfilment of the requirements of the University of Northumbria at Newcastle for the degree of Doctor of Philosophy Research undertaken in the School of Life Sciences and in collaboration with Newcastle University, Newcastle upon Tyne. October 2014 Abstract Sediment cores from Lake Suigetsu, Japan are recognised as a key record of past climate reconstruction because of the finely laminated sediments that provide precise event stratigraphy. Here, we study the relationship between the microbial communities in the lake sediments of Lake Suigetsu during validated episodes of environmental change. We use fossil DNA from the lake sediment and utilising the PCR-DGGE technique, we detected the presence of several taxa. Among the investigated sediment cores, the Acidobacteria community was found to be the most abundant while the Actinobacteria community was the least. The results showed that the overall bacterial community structure and their diversity were significantly affected by sediment depths, rather than the availability of nutrient (i.e. TOC and TN). The first event was the introduction of saline water in Lake Suigetsu. Historical records have described this event occurring during 1664 AD, which equates to the sediment depth of approximately 81.64 cm. A metagenomics study based on selected sediment depths has exhibited a shift in the bacterial taxa, consistent with the transition of lake salinity from freshwater to brackish. Bacillaceae and Clostridiaceae were found to be more predominant in the brackish sediments relative to the freshwater sediments. Evidence of the seawater incursion was found in the sediment depths between 82.16 and 83.16 cm. The second event was a climate event dated back to the Late Quaternary period. The results presented here show that bacterial diversity and species richness become increased when climate changed from a cold to warmer conditions. The metagenomics analysis on the sediment deposits has demonstrated distinctive differentiations in bacterial taxa during the climate transition from the colder to warmer episodes. This observation could be related to the rapid adaptation/tolerance of bacteria to environmental changes, or simply the effect of depth. Although the temperature- dependent δ15N isotope can be strongly correlated to the bacterial communities, the weak selectivity of the δ15N isotope could result in false correlation between the δ15N isotope and the diversity of the bacterial communities. I The application of molecular and culture-dependent techniques was used to characterise bacterial diversity in the sedimentary records of Lake Suigetsu. The culture-based techniques showed a better representation of high GC Actinobacteria while molecular techniques revealed a better profile of Gram negative bacteria. Furthermore, based on a polyphasic approach, several putatively new species have been identified, notably Actinobacteria strains that belong to the genera Dermacoccus, Dietzia, Leifsonia and Rhodococcus. Among the tested strains, a novel Rhodococcus isolate that was recovered from the freshwater sediment, merits recognition of new species status and the name Rhodococcus meromictica sp. nov is proposed. II Acknowledgements During the course of this doctoral research, many individuals have provided their help and it is my pleasure to acknowledge their support. First of all, I would like to thank Prof. Takeshi Nakagawa for his permission to access to Lake Suigetsu SG06 sediment cores for this project as without him this project would not be successful. Takeshi has provided invaluable guidance and a great deal of help during the time of core samplings. I would also like to show my gratitude to Takeshi for his willingness to stay late so that the extraction of sediment samples could be completed. I would also like to thank Dr. Amanda Jones for her patience, unfailing encouragement and guidance that she has provided during the past four years. I would also like to express my gratitude to Prof. Stephen Cummings for his great guidance in sampling strategies and in the preparation of this thesis, especially on molecular chapters. I would also like to thank Prof. John Woodward for his tremendous support and advice primarily on the geographical side of the study. I am indebted to my colleague, Dr. Chris Stewart who has provided an immense support on the metagenomics analysis. To the beautiful ladies, Dr. Meng Zhang and Dr. Qian Yang, I would like to thank them for their great advice and continuous personal support whenever it was needed the most. A special thank also goes to Mr. Adrian Blackburn from GENEIUS, Newcastle who has kindly re-sequenced a few of my samples for free. I would also like to thank him for his generosity in allowing me to access to MicroSeq software for 16S rRNA gene sequencing analysis. It has also been a wonderful time working with all my dear friends in lab A321 and cheers to our friendship. Last but not least, this thesis would not have been possible without the constant support, encouragement and love from my family, especially my dad, mom, brothers and sisters. I am also grateful to my soul mate, Hua Khee Chan who has always been supportive throughout my PhD, thank you. III Declaration I declare that the work contained in this thesis is all my own work and it has never been submitted or approved for any other award by this or any other university. Name: Signature: Date: IV Abbreviations 1D/2D-TLC One/Two dimensional thin layer chromatography AD Anno Domini AMS Accelerator mass spectrometry APS Ammonium persulphate ATP Adenosine triphosphate a.p.s.l. Above present sea level BioSiO2 Biogenic silica BP Before present bp Base pair Bølling Bølling-Allerød BSA Bovine serum albumin BLAST Blast local alignment search tool ca. Circa. (about/approximately) cal Calibrated years/Calendar years before 1950 Ca2+ Calcium ions CaCO3 Calcium carbonate Cl- Chlorine ions CO2 Carbon dioxide - CO3 Carbon trioxide ions 2 CO3 Carbonate 12C Carbon-12 12 CO2 Carbon dioxide-12 14C Carbon-14 CCA Canonical correspondence analysis COMX Cool mixed forest V δ15N delta-N-15 DCA Detrended correspondence analysis DDC Dewey decimal classifcation DGGE Denaturing gradient gel electrophoresis DNA Deoxyribonucleic acid dNTP Deoxynucleotide triphosphate FAMES Fatty acids methyl esters F’ Forward Fe2+ Iron (II) Fe2O3 Iron (III) oxide g Gram(s) × g Gravity GC Guanine-Cytosine GC-MS Gas chromatography-mass spectrometry GI Greenland interstadial H’ Shannon-Wiener Index H2S Hydrogen sulphide - HCO3 Bicarbonate ions INQUA International Union for Quaternary Research INTIMATE Integration of Ice-core, Marine and Terrestrial records K+ Potassium ions ka kiloannum, a unit of time equal to one thousand (103) years Kb Kilobase km kilometre km2 kilometre square Mg2+ Magnesium ions mm millimetre VI Na+ Sodium ions NERC Natural Environment Research Council - NO3 Nitrate + NH4 Ammonia N2 Nitrogen m Metre (s) M Molar MA Marine agar mA Milliamps MgSO4 Magnesium sulphate OTU Operational taxonomic unit 206Pb Lead 206 210Pb Lead 210 3- PO4 Phosphate PC1 Principal Component 1 PC2 Principal Component 2 PCR Polymerase chain reaction PLFA Phospholipid fatty acid PLS-DA Principle Least Square Discriminant Analysis Q-PCR Quantitative PCR 222Rn Radon 222 isotope R’ Reverse RDA Redundancy analysis Rr Species richness rDNA ribosomal DNA RNA Ribonucleic acid rRNA ribosomal RNA VII 2- SO4 Sulphate ions SG Suigetsu SRB Sulphate-reducing bacteria TAE Tris-acetate-EDTA TEDE Temperate deciduous forest TEMED N,N,N',N'-Tetramethylethylenediamine TN Total nitrogen TOC Total organic carbon TPA Trypticase peptone yeast agar T-RFLP Terminal Restriction Fragment Length Polymorphism U Units UV Ultraviolet V Volts v/v Volume per volume w/v Weight per volume WAMX Warm mixed forest yr Year VIII List of Figures Figure 1.1: Location of Lake Suigetsu (Nakagawa et al., 2005; 2012) Figure 1.2: Lake Suigetsu and its surrounding lakes Figure 1.3: Fine annually laminated varves of SG06 core of Lake Suigetsu (Nakagawa et al., 2012) Figure 1.4: Example of overlapping lamina patterns from parallel borehole