Elucidation of the microbial N-cycle in the subsurface – key microbial players and processes Dissertation To Fulfill the Requirements for the Degree of “doctor rerum naturalium” (Dr. rer. nat.) Submitted to the Council of the Faculty of Biology and Pharmacy of the Friedrich Schiller University Jena by M.Sc. M.Tech. Swatantar Kumar born on 21-06-1984 in Baijnath Kangra, India Jena, December 2017 Reviewers: 1. Prof. Dr. Kirsten Küsel (Friedrich Schiller University Jena) 2. Prof. Dr. Erika Kothe (Friedrich Schiller University Jena) 3. Prof. Dr. Marcus Horn (Leibniz University Hannover) Day of the official disputation: 19.04.2018 Zugl. : Dissertation, Friedrich-Schiller-Universität Jena, [2018] TABLE OF CONTENTS ______________________________________________________________________________ TABLE OF CONTENTS LIST OF TABLES vii LIST OF FIGURES viii SUMMARY 1 ZUSAMMENFASSUNG (German) 3 1. INTRODUCTION 6 1.1 The biogeochemical nitrogen cycle 6 1.2 Excess reactive nitrogen and its ecological impacts 8 1.3 Perspective on reactive nitrogen with groundwater 9 1.4 How do karst oligotrophic aquifers respond to reactive nitrogen? 10 1.5 Key microbial players for the attenuation of ammonium and nitrate to dinitrogen 12 1.5.1 Anammox bacteria; a piece in the lithotrophy puzzle 12 1.5.2 Denitrifying microorganisms and their role in nitrogen cycle 16 1.6 Motivation of this thesis: understanding nitrogen loss in oligotrophic carbonate-rock 17 aquifers 1.6.1 Biogeochemical role of chemolithoautotrophic anammox in groundwater 18 1.6.2 Biogeochemical role of denitrifiers in oligotrophic groundwater 19 1.6.3 The Collaborative Research Centre 1076 AquaDiva 21 1.7 Research hypotheses and experimental approach 23 2. MATERIALS AND METHODS 26 2.1 Characterization of the study site 26 2.2 Groundwater sampling and characteristics 27 2.2.1 Groundwater samples for hydrochemistry 27 2.2.2 Groundwater samples for microbial community analysis using molecular methods 28 2.2.3 Groundwater samples for quantification of anammox-specific ladderane lipids 28 2.2.4 Groundwater samples for quantification of anammox and denitrification rates 29 2.2.5 Groundwater samples for microbial cultivation of planktonic denitrifiers 29 2.3 Rock chips sample (passive sampler) for microbial cultivation of attached 29 denitrifiers 2.4 Ladderane lipids quantification 29 2.5 Anammox and denitrification rates measurements from groundwater 30 2.6 Cultivation approaches for planktonic and attached denitrifying bacterial community 30 2.6.1 Groundwater based incubation and growth medium for enrichment of planktonic 30 denitrifiers iii TABLE OF CONTENTS ______________________________________________________________________________ 2.6.1.1 Growth medium for semi solid and solid Gelrite shake dilution and plates 32 2.6.1.2 Gelrite shake dilution method 32 2.6.1.3 Filter paper overlay method 32 2.6.2 Rock chips (passive sampler) based incubations for cultivation of attached 33 denitrifiers 2.7 Denitrification activity of an enriched chemolithoautotrophic consortium 34 2.7.1 Microcosm experiments set up M_I and M_II 34 2.7.2 Analysis of denitrification products by Raman spectroscopy from microcosm set 35 up M_I and M_II 2.7.3 Analysis of reactants, intermediates and products of chemolithoautotrophic 36 denitrification by ion chromatography from microcosm set up M_I and M_II 2.8 Nucleic acid extractions, PCR amplifications and cloning 37 2.8.1 Nucleic acids extractions from groundwater samples 38 2.8.2 Nucleic acids extractions from enrichment consortia 38 2.8.3 DNA extractions from pure denitrifying cultures 39 2.8.4 Polymerase chain reaction assays and clone libraries 39 2.9 Quantitative-PCR and Reverse Transcription-qPCR based analysis of microbial 42 abundances and potential gene transcripts from groundwater samples and enrichment culture 2.10 Sample preparations for Illumina MiSeq amplicon sequencing 43 2.10.1 Illumina MiSeq amplicon sequencing of groundwater derived DNA and RNA 43 2.10.2 Illumina sequencing based monitoring of the microbial community during the 44 microcosm experiments 2.11 Sequence data analysis 45 2.12 Statistical analysis 46 3. RESULTS 47 3.1 Groundwater chemistry of eight wells along the Hainich groundwater observation 47 transect 3.2 Co-occurrence of denitrifiers, anammox and aerobic ammonia oxidizing archaea and 49 bacteria in groundwater reveled by quantitative PCR 3.3 Anammox-hzsA transcriptional activity as an indicator of protein synthesis potential 53 of anammox bacteria in Hainich aquifer assemblages 3.4 Correlation with environmental parameters and observed gene abundances 55 3.5 Ladderane lipids provide further support for the presence of an active anammox 55 population iv TABLE OF CONTENTS ______________________________________________________________________________ 