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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Date
2015
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Massey University
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Abstract
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.
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Keywords
Water, Microbiology, Analysis, Beverages, Fiji