Genes and Microbes Impacting the Geochemistry of Arsenic Mobilised Aquifers in Bangladesh and ...

299 Pages·2017·8.53 MB·English

by  Edwin Gnanaprakasam

Genes and Microbes Impacting the Geochemistry of
Arsenic Mobilised Aquifers in
Bangladesh and Cambodia

A thesis submitted to The University of Manchester for the degree of
Doctor of Philosophy
in the Faculty of Science and Engineering

2017

Edwin Thadheu Gnanaprakasam
School of Earth and Environmental Sciences

0
Table of Contents
List of Figures …………………………………………………………………………………………………………….. 6
List of Tables ……………………………………………………………………………………………………………… 9
List of Abbreviations………………………………………………………………………………………………….. 11
Thesis Abstract …………………………………………………………………………………………………………. . 13
Declaration ……………………………………………………………………………………………………………….. 14
Copyright Statement …………………………………………………………………………………………………. 15
Acknowledgements ………………………………………………………………………………………………….. 16
About the Author ……………………………………………………………………………………………………… 18
Chapter 1 : Introduction ............................................................................................................... 19
1.1. Project context ................................................................................................................. 19
1.2. Aims and objectives ....................................................................................................... 20
1.3. Thesis structure .............................................................................................................. 22
1.4. Paper status and author contributions ......................................................................... 25
Chapter 2 : Literature Review ...................................................................................................... 27
2.1. Arsenic general introduction ......................................................................................... 27
2.2. Arsenic geochemistry .................................................................................................... 30
2.2.1. Arsenic minerals and compounds ......................................................................... 30
2.2.2. Arsenic speciation ................................................................................................... 31
2.3. Arsenic bio-geochemical cycle ...................................................................................... 33
2.4. Geomicrobiology of arsenic mobilisation..................................................................... 35
2.4.1. Extracellular interaction ........................................................................................ 35
2.4.2. Intracellular interaction ........................................................................................ 36
2.4.2.1. Methylation and demethylation ..................................................................... 36
2.4.2.2. As(III) oxidation ............................................................................................. 38
2.4.2.3. As(V) reduction............................................................................................... 38
2.5. Role of other elements in the arsenic biogeochemical cycle ....................................... 40
2.6. Metagenomics of arsenic ............................................................................................... 41
2.6.1. 16S rRNA gene based community analysis ............................................................ 42
2.6.2. Functional genes analysis (targeted amplification and sequencing) .................. 44
2.6.3. Whole genome sequencing (WGS) based community analysis and gene
annotation ....................................................................................................................... 50
2.7. Metatranscriptomic analysis of arsenic mRNAs ........................................................... 51
2.8. Metaproteomic analysis of arsenic proteins ................................................................. 52
2.9. Summary ........................................................................................................................ 54

1
Chapter 3 : Methodology ............................................................................................................. 55
3.1. Introduction ................................................................................................................... 55
3.2. Sampling methods ......................................................................................................... 55
3.3. General methods involved in the molecular analysis................................................... 57
3.3.1. DNA extraction ....................................................................................................... 57
3.3.2. RNA extraction ....................................................................................................... 58
3.3.3. Protein extraction .................................................................................................. 58
3.3.4. Primer design ......................................................................................................... 59
3.3.5. Thermal cycler and polymerase chain reaction (PCR) ......................................... 59
3.3.6. Gel electrophoresis and documentation ............................................................... 59
3.3.7. DNA and RNA quantification ................................................................................ 60
3.3.8. SequalPrep™ normalisation of PCR products ....................................................... 62
3.3.9. Pooling ................................................................................................................... 62
3.3.10. Next generation sequencing ................................................................................. 62
3.3.10.1. Roche/454 (Pyrosequencing) ......................................................................... 63
3.3.10.2. MiSeq and HiSeq (Illumina) .......................................................................... 65
3.3.11. High performance computing (HPC) ................................................................... 66
3.3.12. Bioinformatics tools .............................................................................................. 66
3.4. Metagenomic methods .................................................................................................. 69
3.4.1. 16S rRNA gene based community analysis ............................................................ 70
3.4.1.1. Roche 454 pyrosequencing and QIIME pipeline ............................................ 70
3.4.1.2. MiSeq and the Amplicon analysis pipeline .................................................... 72
3.4.2. WGS based metagenomic analysis ........................................................................ 74
3.5. Metatranscriptomics methods ...................................................................................... 76
3.6. Proteomic methods ....................................................................................................... 77
3.7. Geochemical methods ................................................................................................... 78
3.7.1. pH and Eh ............................................................................................................... 78
3.7.2. Diffuse reflectance spectrum: ................................................................................ 79
3.7.3. X-Ray fluorescence ................................................................................................. 79
3.7.4. EXAFS spectra for iron and arsenic ....................................................................... 79
3.7.5. Analysis of trace elements ...................................................................................... 81
3.8. Statistical methods ........................................................................................................ 82
3.8.1. O2-PLS method (Orthogonal Projection to Latent Structure) ............................. 82
3.8.2. Spearman’s rank correlation ................................................................................. 83

