Masters Degrees (Microbiology)

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The ability of antimicrobial peptides to migrate across the gastrointestinal epithelial and vascular endothelial barriers
(Stellenbosch : Stellenbosch University, 2018-03) Dreyer, Leane; Smith, Carine; Dicks, Leon Milner Theodore; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Antibiotic resistance has become a major threat to humankind, necessitating research and development of alternative antimicrobial compounds. Many bacteria, including lactic acid bacteria, produce small antimicrobial peptides, referred to as bacteriocins. These peptides are generally not toxic, are active at low concentrations and have a narrow spectrum of antimicrobial activity. However, they usually have low in vivo stability due to degradation by proteolytic enzymes. Drug delivery systems are thus required to transport bacteriocins to the site of infection. Probiotic bacteria are an attractive delivery system, since these bacteria normally colonise the gastrointestinal tract and produce bacteriocins. Numerous studies have been done on the probiotic Entiro™ which consists of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA. The bacteriocins produced by these strains, plantaricin 423 and bacST4SA, respectively, are active against a variety of pathogens and are potential alternatives to antibiotics. However, it is unknown whether these bacteriocins are able to migrate across the gastrointestinal epithelium and vascular endothelium in order to enter the bloodstream. The aim of this study was to evaluate the stability, cytotoxicity and permeability of plantaricin 423 and bacST4SA in vitro and evaluate the potential use of these peptides as an alternative to antibiotics. The well-known lantibiotic, Nisin A, produced by Lactococcus lactis subsp lactis, was used as control and Listeria monocytogenes EGDe as target (sensitive) organism. Migration of the lantibiotic, nisin A and class IIa bacteriocins, plantaricin 423 and bacST4SA across simulated models of the vascular endothelial and gastrointestinal epithelial barriers was studied by growing human umbilical vein endothelial cells (HUVEC)- and human colonic adenocarcinoma (Caco-2) cells on transmigration inserts and adding fluorescently labelled nisin A, plantaricin 423 and bacST4SA to the inserts. All three peptides diffused across HUVECs and Caco2 cells. Only 21% nisin A, 11% plantaricin 423 and 12% bacST4SA remained attached to Caco-2 cells and only 6% nisin A and 3% bacST4SA attached to the HUVECs, and plantaricin 423 did not attach. The viability of both cell types remained unchanged when exposed to 50 μM nisin A, 50 μM plantaricin 423 and 50 μM bacST4SA, respectively. Furthermore, little extracellular lactate dehydrogenase (LDH) activity was recorded when cells were exposed to 100 μM of each peptide, suggesting that the peptides are not cytotoxic. The three peptides retained 60% of their antimicrobial activity when 25 μM of each were exposed to 80% human plasma for 24 h. However, at higher concentrations (50 μM) 68% of the original antimicrobial activity was recorded and at 100 μM the peptides retained 79% of their activity. This is the first report of nisin A, plantaricin 423 and bacST4SA migrating across simulated gastrointestinal- and vascular barriers. In vivo studies are required to confirm these findings and determine the effect these peptides may have in the treatment of systemic infections.
Anaerobic digestion application in the treatment of gelatin-manufacturing effluent
(Stellenbosch : Stellenbosch University, 2000-12) Lloyd, Magaretha Hester; Van der Merwe-Botha, M.; Britz, T. J.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: A severely polluted industrial effluent is generated by the local gelatinmanufacturing industry. Due to increasingly stringent restrictions on discharge qualities enforced by the National Water Act of 1998 and National Environmental Management Act of 1998, as well as increasing trade-effluent charges implemented via the Local Municipal Bylaws, the industry is compelled to consider a system to pre-treat the polluted effluent. A study was undertaken to examine the viability of anaerobic treatment of the gelatin-manufacturing effluent, since the anaerobic digestion technology is well recognised for the high success rate in the treatment of high-strength, complex wastewaters. Various laboratory and pilot-scale studies were done, using different hybrid Upflow Anaerobic Sludge Blanket (UASB) and contact designs. Two mesophilic laboratory-scale hybrid UASB digester designs, fitted with polyethylene (AD-1) and polyurethane (AD-2), performed well at a hydraulic retention time (HRT) of 1.0 d. Chemical oxygen demand (COD) removal efficiencies of up to 90% (avg. 53%) for AD-1 and 83% (avg. 60%) for AD-2 at organic loading rates (OLR) of 9.56 and 4.62 kg COD.m-3.d-1, respectively, were obtained. High sulphate (S04) removal efficiencies of up to 96% (avg. 86%) for AD-1 and 98% (avg. 82%) for AD-2 were also achieved, respectively. A maximum total solid (TS) removal of 65% (avg. 25%) for AD-1 and 62% (avg. 28%) for AD-2 was reported. An average methane content of 80% (AD-1) and 79% (AD-2) with average methane yields per COD removed of 2.19 and 1.86 m3. kg CODremoved.df-o1r AD-1 and AD-2 were found, respectively. When the same digesters (AD-1 and AD-2) were combined in a muItiphase series configuration, a total COD removal efficiency of up to 97% (avg. 80%) at an OLR of 8.32 kg COD.m-3.d-1,was achieved. Excellent total S04 removals of 96% (avg. 69%) were accomplished. Up to 82% TS (avg. 29%) was also removed during this study and the biogas consisted of 89% methane (avg. 79%). For this multi-phase combination up to 92% volatile fatty acids (VFA) (avg. 48%) were removed, indicating possible selective phase separation of the respective fatty acid producing/utilising bacterial populations. The use of a laboratory-scale UASB bioreactor with recirculation, resulted in COD removal efficiencies of up to 96% (avg. 51%) at an HRT of 3.0 d, and 95% (avg. 54%) at a HRT of 1.0 d. Low performances were generally found, with average S04 and TS removals of 59% (max. 97%) and 26% (max. 67%), respectively at an HRT of 1.0 d. The biogas production was very low throughout the study (0.05 - 0.63 I,d-1 ). A pilot-scale UASB reactor (300 I) was constructed and performed satisfactory with a 58% average COD removal and maximum of 96%. S04 and TS removals up to 96% (avg. 44%) and 93% (avg. 63%), respectively, were obtained. The methane content of the biogas was 85%. The pilot-scale studies were conducted under actual field conditions, where various shock and organic loads had to be absorbed by the system. The pilot-scale contact configuration (300 I) did not perform satisfactory as a result of continuous blockages experienced in the feed and recirculation lines. Maximum COD, S04, VFA and TS removal efficiencies of 41% (avg. 27%), 62% (avg. 41%), 64% (avg. 27%) and 39% (avg. 21%), respectively, were obtained. The results of all the studies indicated acceptable COD removals with increasing OLR's. Indications of the presence of active methanogenic and sulphate-reducing bacterial populations were apparent throughout the studies. One possibility for the successful start-up and commissioning of the anaerobic reactors was the use of a well-adjusted biomass, which consisted of highly selected and adapted microbial consortium for the specific gelatinmanufacturing effluent. It was clear from this study that gelatin-manufacturing effluent can be treated successfully, especially with the use of the UASB design. A welldefined data base was constructed which could be of great value for further upscaling to a full-scale digester.
Analysis of an 18kb accessory region of plasmid pTcM1 from Acidithiobacillus caldus MNG
(Stellenbosch : University of Stellenbosch, 2009-03) Louw, Lilly-Ann; Rawlings, Douglas E.; University of Stellenbosch. Faculty of Science. Dept. of Microbiology.
