School of Molecular & Cell Biology (ETDs)

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Exploring temporal changes in the malting barley seed microbiome with meta-omics to understand nitrogen content effects
(University of the Witwatersrand, Johannesburg, 2024-10) Tshisekedi, Kalonji Abondance; De Maayer, Pieter; Botes, Angela
Barley (Hordeum vulgare L.) is a critical cereal crop, particularly in beer production, where it plays a significant role in the economy, especially in South Africa. Despite its importance, the barley seed microbiome, which affects seed storage and quality, is not well understood. This research addresses two key questions: (1) how microbial composition and function evolve during storage and (2) how the inherent nitrogen content of the grain affects these dynamics. Using metagenomic and metaproteomic approaches, eight barley samples from the Kadie cultivar, stored for various durations (harvest, three, six, and nine months) with high and low nitrogen content, were analysed. Metagenomic sequencing revealed a predominance of bacterial sequences and minimal fungal presence, with storage time having a greater impact on microbial diversity than nitrogen content. However, specific bacterial genera such as Erwinia, Pantoea, Pseudomonas, and Stenotrophomonas showed nitrogen-dependent prevalence. Metagenome-assembled genomes (MAGs) were reconstructed, representing 26 bacterial genera, with minimal shared orthologues, highlighting taxonomic diversity. Functional analysis identified key metabolic pathways and carbohydrate-active enzymes (CAZymes) essential for microbial adaptation during storage. Metaproteomic analysis further showed the active expression of proteins related to nutrient transport and stress response, indicating functional changes over storage time. Overall, this research enhances the understanding of the barley seed microbiome, providing valuable insights into storage practices that could improve brewing quality and agricultural sustainability.
Metagenome sequencing of the lichen species Flavopunctelia flaventior and Parmotrema tinctorum from Gauteng, South Africa
(University of the Witwatersrand, Johannesburg, 2024-06) Katane, Malebogo Dimpho; Botes, Angela; De Maayer, Pieter
Lichens are defined as a mutualistic association between fungi (mycobiont) and an algal and/or cyanobacterial photobiont. Increasing evidence suggests that lichens comprise more diverse microorganisms than initially thought, where lichens represent an interaction between archaea, bacteria, filamentous fungi, green algae, yeasts, and viruses. Not many comprehensive studies have been done of South African lichen species. The present study employed metagenome sequencing to investigate the lichen microbiomes of Flavopunctelia flaventior and Parmotrema tinctorum sampled from Bryanston, Gauteng province, South Africa. Furthermore, the roles played by the members of the lichen microbiome within symbioses were also studied by functionally annotating the assembled metagenomes of the two lichen species. This study sets the groundwork for future studies on South African lichen species. In Chapter 1, an extensive literature review on lichens, their ecology, taxonomy and biology is discussed. Furthermore, it delves into the existence and shape of the microbiome beyond the mycobiont and the photobiont. Additionally, possible roles that the lichen microbiome may play in sustaining the lichen symbiosis is also discussed. In Chapter 2, the metagenomes of two lichen species were sequenced, the quality of the reads were assessed, and taxonomic classification was performed to elucidate the composition of microorganisms associated with each lichen species. Both microbiomes were dominated by bacteria, with limited fungi, viruses, and archaea. The majority of the identified phyla and genera were found to be common between the two lichen species. Similarities in the core microbiome was accounted for by the fact that F. flaventior and P. tinctorum were sampled from the same location and they are both members of the Parmeliaceae family. In Chapter 3, the metagenomic reads were assembled and functionally annotated using various bioinformatics tools. We demonstrate that the members of the lichen microbiome are involved in the cycling of nutrients such as carbon and nitrogen. We also found differences in carbon fixation pathways, which were attributed to the accessory microbiome. Finally, a summary highlights key results and recommendations on future work that could be undertaken to further provide insight into biological pathways essential to sustain the lichen symbiosis.