Commercial and public health applications of DNA monitoring : a thesis presented in partial fulfillment of requirements of the degree of Doctor of Philosophy in Microbiology and Genetics at Massey University, Palmerston North, New Zealand. EMBARGOED until 1 August 2020

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This thesis reports analyses and findings from microbiology and metagenomic assessments of microorganisms at a beverage processing plant in Fiji. It includes metagenomics analyses from rivers and soils in Western Viti Levu. Chapter 1 provides a general overview of microbiological issues associated with product quality in beverage plants. It outlines regulatory expectations and accepted protocols for product quality assurance. It then introduces methods of comprehensive microbial assessments based on DNA sequencing technologies, some of which were applied in the present study. Chapter 2 addresses the issue of biofilms at the plant, asks and answers the question of whether existing Clean in Place (CIP) protocols are necessary and sufficient to ensure product quality. It was shown that ozonation is a redundant component of the current CIP protocols. Furthermore, it was established that CIPs could be extended from 24 hour intervals to 36 hour intervals without compromising product quality. These findings have led to procedural changes at the plant. This refinement has been estimated to annually save the plant US $11 million. Chapter 3 provides a comprehensive assessment of microorganisms at the plant (storage tanks, buffer tanks and boreholes) and in rivers and tributaries within a 10 km radius of the plant. Whole genome shotgun sequences showed that microbial compositions were similar in the rivers and boreholes. Although water bottled at the plant is drawn from an aquifer, the similarity of microbial compositions suggests that management of water quality at the plant needs to consider surrounding rivers that may affect aquifer water quality. Chapter 4 examines the microbial composition of soils within and outside the plant (farmlands). The microbial composition was found to be similar in these two zones. This suggests that environmental management plans also need to consider the close relationship between land-based activities within and surrounding the plant (farmlands). In both Chapter 3 and 4 a small proportion of reads were reported from organisms that have the potential to impact water quality. This included organisms with pathogenic potential and also organisms whose properties could affect the aesthetic quality of the beverage. Thus, this raises the need for their continuous monitoring and control. Chapter 5 compares two methods of comprehensive assessment (Whole Genome Shotgun Sequencing WGSS and 16S rDNA sequencing) with the Illumina sequencing platform. It was shown that WGSS sequence analysis provided up to 100 orders of magnitude more taxonomic resolution than 16S rDNA. 16S rDNA was nevertheless shown to be useful for drawing conclusions regarding composition differences with some samples. Thus, given its relative low cost in comparison to WGSS, 16S rDNA sequencing is likely to be a useful tool for some applications and questions in environmental microbiology. Chapter 6 reports a bioinformatic approach to characterise bacteria recovered from the beverage product at the plant. The genomes of these isolates contained pathogenicity, virulence and antibiotic resistance loci, raising the issue of the need for their possible future monitoring at the plant. One advantage of the bioinformatics approach adopted for characterisation of bacteria over characterisation using biochemical tests is that the sequence data obtained provide a means to now develop DNA amplification primers to be used in future monitoring. Chapter 7 describes loop mediated amplification (LAMP), and the possibility of using this isothermal DNA amplification approach for future monitoring at the plant. This method could be used to specifically target microorganisms of potential concern identified in comprehensive assessments, such as undertaken for the current project. LAMP methodology is currently seeing widespread uptake in clinical health, environmental monitoring and food safety sectors because of its low cost, robustness and quantitative nature.
Water, Microbiology, Analysis, Beverages, Fiji