Biomining organisms are generally found in metal-rich, inorganic environments such as iron and sulfur containing ores; where they play a vital role in mineralization and decomposition of minerals. They are typically obligatory acidophilic, mesophilic or thermophilic, autotrophic, usually aerobic, iron-or sulfur oxidizing chemolithotrophic bacteria. The most prominent biomining organisms used in bioleaching of metal sulfides are Acidithiobacillus ferrooxidans, At. thiooxidans, At. caldus, Sulfobacillus spp. and Leptospirillum spp. Biomining enables us to utilize low grade ores that would not have been utilized by conventional methods of mining. Research has focused on the backbone features of plasmids isolated from bacteria of biomining environments. The aim of this study is to sequence and analyze an 18 kb region of the 66 kb plasmid pTcM1 isolated from At. caldus MNG, focusing on accessory genes carried by this plasmid. Fifteen putative genes / open reading frames were identified with functions relating to metabolism and transport systems. The genes are located in two divergently located operons. The first operon carries features related to general metabolism activities and consists of a transcriptional regulator (ORF 2), a succinate / fumarate dehydrogenase-like subunit (ORF 3), two ferredoxin genes (ORF 4 and ORF 7), a putative HEAT-like repeat (ORF 6) which is interrupted by an insertion sequence (ORF 5) and a GOGAT-like subunit (ORF 8). The second operon contains an ABC-type nitrate / sulfonate bicarbonate-like gene (ORF 9), a binding protein-dependent inner membrane component-like gene, another ABC sulfonate / nitrate-like gene (ORF 12i and 12ii) which is interrupted by an insertion sequence (ORF 13) and two hypothetical proteins with unknown functions (ORF 14 and ORF 15). Southern hybridization analysis have shown that most of the genes from the two operons are found in other At caldus strains #6, “f”, C-SH12 and BC13 from different geographical locations. Expression of the GOGAT-like subunit and the succinate / fumarate-like subunit was demonstrated in At. caldus MNG showing that these genes are functional and actively transcribed. The transcriptional regulator (ORF 2) has been shown to repress the downstream genes of putative operon 1. The persistence of these genes on plasmids together with the fact that they are being expressed, represents a potential metabolic burden, which begs the question why they have been maintained on the plasmid from geographically separated strains (and perhaps also growing under very different nutrient availability conditions) and therefore what possible role they may play.
Analysis of arsenic resistance in the biomining bacterium, Acidithiobacillus caldus
(Stellenbosch : University of Stellenbosch, 2007-03) Kotze, Andries Albertus; Rawlings, Douglas E.; University of Stellenbosch. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: In this study the chromosomal arsenic resistance (ars) genes shown to be present in all Acidithiobacillus. caldus isolates were cloned and sequenced from At. caldus #6. Ten open reading frames (ORFs) were identified on a clone conferring arsenic resistance, with three homologs to arsenic genes, arsC (arsenate reductase), arsR (regulator) and arsB (arsenite export). This ars operon is divergent, with the arsRC and arsB genes transcribed in opposite directions. Analysis of the putative amino acid sequences of these arsRC and arsB genes revealed that they are the most closely related to the ars genes of Acidithiobacillus ferrooxidans. These ars genes were functional when transformed into an Escherichia coli ars deletion mutant ACSH50Iq, and conferred increased levels of resistance to arsenate and arsenite. ArsC was required for resistance to arsenate, but not for resistance to arsenite. None of the other ORFs enhanced arsenic resistance in E. coli. A transposon located arsenic resistance system (TnAtcArs) has been described for highly arsenic resistant strains of the moderately thermophilic, sulfur-oxidizing, biomining bacterium At .caldus #6. In the latter study it was shown that TnAtcArs confers higher levels of resistance to arsenate and arsenite than the chromosomal operon. TnAtcArs was conjugated into a weakly ars resistant At. caldus strain (C-SH12) and resulted in greatly increased arsenite resistance. RT-PCR analysis revealed that arsR and arsC are co-transcribed. Despite ORF1 (cadmium inducible-like protein) and ORF5 (putative integrase for prophage CP-933R) not being involved in resistance to arsenic, ORF1 was co-transcribed with arsRC and ORF5 with arsB. Using arsR-lacZ and arsB-lacZ fusions it was shown that the chromosomal ArsR-like regulator of At. caldus acts as a repressor of the arsR and arsB promoter expression. Induction of gene expression took place when either arsenate or arsenite was added. The chromosomal located ArsR was also able to repress TnAtcArs, but the transposon-located ArsR was unable to regulate the chromosomal system.
An analysis of glycerol synthesis by Saccharomyces cerevisiae
(Stellenbosch : Stellenbosch University, 2002-12) Cronwright, Garth Rupert, 1974-; Prior, B. A.; Rohwer, J. M.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Glycerol metabolism is paramount to the physiological adaptation by Saccharomyces cerevisiae to hyper-osmotic stress conditions. Glycerol metabolism also -plays a fundamental role in maintaining a redox state favourable for growth under fermentative conditions. All aspects of the relationship between redox balancing and glycerol metabolism are not yet fully defined and attempts to manipulate this relationship, i.e., to increase or decrease glycerol yields from fermentation, result in a redox disturbance that is often detrimental to other aspects of metabolism. Another aspect of glycerol metabolism that is not thoroughly understood, is how the various parameters of the glycerol synthesis pathway, each independently and in conjunction with each other, control the rate at which glycerol is synthesized. Addressing these questions has been the topic of this thesis. In this regard, the theory of metabolic control analysis (MeA) was adopted and calculations were performed with the aid of an experimentally validated kinetic model. To ascertain the in vivo substrate, product, coenzyme and known modifier concentrations of the glycerol synthesis pathway, reliable techniques to halt metabolism, extract and measure these metabolites had to be established. The metabolite concentrations constitute a portion of the parameters of the pathway and are necessary to construct a detailed kinetic model. Measuring the concentration of an intracellular metabolite enzymatically requires the cell extract to have an adequate quantity of the metabolite in question. This may be achieved by concentrating the cells, before extracting the metabolite, by means of rapid filtration. Then by freezing the cells with liquid nitrogen, metabolism can be halted instantly. It was found that when metabolites were measured, yields were largely dependent on the method of extraction, since different metabolites are sensitive to different pH and temperature conditions. Methods of extraction found to be reliable for the metabolites of interest in this study are presented in Chapter 3. Metabolic control coefficients calculated by the model helped identify the parameters that control flux through the glycerol synthesis pathway most rigidly. The first reaction of the pathway, catalyzed by NAO+-dependent glycerol 3- phosphate dehydrogenase, had a flux control coefficient ( c; )of 0.83 to 0.87 and exercises the majority of control of flux through the pathway, while the subsequent reaction, catalyzed by glycerol 3-phosphatase, had far less control (C:2 = 0.13 to 0.17). The response coefficients (RJ ) of various parameter metabolites indicate [x] that flux through the pathway is most responsive to the concentration of the substrate, DHAP (RJ = 0.48 to 0.69), followed by the concentration of the [DHAP] inhibitor, ATP (RJ =-0.21 to -0.5). Interestingly, the model also predicts that [ATP] the pathway responds far more severely to the ATP/ADP ratio than to the NADH/NAD ratio, because of the weak response coefficient attributed to NADH (RJ = 0.03 to 0.08). Thus, the model suggests that the targets most strategic [NADH] for altering glycerol synthesis would be the Vmax of the glycerol 3-phosphate dehydrogenase reaction and the concentrations of DHAP and ATP. Ideally, the approach would entail manipulating each of these parameters to their optimal levels in conjunction with each other, with the least detrimental physiological effect possible.
