The Microbial ecology of groundwater at the Fishermans Bend urban renewal area in Melbourne, Victoria
Creators: name: Shikha, Sneha
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310607
subject: Nanobiotechnology
classificationCode: 401807
subject: Nanomaterials

description: Groundwater is the largest reservoir of accessible freshwater on Earth; nearly 2.5 billion people around the world rely solely on groundwater to fulfill their daily water needs, while around 50% of the global urban population utilise it as their primary source of drinking water. Increasing urbanisation and growing population have resulted in over-exploitation and contamination of groundwater aquifers due to various anthropogenic activities. There is an urgent need to manage our freshwater resources sustainably to ensure safe water is available for the global population, particularly as climate change is threatening to increase the burden on urban and regional water supplies. This urgent requirement was also highlighted by the United Nations in the last General Assembly as one of the sustainable development goals for the 2030 Agenda.

Groundwater ecosystems harbour a vast diversity of microbial communities which help in maintaining groundwater quality by natural attenuation or intrinsic bioremediation and thus play an integral role in providing essential ecosystem services. Environmental contaminants enter groundwater ecosystems due to various overlying activities and often carry the fingerprints of historic land use which impact and directly influence the biogeochemistry of groundwater by altering the native microbial community structure which in turn alters the various biogeochemical cycling processes. The overarching aim of this thesis was to characterise the variations in the structural assembly, diversity, distribution of taxa composition and ecosystem functions of groundwater microbial communities in Australia’s largest urban redevelopment project at the Fishermans Bend precinct in Melbourne, Victoria in relation to the physico-chemical environment which has been shown to be contaminated with elevated concentrations of multiple toxic heavy metals and other co-pollutants due to its complex legacy of extensive industrial manufacturing works for over a century. 

The research objectives of the first results chapter (Chapter 3) were to determine spatial and temporal variations in the various hydrogeochemical parameters including the physicochemical variables, major dissolved ions, and metals dissolved in groundwater. Multivariate statistical analyses including principal component analysis and hierarchical cluster analysis proved to be a powerful tool in deciphering spatial and temporal variations both within (n=3) and between (n=9) the groundwater wells sampled during the Autumn season in 2017 and the Spring season in 2018. Physicochemical factors including electrical conductivity, total dissolved solids, the major dissolved ions, redox potentials, dissolved oxygen, pH and some metals were identified as the key determinants in driving the spatial and temporal variations within and between the wells. A systematic approach was used to identify the distribution of predominant redox processes in each of the groundwater wells based on dissolved oxygen concentrations and identification of probable electron acceptors undergoing the reduction process sequentially in groundwater. Anoxic or mixed (oxic-anoxic) redox conditions were prevalent at the study site with either Fe (III)/SO42- or a combination of intermittent O2 – Fe (III)/SO42- as one of the dominant redox processes in the present study. Wide variations in spatio-temporal patterns and hydrogeochemical parameters were hypothesised to ultimately determine the selection of unique microbial taxa which may have adapted to the distinct spatial and temporal dynamics of the Fishermans Bend groundwater aquifers and were further studied in the second results chapter (Chapter 4). 

The spatial and temporal biogeographic patterns in the structure, diversity, composition, and putative functions of microbial (bacterial and archaeal) communities inhabiting groundwater ecosystems of the Fishermans Bend precinct were investigated by sequencing the 16S rRNA genes (V3-V4 region). The prokaryotic community composition in groundwater ecosystems was dominated largely by bacteria (&gt; 95%) and hence formed the core taxa of groundwater microbiome in the present study, with Proteobacteria as the predominant phyla detected in all the groundwater wells. A prominent seasonal and temporal variation was observed in the relative abundance of Proteobacteria which was nearly 20% higher during the Spring season as compared to Autumn season while the abundance of Chloroflexi, Acidobacteria, Actinobacteria, Bacteroides and Nitrospirae remained relatively stable over time. Substantial seasonal differences were also observed within the different subclasses of Proteobacteria as a 3-fold increase in the dominance of Betaproteobacteria was observed during the Spring season in 2018 compared to the Autumn season in 2017 when the abundance of Alphaproteobacteria was higher. The 16S rRNA gene analysis demonstrated the detection of a subset of both attached (e.g., Parcubacteria) and unattached (e.g., Planctomycetes) phyla in groundwater samples. Further, seasonal variation was also observed in the relative abundance of the attached and unattached microbial communities; the relative abundance of the attached communities was found higher during the Spring season whereas the Autumn season was predominated by the unattached and/or planktonic microbial communities.

