GROUNDWATER CHEMISTRY AND MICROBIOLOGY IN A WET-TROPICS AGRICULTURAL CATCHMENT James Stanley B.Sc. (Earth Science). Submitted in fulfilment of the requirements for the degree of Master of Philosophy School of Earth, Environmental and Biological Sciences, Science and Engineering Faculty. Queensland University of Technology 2019 Page | 1 ABSTRACT The coastal wet-tropics region of north Queensland is characterised by extensive sugarcane plantations. Approximately 33% of the total nitrogen in waterways discharging into the Great Barrier Reef (GBR) has been attributed to the sugarcane industry. This is due to the widespread use of nitrogen-rich fertilisers combined with seasonal high rainfall events. Consequently, the health and water quality of the GBR is directly affected by the intensive agricultural activities that dominate the wet-tropics catchments. The sustainability of the sugarcane industry as well as the health of the GBR depends greatly on growers improving nitrogen management practices. Groundwater and surface water ecosystems influence the concentrations and transport of agricultural contaminants, such as excess nitrogen, through complex bio-chemical and geo- chemical processes. In recent years, a growing amount of research has focused on groundwater and soil chemistry in the wet-tropics of north Queensland, specifically in regard to mobile - nitrogen in the form of nitrate (NO3 ). However, the abundance, diversity and bio-chemical influence of microorganisms in our wet-tropics groundwater aquifers has received little attention. The objectives of this study were 1) to monitor seasonal changes in groundwater chemistry in aquifers underlying sugarcane plantations in a catchment in the wet tropics of north Queensland and 2) to identify what microbiological organisms inhabit the groundwater aquifer environment. This was completed by regular monthly & bi-monthly sampling and analysis of groundwater over 12 months through wet and dry seasons. Soil samples were also collected for microbiological analysis from a plantation paddock in the study area. It was hypothesised that denitrifying bacteria would be identified in soil and groundwater samples as a result of the - widespread application of nitrogen-rich fertilisers and that NO3 concentrations may vary with aquifer depths. - While NO3 concentrations in groundwater consistently remained within acceptable - environmental limits (< 10 mg/L, ANZECC) throughout the study, NO3 was found to occur more frequently and in higher concentrations in some bores than historical data indicates. This - can be attributed to infrequent historical sampling and analysis. NO3 concentrations were also - variable across examined sites with 2 shallow aquifer bores displaying constantly low NO3 - concentrations, indicating that aquifer depth is not necessarily a controlling factor on NO3 concentrations. Analysis of extracted sediment cores from the location of these 2 these bores Page | 2 revealed dense, kaolinite-rich clays overlying and underlying the aquifer material. The clays revealed strong discolouration from sulphur (S2-) and ferric iron (Fe3+) oxidation and X-Ray Diffraction (XRD) analysis showed the presence of goethite (FeO(OH)) and amorphous Fe- oxyhydroxides in the clays. Combined standing water level (SWL) data and groundwater - chemistry data suggest that the clays may be inhibiting the infiltration of NO3 and dissolved oxygen (DO) from the soil to the deeper aquifer material and that variable surface charge - associated with kaolinite clays may result in adsorption of DO and NO3 on clay surfaces. - Measured nitrite (NO2 ) concentrations and slow SWL recessions following rainfall indicate restriction of groundwater flow at one particular clay-rich site. This suggests an increased residence time for groundwater, allowing time for denitrification (DN) to progress and contributing to the creation of a DN “hot-spot”. - Bores with very low dissolved DO and NO3 concentrations consistently displayed relatively 2- 2- 2+ higher concentrations of sulphate (SO4 ), S and Fe . In anerobic conditions, Fe-oxide 2+ minerals may release Fe ions and precipitate pyrite (FeS2) in-situ with available sulphur from 2+ hydrogen-sulphide (H2S). This would account for the high concentrations of Fe in groundwater in some aquifers and the capacity for these particular aquifers to consistently 2- produce relatively higher SO4 concentrations, compared to groundwater sites with higher DO concentrations. This process requires the presence of sufficient S2-, which is most likely sourced from agricultural inputs (through application of gypsum (CaSO4·2H2O) and fertilisers) and to some extent from lithological sources (chemically weathered parent rock material). Metagenomic analysis of 16S ribosomal RNA (16S rRNA) from