An analysis of population structure using microsatellite DNA in twelve Southern African populations of the Mozambique tilapia, Oreochromis mossambicus (Peters)
(Stellenbosch : Stellenbosch University, 2001-12) Hall, Edward G.; Lewis, Rupert; Brink, Danie; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: DNA micro satellite loci express extensive allelic variation making them convenient markers for research in many fields employing population genetic tools, including aquaculture and conservation genetics. Twelve Oreochromis mossambicus populations from wild, captive and introduced sources in Southern Africa were screened for genetic variation at ten CA repeat micro satellite loci. Three of the loci - UNHI04, UNHlll, and UNH123 - were sufficiently well resolved to screen extensively and were interpreted according to a model of Mendelian inheritance. Data was analyzed in terms of genetic structure and levels of genetic variation, the effect of management regime in captivity through successive generations on genetic diversity, and the nature of phylogenetic relationships present between populations. Exact tests, carried out using Monte Carlo type multiple resampling techniques, and F-Statistics were used to detect and quantify genetic structure among the twelve populations. The Exact test X2 (P < 0.001), a FST of 0.27 (P < 0.001), eST of 0.26, RsT of 0.28, and a ST of 0.17 all indicated significant structuring among the populations. The evident genetic structuring endorsed the practice of maintaining the populations as separate genetic stocks, in separate tanks, in order to preserve unique genetic material for aquaculture strain development. Populations also exhibited some significant deviations from Hardy Weinberg equilibrium characterised by an overall reduced heterozygosity across the loci. In microsatellite studies, null alleles are often suggested as major contributors to heterozygote deficits. To test for null alleles, two controlled crosses of 0. mossambicus were made. The progeny from each cross were examined for expected parental allelic ratios at the UNHI04, UNHlll and UNH123 loci. All three loci presented evidence of possible null alleles. Accelerated inbreeding and genetic drift through successive generations in captivity can reduce heterozygosity and gene diversity. To investigate loss of diversity a sample taken from the Bushmans population in 1999 (N = 25) was compared with a Bushmans 2000 sample (N = 36). The comparison highlighted altered allele frequencies, a significant increase in average observed heterozygosity and a non-significant change in average expected heterozygosity using the UNHI04 and UNH123 loci. Calculation of genetic distances and phylogenetic comparisons between the populations provided insight into the degree of management required in conserving genetic diversity in natural populations of Mozambique tilapia. UPGMA and Neighbour-Joining techniques were used to construct phylogenetic trees using Dm and ({)~)2 distance matrices. Clustering of populations appeared to reflect geographic locality of the source populations, however certain populations were not congruent with geography. Mantel tests were used to expose a possible association between genetic distance matrices generated from each individual locus. An association would support a geographic background to population genetic structure. The Mantel tests did not provide conclusive evidence. Mantel tests for association between the combined locus Dm and (81l)2 genetic distance matrices and a geographic distance matrix were similarly non-significant. Multi-dimensional scaling (MDS) plots of Euclidean distance values for Dm and (81l)2 matrices presented a two-dimensional view of the genetic distance data. The degree of similarity with the UPGMA and Neighbour-Joining tree-clustering pattern was higher for the (81l)2 than for the Dm MDS plots. Scatter plots indicated a reliable non-linear correlation between Euclidean distance and genetic distance for the two-dimensional MDS. The micro satellite markers employed in this research provided molecular information needed for complimenting a co-study on quantitative genetic evaluation of the twelve populations. The quantitative co-study provided measures of average length and weight gain indices for the populations based on progeny growth trials. No significant correlation of average heterozygosity (gene diversity) with either average weight or length gain was found. The significant genetic diversity and structure present between the twelve populations provided rationale for implementing strategies to conserve natural 0. mossambicus populations as genetic resources, and manage captive populations for long term maintenance of genetic diversity.
Analysis of the mobilization region of the broad host-range IncQ-like plasmid, pTC-F14, and its ability to interact with a related plasmid, pTF-FC2
(Stellenbosch : Stellenbosch University, 2003-12) Van Zyl, Leonardo Joaquim; Rawlings, D. E.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: The 14.2 kb plasmid pTC-FI4 was isolated from the moderately thermophilic (45°- 50°C), highly acidophilic (pH 1.5 to 2.5), chemolithotrophic bacterium Acidithiobacil/us caldus and has a replicon that is closely related to the promiscuous, broad host-range, IncQ-family of plasmids. The region containing the mobilization genes was sequenced and encoded five Mob proteins and an origin of transfer, which are related to the DNA processing (Tral) region of IncPI plasmids, rather than to the three Mob protein systems of the IncQ-l-group plasmids (e.g. plasmids RSFIOIO or R1162). Plasmid pTC-F14 is the third example of an IncQ family plasmid that has five mob genes, with the others being pTF-FC2 and pRAS3.1. The minimal region that was essential for mobilization included the mobA, mobB and the mobC genes as well as the oriT. The mobD and mobE genes were non-essential, but together enhanced the mobilization frequency by approximately 300-fold. The repB gene increased the mobilization frequency but was not essential for mobilization. Mobilization of pTC-F14 between Escherichia coli strains by a chromosomally integrated RP4 plasmid was more than 3500-fold less efficient than the mobilization ofpTF-FC2. When both plasmids were co-resident in the same E. coli host, pTC-FI4 was mobilized at almost the same frequency as pTF-FC2. This enhanced pTC-FI4 mobilization frequency was due to the presence of a combination of the pTF-FC2 mobD and mobE gene products, the functions of which are still unknown. pTF-FC2 could mobilize the oriT of pTC-FI4 whereas pTC-F14 could only mobilize the pTFFC2 oriT if provided with some of the mobilization genes from the pTC-FC2 mobilization region. Unexpectedly either the mobEDC genes or the mobAB genes would allow the mobilization of the pTF-FC2 oriT by pTC-F14 even though there was no common gene between the two subsets. No evidence for any negative effect on the transfer of one plasmid by the related, potentially competitive plasmid was obtained.
Antimicrobial susceptibility and population dynamics of a defined biofilm community under different nutrient conditions
(Stellenbosch : Stellenbosch University, 2004-03) Garny, Kerstin; Wolfaardt, Gideon M.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Little is known about the impact of nutrient conditions on antimicrobial resistance in biofilms grown under continuous flow conditions. Furthermore, community-level response of biofilms to antimicrobial substances and different nutrient regimes are poorly described. A better understanding of the influence of environmental conditions on biofilm behavior and antimicrobial susceptibility may contribute to the efforts, addressing the problems associated with increased antimicrobial resistance. Therefore, the aim of this study was to evaluate the survival and population dynamics in a defined mixed-species biofilm community grown under different nutrient conditions and when subjected to biocide treatment. Epi-fluorescence microscopy in conjunction with the LIVE/DEAD® BacLight™ viability kit, a conventional cultivation technique (plate counts), and culture-independent techniques (terminal restriction fragment length polymorphism and fluorescent in situ hybridization) were applied to observe biofilm and planktonic antimicrobial susceptibility, as well as population dynamics. A defined mixed-species community, consisting of four bacterial strains, was cultivated and monitored in a flow cell system. Two nutrient types were used: 1) a complex growth medium [tryptone soy broth (TSB)] and 2) a defined synthetic medium [minimal salts supplemented with glucose (MSM + Glucose)]. In addition, these two nutrient types were applied in different concentrations. Biofilm and planktonic community behavior was influenced by the nutrient type and concentration. Species evenness in the planktonic community was influenced by the nutrient conditions, while species richness changed in response to biocide treatment and nutrient conditions. TSB-grown microbial communities were more susceptible directly after biocide treatment than those grown in MSM + Glucose, however, biofilm viability in the latter nutrient condition decreased within 24 h after biocide treatment. Furthermore, a surprising difference in the recovery rate between biofilm and associated planktonic communities was observed. A conceptual model was developed that aimed to explain the observed biofilm-planktonic interactions. This model proposes that the cells found in the outer regions of a biofilm are the primary source of the associated planktonic cells, and that this phenomenon is independent from overall biofilm activity.
