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Title: Microbial quality control management in industry : approaches for assessing bioburden and community composition
Author: Davies, Adrienne
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2015
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Abstract:
Microbial contamination of industrial products and processes can endanger the health and safety of consumers and workers and negatively affect businesses by causing product spoilage and decreases in process efficiency. Major microbial contamination events can result in financial losses from product recalls, unplanned production downtime, loss of sales and litigation. Effective microbial monitoring is essential for detecting the presence of microorganisms and helps businesses control and maintain product quality. This thesis first examines the challenges in achieving accurate assessments of microbial load, which is defined as the quantity of viable microorganisms within a given sample matrix (Chapter 2). Classical culture techniques remain the most widely utilized and approved methods but their major shortcomings have limited their applicability in Quality Management systems employed to control contamination throughout product processing, handling, and distribution. Issues such as the long incubation times to achieve a result, the selective bias of culture conditions on species recovery, and the inability to detect damaged or injured cells are discussed. Rapid microbiological methods (RMMs) offer increased sensitivity and accuracy, can deliver results in near real-time, and could enable more effective and proactive management of microbial contamination problems. Nevertheless, their uptake in industry has been hindered by their higher operating costs and time-consuming proof-of-equivalency studies to the reference culture methods, which are required for approval by the relevant regulatory bodies. An overview of commercially available RMMs is provided and the suitability of each method is application- and industry-dependent. An industrial case study of microbial monitoring methods was performed with Omya AG (Switzerland) the top global producer of calcium carbonate (CC) slurries (Chapter 3). CC slurries are used as manufacturing fillers in a wide range of industries including paper, paints, construction and food. High microbial loads have been shown to adversely effect slurry quality and performance and negatively impact on customers’ finished products (Schwarzentruber, 2003). Quality Assurance (QA) managers rely on culture dip slides for detecting microbial load and directing biocide treatment to manage excessive population growth. RMMs offer Omya the potential for cost savings from effective biocide management and could help QA managers uncover and remedy microbial-related product spoilage sooner. The viability-based RMM CellFacts II (CFII) (CellFacts2014 Ltd., UK) combines electrical flow impedance and viable cell specific fluorescent staining to produce an accurate, real-time measurement of the contaminating population. To improve its usability and desirability to Omya the technology was optimized by streamlining sample preparation steps, the cost per test was reduced, and data presentation and interpretation was simplified. During this study CFII showed increased sensitivity and reliability compared to dip slides and highlighted the differences in preservation efficiency and bioburden levels in slurries preserved with biocide blends or by pHstabilization. Although pH stabilizers are added to slurries during production, Omya still relies upon biocides for managing any uncontrolled microbial growth detected during product storage or upon delivery to customers. The biocide-preserved slurries that were analyzed were maintained at pH 8.5-9.0 while the pH-stabilized slurry were pH 9.5-10.0. It was hypothesized that this 1-log difference in pH impacted upon microbial community composition and it was necessary to ensure that these populations were still susceptible to post-production biocide treatments. A multifaceted approach was used to characterize the communities and assess the coverage and limitations of each method (Chapter 4). Samples were collected and microbial load was determined by plate culture and CFII, and population diversity was elucidated by the recovery of species in culture and by 16S rRNA gene analysis by Terminal-Restriction Fragment Length Polymorphism (T-RFLP) and sequencing on the Illumina MiSeq (USA). A comparison of direct and indirect DNA extraction methods was performed to observe the efficiency and differences in DNA recovery from microbial species. In addition, DNA from the viable population was selectively amplified by treating isolated microbial cells with propidium monoazide (PMA), a dye that enters non-viable cells, binds DNA and inhibits PCR amplification. Biocidepreserved slurry showed lower diversity in culture compared to pH-stabilized slurry but displayed a higher number of operational taxonomic units (OTUs) by T-RFLP and sequence analysis in the microbiome analysis platform Quantitative Insights in Microbial Ecology (QIIME). The microbial communities were significantly different with Pseudomonas spp. associated with biocide-preserved slurry and alkaliphilic Bacillus spp. dominating in pH-stabilized products. The culture media conditions were inadequate for recovering the highly abundant alkaliphilic population in pHstabilized slurry and future culture work must be optimized on alkaliphilic media. 16S rRNA gene analyses produced a more truthful representation of the community and DNA extracted from PMA-treated cells revealed changes in population structure after 48 hours of incubation and in response to biocide addition. Slurry populations are dynamic; regular sample collection at various time points is necessary to achieve accurate detection and monitoring of microbial communities.
Supervisor: Not available Sponsor: Omya International AG
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.687143  DOI: Not available
Keywords: QR Microbiology
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