Disentangling the role of prokaryotes in regulating export flux via suspended and sinking organic matter in the southern ocean
- Authors: Dithugoe, Choaro David
- Date: 2022-10-14
- Subjects: Microbial ecology , Bioinformatics , Biochemistry , Oceanography , Metagenomics , Carbon cycle (Biogeochemistry) , Prokaryotes
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365745 , vital:65782 , DOI https://doi.org/10.21504/10962/365745
- Description: The role of phytoplankton in regulating atmospheric carbon dioxide in the marine environment has been the subject of extensive research. We lack, however, comparative insights regarding the functional contributions of bacteria, archaea, fungi, and viruses (the microbiota) to organic matter export especially in understudied polar marine environments such as the Southern Ocean. This knowledge deficit is in part due to the high levels of microbial diversity which obscures efforts to study the relationship between diversity and ecosystem functions including their roles in the sequestration of carbon and nitrogen. Elucidating their precise contributions to organic matter export may be central to potential ecosystems feedbacks to global climate change. We examined several factors which may influence organic matter export to depth including net primary production, phytoplankton biomass, temperature, and prokaryotic functional capacity in the Southern Ocean. A Marine Snow Catcher was used to collect suspended and sinking material 10 metres below mixed layer depth at Southern Ocean Time Series (SOTS) in autumn (March-April) and in the Atlantic sector of the Southern Ocean in winter (July-August) and spring (October-November) 2019. The suspended and sinking material was used to determine the particulate organic carbon and nitrogen concentrations which were then used to calculate fluxes and export ratio ((e-ratio) - particulate organic carbon flux divided by net primary production). Additionally, genomic DNA was extracted from the suspended and sinking material and sequenced to obtain Shotgun metagenomic data which was employed to reconstruct metagenome assembled genome (MAGs) and their functional capacity using bioinformatic tools such as DRAM. Data from the Atlantic sector of the Southern Ocean, demonstrate that net primary production and temperature were inversely related to the e-ratio which is consistent with previous findings from the northern region of the Southern Ocean. Genomic functional capacity from SOTS suggested that r-strategist (organisms adapted to live in unstable environments) bacteria (e.g., Gammaproteobacteria) were prominent in the suspended pool. By contrast, the sinking particle-pool appeared to be dominated by K- strategists (organisms adapted to stable environment). The opposite was true for the archaea. This finding (i.e., bacteria) differs from a previous study in the northern region of the Southern Ocean, showing that microbes with K-strategists were more abundant in the suspended fraction. K-strategists typically degrade sinking organic matter into suspended organic matter or dissolved organic matter reducing the organic carbon export efficiency. Furthermore, Data from the Atlantic sector of the Southern Ocean revealed that seasonal temperature changes might dictate the rate of regional prokaryotic degradation across the zones. Resulting in rapid degradation at the northerly warmer regions and slow degradation further south. The data further provide evidence of chemolithoautotrophic mechanisms, with prokaryotes harbouring key pathways, required to transform dissolved inorganic carbon into complex organic forms, including recalcitrant dissolved organic carbon. Collectively, the SOTS and Atlantic sector of the Southern Ocean data suggest that shifts in prokaryotic community structure and functional capacity may regulate (either degradation or synthesis of organic matter) carbon export to depth. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Dithugoe, Choaro David
- Date: 2022-10-14
- Subjects: Microbial ecology , Bioinformatics , Biochemistry , Oceanography , Metagenomics , Carbon cycle (Biogeochemistry) , Prokaryotes
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365745 , vital:65782 , DOI https://doi.org/10.21504/10962/365745
- Description: The role of phytoplankton in regulating atmospheric carbon dioxide in the marine environment has been the subject of extensive research. We lack, however, comparative insights regarding the functional contributions of bacteria, archaea, fungi, and viruses (the microbiota) to organic matter export especially in understudied polar marine environments such as the Southern Ocean. This knowledge deficit is in part due to the high levels of microbial diversity which obscures efforts to study the relationship between diversity and ecosystem functions including their roles in the sequestration of carbon and nitrogen. Elucidating their precise contributions to organic matter export may be central to potential ecosystems feedbacks to global climate change. We examined several factors which may influence organic matter export to depth including net