The application of astaxanthin producing bacteria in poultry feed
(Stellenbosch : Stellenbosch University, 2017-03) Conradie, Tersia Andrea; Jacobs, Karin; Pieterse, Elsje; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: In the food industry, the colour of the product is important to the consumer as it gives an indication of the freshness and quality of the product. Hens are not able to produce pigments and absorb pigments through their diet. This has led to a rapidly emerging trend in poultry farming to enhance egg yolk colour as the yolk colour is influenced by the diet of the hen. Over the years, natural or synthetic carotenoids have been added to poultry feed. Several studies have focused on using astaxanthin producing microorganisms, such as the microalga, Haematococcus pluvialis, and yeast, Xanthophyllomyces dendrorhous. However, the production costs are expensive and the thick cell walls of the microalga and yeast limits its whole cell application as a feed additive. Some bacterial species are also able to produce astaxanthin, including the bacterium Paracoccus marcusii, and have previously not been used as a feed additive to enhance yolk colour. The purpose of this study was, therefore, to determine the whole cell application of P. marcusii as a feed additive to enhance egg yolk colour, without the need to homogenise the cells or extract the pigment. In the first experimental chapter (Chapter 2), the growth conditions and astaxanthin production of P. marcusii was optimised. Furthermore, the stability of the astaxanthin molecule under different storage conditions, namely lyophilisation and microencapsulation, was determined. The optimum growth conditions for P. marcusii and for astaxanthin production was at 26 °C in a specialised medium containing yeast extract (5 g/L), bacteriological peptone (10 g/L) and NaCl (3%) at a pH between 6 – 7. Astaxanthin is a valuable compound with several health promoting benefits for humans and animals. However, the molecule is unstable when exposed to oxygen, light and temperature. After lyophilisation in sucrose (10% m/v), there was an 85% loss in astaxanthin concentration after 3 weeks. However, the loss in cell viability was low. When P. marcusii was microencapsulated in calcium alginate beads, cell viability significantly decreased when stored at 20 °C compared to 4 °C. However, only 30% of the total astaxanthin concentration was lost after 3 weeks at both storage temperatures. The microencapsulation significantly improved the stability of astaxanthin under storage. The highest concentration of astaxanthin obtained was 24.25 μg/g dry cell weight. Chapter 3 examined the pigmentation effect of P. marcusii when fed to laying hens to enhance egg yolk colour. Paracoccus marcusii was fed to hens daily either in a sucrose solution (10% m/v) or microencapsulated in calcium alginate beads. After the pilot study, it was clear that a diet free of all pigments was needed to effectively determine the pigmentation effect of P. marcusii. In all the experimental trials there was a significant increase in yolk colour and no negative effect on egg quality, laying rate or hen weight was observed. There was also an increase in whole egg and yolk weight when compared to the control groups. Furthermore, the microbial communities of the duodenum and caeca were investigated after a prolonged feeding of P. marcusii to detect any changes that might have occurred (Chapter 4). The microbial community of the hen’s gastro-intestinal tract (GIT) starts out as a simple community in the small intestines which becomes increasingly diverse and complex further down the intestinal tract. The findings in this study revealed a similar pattern when considering the results obtained from the Shannon diversity index and total number of operational taxonomic units (OTUs). Starting in the duodenum, the index ranged between 2.14 – 2.59 and increased in the caeca to between 2.45 – 3.03. OTUs increased from 21.44 – 28.60 in the duodenum to 28.30 – 38.00 in the caeca. A significant difference was only observed for the OTUs of the experimental group compared to the control groups in both the duodenum and caeca. There was no significant difference observed in the microbial community structure of the duodenum. However, distinct patterns and clusters formed in the caeca between the experimental diet group compared to the control diet groups. Since no mortalities were recorded during the trial and all hens appeared in excellent health, it is safe to assume that the change in microbial community structure of the caeca was not negative. Therefore, P. marcusii is safe to use as a feed additive for laying hens. Finally, Chapter 5 evaluated the costs associated with the small-scale production of P. marcusii microencapsulated in calcium alginate beads and its feasibility in the poultry industry. Based on the economic assessment, the total cost for one month’s production of 210 g calcium alginate beads is estimated at R2912.88. This is too expensive and not practical to be used by poultry farmers. Possible solutions can include the use of inexpensive peptones, production on a larger scale and also increasing the concentration of bacterium encapsulated in the bead.
Application of solar pasteurization for the treatment of harvested rainwater
(Stellenbosch : Stellenbosch University, 2017-03) Reyneke, Brandon; Khan, Wesaal; Khan, Sehaam; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Rainwater harvesting has been earmarked by South African governmental authorities as an intervention strategy that could alleviate the pressures on existing centralised water distribution systems, especially in rural areas and urban informal settlements, where insufficient waste removal and potable water infrastructure are available. However, numerous studies have indicated that harvested rainwater may not be safe to use for all daily water requirements, as numerous chemical and microbial contaminants may be associated with stored tank water. Rainwater treatment technologies, including solar pasteurization (SOPAS), have subsequently been investigated (Chapter 1). In order to determine whether decentralised rainwater harvesting SOPAS systems may be a viable alternative in providing the inhabitants of informal settlements with a supplementary water source, two small- (Sites 1 and 2) and one large-scale (Site 3) rainwater harvesting SOPAS systems were installed in Enkanini informal settlement, Stellenbosch, South Africa (Chapter 2). The microbial and chemical quality of the unpasteurized and pasteurized (produced by the respective systems) rainwater was monitored using conventional water quality monitoring techniques, including the culturing of indicator organisms, screening for selected indigenous rainwater pathogens using the polymerase chain reaction (PCR) and quantitative PCR (qPCR) assays and the monitoring of anion and cation concentrations. Additionally, the operational sustainability of the systems and water usage by the participating households were monitored. Chemical analyses indicated that all anions and cations were within the limits stipulated by various national and international drinking water quality guidelines, with the exception of zinc which contravened the respective guidelines before (mean: 3919 μg/L) and after (mean: 3964 μg/L) pasteurization at both Sites 1 and 2. In addition, the arsenic concentrations measured at Site 3 before (mean: 18.69 μg/L) and after (mean: 18.30 μg/L) pasteurization exceeded the respective drinking water guidelines. The increased zinc concentrations were attributed to the galvanised zinc roofing material installed at Sites 1 and 2, while the increased arsenic concentrations may be attributed to a roofing treatment or paint utilised to cover the catchment area at Site 3. Microbial analyses indicated that pasteurization temperatures of 53 °C (small-scale systems) and 55 °C (large-scale system) were required to reduce Escherichia coli and total and faecal coliforms to below the detection limit [< 1 colony forming units (CFU)/100 mL]. However, minimum pasteurization temperatures of 66 °C (small-scale systems) and 71 °C (large-scale system), were required to reduce the heterotrophic plate count (HPC) to within drinking water limits (1.0 × 104 CFU/100 mL). Of the opportunistic pathogens detected using PCR assays, Legionella spp. was the most prevalent pathogen detected in the small-scale systems [unpasteurized (100%) and pasteurized (91%)] and the large-scale system [unpasteurized (83%) and stored pasteurized tank water (100%)]. Quantitative PCR analysis then indicated that while the gene copies of Legionella spp., Pseudomonas spp. and Salmonella spp. were reduced during SOPAS, the organisms were still detected at the highest pasteurization temperatures analysed for each site (Site 1 – 85 °C; Site 2 – 66 °C; Site 3 – 79 °C). Additionally, the application of a metabolic responsiveness adenosine triphosphate (ATP) assay (BacTiter-GloTM Microbial Cell Viability Assay) indicated the presence of metabolically active cells in all pasteurized rainwater samples analysed. Results also indicated that the systems required limited maintenance and the small-scale systems in particular were able to provide the participating households with an alternative warm water source that could be utilised for numerous domestic purposes. As various limitations have been associated with the use of culture-based analyses for the monitoring of water quality, the aim of Chapter 3 was to compare molecular-based viability assays [ethidium monoazide bromide (EMA)-qPCR, propidium monoazide (PMA)-qPCR and DNase treatment in combination with qPCR] as well as the metabolic responsiveness ATP assay to culturing analysis for their ability to accurately determine cell viability in bacterial monocultures following heat treatment. Three Gram-negative (Legionella spp., Pseudomonas spp. and Salmonella spp.) and two Gram-positive (Staphylococcus spp. and Enterococcus spp.) bacteria commonly associated with water sources were selected as test organisms. Of the various concentrations of EMA and PMA analysed, 6 μM EMA and 50 μM PMA were identified as the optimal dye concentrations as low log reductions were recorded (viable and heat treated samples) in comparison to the no viability treatment control. Comparison of the results obtained for all the molecular viability assays (6 μM EMA, 50 μM PMA and DNase treatment) then indicated that the 6 μM EMA concentration was comparable to both the 50 μM PMA and the DNase treatment for the analysis of most of the test organisms (viable and heat treated). In addition, the results for the culturing analysis (CFU) of the viable S. typhimurium as well as the viable and heat treated samples of L. pneumophila and P. aeruginosa were comparable to the gene copies detected using molecular-based viability assays. However, the CFU in the heat treated samples of S. typhimurium were significantly lower than the gene copies detected using DNase in combination with qPCR, with no gene copies or CFU detected in the heat treated samples of S. aureus and E. faecalis. In contrast, while the ATP assays indicated the presence of metabolically active cells in the viable and heat treated samples, the ATP assay also indicated the presence of metabolically active cells in samples that had been autoclaved (negative viability control). It was thus concluded that molecular-based assays may be used to supplement culture based analysis for the comprehensive identification of the viable microbial population in water samples (before and after treatment).
