Doctoral Degrees (Microbiology)

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The abundance and diversity of Acidobacteria in fynbos soil: a closer look at culturability and function
(Stellenbosch : Stellenbosch University, 2021-04) Conradie, Tersia Andrea; Jacobs, Karin; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH SUMMARY: The Acidobacteria are considered to be one of the most widespread and highly abundant soil bacterial phyla. This phylum was first described in 1997 with only three cultured representatives. Currently, the Acidobacteria is divided into 15 class-level subdivisions, of which only 5 subdivisions contain the 62 successfully cultured and fully described species in the Acidobacteria. The reason for their low representation in culture collections, is partly due to their unculturable, or difficult to culture nature. The application of 16S rRNA gene techniques has revealed that this phylum can represent almost 50% of the soil bacterial community, averaging around 10-20% of the global soil bacteria. Their proliferation in soils suggests that the Acidobacteria play an important role in biogeochemical processes. Microorganisms are an essential part of the terrestrial environment and are important in maintaining ecological functions. These functions are especially important in biomes where nutrient availability is low, and plants depend on their symbiotic relationships with the soil microbiome. One example of such an environment include the fynbos biome in the Cape Floristic Region (CFR) of South Africa. Despite the dominant presence of the Acidobacteria in several habitats, little is still known about their diversity and distribution in the fynbos biome. The aim of this study, therefore, was to explore the Acidobacterial communities in fynbos soils with the use of 16S rRNA gene sequencing, and how they respond to environmental change, as well as agricultural practices. Further, we aimed to isolate as many species as possible from the fynbos biome, and taxonomically characterise novel species. In Chapter 2, we explored the distribution patterns of the Acidobacteria in different fynbos soils from native conservation areas, and in Chapter 3 how the Acidobacteria responds to seasonal changes and the cultivation of Aspalathus linearis (rooibos) and Cyclopia spp. (honeybush), two indigenous plants used in commercial agriculture. A total of 27 soil samples were collected at three nature reserves, namely Jonkershoek, Hottentots Holland, and Kogelberg. In addition, data from two previous studies from our research group, with GenBank accession numbers DRA003953 for Cyclopia spp. (honeybush) and DRA004000 for Aspalathus linearis (rooibos), were included in our analysis. A total of 33 acidobacterial operational taxonomic units (OTUs) were identified in the nature reserve samples, and a total of 32 and 31 OTUs were identified for honeybush and rooibos, respectively. The majority of OTUs in all samples were classified as representatives of subdivisions 1, 2, and 3. Significant differences were observed in the distribution and composition of these OTUs between nature reserves, between seasons for both honeybush and rooibos, as well as between the agricultural practices in some cases. Several OTUs and subdivisions correlated significantly with soil pH, potassium, phosphorus, and in some instances carbon and calcium. In Chapter 4, we successfully isolated two novel species, both within subdivision 1. We proposed the classification of strain HDX4T as the type strain of Edaphobacter sabuleum nom. prov., and strain ADX1T as the type strain of Terriglobus capensi nom. prov. Based on the genome sequences, both strains had the genomic potential for several complete carbohydrate metabolic pathways, organic nitrogen metabolism, as well as several survival mechanisms that contributes to their survival in the soil environment. In short, this study has contributed greatly to our knowledge of the Acidobacteria and their distribution and diversity in the fynbos biome. The successful isolation of two novel species were added to the list of cultivated Acidobacteria from around the globe.
Antibiotic resistance in surface waters and biofilm-response to environmental contaminants
(Stellenbosch : Stellenbosch University, 2021-12) Tucker, Keira; Wolfaardt, Gideon M.; Botes, Marelize; Feil, Edward; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Ensuring water security for the future has become important due to rapid urbanisation and diminishing freshwater resources. South Africa’s water resources are scarce and as a result, reclamation of alternative freshwater resources such as treated wastewater is being investigated. There is growing evidence that drinking and wastewater treatment is either non- compliant to quality standards or lacking in certain communities. In areas with no infrastructure for wastewater removal, open sewers create a health risk for humans, animals, and the environment. Poor antimicrobial stewardship, over-use and incorrect disposal has led to increased resistance to antibiotics, rendering some bacterial infections untreatable. There is a concern that sub- inhibitory concentrations of antibiotics create a selection pressure that promotes horizontal gene transfer and emergence of bacterial communities that are resistant to antibiotics. Antibiotics, antibiotic resistant bacteria (ARB), as well as other contaminants that have been shown to promote antimicrobial resistance (AMR) such as heavy metals, enter surface waters and wastewater treatment works (WWTW) in trace concentrations via multiple pathways. As a result, WWTW are deemed hotspots for the emergence and dissemination of AMR. In addition, environmental waters are home to various matrices, including biofilms that are especially problematic in a clinical setting due to their antibiotic resistant and persistent nature. The research presented in this dissertation aimed to contribute to the knowledge surrounding the abundance of ARB in WWTW and surface waters in a South African context. Although ARB and antibiotic resistance genes (ARG) were detected in WWTW effluent, the abundance of both were reduced compared to the influent, suggesting that WWTW played a role in reducing AMR in receiving waters, while exposure to sub-inhibitory concentrations of antibiotics did not result in a significant change in the number of target ARG in isolates selected as representatives of a cultured population. This was emphasised in an expanded study that monitored various regions over a year. In addition, it was shown that surface waters, biofilms and sediments influenced by anthropogenic activities from residential and industrial sectors had higher prevalence of ARB compared to samples influenced by agricultural activity. Metagenomic analysis revealed that ARG relating to efflux pumps were the most common compared to those specific for target antibiotics. Due to heavy-metals and antibiotics being present in the environment in trace concentrations, exposure of mixed-community biofilms to sub-inhibitory concentrations of these contaminants was investigated. AMR in the biofilms did not increase, but it was suggested that the sub-inhibitory exposure promoted the development of persistent mixed community biofilms. Treatment interventions are crucial for removing pollutants and AMR already present in the environment. However, with due recognition of the complexity involved when considering humans, animals, the environment and a diverse pool of contaminants, this dissertation argues the need to expand the approach for mitigation of emergence or dissemination of AMR in the environment by incorporating greater emphasis on antibiotic stewardship, policies around antibiotic usage in all sectors, and overall public awareness.
Assessing the occurrence and mechanisms of horizontal gene transfer during wine making
(2009-12) Barnard, Desire; Bauer, Florian; Wolfaardt, Gideon M.; University of Stellenbosch. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Saccharomyces cerevisiae is the most commonly used organism in many fermentation-based industries including baking and the production of single cell proteins, biofuel and alcoholic beverages. In the wine industry, a consumer driven demand for new and improved products has focussed yeast research on developing strains with new qualities. Tremendous progress in the understanding of yeast genetics has promoted the development of yeast biotechnology and subsequently of genetically modified (GM) wine yeast strains. The potential benefits of such GM wine yeast are numerous, benefitting both wine makers and consumers. However, the safety considerations require intense evaluation before launching such strains into commercial production. Such assessments consider the possibility of the transfer of newly engineered DNA from the originally modified host to an unrelated organism. This process of horizontal gene transfer (HGT) creates a potential hazard in the use of such organisms. Although HGT has been extensively studied within the prokaryotic domain, there is an urgent need for similar studies on their eukaryotic counterparts. This study was therefore undertaken to help improve our understanding of this issue by investigating HGT in a model eukaryotic organism through a step-by-step approach. In a first step, this study attempted to determine whether large DNA fragments are released from fermenting wine yeast strains and, in a second step, to assess the stability of released DNA within such a fermenting background. The third step investigated in this study was to establish whether “free floating” DNA within this fermenting environment could be accepted and functionally expressed by the fermenting yeast cultures. Finally, whole plasmid transfer was also investigated as a unified event. Biofilms were also incorporated into this study as they constitute a possibly conducive environment for the observation of such HGT events. The results obtained during this study help to answer most of the above questions. Firstly, during an investigation into the possible release of large DNA fragments (>500 bp) from a GM commercial wine yeast strain (Parental strain: Vin13), no DNA could be detected within the fermenting background, suggesting that such DNA fragments were not released in large numbers. Secondly, the study revealed remarkable stability of free “floating DNA” under these fermentation conditions, identifying intact DNA of up to ~1kb in fermenting media for up to 62 days after it had been added. Thirdly, the data demonstrate the uptake and functional expression of spiked DNA by fermenting Vin13 cultures in grape must. Here, another interesting discovery was made, since it appears that the fermenting natural grape must favours DNA uptake when compared to synthetic must, suggesting the presence of carrier molecules. Additionally, we found that spiked plasmid DNA was not maintained as a circular unit, but that only the antibiotic resistance marker was maintained through genomic integration. Identification of the sites of integration showed the sites varied from one HGT event to the next, indicating that integration occurred through a process known as illegitimate recombination. Finally, we provide evidence for the direct transfer of whole plasmids between Vin13 strains. The overall outcome of this study is that HGT does indeed occur under the conditions investigated. To our knowledge, this is the first report of direct horizontal DNA transfer between organisms of the same species in eukaryotes. Furthermore, while the occurences of such events appears low in number, it cannot be assumed that HGT will not occur more frequently within an industrial scenario, making industrial scale studies similar to this one paramount before drawing further conclusions.