groundwater and soil samples showed that 99.58% of all classified organisms in the groundwater are bacteria, dominated by the phyla Proteobacteria, Firmicutes, Actinobacteria, Acidobacteria and Bacteroidetes, with archaea representing only ~0.42 %. The analysis was successful in classifying >80% bacteria in groundwater and soil to genus level (average: 86.71%), while an average of 38.96% of bacteria were classified to species level. This means that a large percentage of organisms remain unclassified to genus and species level. The analysis revealed a regular consortium of most-abundant bacteria across the whole catchment that are identified as being associated with common soil and plant process, dominated by the families Oxalobacteraceae, Comamondacea, Acetobacteraceae, Thermoanaerobacteraceae and the genera Janthinobacterium, Acetobacter, Curvibacter and Thermoanaerobacter). This indicates that broadscale agriculture has a direct influence on groundwater microbiota, assisted by considerable recharge from tropical rainfall. These bacterial species were present in both shallow (<10 m depth) and deep (>30 m depth) Page | 3 aquifers, suggesting common connectivity between soil water and groundwater. Within the communities of groundwater bacteria were also identified chemo-lithotrophic organisms who 2- 2- 2- are known to utilise S , SO4 or S2O3 (thiosulphate). These chemical metabolites are sourced from anaerobic subsurface environments, through the interaction of clay mineralogy and - - agricultural chemical inputs. Bacteria associated with reduction of NO3 and NO2 occurred in lower abundances in both groundwater and soil samples. Given that a large percentage of the bacteria species remain “unclassified”, there is still uncertainty regarding the biochemical influence of groundwater and soil organisms on the process of DN. Further metagenomic research incorporating the technique of identifying the abundance of gene coding for the production of nitrite-reductases enzymes as a marker for DN would provide more useful information regarding the DN potential of specific sites. The most abundant bacteria present in the soil samples from the paddock differed to those in groundwater samples and a marked difference was observed between most abundant soil bacteria species at different soil horizons (25 cm vs 65 cm depth). Less diversity in genus and species was observed at 65 cm depth, where the B soil horizon has a distinctly higher clay content. This indicates that variations in clay content, hence variations in permeability and capacity for gas-exchange and ion-exchange, influence bacteria species abundance and diversity. There are also indications that certain spikes in bacteria species abundance during the study period were linked to increases in dissolved chemical element concentrations in groundwater (e.g. Ca2+ & Mg2+ ) as a result of catchment-wide agricultural practices (such as the application of gypsum as a soil conditioner) following significant rainfall. It is also possible that the most abundant species of bacteria, C. lanceolatus, is having a direct effect on groundwater chemistry by means of biological precipitation of calcium-carbonate (calcite; CaCO3). This is the first study conducted in the Silkwood region, north Queensland, which provides a detailed look at seasonal changes in groundwater chemistry in the catchment and the first to produce any information about groundwater & soil microbiology in the region. The data from this research provides an important contribution to the emerging transdisciplinary field of groundwater microbiology. This enhances our understanding of groundwater contaminant dynamics in regions of intensive agriculture, both locally and globally. Page | 4 KEYWORDS Groundwater - Microbiology - Chemistry - Nitrate- Aquifer - Denitrification - Metagenomics Bacteria- Tropics - Sugarcane - Sulphate - Queensland - DNA Page | 5 TABLE OF CONTENTS Abstract ........................................................................................................................2. Keywords .....................................................................................................................5. Table of Contents .........................................................................................................6. List of Figures ..............................................................................................................7. List of Tables .............................................................................................................11. List of Abbreviations .................................................................................................12. Statement of Original Authorship ..............................................................................14.