Arbuscular mycorrhizal root colonisation and the subsequent host plant response in young grapevines in a South African commercial vineyard
(Stellenbosch : Stellenbosch University, 2003-03) Meyer, Andre Harold; Botha, Alfred; Valentine, A. J.; Archer, E.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH SUMMARY: Arbuscular mycorrhizal (AM) fungi facilitate the uptake of nutrients, improve growth and alleviate drought stress in grapevines. Consequently, AM fungal root colonisation contributes to the optimum performance of grapevines. It is for this reason that young grapevines are sometimes inoculated with commercial AM fungal strains to reduce environmental stresses during transplant. In the past, soil fumigation has often been considered as a prerequisite for soil conditioning with commercial AM fungal strains. However, grape growers opting to inoculate with these fungal strains will have to do so in unfumigated soils, since the use of fumigants in South African agricultural soils is currently being phased out. Since little is known about the nature and scope of indigenous AM fungi that may be present in SA vineyard soils, it is difficult to predict the grapevine's response to artificial inoculation in soils already containing adequate concentrations of these fungi. In the first part of the study, commercially available AM inocula were tested under field conditions that would prevail on a typical farm. This entailed measuring vine growth, nutrition, drought stress resistance and percentage root colonisation, over two consecutive seasons, from the onset of planting new commercial grapevines. The field trial carried out at Groenland, a commercial farm in the Stellenbosch Region. Merlot grafted onto 101-14 Mgt, 110 Richter (110 R) and 99 Richter (99 R), was planted in December 1998. These rootstocks were selected to accommodate different soil forms: 101-14 Mgt and 110 R on a Westleigh soil form, which was ridged and 99 R on an unridged Fernwood soil form. Vine roots were inoculated during planting with different AM inocula, i.e. Biocult®, Vaminoc" and Glomus sp. 1054. One treatment was left uninoculated and treated with a combination of the fungicides Benlate" (active ingredient: benomyl) and Rovral Flo® (active ingredient: iprodione). The control received neither fungicides nor inocula. Microscopic examination of the vine roots revealed that, apart from a significantly higher level of root colonisation observed in Biocult-treated 99 R vines during the first season, the level of AM root colonisation was similar in both the uninoculated (control) and inoculated vines. Infected control vines indicated that indigenous AM fungi were present in the vineyard soil. This level ranged between 40% and 85%, and 70% and 90% in the first and second season, respectively. Apart from the significant growth improvement observed in 110 R vines inoculated with Glomus sp. 1054 during the first season, no growth improvement was observed for the other rootstocks or treatments. Furthermore, generally no alleviation of water stress and nutritional benefits could be detected for both the seasons. Despite this, less than 1% dieback was recorded for the vines. In the second part of the study, additional information on the diversity of indigenous AM fungal species was obtained, which included the quantification and identification of these fungi present in the soil. The AM fungal spore numbers in the vineyard soil ranged from 1000 to 3779 spores/100 g dry soil. The results confirmed that the majority of AM fungal species found in the soil was not part of the commercial inocula, but originated either from the vineyard and/or the nursery where the vines were obtained. The uncovered AM fungal species belong to the genera Acaulospora, Gigaspora, Glomus, Sclerocystis and Scutellospora. This is similar to the AM fungal genera recorded in vineyards by other workers. To the best of our knowledge, this study provided the first documented evidence on the diversity of indigenous AM fungi present in SA vineyard soils. Although it may be preliminary in nature, the results clearly showed that a wide diversity and abundance of indigenous AM fungal populations may occur in a typical SA vineyard. Depending on the superiority and possible masking effects on the part of the indigenous AM fungal populations, positive responses to inoculation with commercial AM fungal strains in grapevines grown in such vineyard soils may consequently be unlikely. Thus, before reconditioning of vineyard soils with these fungi can commence, it is essential for farmers to first assess the mycorrhizal status of their soils and nursery vines. Since the majority of SA farmers are not yet familiar with inoculation practices and are still unacquainted with the mycorrhizal status of their soils, the findings from this study could be of great benefit to particularly wine grape growers opting to inoculate with commercial AM fungal strains on a large-scale.
Assessing the capacity for micropollutants to induce changes in the biofilm EPS composition and yield
(Stellenbosch : Stellenbosch University, 2021-12) Rossouw, Johann Herman; Wolfaardt, Gideon M.; Stone, Wendy; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: A consequence of widespread chemical manufacturing and usage is the increasing presence of a new class of contaminant: micropollutants. Despite the investment of significant resources into the development of novel approaches to wastewater treatment, the removal efficiency of micropollutants has been varied and conflicting between different studies. A notable gap in current research efforts is assessing the capacity for chronic micropollutant exposure to alter the main mechanisms contributing to their removal in secondary wastewater treatment. The aim of this study was three-fold. First, to investigate the comparative homogeneity of the Slime-EPS matrix composition for multi- versus single-species biofilms. Secondly, to attempt to quantify the adsorptive capacity of the Slime-EPS fragment utilizing a published dye-probing analysis protocol. Finally, to assess the capacity for chronic (7-day) micropollutant exposure to influence the composition and yield of the Slime-EPS fragment for a known, biofilm producing Pseudomonas species. Single-species biofilms exhibited a more consistent Slime-EPS composition in terms of their protein: carbohydrate ratio. Dye-probing analysis efforts indicated the capacity for toluidine blue dye to exhibit altered spectral absorbance as a result of increased dimerization – which was found to be influenced by both the introduction of the Slime-EPS itself and increasing concentrations of NaCl. Increasing concentrations of TB dye was shown to induce hypsochromic (blue-) spectral shifts. Ciprofloxacin was found to significantly reduce the biofilm Slime-EPS yield (p < 0.05) following 7 days of continuous exposure, whereas exposure to diclofenac for the same interval had no significant effect on Slime-EPS yield. Neither ciprofloxacin nor diclofenac had a significant effect on the Slime-EPS protein: carbohydrate ratio following 7 days of exposure.