Binary interactions between bacteria and Candida albicans
(Stellenbosch : Stellenbosch University, 2017-03) Benade, Eliska; Botha, Alfred; Mouton, Marnel; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: The primary habitat of the ascomycetous yeast Candida albicans is thought to be the mammalian gastrointestinal (GI) tract. This opportunistic pathogen however, was also found to be capable of sustainable growth in the anaerobic zones of fecal contaminated rivers. Although it is known that bacteria may impact the survival of C. albicans under aerobic conditions, the effect of an anaerobic environment on these bacteria/yeast interactions has never been explored. Therefore, using both aerobic and anaerobic liquid co-cultures, binary interactions between C. albicans and a number of environmental bacterial isolates were studied at 26ºC. The bacteria represented Aeromonas hydrophila, Bacillus cereus, Bacillus subtilis, Clostridium, Enterobacter, Klebsiella pneumoniae, Kluyvera ascorbata and Serratia marcescens. Using plate counts it was found that bacterial growth inhibits the yeast’s growth under aerobic conditions; however, the inhibitory effect was ameliorated under anaerobic conditions. Bacterial enzymes capable of degrading yeast cell walls, including chitinases and mannanases, were produced by the majority of the bacteria. In contrast, except for A. hydrophila, these enzymes were not detected in anaerobic bacterial cultures, nor was the antimicrobial compound, prodigiosin found in anaerobic cultures of S. marcescens. Crude extracellular enzyme preparations from K. pneumoniae and S. marcescens had no effect on the yeast’s growth, but were found to enhance the toxicity of prodigiosin towards the yeast, especially in combination with mannanases. The above-mentioned in vitro methods were also used to study binary interactions at 37ºC between C. albicans and selected GI bacteria. The latter included Bacteroides fragilis, Bacteroides vulgatus, Clostridium perfringens, Escherichia coli, K. pneumoniae, Lactobacillus acidophilus and Lactobacillus plantarum. Using live/dead yeast viability staining in combination with epifluorescence microscopy it was demonstrated that the aerobic filamentous phase of C. albicans, which is usually induced at 37ºC, was either killed or inhibited by the bacteria; however, the bacteria had no effect on anaerobic yeast growth. Mannanase containing crude enzyme preparations from K. pneumoniae and B. fragilis also had no effect on anaerobically cultured yeast cells. Also, the enzyme preparations did not enhance the antagonistic effect of antimicrobials, such as Amphotericin B and prodigiosin, towards the yeast. Generally, bacterial growth was unaffected by the presence of C. albicans, neither at 26 or 37ºC, nor under aerobic or anaerobic conditions. Under anaerobic conditions at 37ºC however, the presence of either C. albicans or the model ascomycete Saccharomyces cerevisiae, significantly increased the numbers of mannan utilizing B. fragilis and B. vulgatus. Conversely, this increase was not as pronounced in the presence of engineered S. cerevisiae strains with compromised cell wall mannan layers. Treating monocultures of the two Bacteroides species with intact dead yeast cells also resulted in significant increases in bacterial numbers, while removal of the cell wall mannan layers from these dead yeasts resulted in no increase in Bacteroides numbers. These findings indicated that mannan utilizing Bacteroides strains may form commensalistic interactions with C. albicans. Overall, our findings suggest that the symbioses between C. albicans and bacteria may differ depending on the presence or absence of oxygen.
The biology, diversity and evolution of the broad host-range, promiscuous INCQ plasmids, with an emphasis on the INCQ2 sub-family
(Stellenbosch : Stellenbosch University, 2014-12) Rawlings, Douglas Eric; Van Zyl, Willem Heber; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Plasmids belonging to the IncQ family have an exceptionally broad host-range and are highly mobilizable in the presence of the self-transmissible IncP plasmids. All IncQ plasmids identified to date have certain features in common. The feature that distinguishes them most from all other plasmids is that they have a unique mechanism of replication. Their replicons consist of repA, repB and repC genes encoding a replicase, primase and DNA-binding proteins respectively. All IncQ plasmids contain at least three 22-bp iterons (or 20-bp iterons with 2-bp spacers) that are identical in sequence and to which the RepC DNA-binding protein binds. They replicate by means of a unique strand-displacement mechanism that is considered to place a limit on their size. Replication proceeds by a partially single-stranded intermediate that is believed to result in an increased likelihood of structural instability with an increase in plasmid size. The most compact backbone of IncQ plasmids is approximately 5.9-kb and the largest natural IncQ plasmid reported is 14.2-kb. Although the mobilization regions of IncQ plasmids are not as unique as the replicons, they are all characterized by the primase of the replicon being fused to the relaxase of the mobilization genes. The remainder of the mobilization genes may vary substantially in number and sequence between plasmids and have been subdivided into at least four distinct lineages. This dissertation consists of twenty one manuscripts published during the period 1984 to 2012. The focus is almost entirely on the IncQ plasmid subfamily known as IncQ2. Most of the earlier work was on determining the nature and extent of the replicons, mobilization genes and the toxin-antitoxin plasmid stability system. A strong theme in the latter work focussed on using the natural variation among the IncQ2 plasmids as a means to understand IncQ plasmid evolution. The collection of articles comprises a combination of original research and reviews.
Bioprospecting for novel biosurfactants and biosurfactant producing bacteria in wastewater
(Stellenbosch : Stellenbosch University, 2017-03) Ndlovu, Thando; Khan, Wesaal; Khan, Sehaam; Rautenbach, Marina; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Biosurfactants are surface active amphiphilic compounds, synthesised by numerous bacteria, fungi and yeast. They are known to exhibit broad spectrum antimicrobial activity and are currently applied as antimicrobial agents, antiadhesives, foaming agents, emulsifiers etc. in the cosmetic, food, pharmaceutical and biotechnology industries. The primary aim of the study was thus to bioprospect for novel biosurfactants and biosurfactant-producing bacteria in a wastewater treatment plant (WWTP). Wastewater was selected as it is a suitable environment for the growth of diverse microorganisms and the presence of numerous organic and inorganic contaminants were postulated to enable the flourishing of biosurfactant-producing microorganisms. Chapter 1 then outlined literature pertaining to biofurfactants, their characterisation and mode of action, amongst many other topics. Chapter 2 of this study focused on the distribution and diversity of biosurfactant-producing bacteria isolated from wastewater. Wastewater samples were collected from various points of the Stellenbosch WWTP and culturable isolates were screened for possible biosurfactant production using the oil spreading and drop collapse methods. Surface tension and emulsification activities were then used for the partial characterisation of the produced biosurfactant compounds. Thirty-two of the 667 bacterial isolates were regarded as biosurfactant producers and were classified into the Aeromonadaceae, Bacillaceae, Enterobacteriaceae, Gordoniaceae and the Pseudomonadaceae families using 16S rRNA analysis. Bacillus and Pseudomonas were among the most dominant genera, which constituted 21.8% (7/32) and 12.5% (4/32) of all isolates, respectively. High surface tension reduction of the growth medium (71.1 mN/m) was also observed for the Bacillus ST34 (34.4 mN/m) and the Pseudomonas ST5 (32.3 mN/m) isolates. In addition, the Bacillus ST34 and Pseudomonas ST5 isolates tested positive for the sfp and rhlB genes involved in the biosynthesis of surfactin and rhamnolipid biosurfactants. While numerous studies have reported on the isolation of biosurfactant-producing bacteria from contaminated soil and terrestrial environments, the current study indicated that municipal wastewater could be exploited for the isolation of diverse biosurfactant-producing bacterial strains. In chapter 3, 32 biosurfactant-producing isolates were then genotypically differentiated utilising repetitive element PCRs (rep PCRs) [targeting the repetitive extragenic palindromic (REP) and the BOX element sequences]. This molecular differentiation was performed as the genetic diversity amongst bacterial species is known to produce different concentrations and proportions of various homologues of biomolecules such as biosurfactants and antibiotics. With the use of the conventional PCR assays, some of the isolates were identified as Bacillus subtilis (n = 4), Aeromonas hydrophila (n = 3) and Bacillus amyloliquefaciens (n = 2), amongst others. These bacterial species were genotypically differentiated into four, three and two sub-species (strains), respectively, utilising rep PCRs. The BOX AIR and REP primers utilised for rep PCR in the current study thus provided a powerful tool to discriminate between biosurfactant-producing bacterial isolates identified as the same species. Chapter 4 focused on the characterisation and antimicrobial activity of the biosurfactant extracts produced by the isolates B. amyloliquefaciens ST34 and Pseudomonas aeruginosa ST5. Crude biosurfactants from ST34 and ST5 culture broth were extracted using solvent extraction based methods. Thereafter, the high resolution ultra-performance liquid chromatography (UPLC) coupled to electrospray ionisation mass spectrometry (ESI-MS) method, developed in the current study, was utilised to characterise the produced compounds. Results indicated that B. amyloliquefaciens ST34 primarily produced the C13, C14, C15 and C16 surfactin analogues when grown on mineral salt medium (MSM) supplemented with glycerol. For P. aeruginosa ST5, high resolution ESI-MS linked to UPLC confirmed the presence of dirhamnolipid congeners, specifically Rha-Rha-C10-C10 as well as monorhamnolipid congeners, specifically Rha-C10-C10. The crude surfactin and rhamnolipid extracts were also assessed for their antimicrobial activities and displayed significant antimicrobial activity against a broad spectrum of opportunistic and pathogenic microorganisms, including antibiotic resistant Staphylococcus aureus and Escherichia coli strains. The quantitative and qualitative effects of various substrates utilised for the surfactin and rhamnolipid production by B. amyloliquefaciens ST34 and P. aeruginosa ST5 strains, respectively, were assessed in chapter 5. For B. amyloliquefaciens ST34, maximum biosurfactant production was observed in the MSM supplemented with fructose (28 mg/L). In addition, four surfactin analogues were produced by B. amyloliquefaciens ST34 using the different substrates, however, the Srf2-4 (C13-15 surfactins) were the most dominant in all the B. amyloliquefaciens ST34 extracts. For P. aeruginosa ST5, maximum biosurfactant production was observed in the MSM supplemented with glucose (307 mg/mL). In addition, six rhamnolipid congeners were produced by P. aeruginosa ST5 using the different substrates, however, similar to results obtained in Chapter four, the dRL2 (Rha-Rha-C10-C10) and mRL2 (Rha-C10-C10) were the most abundant compounds produced in all P. aeruginosa ST5 extracts.