3.6 Nitrogen loss from pristine carbonate-rock aquifers is primarily driven by 56 chemolithoautotrophic anammox processes 3.7 Presence of rRNA as indicative of protein synthesis potential of Planctomycetes 57 organisms in limestone aquifers 3.8 Candidatus Brocadia fulgida dominated the anammox bacterial community of the 60 Hainich aquifer assemblages 3.9 Denitrifying community composition based on nirS-genes revealed dominance of a 62 chemolithotrophic community utilizing sulfur, hydrogen and iron as electron donors 3.10 Taxonomic identification of denitrifying enrichments and bacterial cultures 64 originating from the Hainich aquifer assemblages 3.10.1 Community structure of a chemolithoautotrophic denitrifying bacterial 64 enrichment originating from groundwater 3.10.2 Chemolithotrophic bacterial enrichment originating from rock-chips and gelrite 66 shake dilution (groundwater based) 3.11 Chemolithoautotrophic denitrification coupled to the oxidation of thiosulfate and 67 hydrogen 3.11.1 Microcosm experiment M_I: turnover of nitrogen and sulfur compounds by 67 enriched consortium 1E 15 3.11.2 Microcosm experiment M_II: complete denitrification ( N2 production) and 67 hydrogen utilization was witnessed by Cavity Enhanced Raman Spectroscopy (CERS) 3.11.3 Transcriptional activity of the enriched consortium during the two microcosm 69 experiments 3.12 Thiobacillus denitrificans as key transcriptionally active denitrifier in the 72 consortium 3.13 Shifts in community composition during the microcosm experiments 73 3.14 Taxonomic classification of pure mixotrophic and heterotrophic denitrifying 75 isolates 3.14.1 Heterotrophic denitrification by pure culture of strain 2_Acidovorax defluvii 77 4 DISCUSSION 78 4.1 Nitrogen loss is primarily driven by anammox processes in carbonate-rock aquifers 79 of the Hainich CZE 4.2 Co-occurrence of gene transcripts of aerobic and anaerobic ammonium oxidizers 81 suggest potential coupling of aerobic and anaerobic ammonia oxidation in suboxic groundwater 4.3 Transcriptionally active anammox bacteria in oxic and anoxic groundwater of the 82 two aquifer assemblages v TABLE OF CONTENTS ______________________________________________________________________________ 4.4 Low denitrification rates in suboxic groundwater of Hainich aquifer assemblages 83 4.5 Reoccurring large fractions of potential autotrophic denitrifiers oxidizing reduced 84 sulfur compounds, hydrogen, or reduced iron in suboxic groundwater of Hainich aquifer assemblages 4.6 Chemolithoautrophic enrichment culture obtained from groundwater representing 85 key metabolic features of the natural groundwater denitrifier communities 4.7 Denitrification by Chemolithotrophic consortium 1E, coupled to the oxidation of 88 thiosulfate and hydrogen 4.8 Growth of chemolithoautotrophic denitrifiers complemented with nirS- and nosZ- 89 transcriptional activity during microcosm experiments 4.9 Verification of Hypotheses and conclusions 91 4.10 Strengths of the current study 93 4.11 Limitations of study and considerations for future investigations 93 5. CONCLUSIONS 95 5.1 Anammox versus denitrification in carbonate-rock aquifers: Nitrogen loss from 95 pristine carbonate-rock aquifers of the Hainich Critical Zone Exploratory (Germany) is primarily driven by chemolithoautotrophic anammox processes 5.2 Physiological experiments with denitrifying microorganisms: Chemolithotrophic 96 consortium provided insights into the complexities of denitrifiers in oligotrophic groundwater and their nitrate attenuation capacity REFERENCES 98 APPENDIX 117 ACKNOWLEDGEMENTS 126 DECLARATION OF AUTHORSHIP 128 PUBLISHED ARTICLE 129 vi LIST OF TABLES ___________________________________________________________________________ LIST OF TABLES Table Page Table 1 | Stoichiometric reactions of anammox and denitrification. 12 Table 2 | PCR Primers used in this study for gene detection and quantification. 39-40 Table 3 | Detection of denitrification genes in groundwater isolates 75 Table 4 | Anammox and denitrification rates in marine and freshwater 80 environments. Table 5 | Composition of vitamin and trace element solution used in growth 117 media (NTC). Table 6 | Physicochemical parameters of groundwater samples from eight wells 118 of the Hainich aquifer assemblages. Table 7 | Correlation analysis (Spearman rank correlation coefficient) between 118 physicochemical parameters and gene abundances of groundwater samples Table 8 | Results of MiSeq Illumina amplicon sequencing of bacterial 16S rRNA 119 genes (DNA-based). Table 9 | Results of MiSeq Illumina amplicon sequencing of bacterial 16S rRNA 120 genes (RNA-based). Table 10 | Results of MiSeq Illumina amplicon sequencing of nirS genes in the 121 groundwater of eight wells across the two aquifer