2
Chapter 4 : Paper I - The microbial community structure and arsenic biogeochemistry in
two arsenic impacted aquifers in Bangladesh…………………………… .............................................. 84
4.1. Introduction ................................................................................................................... 85
4.2. Materials and methods .................................................................................................. 89
4.2.1. Study site ................................................................................................................ 89
4.2.2. Sample collection ................................................................................................... 90
4.2.3. Sediment chemistry ................................................................................................ 91
4.2.3.1. X-Ray fluorescence ........................................................................................... 91
4.2.3.2. EXAFS spectra for iron and arsenic ................................................................ 92
4.2.4. Aqueous chemistry ................................................................................................ 94
4.2.5. Microbial Ecology .................................................................................................. 94
4.2.5.1. 16S rRNA Based Bacterial Community Analysis ............................................. 94
4.2.5.2. Functional Gene Analyses .............................................................................. 95
4.2.6. Statistical Analyis: ................................................................................................. 95
4.3. Results ............................................................................................................................ 96
4.3.1. Site F........................................................................................................................ 96
4.3.1.1. Sediment chemistry ......................................................................................... 96
4.3.1.2. Aqueous chemistry .......................................................................................... 98
4.3.1.3. Microbial ecology ............................................................................................ 98
4.3.1.3.1. 16S rRNA gene based community analysis ............................................... 98
4.3.1.3.2. Iron-reducing bacteria ............................................................................. 100
4.3.1.3.3. Arsenic-metabolising bacteria .................................................................101
4.3.1.3.4. Nitrate/Nitrite-metabolising bacteria .................................................... 102
4.3.1.3.5. Sulphate-reducing bacteria ..................................................................... 102
4.3.1.3.6. PCR confirmation of functional genes ................................................... 103
4.3.2. Site B ...................................................................................................................... 103
4.3.2.1. Sediment chemistry ........................................................................................ 103
4.3.2.2. Aqueous chemistry ........................................................................................ 105
4.3.2.3. Microbial ecology ........................................................................................... 105
4.3.2.3.1. 16S rRNA community analysis ................................................................ 105
4.3.2.3.2. Iron-metabolising bacteria ..................................................................... 107
4.3.2.3.3. Arsenic-metabolising bacteria ................................................................ 107
4.3.2.3.4. Nitrate/nitrite-metabolising bacteria .................................................... 108
4.3.2.3.5. Sulphate reducing bacteria ..................................................................... 108
4.3.2.3.6. PCR confirmation of functional genes ................................................... 109
4.3.2.4. Correlation between the microbial and mineral interphase........................ 109
4.4. Discussion ...................................................................................................................... 111
Supporting Information …………………………………………………………………………………………… 116