primary production, phytoplankton biomass, temperature, and prokaryotic functional capacity in the Southern Ocean. A Marine Snow Catcher was used to collect suspended and sinking material 10 metres below mixed layer depth at Southern Ocean Time Series (SOTS) in autumn (March-April) and in the Atlantic sector of the Southern Ocean in winter (July-August) and spring (October-November) 2019. The suspended and sinking material was used to determine the particulate organic carbon and nitrogen concentrations which were then used to calculate fluxes and export ratio ((e-ratio) - particulate organic carbon flux divided by net primary production). Additionally, genomic DNA was extracted from the suspended and sinking material and sequenced to obtain Shotgun metagenomic data which was employed to reconstruct metagenome assembled genome (MAGs) and their functional capacity using bioinformatic tools such as DRAM. Data from the Atlantic sector of the Southern Ocean, demonstrate that net primary production and temperature were inversely related to the e-ratio which is consistent with previous findings from the northern region of the Southern Ocean. Genomic functional capacity from SOTS suggested that r-strategist (organisms adapted to live in unstable environments) bacteria (e.g., Gammaproteobacteria) were prominent in the suspended pool. By contrast, the sinking particle-pool appeared to be dominated by K- strategists (organisms adapted to stable environment). The opposite was true for the archaea. This finding (i.e., bacteria) differs from a previous study in the northern region of the Southern Ocean, showing that microbes with K-strategists were more abundant in the suspended fraction. K-strategists typically degrade sinking organic matter into suspended organic matter or dissolved organic matter reducing the organic carbon export efficiency. Furthermore, Data from the Atlantic sector of the Southern Ocean revealed that seasonal temperature changes might dictate the rate of regional prokaryotic degradation across the zones. Resulting in rapid degradation at the northerly warmer regions and slow degradation further south. The data further provide evidence of chemolithoautotrophic mechanisms, with prokaryotes harbouring key pathways, required to transform dissolved inorganic carbon into complex organic forms, including recalcitrant dissolved organic carbon. Collectively, the SOTS and Atlantic sector of the Southern Ocean data suggest that shifts in prokaryotic community structure and functional capacity may regulate (either degradation or synthesis of organic matter) carbon export to depth. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2022
- Full Text:
- Date Issued: 2022-10-14
An investigation into the bacterial biosynthetic origins of bioactive natural products isolated from South African latrunculid sponges
- Authors: Waterworth, Samantha Che
- Date: 2018
- Subjects: Marine biodiversity , Metagenomics , Sponges Biotechnology , Spirochetes , Natural products Biotechnology
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/61826 , vital:28065
- Description: Several pyrroloiminoquinone alkaloids exhibiting cytotoxic, anti-tumour activity have been isolated from sponges within the Latrunculiidae family that are endemic to the South African coastline. Other, structurally similar pyrroloiminoquinone compounds have been isolated from geographically distant and phylogenetically distinct marine sponges, as well as terrestrial myxomycetes which suggested that sponge-associated bacteria may be the true biosynthetic origin of pyrroloiminoquinone compounds. Previous studies have shown that there is conservation of spirochete and betaproteobacterial species in the bacterial communities associated with South African Latrunculiidae sponges and it was proposed that these conserved bacteria represented candidate pyrroloiminoquinone-producers. This study aimed to confirm the conserved dominance of betaproteobacteria and spirochetes within bacterial communities associated with South African latrunculid sponges and employed a shotgun metagenomic approach to assess the functional and biosynthetic potential of associated microbiota in Tsitsikamma favus sponges. Clustering of assembled contigs revealed twenty-three putative bacterial genomes, of which, two were identified as representatives of the conserved betaproteobacteria and spirochete species previously identified in Tsitsikamma sponges. It was shown that the spirochete was most likely an obligate symbiont that benefitted the host sponge through possible defence against pathogenic bacteria and/or nutrient acquisition. The putative genome representing the conserved betaproteobacteria was found to be heavily contaminated and further sequencing is required to accurately resolve the genome for functional characterization. Several biosynthetic gene clusters were identified and demonstrated the bioactive potential of Tsitsikamma favus-associated bacteria. A biosynthetic gene cluster was identified on an unclustered contig that included several genetic features that were indicative of possible pyrroloiminoquinone production.