Assessment of wood degradation by Pycnoporus sanguineus when co-cultured with selected fungi
(Stellenbosch : Stellenbosch University, 2007-03) Van Heerden, Andrea; Botha, Alfred; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: It is commonly known that a diversity of fungi, including yeasts, may occur on plant surfaces. Similarly, on fallen trees an ecological succession of different fungal species is known to occur during wood degradation. Some of these fungi may be pioneer fungi contributing to the initial degradation process, while others may be yeasts associated with the fruiting bodies of macro-fungi which in turn are able to utilize the more recalcitrant polymers in wood. Previously, it was revealed that an increase occurs in the wood degradation rate of certain white-rot fungi when co-cultured with selected yeast species. A well known inhabitant of decomposing trees is the white rot fungus Pycnoporus sanguineus. It was found by some that this fungus is capable of selective delignification while growing on the wood of poplar trees, while other authors found a simultaneous delignification pattern on Eucalyptus grandis trees. In the latter case cellulose and lignin are degraded simultaneously. We were interested in how yeasts occurring on the surface of P. sanguineus fruiting bodies, and the pioneer fungus Aspergillus flavipes, impact on wood degradation by this white-rot fungus. Restriction Fragment Length Polymorphisms (RFLP) analyses were used to obtain an indication of the species composition of the culturable yeast community associated with fruiting bodies of P. sanguineus. The impact of the most dominant of these yeasts species, i.e. Pichia guilliermondii and Rhodotorula glutinis, as well as A. flavipes, on wood degradation by P. sanguineus was then determined by analyzing the major wood components after growth of co-cultures on hot water washed E. grandis wood chips. Co-cultures of P. sanguineus with the other fungi were prepared by inoculating the wood chips, contained in solid state bioreactors and supplemented with molasses and urea, with the an appropriate volume of fungal inoculum, resulting in an initial moisture content of 60%. After two weeks of incubation at 30°C with constant aeration, the chips were harvested. Standard protocol (TAPPI Standard Methods), commonly used by the paper and pulp industry, were then employed to determine the percentage cellulose, Klason Lignin, as well as polar and solvent-borne extractives in the chips. The resulting data were analyzed using box plots, as well as biplots. No degradation of Klason lignin was observed, while the percentage cellulose did decrease during fungal degradation. Taking into account the inherent shortcomings of the Klason Lignin determination, the results supported the findings of others that P. sanguineus shows a simultaneous delignification pattern while growing on E. grandis wood. In addition, it was found that the yeasts played no significant role in the degradation ability of P. sanguineus, while A. flavipes showed an antagonistic effect on P. sanguineus with respect to cellulose degradation. However, it was clear that the analytical methods used in this study were inadequate to accurately determine fungal degradation of wood. In addition, it was obvious that the methods used did not distinguish between fungal biomass and wood components. Nevertheless, the methods provided us with a fingerprint of each culture growing on E. grandis wood, allowing us to compare the chemical composition of the different cultures and the un-inoculated hot water washed wood chips. The question, therefore, arose whether the effect of a particular coculture, on the chemical composition of wood, differs between tree species. Consequently, chemical alterations in different tree species, induced by a P. sanguineus / A. flavipes co-culture, were investigated in the next part of the study. Wood chips originating from four tree species, i.e. Acacia mearnsii, Eucalyptus dunnii, E. grandis, and Eucalyptus macarthurii, were inoculated with this co-culture. The culture conditions and subsequent analyses of the wood components were the same as in the first part of the study. From the box- and biplots constructed from the resulting data, it was clear that the chemical composition of each tree species were altered in a different manner by the coculture. Lignin content showed an apparent increase in A. mearnsii, while E. dunnii showed a decrease in cellulose content. The results indicate that wood of different tree species are degraded in a different manner and this phenomenon should be taken into account in selecting fungi for biopulping.
Binary interactions between Candida albicans and microorganisms associated with the human body, under aerobic and anaerobic conditions
(Stellenbosch : Stellenbosch University, 2019-04) Valentine, Marisa; Botha, Alfred; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: The yeast Candida albicans forms part of the microbiota of healthy human individuals and colonises many body sites, including the skin and gut. This opportunistic fungus can, however, cause candidiasis in immunocompromised individuals and can interact with other potential pathogens. Since such interactions are still largely unexplored, the first aim of this study was to prepare anaerobic bacteria/ yeast co-cultures with a quarter-strength brain heart infusion (¼ BHI; 9.25 g/l) broth to investigate binary interactions between C. albicans and potentially pathogenic representatives of the gut-associated bacterial genus Bacteroides, i.e. Bacteroides fragilis NCTC 9343 and Bacteroides vulgatus ATCC 8482. Compared to growth in monocultures, yeast growth was largely unaffected by the presence of B. fragilis or B. vulgatus. In contrast, growth of both bacteria was enhanced in the presence of C. albicans. Supplementation of Bacteroides monocultures with dead Candida albicans CAB 392 cells, containing intact outer cell wall mannan layers, resulted in increased bacterial concentrations. Culturing of Bacteroides in a liquid minimal medium supplemented with candidal mannan demonstrated that B. vulgatus ATCC 8482, unlike B. fragilis NCTC 9343, utilised the mannan. Furthermore, by reducing initial oxygen levels in monocultures prepared with ¼ BHI broth by supplementation with L-cysteine hydrochloride, bacterial numbers were higher compared to in monocultures prepared with ¼ BHI broth not supplemented with the reducing agent. Suggesting that C. albicans stimulated Bacteroides growth via aerobic respiration and/or antioxidant production. Bacterial growth-promoting metabolite(s) was likely secreted by Candida, since the cell-free supernatant (spent medium) of 24-h-old C. albicans CAB 392 monocultures promoted Bacteroides growth in monocultures. Since C. albicans also colonises the skin of human individuals, the second aim of this study was to investigate binary interactions between C. albicans CAB 392 and a representative of a skin-colonising yeast, Malassezia pachydermatis CAB 820. Enumerating C. albicans CAB 392 and M. pachydermatis CAB 820, after incubation in a liquid artificial sweat medium under different pH and nutrient conditions, revealed that yeast concentrations in monocultures did not differ from yeast concentrations in co-cultures. However, in co-cultures prepared with artificial sweat medium supplemented with 0.05 g/l yeast extract, M. pachydermatis CAB 820 was outcompeted by C. albicans CAB 392 after 24 h of incubation. Additionally, C. albicans CAB 392 numbers were greater when grown in the spent medium of 9-h-old M. pachydermatis CAB 820 monocultures, prepared with the sweat medium supplemented with 0.05 g/l yeast extract, compared to in freshly prepared supplemented sweat medium. Therefore, while this Malassezia species may secrete Candida growth-promoting factor(s), a potential competitive interaction can exist between C. albicans and M. pachydermatis in the presence of certain nutrient levels or types. It was concluded that environmental conditions govern binary interactions between representatives of C. albicans and Bacteroides or Malassezia. Furthermore, it was observed that the interactions of C. albicans and Bacteroides were species- and strain-specific. Since the planktonic phase of bacterial and yeast cells was studied in liquid cultures, future studies should investigate interactions between C. albicans and Bacteroides or M. pachydermatis in dual-species biofilms.
Biodegradability of organic carbon following hydrothermal carbonization treatment
(Stellenbosch : Stellenbosch University, 2021-12) Mangashena, Hazel; Wolfaardt, Gideon M.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Hydrothermal carbonization (HTC) is a ‘wet pyrolysis process’ that opens up a field of potential for feedstocks such as the non-traditional renewable and abundant wet agricultural residues, and municipal wastes for char production. It reduces waste and substitutes primary fuels as a source of energy. The product, known as hydrochar, has received attention because of its potential as precursors of activated carbon in wastewater remediation, soil remediation applications, solid fuels, and other carbonaceous materials. However, persistent priority and emerging micropollutants are consistently detected in numerous wastewater treatment plants that could potentially serve as feedstock for the process- this is a cause for concern. The goal of this study was to assess the impact of HTC on organic micropollutants. The working hypothesis was that the combination of high temperature and pressure would sterilize the biomass and transform micropollutants into benign products, while any residue would be readily biodegradable. Experiments indicated that char resulting from belt press sludge carbonized at 240 (with a corresponding°C pressure of 3,3MPa) was sterile, exhibiting a high fuel ratio of 0,72 and HHV 21,40 Mj/kg. Subsequently, 4 test compounds that are; carbamazepine, chloramphenicol, methylparaben, and bisphenol-A were studied for their biodegradability following HTC. Biodegradation of the test compounds extracted from the hydrochar ranged between 84% and 100% while only methylparaben indicated complete biodegradability from the liquid phase. Results of the study prove that with optimization, the question presented by residual micropollutants persisting in the natural environment following HTC technologies can be mitigated.