Multivariate statistical analyses were used to link the microbiology of groundwater aquifers with the physical and chemical variables in a single study to help identify the potential drivers for each well. The most abundant microbial genera were further correlated with the groundwater geochemistry in Principal Component Analysis which helped identify the different microbial functional guilds known to perform nitrogen cycling, iron cycling, sulphur-oxidising, and methane-metabolising functions. As vital ecosystem functions of groundwater microbiota based on phylogenetic descriptors alone (from the perspective of taxonomy) are insufficient and rather speculative, their potential functional and metabolic profiles were also predicted using computational bioinformatics prediction tools including PICRUSt, KEGG orthologs and LEfSe analysis to estimate metabolic functions based on NGS microbiome data to understand their important ecological functions. Most functional modules identified within these metabolic pathways were essential for housekeeping functions and were necessary for sustaining microbial life within the subsurface aquifers. However, the diverse metabolisms predicted in groundwater ecosystems detected various pathway modules for carbohydrate metabolism, carbon-fixation, methane metabolism, nitrogen metabolism, sulfur metabolism in addition to the degradation of various aromatics and xenobiotics including the degradation of nicotine, toluene, xylene, benzene, naphthalene, cymene, benzoate, biphenyls and phthalates degradation.
Together with predictive functional analysis, carbon utilisation profiles using Biolog EcoPlates (at the community level) were used as a rapid and convenient approach to measure the functional response and metabolic potential of groundwater microbiome for 31 different carbon substrates. Carbon use efficiency was determined for the six guilds of carbon substrates including carbohydrates, carboxylic acids, polymers, amino acids, and amines in the third results chapter (Chapter 5). Phenol was identified as the most commonly used carbon substrate in all nine groundwater wells under aerobic as well as anaerobic conditions while amino acids were the least preferred carbon substrates. It was interesting to note that similar results were also predicted in the previous chapter which indicated contamination from similar aromatic compounds (e.g., benzene, toluene) in the predictive metabolic profile analysis. Together with predictive functional analysis, carbon utilisation profiles (at the community level) proved to be a useful tool in generating a metabolic fingerprint of groundwater microbial communities. 
Rehabilitation and restoration of polluted groundwater aquifers is inherently complex and requires foundational knowledge of microbial ecological baseline information particularly of the microbial community's structure, diversity, and taxonomic composition in regional groundwater. Any shifts or alterations in these microbial signatures may be used as reference information to indicate the rise in potential contaminants of concern in groundwater or may signify that recovery is currently in progress from past pollution. Community and functional profile analysis of the groundwater microbiome is essential to provide early warnings of deterioration in groundwater quality and ecosystem health as the functional metabolic activities of dominant microbial taxa may help identify the type of contaminants undergoing degradation. It is practically impossible to detect and determine the virtually limitless list of contaminants present in groundwater as the quantity of emerging contaminants are increasing exponentially with time due to increasing urbanisation. However, predicting the function of resident groundwater microbial communities may help indicate the specific contaminants of concern that are undergoing degradation or transformation and may potentially help control expenditures in detecting and targeting the contaminants of concern in groundwater. The novel insights revealed in the present study will assist in developing a future blueprint to help promote groundwater sustainability particularly, in times of climate change, thus contributing to risk mitigation.