3
Chapter 5 : Paper II - Application of two block latent variable regression analysis (O2-PLS)
of microbial and geochemical data to identify potential arsenic cycling microbes in
Cambodian aquifers .................................................................................................................... 124
5.1. Introduction .................................................................................................................. 125
5.2. Materials and methods ................................................................................................. 128
5.2.1. Study site ................................................................................................................ 128
5.2.2. Sample collection .................................................................................................. 129
5.2.3. Geochemistry ........................................................................................................ 129
5.2.4. Molecular (DNA) analysis .................................................................................... 130
5.2.4.1. DNA extraction and library preparation for MiSeq ...................................... 130
5.2.4.2. 16S rRNA gene analysis pipeline .................................................................... 131
5.2.4.3. The analysis of arrA gene ............................................................................... 131
5.2.5. Uni- and multi-variate analyses ........................................................................... 132
5.2.5.1. O2-PLS method (Two-way Orthogonal Projection to Latent Structure) ..... 132
5.2.5.2. Spearman’s rank correlations ........................................................................ 133
5.3. Results ........................................................................................................................... 133
5.3.1. Geochemistry ......................................................................................................... 133
5.3.2. Molecular ecology ................................................................................................. 135
5.3.3. arrA marker gene analysis .................................................................................... 139
5.3.4. Uni- and multi-variate analyses of geochemistry and microbes......................... 140
5.3.4.1. Spearman’s rank correlations ......................................................................... 140
5.3.4.2. O2-PLS model ................................................................................................. 141
5.4. Discussion ..................................................................................................................... 144
Supporting Information ………………………………………………………………………………………. ... 148
Chapter 6 : Paper III: Metagenomic and metatranscriptomic studies reveal the diversity of
microbes and their functional genes that could influence the arsenic redox cycle in
aquifers in Bangladesh ................................................................................................................ 156
6.1. Introduction .................................................................................................................. 157
6.2. Materials and Methods .................................................................................................161
6.2.1. Site description .......................................................................................................161
6.2.2. Sample collection and processing ........................................................................ 162
6.2.3. Metagenomics Methods ....................................................................................... 163
6.2.3.1. 16S rRNA based community analysis ............................................................. 163
6.2.3.2. Whole genome sequencing (WGS) based metagenome analysis ................ 164
6.2.4. Metatranscriptomics methods ............................................................................. 165
6.2.5. Geochemical methods .......................................................................................... 166
6.2.5.1. Analysis of trace elements in sediment ......................................................... 166
6.2.5.2. Analysis of trace elements in water .............................................................. 167

4
6.3. Results ........................................................................................................................... 167
6.3.1. XRF and ICP-MS based As, Fe and S quantification ............................................ 167
6.3.2. 16S rRNA based microbial community analysis (Site F and Site B) .................... 169
6.3.3. 16S rRNA gene based community analysis of two contrasting aquifers ............. 173
6.3.4. WGS based Metagenome ..................................................................................... 174
6.3.4.1. Reads, scaffolds and bins ............................................................................... 174
6.3.4.2. Prokaryotic communities .............................................................................. 175
6.3.4.3. Arsenic Genes ................................................................................................ 176
6.3.5. Arsenic related metatranscriptomics ................................................................... 178
6.4. Discussion ..................................................................................................................... 179
Supporting Material ………………………………………………………………………………………. ..........183
Chapter 7 : Conclusions and future work directions ................................................................. 192
7.1. Conclusions ................................................................................................................... 192
7.2. Future work directions ................................................................................................. 194
List of References ……………………………………………………………………………………..………………196
Appendix I: Supporting Information-2 for Chapter 4 ............................................................. 226
Appendix-II: A laboratory experiment on As(V) respiration and dissimilatory
arsenate reductase protein expression by Shewanella sp. ANA -3 …………………………235

5
List of Figures
Chapter 1 : Introduction ............................................................................................................... 19
Figure 1-1. The context and approaches of the research presented in the thesis ............ 20
Chapter 2 : Literature Review ..................................................................................................... 27
Figure 2-1. The geographic occurrence of groundwater arsenic pollution……………….. .... 29
Figure 2-2. The Eh-pH diagram for As at 25oC and one atmosphere pressure with
total arsenic 10-5 mol. l-1 and total sulphur 10-3 mol.L-1 ................................................. 32
Figure 2-3. Arsenic bio-geochemical cycle mediated by microorganisms………………….. .. 33
Figure 2-4. Mechanisms of the microbial transformations of arsenic in the
environment. Source: (Lloyd and Oremland, 2006) ........................................................ 39
Figure 2-5. Schematic depiction of the various aspects of metagenomic analysis .......... 42
Figure 2-6. Arsenic gene clade found in different dissimilatory arsenic respiring
bacteria ............................................................................................................................... 45
Chapter 3 : Methodology ............................................................................................................. 55
Figure 3-1. Summary of instruments, techniques and bioinformatics tools used in
this project…………………………………………………………………………………………………………………… 55
Figure 3-2. Field photos on sediment and water sampling for metaomic analysis…… .... 56
Figure 3-3. Bioanalyser-Agilent high density nucleic acid chips………………………………… ... 61
Figure 3-4. The next generation sequencing (NGS) machines used in the project…… ... 63
Figure 3.5. An illustration of a cycle in a pyrosequencing reaction…………………………… ... 64
Figure 3-6. An illustration of the steps involved in sequencing by means of the
synthesis method used in Illumina MiSeq and HiSeq ..................................................... 65
Figure 3-7. A diagrammatic depiction of the primer structure used for the
pyrosequencing PCR .......................................................................................................... 70
Figure 3-8. The workflow of the 16S rRNA gene based community analysis using the
454 sequencing platform and QIIME downstream analysis……………………………… ............ 71
Figure 3-9. A diagrammatic depiction of the primer structure used for the MiSeq
indexing PCR………………………………………….. ........................................................................ 73
Figure 3-10. The workflow of the 16S rRNA gene based community analysis using the
MiSeq sequencing platform and amplicon analysis pipeline ........................................... 74
Figure 3-11. The work flow of the Whole Genome Sequence (WGS) analysis using the
HiSeq sequencing platform and metagenomic pipeline .................................................. 75
Figure 3-12. The schematic workflow of the metatranscriptomic analysis using the
HiSeq sequencing platform ............................................................................................... 76
Figure 3-13. Proteomics workflow used to analyse the expression of Arsenate
Reductase (ARR) in Shewanella sp. ANA-3 ....................................................................... 78
Figure 3-14. The O2-PLS method used for the multivariate analysis of geochemical
and molecular data……………………………………………………………………………………………………… . 83