Biodegradation of winery wastewater
(Stellenbosch : University of Stellenbosch, 2003-04) Malandra, Lida; Bloom, M.; Wolfaardt, Gideon M.; University of Stellenbosch. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: Large volumes of wastewater are generated annually during the grape harvest season from various processing and cleaning operations at wineries, distilleries and other wine-related industries. South African regulatory bodies dictate that wastewater should have a pH of 5.5 to 7.5 and a chemical oxygen demand (COD) lower than 75 mg/L. However, winery wastewater has a typical pH of 4 to 5 and a COD varying between 2 000 and 12 000 mg/L. Urban wineries channel the wastewater to local sewage treatment facilities and are often heavily fined for exceeding governmental requirements. Rural wineries usually have little or no treatment operations for their wastewater and it is often irrigated onto crops, which may result in environmental pollution and contamination of underground water resources. Various criteria are important in choosing a wastewater treatment system, such as an ecofriendly process that is flexible to withstand various concentration loads and characteristics, requiring low capital and operating costs, minimal personal attention and do not require too much land. In this study, a large variation in COD, pH and chemical composition of the winery wastewater was observed that could be related to varying factors such as the harvest load, operational procedures and grape variety. Wastewater from destemming and pressing operations contained higher concentrations of glucose, fructose and malic acid, which originated from the grape berries. The fermentable sugars (glucose and fructose) contributed to almost half of the COD with a smaller contribution from ethanol and acetic acid. The low pH can be ascribed to relative high concentrations of organic acids in the wastewater. The efficacy of biological treatment systems depends strongly on the ability of microorganisms to form biofilm communities that are able to degrade the organic compounds in the wastewater. Preliminary identification of microorganisms that naturally occur in winery wastewater indicated the presence of various bacterial and yeast species that could be effective in the biological treatment of the wastewater. When evaluated as pure cultures under aerobic conditions, some of the yeast isolates effectively reduced the COD of a synthetic wastewater, whereas the bacterial isolates were ineffective. The most effective yeast isolates were identified as Pichia rhodanensis, Kloeckera apiculata, Candida krusei and Saccharomyces cerevisiae. Our search for cost-effective biological treatment systems led to the evaluation of a Rotating Biological Contactor (RBC) for the treatment of winery wastewater. The RBC was evaluated on a laboratory scale with 10% (v/v) diluted grape juice and inoculated with a mixed microbial community isolated from winery wastewater. The results showed a reduction in the COD that improved with an extended retention time. Evaluation of the RBC on-site at a local winery during the harvest season resulted on average in a 41% decrease in COD and an increase of 0,75 pH units. RFLP analysis of the biofilm communities within the RBC confirmed a population shift in both the bacterial and fungal species during the evaluation period. The most dominant yeast isolates were identified with 18S rDNA sequencing as Saccharomyces cerevisiae, Candida intermedia, Hanseniaspora uvarum and Pichia membranifaciens. All these species are naturally associated with grapes and/or water and with the exception of Hanseniaspora uvarum, they are able to form either simple or elaborate pseudohyphae.
Biodiversity in the genus Penicillium from coastal fynbos soil
(Stellenbosch : Stellenbosch University, 2008-12) Visagie, Cobus M.; Jacobs, Karin; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
Penicillium is a well‐known cosmopolitan genus with more than 225 accepted species. Species from this diverse genus, in general, are considered to primarily be soil fungi, with decomposition as its main function. Therefore, together with its ubiquitous nature, these species are of great importance in ecosystems, agriculture and biotechnology. However, in South Africa, very little research has been done on this complex genus, as species identification were often found to be problematic, even for experienced taxonomists. This lead to a number of South African studies only mentioning that a Penicillium spp. were isolated, without making any attempt of showing the extent of diversity within the genus from the unique habitats. The present study set out to explore the extent of the species diversity in Penicillium isolated from the Cape Floristic Region, specifically focusing on coastal fynbos soil. Soil samples were collected from this region, at sites situated outside Malmesbury. Four hundred and thirty four Penicillium strains were isolated from soil‐dilutions. The strains were characterized using morphological characters and subsequently placed into 24 morphological groups. There were, also, more or less 40 strains that could not be grouped with any other isolates. Groupings were made according to conidiophore branching patterns which divided the strains into their respective subgenera. Eight species from subgenus Aspergilloides, seven from subgenus Furcatum, eight from subgenus Biverticillium and one from subgenus Penicillium were isolated. The species were further characterized in subsequent chapters. In the second chapter of this thesis, one of the taxonomic groups in subgenus Biverticillium, isolated from coastal fynbos soil, Protea infructescences and on moth‐damaged Riesling grapes in Canada, was examined. This species was characterized using morphology and were found to have several unique characters, such as the very short synnema produced after prolonged incubation. These characters did not conform to descriptions of previously described species. Its novelty was confirmed by an ITS phylogeny and the strains were subsequently described as Penicillium ramulosum prov. nom. with P. cecidicola and P. dendriticumas its sister taxa. In chapter three, a further seven groups belonging to Penicillium subgenus Biverticillium were characterized. These strains were identified as P. minioluteum, P. verruculosum and P. rugulosum‐like, respectively. Four of the groups showed unique morphological characters, with the ITS phylogeny resolving the fynbos strains separate from all previously described species. The strains were, therefore, considered to be new to science and described as P. solicola prov. nom., P. ptychoconidium prov. nom., P. occultum prov. nom. and P. chloroloma prov. nom., respectively. A key to species from subgenus Biverticilliumcluded. is also inPenicillium subgenus Furcatum was the subject of the fourth chapter of this thesis. Our survey found that although the species diversity in this group was not as high as for the other subgenera, it was the group most often isolated in this study. Species were identified as P. janczewskii, P. canescens, P. melinii, P. corylophilum and P. citrinum using morphological characters. One species belonging to subgenus Penicillium, P. expansum, was also isolated and compared to species recorded during a previous survey. Amongst the identified species, were two groups that could not be identified using published keys, with their novelty confirmed by an ITS phylogeny. They are, therefore, described here as P. subturcoseum prov. nom. and P. hemitrachum prov. nom. A key to species in this subgenus is also provided. In Chapter 5 the presence of Penicillium subgenus Aspergilloides, which is characterized by monoverticillate conidiophores, were investigated. Species were identified as P. roseopurpureum, P. restrictum, P. hirayamae and P. toxicarium. Amongst the identified species, were four groups that did not conform to previously described species and are described here as P. brachycaulon prov. nom., P. malacosphaerula prov. nom., P. cumulacinatum prov. nom. and P. vulgaris prov. nom., respectively. The newly described species have been included in a key, together with closely related species and the other species of subgenus Aspergilloides from the fynbos soil. Species identifications in Penicillium is often problematic and South African taxonomists have often not attempt to identify strains down to species level. During this study, Penicillium was found to be well represented in the soil, with a large proportion being previously undescribed. For this reason, a dichotomous and synoptic key to species isolated during this study are provided in the final chapter. This study should thus serve as a basis for further explorations into the diversity and ecological role of this group of organisms in this ecologically mportant biome.