Carnitine in yeast and filamentous fungi
(Stellenbosch : Stellenbosch University, 2003-12) Swiegers, Jan Hendrik; Bauer, Florian; Pretorius, I. S.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: In the yeast Saccharomyces cerevtstee, two biochemical pathways ensure that activated cytoplasmic or peroxisomal acetyl-groups are made available for mitochondrial energy production when the cells utilise non-fermentable carbon sources. The first pathway is the glyoxylate cycle, where two activated acetyl-groups are incorporated into each cycle, which releases a C4 intermediate. This intermediate is then transported to the mitochondria where it can enter the tricarboxylic acid cycle. The second pathway is the carnitine shuttle. Activated acetyl-groups react with carnitine to form acetylcarnitine, which is then transported to the mitochondria where the acetyl group is transferred. In this study it was shown that the deletion of the glyoxylate cycle specific citrate synthase, encoded by CIT2, results in a strain that is dependent on carnitine for growth on non-fermentable carbon sources. Using a /::"cit2 strain, mutants affected in carnitine-dependent metabolic activities were generated. Complementation of the mutants with a genomic library resulted in the identification of four genes involved in the carnitine shuttle. These include: (i) the mitochondrial and peroxisomal carnitine acetyltransferase, encoded by CAT2; (ii) the outer-mitochondrial carnitine acetyltransferase, encoded by YA T1; (iii) the mitochondrial carnitine translocase, encoded by CRC1; and (iv) a newly identified carnitine acetyltransferase, encoded by YAT2. All three carnitine acetyltransferases are essential in a carnitine-dependent strain. The dependence on exogenous carnitine of the /::"cit2 strain when grown on nonfermentable carbon sources suggested that S. cerevisiae does not biosynthesise carnitine. Measurements using electrospray mass spectrometry confirmed this hypothesis. As a result an investigation was initiated into carnitine biosynthesis in order to genetically engineer a S. cerevisiae strain that could endogenously biosynthesise carnitine. The filamentous fungus, Neurospora crassa, was one of the first organisms used in the seventies to identify the precursor and intermediates of carnitine biosynthesis. However, it was only about twenty years later that the first genes encoding these enzymes where characterised. Carnitine biosynthesis is a four-step process, which starts with trimethyllysine as precursor. Trimethyllysine is converted to hydroxytrimethyllysine by the enzyme trimethyllysine hydroxylase (TMLH). Hydroxytrimethyllysine is cleaved to trimethylamino-butyraldehyde by the hydroxytrimethyllysine aldolase (HTMLA) releasing glycine. Trimethylaminobutyraldehyde is dehydrogenated to trimethylamino-butyrate (y-butyrobetaine) by trimethylamino-butyraldehyde dehydrogenase (TMABA-DH). In the last step, ybutyrobetaine is converted to t-carnltine by y-butyrobetaine hydroxylase (BBH). The N. crassa TMLH homologue was identified in the genome database based on the protein sequence homology of the human TMLH. Due to the high amount of introns predicted for this gene, the cDNA was cloned and subjected to sequencing, which then revealed that the gene indeed had seven introns. Functional expression of the gene in S. cerevisiae and subsequent enzymatic analysis revealed that the gene coded for a TMLH. It was therefore named cbs-1 for "carnitine biosynthesis gene no. 1JJ. Most of the kinetic parameters were similar to that of the human TMLH enzyme. Following this, a genomic copy of the N. crassa BBH homologue was cloned and functionally expressed in S. cerevisiae. Biochemical analysis revealed that the BBH enzyme could biosynthesise L-carnitine from y-butyrobetaine and the gene was named cbs-2. In addition, the gene could rescue the growth defect of the carnitinedependent Scii? strain on non-fermentable carbon sources when y-butyrobetaine was present. This is the first report of an endogenously carnitine biosynthesising strain of S. cerevisiae. The cloning of the remaining two biosynthesis genes presents particular challenges. To date, the HTMLA has not been characterised on the molecular level making the homology-based identification of this protein in N. crassa impossible. Although the TMABA-DH has been characterised molecularly, the protein sequence is conserved for its function as a dehydrogenase and not conserved for its function in carnitine biosynthesis, as in the case of TMLH and BBH. The reason for this is probably due to the fact that the enzyme is involved in other metabolic processes. The use of N. crassa carnitine biosynthesis mutants would probably be one way in which to overcome these obstacles. The !1cit2 mutant proved useful in studying carnitine related metabolism. We therefore searched for suppressors of !1cit2, which resulted in the cloning of RAS2. In S. cerevisiae, two genes encode Ras proteins, RAS1 and RAS2. GTP-bound Ras proteins activate adenylate cyclase, Cyr1 p, which results in elevated cAMP levels. The cAMP molecules bind to the regulatory subunit of the cAMP-dependent kinase (PKA), Bcy1 p, thereby releasing the catalytic subunits Tpk1 p, Tpk2p and Tpk3p. The catalytic subunits phosphorylate a variety of regulators and enzymes involved in metabolism. Overexpression of RAS2 could suppress the growth defect of the Sclt? mutant on glycerol. In general, overexpression of RAS2 enhanced the proliferation of wild-type cells grown on glycerol. However, the enhancement of proliferation was much better for the !1cit2 strain grown on glycerol. In this respect, the retrograde response may play a role. Overexpression of RAS2 resulted in elevated levels of intracellular citrate and citrate synthase activity. It therefore appears that the suppression of !1cit2 by RAS2 overexpression is a result of the general upregulation of the respiratory capacity and possible leakage of citrate and/or citrate synthase from the mitochondria. The phenotype of RAS2 overexpression contrasts with the hyperactive RAS2val19 allele, which causes a growth defect on glycerol. However, both RAS2 overexpression and RAS2val19activate the cAMP/PKA pathway, but the RAS2val19dependent activation is more severe. Finally, this study implicated the Ras/cAMP/PKA pathway in the proliferation effect on glycerol by showing that in a Mpk1 strain, the growth effect is blocked. However, the enhanced proliferation was still observed in the Mpk2 and Mpk3 strains when RAS2 was overexpressed. Therefore, it seems that Tpk1 p plays an important role in growth on non-fermentable carbon sources, a notion that is supported by the literature.
Characterisation of L-malic acid metabolism in strains of Saccharomyces and the development of a commercial wine yeast strain with an efficient malo-ethanolic pathway
(Stellenbosch : Stellenbosch University, 2002-12) Volschenk, Heinrich; Van Vuuren, H. J. J.; Bloom, M.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: L-Malic and tartaric acid are the most prominent organic acids in wine and playa crucial role in winemaking processes and wine quality, including the organoleptic quality and the physical, biochemical and microbial stability of wine. The production of premium wines depends on the oenologist's skill to accurately adjust wine acidity to obtain the optimum balance with other wine components to produce wine with optimum colour and flavour. Strains of Saccharomyces, in general, rarely degrade L-malic acid completely in grape must during alcoholic fermentation, with relatively minor modifications in total acidity during vinification. The degree of L-malic acid degradation, however, varies from strain to strain. Some strains of Saccharomyces are known to be able to degrade a higher percentage of L-malic acid, but the underlying reason for this phenomenon is unknown. The underlying mechanisms of this phenomenon have been partially revealed during preliminary transcriptional regulation research during this study. In contrast, S. pombe cells can effectively degrade up to 29 gil L-malic acid via the malo-ethanolic pathway that converts L-malic acid to pyruvate and CO2, and ultimately to ethanol under fermentative conditions. A number of reasons for the weak degradation of L-malic acid in Saccharomyces cerevisiae have been postulated. Firstly, S. cerevisiae lacks the machinery for the active transport of L-malic acid found in S. pombe and relies on rate-limiting simple diffusion for the uptake of extracellular L-malic acid. Secondly, the malic enzyme of S. cerevisiae has a significantly lower substrate affinity for L-malic acid (Km = 50 mM) than that of S. pombe (Km = 3.2 mM), which contributes to the weaker degradation of L-malic acid in S. cerevisiae. Lastly, the mitochondrial location of the malic enzyme of S. cerevisiae, in contrast to the cytosolic S. pombe malic enzyme, suggests that the S. cerevisiae malic enzyme is inherently subject to the regulatory effects of fermentative metabolism. The malate permease gene tmael) and the malic enzyme gene (mae2) of S. pombe was therefore cloned and co-expressed in single or multi-copy under regulation of the constitutive S. cerevisiae 3-phosphoglycerate kinase (PGK1) promoter and terminator sequences in a laboratory strain of S. cerevisiae. This introduced a strong malo-ethanolic phenotype in S. cerevisiae where L-malic acid was rapidly and efficiently degraded in synthetic and Chardonnay grape must with the concurrent production of higher levels of ethanol. Functional expression of the malo-ethanolic pathway genes of S. pombe in a laboratory strain of S. cerevisiae paved the way for the genetic modification of industrial wine yeast strains of Saccharomyces for commercial winemaking. A prerequisite for becoming an inherited component of yeast is the stable integration of the malo-ethanolic genes into the genome of industrial wine yeast strains. Genetic engineering of wine yeasts strains of Saccharomyces is, however, complicated by the homothallic, multiple ploidy and prototrophic nature of industrial strains of Saccharomyces. Transformation and integration of heterologous genes into industrial strains of Saccharomyces require the use of dominant selectable markers, i.e. antibiotic or toxic compound resistance markers. Integration of these markers into the yeast genome is, however, not acceptable for commercial application due to the absence of long-term risk assessment and consumer resistance. A unique strategy for the integration of the S. pombe mae} and mae2 expression cassettes without the incorporation of any non-yeast derived DNA sequences was. The malo-ethanolic cassette, containing the S. cerevisiae PGK} promoter and terminator regions together with the S. pombe mae] and mae2 open reading frames, was integrated into the VRA3 locus of an industrial strain of Saccharomyces bayanus EC 1118 during co-transformation with a phleomycin-resistance plasmid, pUT332. After initial screening for phleomycin resistance, S. bayanus EC1118 transformants were cured of the phleomycin-resistance plasmid, resulting in the loss of non-yeast derived DNA sequences. After correct integration of the mae] and mae2 expression cassettes was verified, small-scale vinification in synthetic and Chardonnay grape must with stable transformants resulted in rapid and complete degradation of L-malic acid during the early stages of alcoholic fermentation. Integration and expression of the malo-ethanolic genes in S. bayanus ECll18 had no adverse effect on the fermentation ability of the yeast, while sensory evaluation and chemical analysis of the Chardonnay wines indicated an improvement in wine flavour compared to the control wines, without the production of any off-flavours.