6
Chapter 4 : Paper I - The microbial community structure and arsenic biogeochemistry in
two arsenic impacted aquifers in Bangladesh ………………………………………………………… ............... 84
Figure 4-1. The geolocation of the study sites with the arsenic concentration………… .... 90
Figure 4-2. Chemical and microbial ecology of sediments and water fr0m Site F
aquifer ................................................................................................................................. 97
Figure 4-3. Chemical and microbial ecology analysis of sediments and water from
Site B aquifer……………………………………………………………………………………………………………….. 104
Figure 4-3. Top Spearman rank correlation (p=0.01) plots relating microbial
communities to mineral species ........................................................................................110
Figure 4-S1-1. PCR based confirmation of functional genes………………………………………… . 117
Figure 4-S1-3. Top 9 correlation of bacteria with arsenic at site F…………………………….. ... 121
Figure 4-S1-3. Top 9 correlation of bacteria with arsenic at site F…………………………….. ... 121
Figure 4-S1-4. Top 9 correlation of bacteria with Fe and As at site B………………………….. 122
Figure 4-S1-5. Top 9 correlation of bacteria with arsenic at site B…………………………….. . 122
Figure 4-S1-6. Top 9 correlation of bacteria with Fe and As at site F and B……………… .. 123
Figure 4-S1-7. Top 9 correlation of bacteria with Arsenic at site F and site B…………… .. 123
Chapter 5 : Paper II - Application of two block latent variable regression analysis (O2-PLS)
of microbial and geochemical data to identify potential arsenic cycling microbes in
Cambodian aquifers .................................................................................................................... 124
Figure 5-1. Sampling transects of T-Sand (along the Bassac river) (Sample sites LR-01
through LR-09) and T-Clay (along the Mekong river) (Sample sites LR-10 through
LR-14) ................................................................................................................................ 128
Figure 5-2. A schematic diagram of the orthogonal projection to latent structure
(O2-PLS) model used to analyse the correlation between geochemistry and microbes
in the aquifer system ........................................................................................................ 132
Figure 5-3. Box chart representing summary geochemical statistics on a logarithmic
scale for the composition of Cambodian groundwater and surface water ..................... 134
Figure 5-4. Bar diagrams summarising the number of reads and OTUs obtained in
each sample in 16S rRNA community analysis using MiSeq ........................................... 135
Figure 5-5. Weighted principle coordinate analysis (PCoA) plot of betadiversity
among the samples ........................................................................................................... 136
Figure 5-6. Percent abundance of bacteria belonging to different phyla and classes,
and the micro molar concentration of arsenic ................................................................ 137
Figure 5-7. A bar diagram representing the total number of prokaryotes correlating
to various geochemical parameters through Spearman’s rank correlation .................... 140
Figure 5.8. Joint component scores plot showing the similarity between geochemical
and microbial data ............................................................................................................. 141
Figure 5.9. The joint loadings of geochemistry and microbial blocks ............................ 142
Figure 5-10. Arsenic prediction using the PLS-R-RFE algorithm .................................... 143