Biodiversity of yeasts associated with mosquito larvae from different water habitats
(Stellenbosch : Stellenbosch University, 2016-03) Steyn, Annica; Botha, Alfred; Roets, Francois; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: The success of mosquitoes in nature has been linked to their microbiota and bacteria in particular. Yet, knowledge on their symbioses with yeasts is lacking. To explore possible associations, culturable yeasts were isolated from wild Culex larvae, and their habitat water, from sites that differed in land-use type such as natural forests, plantations, pristine river pools and urban sites. Isolated yeasts were classified using restriction fragment length polymorphism (RFLP) analyses and identified by sequencing the D1/D2 region of the 26S rRNA gene. Fungal representative strains of Aureobasidium, Candida, Cryptococcus, Debaryomyces, Galactomyces, Hannaella, Hypocreales, Lecythophora, Meyerozyma, Pichia, Rhodosporidium, Rhodotorula, Trichosporon and Wickerhamomyces were isolated from the larvae. These were the first records of the yeast microbiota from wild mosquito larvae and showed that they may harbour potential clinically relevant yeast species, including the well- known opportunistic human pathogen Candida albicans. Water quality of sites was analyzed for different physicochemical parameters and related to yeast assemblages in water and in larvae. The effects of physicochemical characteristics were more pronounced on yeast assemblages occurring in the habitat waters than on the endogenous yeasts occurring within the larvae. Larvae also harboured a higher than expected abundance of ascomycetous, fermentative and non-pathogenic yeasts in their gut, in relation to water from their habitat, suggesting some sort of selection pressure on yeast gut symbionts. Significant influences of land-use types on yeast assemblages were only detected for water-associated yeasts. These results indicated that larvae may act as the dominant factor that determines their endogenous yeast assemblages. Selected yeast strains of C. albicans, Candida glabrata, Candida pseudolambica, Cryptococcus gattii, Metschnikowia bicuspidata, Saccharomyces cerevisiae and Wickerhamomyces anomalus were also used as sole feed in a 21 day feeding experiment, during which the effect of each was tested on the ontogeny of Culex pipiens. Although most yeasts supported larval growth in a similar manner to the positive control S. cerevisiae strain, the different yeast strains impacted differently on Cx. pipiens ontogeny. Notably, survival and pupation of larvae were negatively impacted by a representative strain of the primary pathogen C. gattii, signifying that some yeasts are natural antagonists of mosquitoes. In addition, the microbiota of newly emerged adults was examined and the results supported the hypothesis of microbial reduction/ elimination during adult emergence for this species, thereby questioning the role of mosquitoes in yeast dispersal amongst different habitats.
Biological indicators of copper-induced stress in soil
(Stellenbosch : Stellenbosch University, 2002-03) Du Plessis, Keith R. (Keith Roland); Botha, Alfred; Wolfaardt, Gideon M.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: The concentrations of copper (Cu) in vineyard soils of the Western Cape range from 0.1 to 20 ppm. However, more than 160 tons of the fungicide copper oxychloride are annually being sprayed on these vineyards. This has raised concerns that Cu may accumulate in these soils, resulting in a negative impact on the soil biological processes, especially since the soils in the Western Cape are slightly acidic, making Cu more mobile and available for soil organisms than would have been the case in alkaline soils. The goal of the initial part of this study was therefore to identify those soil microbial communities indigenous to the Western Cape, which are most susceptible to Cu-induced stress as a result of the addition of copper oxychloride. These potential bioindicators of Cu-induced stress were first searched for in uncultivated agricultural soil from Nietvoorbij experimental farm. Consequently, a series of soil microcosms was prepared by adding various concentrations of Cu as a component of copper oxychloride, to each of eight aliquots of soil: 0 (control), 10, 20, 30, 40, 50, 100, 500 and 1000 ppm. The resulting concentrations of exchangeable Cu in these microcosms were found to be 2 (control), 12,23,34,42,59, 126,516 and 1112 ppm. Selected microbial communities in each microcosm were subsequently monitored over a period of 245 days. It was found that the culturable microbial numbers did not provide a reliable indication of the effect of Cu on community integrity. However, analyses of terminal-restriction fragment length polymorphism (T-RFLP) community fingerprints and especially analyses of the whole community metabolic profiles, revealed that shifts in the soil microbial communities took place as the Cu concentration increased. Direct counts of soil protozoa also revealed that the addition of Cu to the soil impacted negatively on the numbers of these eukaryotes. To confirm these findings in other soil ecosystems, the impact of copper oxychloride on whole community metabolic profiles and protozoan numbers were investigated in soils from Koopmanskloof commercial farm and Nietvoorbij experimental farm. These potential bioindicators were subsequently monitored in a series of soil microcosms prepared for each soil type by adding the estimated amounts of 0 (control), 30, 100 and 1000 ppm Cu as a component of copper oxychloride to the soil. The results confirmed the fmdings that elevated levels of copper impact negatively on the metabolic potential and protozoan numbers of soil. Consequently, it was decided to investigate a combination of protozoan counts and metabolic profiling as a potential bioindicator for Cu-induced stress in soil. Data collected from all the microcosms containing exchangeable Cu concentrations ranging from 1 ppm to 1112 ppm was used to construct a dendrogram using carbon source utilization profiles in combination with protozoan counts. It was found that the microcosms grouped into clusters, which correlated with the concentration of exchangeable Cu in the soil. Under the experimental conditions used in this study, the combination of protozoan counts and metabolic profiling seemed to be a reliable indicator of Cu-induced stress. However, this bioindicator must be further investigated in other soil types using other types of stress inducing pollutants. In addition to the above fmdings it was also found that the numbers of soil protozoa was particularly susceptible to Cu-induced stress in soils with a low soil pH. This is in agreement with the fmdings of others on the bio-availability of heavy metals in low pH soils. In these soils, nutrient cycling as a result of protozoan activity, may therefore be particularly susceptible to the negative impact of copper to the soil.
The biological sulphate removal process
(Stellenbosch : Stellenbosch University, 2001-12) Greben, Harma; Wolfaardt, Gideon M.; Maree, J. P.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
ENGLISH ABSTRACT: South Africa is one of the world's major coal producers, resulting in the second highest foreign exchange earner for South Africa. However, the mining industry contributes negatively to (ground) water pollution, due to the formation of acid mine drainage (AMD). AMD originates from the bacterial oxidation (Thiobacillus ferrooxidans) of pyrite (FeS) and contains high levels of sulphate and metals. Sulphate rich waters can be treated applying the biological sulphate removal technology. This study concentrated on biologically removing sulphate from synthetic feed- and mine water, using the single-stage completely-mixed reactor system. The advantage of using this reactor system is that except for removing sulphate from about 2000 to less than 200 mg/t', it can also partly biologically remove the formed sulphides. It was established that both ethanol and sugar can be used, as the carbon and energy source, however ethanol is more cost effective than sugar. Ethanol dosage and Hydraulic Retention Time (HRT) studies were undertaken to investigate at what concentration, the highest sulphate and sulphide removal rates were achieved. It was found that the highest sulphate reduction rates were obtained when using 1mf ethanol/f feed and that the removal rates were dependent on the HRT: the lower the HRT, the higher the sulphate reduction rate. The highest sulphide oxidation rate was achieved at the HRT of 6 h. It was, furthermore shown that the single stage completely-mixed reactor system could successfully be used to remove sulphate from Schoongezicht mine effluent, not only removing the sulphate, but also most of the metals, thereby increasing the mine effluent pH from 2.5 to 7. The conclusion of this study was that a completely-mixed reactor system, as described in this thesis, can successfully be applied to treating acid mine drainage using ethanol (1 m.e etanol/f feed water) as the carbon and energy source at a hydraulic retention time as low as 4 hours. This technology has great potential for pilot- and full-scale treatment of sulphate rich effluents such as acid mine drainage.

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