Characterisation, cloning and heterologous expression of the α-glucuronidase from Aureobasidium pullulans
(Stellenbosch : Stellenbosch University, 2004-03) De Wet, Barend Johannes Marthinus; Prior, B. A.; Van Zyl, Willem Heber; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Xylanolytic accessory enzymes produced by the endo-p-l,4-xylanase overproducing, colour-variant strain of the euascomycetous fungus Aureobasidium pullulans, NRRL Y-2311-1, were studied. a- Glucuronidase activity was only induced during cultivation on carbon sources containing both xylose and glucuronic acid. An a-glucuronidase was partially purified from the supernatant of A. pullulans cultivated on birchwood glucuronoxylan. The enzyme had an apparent mobility on SDS-PAGE of 170 kDa, and after deglycosylation its mobility shifted to 118 kDa, indicating an extensively decorated protein. Maximal activity was measured at pH 3 in McIlvaine's phosphate-citrate buffer and at 40°C, and the enzyme was stable for 3 h at 40°C. The enzyme displayed substrate inhibition, and Km- and Kj-values were calculated as 3.3 ± 0.29 mM and 9.8 ± 3.8 mM for aldotriouronic acid and 29.5 ± 7.6 mM and 29.0 ± 7.8 for aldobiouronic acid respectively. PCR methods were used to clone the genes encoding an a-glucuronidase and an a-Larabinofuranosidase of A. pullulans NRRL Y-2311-1. The deduced amino acid sequence of the aglucuronidase encoding gene, aguA, shared greater than 60% identity with fungal glucuronidases and between 34% and 42% identity with bacterial a-glucuronidases, and it is member of family 67 of the glycoside hydrolases. The aguA gene encodes a protein of 836 amino acids with a putative secretion signal of 15 amino acids, resulting in a mature protein with a predicted molecular weight of 91 kDa. The gene was expressed in S. cerevisiae Y294 under control of the ADH2 promoter and terminator. The heterologous a-glucuronidase was purified to homogeneity using Ni-chelate affinity chromatography, and it had an electrophoretic mobility of 120 kDa on SDS-PAGE. The enzyme was maximally active at 65°C and between pH 5 and pH 6. The enzyme was stable at 45°C, lost half of its activity after 22.5 minutes at 55°C, and had a half-life of 5.6 min at 65 °C. It was stable at pH 4 and pH 6, and had a half-life of 17 min at pH 8. The enzyme had Km-values in the millimolar range for the series from aldobiouronic acid to aldopentaouronic acid. It had the highest catalytic efficiency on aldobiouronic acid and the catalytic efficiency decreased with increasing chain-length of the oligosaccharide substrate. The deduced amino acid sequence of the a-L-arabinofuranosidase gene, ab/A, shared between 69% and 76% identity with family 54 c-arabinofuranosidases. The gene encodes a polypeptide of 498 amino acids with a putative signal peptide of 20 amino acids resulting in a mature protein with a calculated molecular weight of 49.9 kDa. It was expressed in S. cerevisiae Y294 and the heterologous enzyme was purified to homogeneity by gel filtration. It's size estimated by gel filtration was 36 kDa, and it had an apparent mobility of 49 kDa on SDS-PAGE. It showed maximal activity at 55°C and between pH 3.5 and pH 4. It was stable at 50°C and between pH 4 and pH 5. The enzyme had a Km for p-nitrophenyl c-arabinofuranoside of 3.7 ± 0.36 mM and a Vrnax of 34.8 ± 1.1 U/mg protein. It displayed 0.2 U/mg activity against p-nitrophenyl ~-xylopyranoside.
Characterization and evaluation of indigenous Saccharomyces cerevisiae strains isolated from South African vineyards
(Stellenbosch : Stellenbosch University, 1999-12) Van der Westhuizen, Theunes Johannes; Pretorius, I. S.; Augustyn, O. P. H.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH SUMMARY: The bioconversion of grape juice into wine by simply allowing the yeasts, associated with grape berries and winery equipment, to ferment the sugars to ethanol, carbon dioxide and other minor, but important metabolites, is an ancient process. The art and science of winemaking has been extensively studied since the time when Louis Pasteur demonstrated, for the first time, the relationship between yeast and alcoholic fermentation. It is now recognized that the fermentation of grape must and production of premium quality wines is a complex ecological and biochemical process involving the sequential development of microbial species, as affected by a particular environment. This complex heterogeneous microbiological process includes the interaction of many microbial species, represented by fungi, yeasts, lactic acid bacteria and acetic acid bacteria, as well as the mycoviruses and bacteriophages affecting these grape-associated microorganisms. However, of all these different microbes and viruses, yeast represent the heart of the harmonious biochemical interaction with the musts derived from the various varieties of Vitis species which, in turn, are largely products of their respective genetic make-ups and the terroir. These yeasts are significant in winemaking because they not only conduct the alcoholic fermentation, but can also spoil wine during conservation in the cellar and after packaging, and they affect wine quality through the production of fermentation metabolites and through autolysis. A sound understanding of yeast systematics, biogeography and ecology is therefore essential to endeavours to preserve and exploit the hidden oenological potential of the untapped wealth of yeast biodiversity in our wine-producing regions. One of the main thrusts of this kind of eco-taxonomic survey is to determine the actual contribution of the indigenous strains of the so-called wine yeast (Saccharomyces cerevisiae) and wild yeasts (non-Saccharomyces species) to the sensory properties of wines and to eventually develop new starter culture strains for guided fermentations, including mixed starter cultures tailored to reflect the characteristics of a given wine region. Against this background, a comprehensive, long-term biogeographical survey and strain development programme was launched. This dissertation represents the first phase of this long-overdue research programme aimed to systematically catalogue yeasts in different climatic zones of the 350-year-old wine-producing regions of the Western Cape and to develop new yeast starter cultures that would further increase the quality of South African wine. The specific aims of this dissertation included (i) the evaluation of yeast fingerprinting techniques for their suitability to accurately and rapidly differentiate amongst S. cerevisiae strains; (ii) the isolation and characterization of S. cerevisiae strains from the coastal regions of the Western Cape; (iii) to determine the natural population dynamics of S. cerevisiae strains in selected vineyards over a four-year period; (iv) to make a preliminary determination of the possible effect that these indigenous S. cerevisiae isolates may have on wine flavour, and (v) to breed new starter culture strains with improved characteristics. Eighteen strains of S. cerevisiae used for commercial production of wine in South Africa were characterized by means of long-chain fatty acid analysis, randomly amplified polymorphic DNA (RAPD-PCR) and electrophoretic karyotyping (CHEF-DNA analysis). Variations in DNA profiles of the strains were apparent in the number, position and intensity of bands. It was found that electrophoretic karyotyping, as a single technique, seemed to be the most useful method to be used for routine fingerprinting. However, it was proposed that the combined use of these three techniques would provide the most reliable means of differentiating amongst wine yeast strains. Two of these fingerprinting techniques, CHEF-DNA and RAPD-PCR analysis, were used to determine the geographic distribution of indigenous S. cerevisiae strains isolated from local vineyards. Grapes were aseptically harvested from 13 sites in five areas in the coastal regions of the Western Cape during 1995. These sites were Groot Constantia and Buitenverwachting in the Constantia area; Jordan, Lievland, Mont Fleur and Nietvoorbij in the Stellenbosch area; Vergelegen in Somerset West; De Rust, Oak Valley, White Hall and Wildekrans in the Elgin/Bot River area; and Bouchard Finlayson and Hamilton Russel in the Hermanus area. After fermentation, 30 yeast colonies per sample were isolated and examined for the presence of S. cerevisiae. Five sampling sites yielded no S. cerevisiae strains. Electrophoretic karyotyping revealed the presence of 46 unique karyotypes in eight of the remaining sites. No dominant strain was identified and each site had its own unique collection of strains. The number of strains per site varied from two to 15. Only in four cases did one strain appear at two sites, while only one instance of a strain occurring at three sites was recorded. All sites contained killer and sensitive strains, however, killer strains did not always dominate. Commercial strains were recovered from three sites. Although commercial yeasts dominated the microflora at two sites, it appears that fears of commercial yeasts ultimately dominating the natural microflora seem to be exaggerated. As an extension of the 1995 survey samples were taken from the same locations at Groot Constantia, Buitenverwachting, Jordan, Lievland, Mont Fleur, Vergelegen, Bouchard Finlayson and Hamilton Russel during 1996 to 1998. This was done in