- Full Text:
- Date Issued: 2018
- Authors: Waterworth, Samantha Che
- Date: 2018
- Subjects: Marine biodiversity , Metagenomics , Sponges Biotechnology , Spirochetes , Natural products Biotechnology
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/61826 , vital:28065
- Description: Several pyrroloiminoquinone alkaloids exhibiting cytotoxic, anti-tumour activity have been isolated from sponges within the Latrunculiidae family that are endemic to the South African coastline. Other, structurally similar pyrroloiminoquinone compounds have been isolated from geographically distant and phylogenetically distinct marine sponges, as well as terrestrial myxomycetes which suggested that sponge-associated bacteria may be the true biosynthetic origin of pyrroloiminoquinone compounds. Previous studies have shown that there is conservation of spirochete and betaproteobacterial species in the bacterial communities associated with South African Latrunculiidae sponges and it was proposed that these conserved bacteria represented candidate pyrroloiminoquinone-producers. This study aimed to confirm the conserved dominance of betaproteobacteria and spirochetes within bacterial communities associated with South African latrunculid sponges and employed a shotgun metagenomic approach to assess the functional and biosynthetic potential of associated microbiota in Tsitsikamma favus sponges. Clustering of assembled contigs revealed twenty-three putative bacterial genomes, of which, two were identified as representatives of the conserved betaproteobacteria and spirochete species previously identified in Tsitsikamma sponges. It was shown that the spirochete was most likely an obligate symbiont that benefitted the host sponge through possible defence against pathogenic bacteria and/or nutrient acquisition. The putative genome representing the conserved betaproteobacteria was found to be heavily contaminated and further sequencing is required to accurately resolve the genome for functional characterization. Several biosynthetic gene clusters were identified and demonstrated the bioactive potential of Tsitsikamma favus-associated bacteria. A biosynthetic gene cluster was identified on an unclustered contig that included several genetic features that were indicative of possible pyrroloiminoquinone production.
- Full Text:
- Date Issued: 2018
Characterization of termite Trinervitermes trinervoides metagenome-derived glycoside hydrolases, the formulation of synergistic core enzyme sets for effective sweet sorghum and corncob saccharification, and their potential industrial applications
- Authors: Mafa, Mpho Stephen
- Date: 2019
- Subjects: Termites , Metagenomics , Glucosides , Hydrolases , Enzymes , Feedstock
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/72414 , vital:30044 , DOI https://doi.org/10.21504/10962/72414
- Description: The current study investigated the biochemical properties of endo-glucanase (GH5E), exo-glucanase (GH5D), xylanase (GH5H) and endo-glucanase/xylanase (GH45), derived from the hindgut bacterial symbionts of a termite (Trinervitermes trinervoides) for their potential role in the biotechnology industry. All these enzymes, except GH5D, exhibited activities on cellulosic and xylan-rich polymeric substrates, which only displayed activity on p-nitrophenyl cellobioside. GH5D, GH5E, GH5H and GH45 enzymes retained more than 80% of their activities at pH 5.5 and also retained more than 80% of their activities at 40ºC. Furthermore, these enzymes were thermostable at 37ºC for 72 hours. GH5E, GH5H and GH45 were generally stable over a range of metal-ion. The kinetic parameters for GH5E were 5.68 mg/ml (KM) and 34.36 U/mg protein (Vmax). GH5D activity did not follow classical Michaelis-Menten kinetics, suggesting product inhibition. GH5H displayed KM values of 5.53, 95.03 and 2.10 mg/ml and Vmax values of 112.36, 144.45 and 180.32 U/mg protein on beechwood xylan, CMC, and xyloglucan, respectively. GH45 displayed a KM of 6.94 mg/ml and a Vmax of 12.30 U/mg protein on CMC. GH5D [cellobiohydrolase (CBH)] and a commercial CBHII (GH6) enzyme outperformed a commercial CBHI (GH7) enzyme when these enzymes hydrolysed β-glucan. GH5D and CBHII also displayed a higher degree of synergy on β-glucan but