7
Figure 5-S-1. Sampling transects of T-Sand (along the Bassac river) and T-Clay (along
the Mekong river) ............................................................................................................ 148
Figure 5-S-2. 16S rRNA gene based community analysis workflow…………………………… .. 149
Figure 5-S-3. A diagrammatic depiction of the primer structure used for the MiSeq
indexing PCR ..................................................................................................................... 150
Figure 5-S-4. Gel documentation of PCR based amplification of arrA gene. The
second stage of nested PCR yielded 625 base pair amplified product………………………. .. 150
Chapter 6 : Paper III: Metagenomic and metatranscriptomic studies reveal the diversity of
microbes and their functional genes that could influence the arsenic redox cycle in
aquifers in Bangladesh ................................................................................................................ 156
Figure 6-1. The sampling sites of Site F, Site B and CW-CAT ..........................................161
Figure 6-2. As, Fe and S concentration in sediment and groundwater samples ............ 168
Figure 6-3. Bar diagrams summarising the number of reads and OTUs obtained in
each sample in the 16S rRNA community analysis using MiSeq ..................................... 170
Figure 6.4. Weighted and unweighted principle coordinate analysis (PCoA) plots of
betadiversity among the sediment and water samples from Site-F and Site-B ............... 171
Figure 6-5. Relative abundance of bacteria belonging to different phyla and
classes………….. ................................................................................................................... 172
Figure 6-6. Close relatives of the bacterial species and their relative abundance in
the two contrasting aquifers CAT and BA14-3, revealed through 16S rRNA gene based
community analysis .......................................................................................................... 173
Figure 6-7. Phylogenetic tree for samples BA14-3 and CAT ............................................ 176
Figure 6-8. Number of bins containing translated genes ................................................ 177
Figure 6-S-1. The workflow of the 16S rRNA gene based community analysis using
the MiSeq sequencing platform and amplicon analysis pipeline.................................... 183
Figure 6-S-2. Workflow for the whole genome sequencing (WGS) based gene and
community analysis using the HiSeq sequencing platform and metagenomic
pipeline .............................................................................................................................. 184
Figure 6-S-3. Schematic workflow of the metatranscriptomic analysis using the
HiSeq sequencing platform .............................................................................................. 185

8
List of Tables

Chapter 2 : Literature Review ...................................................................................................... 27
Table 2-1. Functional genes involved in the arsenic redox cycle and in arsenic
metabolism ......................................................................................................................... 46
Table 2-2. Targeted amplification of the arrA gene from the metagenome……………… ... 47
Table 2-3. Targeted amplification of the As(III) oxidase gene from the metagenome. .. 48
Table 2-4. Targeted amplification of the As(V) reductase gene (detox) from the
metagenome ....................................................................................................................... 49
Table 2-5. Arsenic related transcriptomic works ............................................................... 51
Table 2-6. Respiratory arsenate reductase from different bacteria…………………………… ... 53
Chapter 3 : Methodology ............................................................................................................. 55
Table 3-1. The differences between MiSeq and HiSeq2500………………………………………… .. 66
Table 3-2. The bioinformatics tools used in this research………………………………………….. .. 67
Chapter 4 : Paper I - The microbial community structure and arsenic biogeochemistry in
two arsenic impacted aquifers in Bangladesh………………… ......................................................... 84
Table 4-S1-1. Chemical and molecular ecology analysis of sediments and water from
the Site F aquifer*……………………………………………………………………………………………………….. .118
Table 4-S1-2. Chemical and molecular ecology analysis of sediments and water from
the Site B aquifer*……………………………………………………………………………………………………….. .119
Table 4-S1-3. Correlations between mineralogy and molecular data……………………….. .. 120
Chapter 5 : Paper II - Application of two block latent variable regression analysis (O2-PLS)
of microbial and geochemical data to identify potential arsenic cycling microbes in
Cambodian aquifers………………………………………………………. ......................................................... 124
Table 5-S-1. The extract of relevant data of geochemistry previously published by
Richards, Magnone, et al., (2017)………………………………………………………………………………… . 151
Table 5-S-2. The extract of relevant data of arsenic speciation previously published
by Richards, Magnone, et al., (2017)…………………………………………………………………………… . 152
Table 5-S-3. Significant microbial variables correlating arsenic based on PLS-RFE…. .. 153
Table 5-S-4. Microbes representing arrA genes in sample LR14-30…………………………….. 154
Chapter 6 : Paper III: Metagenomic and metatranscriptomic studies reveal the diversity of
microbes and their functional genes that could influence the arsenic redox cycle in
aquifers in Bangladesh ................................................................................................................ 156
Table 6-S-1. Summary of the geochemical analysis for Site F used in the analysis….. ... 186
Table 6-S-2. Summary of the geochemical analysis for Site B used in the analysis…. ... 186
Table 6-S-3. Two contrasting aquifers………………………………………………………………………… 186
Table 6-S-4. Total percentage of reads representing different families of prokaryotes
from a total of 32 samples………………………………………………………………………………………….. . 187

9