an effort to assess how the natural population dynamics of S. cerevisiae are affected over the long term by abiotic factors. Thirty colonies per site were isolated and the S. cerevisiae strains were characterized by electrophoretic karyotyping. The identity of strains appearing at more than one site in the same, or different years, was confirmed by RAPD-PCR analysis. Strain numbers per site varied over the four-year study period. Weather conditions resulting in severe fungal infestations and heavy applications of chemical sprays during 1996 and early 1997 dramatically reduced the numbers of S. cerevisiae strains recovered during 1997. A return to normal weather patterns during mid 1997 resulted in a gradual recovery of the indigenous population as noted during the 1998 harvest. Indications are that some of the strains isolated are widespread in the study area and may represent yeasts typical of the area. Again, commercial wine yeast strains were recovered in only a few instances and the likelihood that commercial yeasts will eventually replace the natural yeast microflora in vineyards therefore seems remote. As a preliminary study to determine the possible effect of these indigenous S. cerevisiae strains on wine flavour, 33 of the indigenous yeasts were allowed to ferment Chenin blanc wine in laboratory fermentations. The juice was analyzed. The ability to form esters, fatty acids and higher alcohols was compared to that of two local commercial yeasts. None of the indigenous strains were found to be suitable for fermenting white must at 15°C. Their ability to ferment red musts at much higher temperatures still needs to be assessed. Furthermore, differences noted indicate that some of these strains show potential to be included in our extensive yeast-breeding programme as this would broaden the genetic pool. In parallel with the search to isolate and identify indigenous S. cerevisiae strains with good oenological potential, an extensive selection and breeding programme with cultures from our strain collection was undertaken. The aim of this programme was to generate new strains that are better suited to New World winemaking styles and conditions prevailing in South Africa. As a result, 145 hybrids, differentiated by elecrophoretic karyotyping and long-chain fatty acid analysis, were produced. Fifty-eight of the hybrids were able to ferment juice to dryness at 15°C in less than 21 days during microvinification trials. Five of the strains were released for commercial use after extensive industrial-scale evaluation. Based on the success of these interstrain hybridizations, the breeding programme will now be expanded to include some of the indigenous S. cerevisiae strains. In conclusion, it is only when we have a much better understanding of yeast biodiversity, biogeography, ecology and the interaction within yeast communities that we will be able to optimally harness the genetic pool in our strain development programme, aimed to benefit both the wine producer and the consumer.
Characterization of bacteriocin 423 produced by Lactobacillus pentosus
(Stellenbosch : Stellenbosch University, 2000-12) Van Reenen, Carol A. (Carol Ann); Dicks, Leon Milner Theodore; Van Zyl, Willem Heber; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Worldwide, bacteriocins, particularly those produced by food-related lactic acid bacteria, are receiving attention due to the possible use of these peptides as natural preservatives in food, replacing potentially harmful chemical preservatives. Bacteriocins are ribosomally synthesized proteins or peptides that inhibit closely related microorganisms. Most bacteriocins produced by lactic acid bacteria are small, heat resistant peptides that inhibit other Gram-positive bacteria, including food-borne pathogens such as Listeria monocytogenes, Bacillus cereus, Clostridium perfringens and Staphylococcus aureus, but do not inhibit Gram-negative bacteria, molds or fungi. Bacteriocins are produced as inactive prepeptides that become active after the N-terminal leader peptide is cleaved off. Small heat resistant bacteriocins are either lantibiotics (Class I), containing unusual posttranslationally modified amino acids, or peptides that are non-Ianthionines (Class II). The Class II bacteriocins are further divided into four different groups: Class lIa, the anti-listerial bacteriocins containing the YGNGV consensus sequence in the N-terminal of the protein, Class lib, bacteriocins consisting of two peptides, Class IIc, bacteriocins that are secreted via the sec pathway, and Class lid, bacteriocins that do not belong in the previous three subgroups. A bacteriocin producing lactic acid bacterium was isolated in our laboratory from traditionally home fermented South African sorghum beer. The producing bacterium was found to be a facultative heterofermentative Lactobacillus sp. and was identified as Lactobacillus plantarum or Lactobacillus pentosus by using the API 50 CHL carbohydrate fermentation system and numerical analysis of total soluble cell protein patterns. RAPD-PCR analysis identified the strain as L. plantarum, but 16S rRNA sequencing confirmed its identification as L. pentosus. The bacteriocin, first designated plantaricin 423 and later bacteriocin 423, was identified as a Class lIa small heat resistant anti-listerial bacteriocin containing the YGNGV consensus motif. Bacteriocin 423 inhibited a variety of Gram-positive bacteria, including Lactobacillus spp., Leuconostoc spp., Oenococcus oeni, Pediococcus spp., Enterococcus spp., Propionibacterium spp., Staphylococcus spp., Bacillus spp., Clostridium spp. and Listeria spp. The bacteriocin was inactivated by proteolytic enzymes and active over a wide pH range (pH 1-10). Bacteriocin 423 lost 50 % of its activity after autoclaving for 15 min at 121°C, but was not affected by lesser heat treatments. Bacteriocin production was increased by optimizing the growth medium, which consisted of glucose, tryptone, yeast extract, potassium phosphate, sodium acetate, ammonium citrate, manganese sulphate, Tween 80 and casamino acids. The bacteriocin was found to be plasmid-encoded. Genetic analysis of the bacteriocin operon indicated a high percentage of homology to the operon of another Class lIa bacteriocin, pediocin PA-1, although the structural genes of the two bacteriocins were markedly different. The structural gene of bacteriocin 423 was amplified by PCR and cloned into a yeastJE. coli vector between the ADH1 promoter and terminator sequences and fused in-frame to the MFa1 secretion signal sequence. Saccharomyces cerevisiae transformed with this plasmid expressed the bacteriocin. The sequence of prebacteriocin 423 (MMKKIEKL TEKEMANIIGGKYYGNGVTCGKHSCSVN WGOAFSCSVSHLANFGHGKC) is similar, but not identical to any other reported Class lIa anti-listeria I peptide.
Characterization of nisin F and its role in the control of respiratory tract and skin infections
(Stellenbosch : University of Stellenbosch, 2009-03) De Kwaadsteniet, Michele; Dicks, Leon Milner Theodore; University of Stellenbosch. Faculty of Science. Dept. of Microbiology.
Multidrug resistant strains of Staphylococcus aureus is presenting an increasing threat, especially immune compromised individuals. Many of these strains have developed resistance to newly approved drugs such as quinupristin-dalfopristin, linezolid and daptomycin. The search for alternative treatment, including bacteriocins (ribosomally synthesized antimicrobial peptides) of lactic acid bacteria is increasing . Lactococcus lactis subsp. lactis F10, isolated from freshwater catfish, produced a new nisin variant active against clinical strains of S. aureus. The operon encoding nisin F is located on a plasmid and the structural gene has been sequenced. The lantibiotic is closely related to nisin Z, except at position 30 where valine replaced isoleucine. The antimicrobial activity of nisin F against S. aureus was tested in the respiratory tract of Wistar rats. Non-immunosuppressed and immunosuppressed rats were intranasally infected with S. aureus K and then treated with either nisin F or sterile physiological saline. Nisin F protected immunosuppressed rats against S. aureus, as symptoms of an infection were only detected in the trachea and lungs of immunosuppressed rats treated with saline. The safety of intranasally administered nisin F was also evaluated and proved to have no adverse side effects. The potential of nisin F as an antimicrobial agent to treat subcutaneous skin infections was evaluated by infecting C57BL/6 mice with a bioluminescent strain of S. aureus (Xen 36). Immunosuppressed mice were treated with either nisin F or sterile physiological saline 24 h and 48 h after infection with subcutaneously injected S. aureus Xen 36. Histology and bioluminescence flux measurements revealed that nisin F was ineffective in the treatment of deep dermal staphylococcal infections. Non-infected and infected mice treated with nisin F had an influx of polymorphonuclear cells in the deep stroma of the skin tissue. This suggested that nisin F, when injected subcutaneously, may have modulated the immune system. Nisin F proved an effective antimicrobial agent against S. aureus-related infections in the respiratory tract, but not against subcutaneous infections. The outcome of nisin F treatment thus depends on the route of administration and site of infection.
