Browsing by Author "Steyn, Annica"
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- ItemBiodiversity 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.
- ItemProducing organic acids from pomace wastes – a biorefinery concept(Stellenbosch : Stellenbosch University, 2022-04) Steyn, Annica; Van Zyl, Willem Heber; Viljoen-Bloom, Marinda; Stellenbosch University. Faculty of Science. Dept. of Microbiology.ENGLISH SUMMARY: The production of energy and valuable products via petroleum refineries could be partially replaced by biorefineries that are utilising renewable biomass as substrate. For example, an important metabolite such as malic acid in fruit wastes, like apple and grape pomace, could serve as a substrate for microbial conversion to organic acids and other green chemicals. Specific waste sources were investigated to isolate native yeast strains that may have acquired improved malic acid degradation abilities. The 98 new isolates from grape, apple and plum waste were screened for their ability to degrade extracellular malic acid relative to 50 known strains. Most (94%) of the new isolates degraded more than 50% of the malic acid in both the presence and absence of glucose, whereas only 14% of the known strains could do so, thus confirming the value of exploring and exploiting natural biodiversity for new candidates. The eight best isolates were evaluated in synthetic media, with two strains showing potential for the production of ethanol and acetic acid during aerobic and oxygen-limited growth on apple and grape pomace. The screening of yeasts led to the identification of Saccharomyces cerevisiae strain 61 that showed stronger malic acid-degrading capabilities than expected for this species. Preliminary characterisation revealed that strain 61 degraded malic acid in synthetic media more efficiently (potential for application in the wine industry), produced significantly higher biomass (potential for recombinant protein expression in S. cerevisiae) and is more heat-resistant (potential for consolidated bioprocessing) than commercial wine yeasts. Recombinant S. cerevisiae strains were constructed to express either the Candida krusei (Ckr_fum) or the codon-optimised Escherichia coli (Eco_fum) fumarase gene with or without the XYNSEC secretion signal of Trichoderma reesei xylanase 2. These strains were further engineered to co-express the Schizosaccharomyces pombe transporter (mae1) gene for the active uptake of malic acid. Both strains 5A and 31B(p1), expressing the Ckr_fum and mae1 genes [31B(p1) also contains the XYNSEC secretion signal], produced fumaric acid from extracellular malic acid. Our findings illustrate that disruption of the natural FUM1 gene in S. cerevisiae strains is beneficial when using malic acid as a substrate for fumaric acid production. The inclusion of a transporter allowed for better malic acid degradation and quicker fumaric acid production, and the yeast fumarase was more effective than the bacterial fumarase for fumaric acid production. Strain 5A was able to produce 0.065 g/L fumaric and 2.55 g/L ethanol after 72 h on grape pomace, suggesting that this strain may have potential application for a fruit waste biorefinery following optimisations. This study resulted in the isolation of a number of yeast strains with the ability to utilise malic acid, including S. cerevisiae strain 61. After unravelling the reason(s) for its enhanced malic acid utilisation, this strain could be a promising candidate for the development of a fumaric acid-producing biorefinery host strain. However, this would require the construction of a recombinant strain expressing an effective fumarase and dicarboxylic acid transporter, with the Ckr_fum and mae1 genes as potential candidates.