Characterization of the adhesion genes of probiotic lactic acid bacteria
(Stellenbosch : Stellenbosch University, 2008-03) Ramiah, Kamini; Dicks, Leon Milner Theodore; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
One of the key selection criteria for potential probiotics is the ability to adhere and colonise the host gastrointestinal tract (GIT). Probiotics compete for receptor sites at the host intestinal surface, preventing the colonisation of pathogens, thereby protecting the host from infection. In addition, several important intestinal functions are mediated by the binding of probiotics to host tissue. However, the molecular mechanisms and genotypic characterization of adhesive elements have not received as much attention as other aspects of probiotic research. The present study aims to contribute to this area of research. The first part of the study focused on monitoring the expression of mucus adhesion genes mub, mapA, adhesion-like factor EF-Tu and bacteriocin gene plaA of Lactobacillus plantarum 423, as well as mub, surface layer protein (slp) and EF-Tu of Lactobacillus acidophilus ATCC 4356 when grown in the presence of mucin, bile, pancreatin and at low pH. Real time PCR was used. mub, mapA and EF-Tu of strain 423 were up-regulated in the presence of mucus and expression increased under increasing concentrations of mucus. Expression of mapA was up-regulated under normal gut conditions (0.3%, w/v, bile; 0.3%, w/v, pancreatin; pH 6.5) and at higher levels of bile (1.0%, w/v) and pancreatin (1.0%, w/v). Expression of mub was downregulated in the presence of bile and pancreatin at pH 6.5, whilst the expression of EFTu and plaA remained unchanged. At pH 4.0, the expression of mub and mapA remained unchanged, whilst EF-Tu and plaA were up-regulated. Expression of mapA was down-regulated in the presence of 0.1% (w/v) cysteine, suggesting that the gene is regulated by a mechanism of transcription attenuation that involves cysteine. In the case of L. acidophilus ATCC 4356, none of the genes were up-regulated under increasing concentrations of mucin, whilst only slp and EF-Tu were up-regulated under normal and stressful gut conditions in vitro. In the second part of the study, male Wistar rats were used to evaluate which section of the gastrointestinal tract are colonised by L. plantarum 423 and Enterococcus mundtii ST4SA and determine the effect of adhesion. Fluorescent in situ hybridization (FISH) incorporating strain specific oilgonucleotide probes indicated strong fluorescent signals for L. plantarum 423 along the intestinal lining of the ileum and the cecum. L. plantarum 423 did not colonise the colon as indicated by real timePCR. Fluorescent signals were recorded for E. mundtii ST4SA across the epithelial barrier of cecum and colonic tissue, suggesting that translocation took place. Real time PCR revealed highest cell numbers of strain ST4SA in the cecum and the colon. Haemotoxylin eosin staining of rat tissue revealed no change in morphology or any toxic effects induced upon adhesion of the strains. 16S rDNA PCR and denaturing gradient gel electrophoresis (DGGE) revealed a decrease in enterobacterial species whilst the lactic acid bacterial content remained unchanged. Strains 423 and ST4SA agglutinated yeast cells in vitro, indicating the possible presence of mannose receptors. It is well known that these receptors play a crucial role in the elimination of type 1 fimbriated strains of E. coli. It is thus safe to speculate that mannose receptors may have played a role in diminishing the enterobacterial content in the gut. The third part of the study encompassed characterization of cell surface proteins of L. plantarum 423 and their role in adhesion to Caco-2 cell lines. The strain lacks the typical surface layer protein whilst a multifunctional “intracellular” protein, elongation factor Tu (EF-Tu) and glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI) were detected. Removal of surface proteins reduced adherence of strain 423 to Caco-2 cell lines by 40%, suggesting that these proteins play a role in adhesion. The ability of strain 423 to competitively adhere, exclude and displace Clostridium sporogenes LMG 13570 and Enterococcus faecalis LMG 13566 from Caco-2 cell lines, was studied. Adhesion of C. sporogenes LMG 13570 and E. faecalis LMG 13566 was inhibited by 70% and 90%, respectively. Strain 423 excluded C. sporogenes LMG 13570 from Caco-2 cells by 73% and displaced the pathogen by 80%. E. faecalis LMG 13566 was excluded by 60% and displaced from Caco-2 cells by 90%. Despite removal of the surface proteins, L. plantarum 423 was still capable of competitively adhering to Caco-2 cells and reduced adherence of C. sporogenes LMG 13570 by 50% and E. faecalis LMG 13566 by 70%.
A comparative assessment of water quality of two reservoirs in an ephemeral river in the desert climate of Namibia: Measures to control, and factors affecting growth of toxic cyanobacteria
(Stellenbosch : Stellenbosch University, 2022-12) Sirunda, Jaime Johannes; Wolfaardt, Gideon M.; Oberholster, Paul; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH SUMMARY: Globally, there is an increasing demand for freshwater of suitable quality. Satisfying the water requirement for ecosystem services and anthropogenic activities is challenging and may prove difficult in the context of climate change, growing population, and an increase in pollution from land use activities. With the rapid increase in human population in catchment areas, sustaining the associated human needs will often result in the generation of waste which will end up in water sources downstream causing eutrophication resulting in the proliferation of toxin-producing phytoplankton. The overall aim of the research was to carry out a comparative assessment of the water quality deterioration of ephemeral river connected dams with desert climate conditions, found in the Omaruru-Swakop catchment associated with a mixture of land use activities. The Swakoppoort (SWP) and Von Bach (VB) dams as the major dams in the Omaruru-Swakop catchment found on the Swakop River were selected and assessed seasonally for a number of years. Water samples were analyzed for various constituents, firstly to assess the water quality status of the two dams receiving a mixture of pollutants over space and time using the combination Water Quality Index (WQI). Secondly, to describe the vertical and temporal dynamics of phytoplankton communities in the two dams. Thirdly to investigate the effect of prolonged drought on phytoplankton biomass measured as Chlorophyll a (Chl a) and cyanobacteria. Lastly, a case study to compare the effectiveness of two phytoplankton control measures employed in the SWP Dam was conducted. The two dams were found to be impacted by nutrient, salinity, and particulate matter pollution. The WQIs showed poor water quality conditions in both dams for 17 years. Given the poor water quality of the two dams, Microcystis dominated the vertical and temporal dynamics, followed by Dolichospermum. In the dry seasons, higher cyanobacteria cell numbers were observed in comparison to the rainy season in both dams. In the SWP Dam, the preferable depth ranges for toxic cyanobacteria species were 5 to 10 m while in the VB Dam at 0 to 5 m range. Higher frequencies of prolonged drought years were recorded in the VB Dam in comparison to SWP Dam. The influence of a decrease in vol % on the phytoplankton biomass was observed in the SWP Dam but not in the VB Dam. However, the pattern and magnitude of the statistically significant responses (t-test, p < 0.05) varied among the drought and rainy years. Furthermore, the results showed that, the Solar Powered Circulation (SPC) and Phoslock® as the two phytoplankton control measures employed in the SWP Dam had no effect on cyanobacteria cells. It was evident that the two control measures were ineffective in reducing cyanobacterial cells. Incorporation of the current study research outputs in the utility surface water quality assessment and control program, water withdrawal for treatment and transfer plans, wastewater discharge regulations, and the revision of the integrated water resources management plan in Namibia, may ultimately safeguard the scarce water resources to improve resilience to climate change.
Comparative proteomic and genomic analysis of Flavobacterium johnsoniae-like biofilm, planktonic and agar surface-associated cells
(Stellenbosch : University of Stellenbosch, 2010-03) Flemming, Leonard; Chenia, H. Y.; Rawlings, D. E.; University of Stellenbosch. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Pathogenic Flavobacterium spp. cause serious disease outbreaks in a variety of farmed fish, which lead to large economic losses in the aquaculture industry on an annual basis. The ability of Flavobacterium johnsoniae-like isolates to grow as surface-associated communities (biofilms) in aquaculture systems poses a threat to fish health over extended periods of time. The biofilmforming ability of 28 F. johnsoniae-like isolates obtained from diseased fish were correlated with their chitin-degrading abilities and extracellular carbohydrate complexes (ECC) and their pulsed-field gel electrophoresis (PFGE) genotypes. Physiological changes in the proteome of 5 day planktonic, biofilm and agar surface-associated cultures of F. johnsoniae-like isolates YO12 and YO64 were analyzed by two-dimensional (2-D) gel electrophoresis and 17 differentially expressed and 14 uniquely expressed proteins were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Thirty-two differentially expressed genes in 5 day biofilm and agar surface-associated cultures of F. johnsoniae-like isolates YO12 and YO64 were identified using suppression subtractive hybridization (SSH). Significant negative correlations were observed between the chitin-degrading abilities and ECC and the biofilmforming capacity of 24 h biofilm cultures of F. johnsoniae-like isolates. Genetic heterogeneity was displayed by the F. johnsoniae-like isolates following PFGE. A significant positive correlation was observed between PFGE types and fish host species. Differentially and uniquely expressed proteins identified in planktonic, biofilm and agar surface-associated phases by 2-D/MS as well as differentially expressed genes identified in the biofilm and agar surface-associated phases by SSH were categorized as being involved in adaptation/protection, metabolic processes, membrane/transport/ motility and transcription/ translation. As far as we know, this is the first report on the characterization of differentially expressed genes and gene products of F. johnsoniae-like isolates obtained from diseased fish in South Africa.
Development of an antimicrobial wound dressing by co-electrospinning bacteriocins of lactic acid bacteria into polymeric nanofibers
(Stellenbosch : Stellenbosch University, 2012-12) Heunis, Tiaan de Jager; Dicks, Leon Milner Theodore; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Skin is the largest organ in the human body and serves as a barrier that protects the underlying tissue of the host from infection. Injury, however, destroys this protective barrier and provides a perfect opportunity for microorganisms to invade the host and cause infection, thereby affecting the normal wound healing processes. Furthermore, the ability of microbial pathogens to rapidly develop resistance towards a variety of antimicrobial compounds hampers the effective treatment and control of infections. Antimicrobial-resistant pathogens are increasingly being isolated from patients, placing a huge burden on the health care sector. The search for new and novel antimicrobial agents and treatments is thus of utmost importance and will continue to play an integral role in medical research. Antimicrobial peptides (AMPs) may serve as possible alternatives to antibiotics, or may be used in combination with antibiotics to reduce the risk of antimicrobial resistance. AMPs play a role in innate defence and are produced by a variety of mammals, plants, reptiles, amphibians, birds, fish and insects. The AMPs of bacteria (bacteriocins), especially those of lactic acid bacteria (LAB), are receiving increased attention as antimicrobial agents to treat bacterial infections. Electrospun nanofibers have characteristics that make them suitable as wound dressings, i.e. high oxygen permeability, variable pore size, high surface area to volume ratio and nanofibers are morphologically similar to the extracellular matrix. The ability to incorporate of a variety of biologically active compounds into nanofibers increases their potential as wound dressings. A novel approach would be to incorporate bacteriocins from LAB into nanofiber scaffolds to generate antimicrobial wound dressings. In this study, the feasibility of co-electrospinning bacteriocins from LAB into nanofibers was investigated. Plantaricin 423, produced by Lactobacillus plantarum 423, was successfully co-electrospun into poly(ethylene oxide) (PEO) nanofibers. Plantaricin 423 retained activity after the electrospinning process and continued to inhibit the growth of Lactobacillus sakei DSM 20017T and Enterococcus faecium HKLHS. Viable cells of L. plantarum 423 were also successfully co-electrospun into PEO nanofibers, albeit with a slight reduction in viability. A nanofiber drug delivery system was developed for plantaricin 423 and bacteriocin ST4SA, produced by Enterococcus mundtii ST4SA, by blending PEO and poly(D,L-lactide) (PDLLA) in a suitable solvent before electrospinning. Nanofibers were produced that released the bacteriocins over an extended time period. The PEO:PDLLA (50:50) nanofiber scaffold retained its structure the best upon incubation at 37 °C and released active plantaricin 423 and bacteriocin ST4SA. Nisin A was also successfully co-electrospun into a PEO:PDLLA (50:50) nanofiber scaffold and nisin A, released from the nanofibers, inhibited the growth of Staphylococcus aureus in vitro. Nisin A-containing nanofiber scaffolds significantly reduced viable S. aureus cells in infected skin wounds and promoted wound healing in non-infected wounds. As far as we could determine we are the first to show that bacteriocin-eluting nanofiber scaffolds can be used to treat skin infections and influence wound healing.
Development of improved α-amylases
(Stellenbosch : University of Stellenbosch, 2005-03) Ramachandran, Nivetha; Cordero Otero, Ricardo R.; Pretorius, Isak S.; University of Stellenbosch. Faculty of Science. Dept. of Microbiology.
The technological advancement of modern human civilisation has, until recently, depended on extensive exploitation of fossil fuels, such as oil, coal and gas, as sources of energy. Over the last few decades, greater efforts have been made to economise on the use of these nonrenewable energy resources, and to reduce the environmental pollution caused by their consumption. In a quest for new sources of energy that will be compatible with a more sustainable world economy, increased emphasis has been place on researching and developing alternative sources of energy that are renewable and safer for the environment. Fuel ethanol, which has a higher octane rating than gasoline, makes up approximately two-thirds of the world’s total annual ethanol production. Uncertainty surrounding the longterm sustainability of fuel ethanol as an energy source has prompted consideration for the use of bioethanol (ethanol from biomass) as an energy source. Factors compromising the continued availability of fuel ethanol as an energy source include the inevitable exhaustion of the world’s fossil oil resources, a possible interruption in oil supply caused by political interference, the superior net performance of biofuel ethanol in comparison to gasoline, and a significant reduction in pollution levels. It is to be expected that the demand for inexpensive, renewable substrates and cost-effective ethanol production processes will become increasingly urgent. Plant biomass (including so-called ‘energy crops’, agricultural surplus products, and waste material) is the only foreseeable sustainable source of fuel ethanol because it is relatively low in cost and in plentiful supply. The principal impediment to more widespread utilisation of this important resource is the general absence of low cost technology for overcoming the difficulties of degrading the recalcitrant polysaccharides in plant biomass to fermentable sugars from ethanol can be produced. A promising strategy for dealing with this obstacle involves the genetic modification of Saccharomyces cerevisiae yeast strains for use in an integrated process, known as direct microbial conversion (DMC) or consolidated bioprocessing (CBP). This integrated process differs from the earlier strategies of SHF (separate hydrolysis and fermentation) and SSF (simultaneous saccharification and fermentation, in which enzymes from external sources are used) in that the production of polysaccharide-degrading enzymes, the hydrolysis of biomass and the fermentation of the resulting sugars to ethanol all take place in a single process by means of a polysaccharidefermenting yeast strain. The CBP strategy offers a substantial reduction in cost if S. cerevisiae strains can be developed that possess the required combination of substrate utilisation and product formation properties. S. cerevisiae strains with the ability to efficiently utilise polysaccharides such as starch for the production of high ethanol yields have not been described to date. However, significant progress towards the development of such amylolytic strains has been made over the past decade. With the aim of developing an efficient starch-degrading, high ethanol-yielding yeast strain, our laboratory has expressed a wide variety of heterologous amylase-encoding genes in S. cerevisiae. This study forms part of a large research programme aimed at improving these amylolytic ‘prototype’ strains of S. cerevisiae. More specifically, this study investigated the LKA1- and LKA2-encoded α-amylases (Lka1p and Lka2p) from the yeast Lipomyces kononenkoae. These α-amylases belong to the family of glycosyl hydrolases (EC 3.2.1.1) and are considered to be two of the most efficient raw-starch-degrading enzymes. Lka1p functions primarily on the α-1,4 linkages of starch, but is also active on the α-1,6 linkages. In addition, it is capable of degrading pullulan. Lka2p acts on the α-1,4 linkages. The purpose of this study was two-fold. The first goal was to characterise the molecular structure of Lka1p and Lka2p in order to better understand the structure-function relationships and role of specific amino acids in protein function with the aim of improving their substrate specificity in raw starch hydrolysis. The second aim was to determine the effect of yeast cell flocculence on the efficiency of starch fermentation, the possible development of high-flocculating, LKA1-expressing S. cerevisiae strains as ‘whole-cell biocatalysts’, and the production of high yields of ethanol from raw starch. In order to understand the structure-function relationships in Lka1p and Lka2p, standard computational and bioinformatics techniques were used to analyse the primary structure. On the basis of the primary structure and the prediction of the secondary structure, an N-terminal region (1-132 amino acids) was identified in Lka1p, the truncation of which led to the loss of raw starch adsorption and also rendered the protein less thermostable. Lka1p and Lka2p share a similar catalytic TIM barrel, consisting of four highly conserved regions previously observed in other α-amylase members. Furthermore, the unique Q414 of Lka1p located in the catalytic domain in place of the invariant H296 (TAKA amylase), which offers transition state stabilisation in α-amylases, was found to be involved in the substrate specificity of Lka1p. Mutational analysis of Q414 performed in the current study provides a basis for understanding the various properties of Lka1p in relation to the structural differences observed in this molecule. Knowing which molecular features of Lka1p contribute to its biochemical properties provides us with the potential to expand the substrate specificity properties of this α-amylase towards more effective processing of its starch and related substrates. In attempting to develop ‘whole-cell biocatalysts’, the yeast’s capacity for flocculation was used to improve raw starch hydrolysis by S. cerevisiae expressing LKA1. It was evident that the flocculent cells exhibited physicochemical properties that led to a better interaction with the starch matrix. This, in turn, led to a decrease in the time interval for interaction between the enzyme and the substrate, thus facilitating faster substrate degradation in flocculent cells. The use of flocculation serves as a promising strategy to best exploit the expression of LKA1 in S. cerevisiae for raw starch hydrolysis. This thesis describes the approaches taken to investigate the molecular features involved in the function of the L. kononenkoae α-amylases, and to improve their properties for the efficient hydrolysis of raw starch. This study contributes to the development of amylolytic S. cerevisiae strains for their potential use in single-step, cost-effective production of fuel ethanol from inexpensive starch-rich materials.
The effect of different soil yeasts on the growth and physiology of lupin and wheat
(Stellenbosch : Stellenbosch University, 2018-03) Moller, Leandra; Botha, Alfred; Valentine, Alexander J.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Leguminous plants are often used in break-crop and crop rotation systems to fix atmospheric di-nitrogen (N2), thereby reducing the need for nitrogen (N) fertiliser input during cereal production. In these systems, wheat (Triticum aestivum L.) yield increases following blue lupin (Lupinus angustifolius L.) cultivation. Yields can also be increased by plant growth promoting microorganisms such as yeasts. These plant growth promoting yeasts (PGPY) enhance seed germination, produce plant growth promoting (PGP) factors, and partake in beneficial tripartite and quadripartite symbioses with their host plant and its root symbionts. Despite the importance of blue lupin in agriculture, it was unclear how tripartite and quadripartite interactions involving PGPY affect this legume’s biological N2 fixation (BNF) and growth. In addition, the effect of a single PGPY on germination and growth of plants used in break-crop or crop rotation systems, such as blue lupin and wheat, was unknown. To address these aspects, the impact of a rhizosphere yeast and mycorrhizal fungi on growth and nutrition of nodulated blue lupin was firstly evaluated. The ability of this rhizosphere yeast to affect a break-crop or crop rotation system was subsequently assessed by studying the yeast’s influence on the germination and developmental physiology of blue lupin and wheat. Papiliotrema laurentii (syn. Cryptococcus laurentii) was isolated from the rhizosphere of blue lupin. Nodulated blue lupin seedlings, treated with either P. laurentii CAB 91 (PL), or mycorrhizal fungi (MF), or with both symbionts (PLMF), were then cultivated in a glasshouse, after which the plants’ biomass parameters, symbiotic colonisation, and mineral nutrition were analysed. The PGP traits of PL were subsequently evaluated by comparing them to that of two other rhizosphere yeasts, i.e. Hannaella zeae CAB 1119 (HZ) and Saitozyma podzolica CAB 1199 (SP). The yeasts were used to coat seeds of blue lupin and wheat, where after their influence on the germination of these seeds was assessed under controlled conditions. The cold test was used to evaluate the influence of the three yeast strains on the vigour of both plants. To determine the effect of HZ, PL, and SP on growth and photosynthesis of nodulated blue lupin and wheat, plants were cultivated under glass house conditions. It was found that the MF treatment had no effect on blue lupin seedlings under glass house conditions. Improved growth of PL treated seedlings was underpinned by increased BNF efficiency, while greater nodulation and efficient growth on N resources supported the increased biomass of PLMF treated plants. Subsequent trials with the three rhizosphere yeasts mentioned above, indicated that of these three yeasts only PL can be used to coat the seeds of both blue lupin and wheat, since this yeast increased the germination of blue lupin and the vigour of wheat. Additionally, under glass house conditions PL promoted higher relative growth rates during the early developmental stages of both plants. The latter coincided with enhanced photosynthetic metabolism and water relations. These findings indicate that PL may potentially serve as an efficient bio-fertiliser of blue lupin and wheat to benefit breakcrop and crop rotation systems.
The effects of native Saccharomyces cerevisiae snare gene over expression on Heterologous cellulase secretion
(Stellenbosch : Stellenbosch University, 2015-12) Van Zyl, J. H. D.; Van Zyl, Willem Heber; Den Haan, Riaan; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: The budding yeast Saccharomyces cerevisiae has been successfully utilized in several industrial sectors and has over the last decade emerged as a promising host for the production of valuable heterologous proteins. As with the development of most biologicallybased production systems, there are invariably hurdles to overcome, the most pressing being the sub-optimal production yields for many heterologous proteins. The low protein secretion capacity of S. cerevisiae has been attributed to a great number of factors including various unknown secretory bottlenecks within the secretion pathway that collectively result in secretory titers that are often lower than 1% of the theoretical estimates. Increased secretory titers for the industrially significant fungal cellulases in the S. cerevisiae protein production host would greatly contribute to the economic feasibility of second generation bioethanol production. Improved titers will also benefit the production of commercially important biopharmaceutical proteins. SNAREs (Soluble NSF (N-ethylmaleimide-sensitive factor) Attachment REceptor proteins) represent a class of membrane proteins that are required for the majority of membrane fusion events in the cell, including fusion of the protein secretory vesicles with the cis-Golgi and the plasma membrane. In this study, we attempted to elucidate whether the overproduction of some of these SNARE components at the cis-Golgi interface (BOS1, BET1, SEC22 and SED5) and at the plasma membrane (SNC1, SNC2, SSO1, SSO2 and SEC9) could increase the efficiency of the protein secretion process in S. cerevisiae for two industrially significant fungal cellulases – the Saccharomycopsis fibuligera Cel3A (β-glucosidase) and the Talaromyces emersonii Cel7A (cellobiohydrolase I). Our investigation further attempted to elucidate other physiological effects that these genetic modifications could bring about, both in terms of growth vigor and response to secretory stress. The exocytic t-SNARE Sso1p yielded the most improved secretory phenotype for Sf-Cel3A, with an improvement of approximately 43%, whilst the Snc1p v-SNARE component yielded the largest improvement in Te-Cel7A secretion of 71% (relative to the parental strain). The improvements for this reporter protein could be semi-quantitatively illustrated using SDSPAGE and densitometry analysis. Simultaneous overexpression of exocytic SNARE genes led to a moderate improvement of 52% and 48% for the secretion of Te-Cel7A and Sf-Cel3A, respectively, whilst simultaneous SNARE-overexpression in the strains producing the Sf-Cel3A led to measurable decreases in ethanol and osmotolerance, as well as a decreased growth vigor. For the Endoplasmic Reticulum (ER)-to-Golgi SNAREs, it was the t-SNARE Sed5p that yielded the biggest improvements in the secretion of Sf-Cel3A (22%) and Te-Cel7A (68%). However, overexpression of Sed5p did lead to decreases in ethanol and osmotolerance for strains harboring either of the heterologous cellulases expressed on episomal plasmids, in addition to slight decreases in growth vigor. Simultaneous ER-to-Golgi SNARE overexpression led to less significant secretory improvements for Te-Cel7A and decreased secretory titers for Sf-Cel3A, whilst the yeast could not maintain cell viability upon simultaneous overexpression of the ER-to-Golgi SNAREs in the presence of the beforementioned reporter protein. Co-overexpression of the most promising ER-to-Golgi and exocytic SNARE components identified for the improvement of Sf-Cel3A secretion (SED5 and SSO1, respectively) led to a significant improvement in extracellular activity of 130%. The production of Sf-Cel3A led to a measurably increased unfolded protein response (UPR), a mechanism proportionately induced by the buildup of folded and misfolded proteins in the ER. When Sed5p, which led to an improved secretion phenotype for Sf-Cel3A, was overexpressed in conjunction with the aforementioned reporter protein, the UPR activation was notably diminished. This suggests that a higher dosage of Sed5p may improve ER-to- Golgi protein transport to such an extent that the UPR response diminished. Overexpression of the exocytic SNAREs proved more effective for the improvement of native invertase secretion, with Sso1p and Snc1p leading to improvements of 53% and 32%, respectively. However, Sed5p only yielded a 15% improvement. This study suggests that SNAREs fulfill a prominent role within a larger cascade of secretory pathway components that hold potential as secretory-enhancing factors for the S. cerevisiae heterologous protein production host. The positive effects that overexpression of SNAREs introduced for the secretion of heterologous and native proteins (such as invertase) indicate that these components may be implicated in secretory bottlenecks at the cis-Golgi and/or plasma membrane interface.
Elucidating the duality of biofilms as both a proliferation and survival strategy using novel IoT technology
(Stellenbosch : Stellenbosch University, 2023-12) Klopper, Kyle Brent; Wolfaardt, Gideon M.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Microbes rarely exist and proliferate as individual cells, but rather form complex community structures known as biofilms. Biofilm formation ensures that microbial communities are ubiquitous throughout both natural and man-made environments, where the presence of microbial biofilms can be advantageous or detrimental, depending on the context. Biofilm formation is traditionally viewed as a mechanism for microbial survival, since it provides protection from adverse environmental challenges, including starvation, desiccation, predation and antimicrobials. However, a more contemporary perspective also takes the role of biofilms in microbial proliferation into consideration. The reductionist approach, which classifies a biofilm as either a survival or a proliferation mechanism often leads to poor outcomes when the aim is to transition fundamental biofilm research to the real-world and its associated problems. Owing to the inherently complex and dynamic nature of biofilms, a multi-parameter approach is required to better understand its form-function relationship. The development of the novel BioSpec system provides a simple, reliable, and cost-effective tool to monitor biofilm biomass at high- resolution and in real-time, without disturbing the biofilm structure. Coupling of the BioSpec with the carbon dioxide evolution monitoring system (CEMS), facilitates the simultaneous measurement of two important interrelated biofilm metrics; namely biofilm biomass and metabolic activity. The ability to monitor these parameters non-destructively in real-time provides an unprecedented level of insight into biofilm dynamics, as demonstrated here when challenged with biocides at sub-inhibitory and inhibitory concentrations. The inherent complexity of biofilms requires biofouling mitigation strategies that are based on well-informed chemical treatment dosing regimens and the right combination of biocides and dispersants. In industrial applications, the rapid response of microbial respiration rates to chemical treatment offers a strong utility to monitor the success of antimicrobial application, whereas biofilm biomass measurement shows promise as a direct measure of dispersal or removal efficacy. The role of biofilms as ‘planktonic cell production factories’, even when challenged with antimicrobials, was again highlighted in this thesis and emphasises the need for a change in perspective. Microbial proliferation is of critical importance to multiple facets of society ranging from the food, to the medical and industrial sectors. For too long, the emphasis has been placed on the survival attributes of biofilms, thereby neglecting the equally important role of biofilms as mechanism of proliferation and dissemination. The latter has important implications for sanitation regimes in food and beverage processing facilities, where planktonic cell dissemination and secondary biofilm formation at distant locations within a facility, pose serious challenges and risks.

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