failed to show synergy on Avicel. We therefore concluded that GH5D and CBHII are β-glucan-specific cellobiohydrolases. The corncob (CC) and sweet sorghum bagasse (SSB) substrates were pretreated with lime, NaOH and NaClO2. Subsequent to pretreatment, these substrates were used to investigate if GH5D, GH5E, GH5H and GH45 could operate in synergy. Results revealed that out of 12 possible core enzyme sets constructed, only two (referred to as CES-E and CES-H) displayed higher activities on pretreated CC or SSB. Simultaneous synergy was generally the most effective mode of synergy during hydrolysis of alkaline pretreated SSB and CC samples by both CES-E and CES-H. Both core enzyme sets did not display synergy on oxidative pretreated substrates. These findings suggest that lime and NaOH are more effective pretreatments for CC and SSB substrates. We used PRotein Interactive MOdeling (PRIMO) software to demonstrate that GH5D protein structure is an (α/β)8 barrel with a tunnel-like active site. Enzymes with this type of protein structure are able to perform transglycosylation, a process in which GH5D produced methyl, ethyl and propyl cellobiosides. We concluded that the GH5D, GH5E, GH5H and GH45 enzymes possess novel biochemical properties and that they form synergy during the hydrolysis of complex substrates (SSB and CC). GH5D transglycosylation could be used to produce novel biodegradable chemicals with special properties (e.g. anti-microbial properties). In conclusion, our findings suggest that GH5D, GH5E, GH5H and GH45 can potentially be used to improve biorefinery processes. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2019
- Full Text:
- Date Issued: 2019
- Authors: Mafa, Mpho Stephen
- Date: 2019
- Subjects: Termites , Metagenomics , Glucosides , Hydrolases , Enzymes , Feedstock
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/72414 , vital:30044 , DOI https://doi.org/10.21504/10962/72414
- Description: The current study investigated the biochemical properties of endo-glucanase (GH5E), exo-glucanase (GH5D), xylanase (GH5H) and endo-glucanase/xylanase (GH45), derived from the hindgut bacterial symbionts of a termite (Trinervitermes trinervoides) for their potential role in the biotechnology industry. All these enzymes, except GH5D, exhibited activities on cellulosic and xylan-rich polymeric substrates, which only displayed activity on p-nitrophenyl cellobioside. GH5D, GH5E, GH5H and GH45 enzymes retained more than 80% of their activities at pH 5.5 and also retained more than 80% of their activities at 40ºC. Furthermore, these enzymes were thermostable at 37ºC for 72 hours. GH5E, GH5H and GH45 were generally stable over a range of metal-ion. The kinetic parameters for GH5E were 5.68 mg/ml (KM) and 34.36 U/mg protein (Vmax). GH5D activity did not follow classical Michaelis-Menten kinetics, suggesting product inhibition. GH5H displayed KM values of 5.53, 95.03 and 2.10 mg/ml and Vmax values of 112.36, 144.45 and 180.32 U/mg protein on beechwood xylan, CMC, and xyloglucan, respectively. GH45 displayed a KM of 6.94 mg/ml and a Vmax of 12.30 U/mg protein on CMC. GH5D [cellobiohydrolase (CBH)] and a commercial CBHII (GH6) enzyme outperformed a commercial CBHI (GH7) enzyme when these enzymes hydrolysed β-glucan. GH5D and CBHII also displayed a higher degree of synergy on β-glucan but failed to show synergy on Avicel. We therefore concluded that GH5D and CBHII are β-glucan-specific cellobiohydrolases. The corncob (CC) and sweet sorghum bagasse (SSB) substrates were pretreated with lime, NaOH and NaClO2. Subsequent to pretreatment, these substrates were used to investigate if GH5D, GH5E, GH5H and GH45 could operate in synergy. Results revealed that out of 12 possible core enzyme sets constructed, only two (referred to as CES-E and CES-H) displayed higher activities on pretreated CC or SSB. Simultaneous synergy was generally the most effective mode of synergy during hydrolysis of alkaline pretreated SSB and CC samples by both CES-E and CES-H. Both core enzyme sets did not display synergy on oxidative pretreated substrates. These findings suggest that lime and NaOH are more effective pretreatments for CC and SSB substrates. We used PRotein Interactive MOdeling (PRIMO) software to demonstrate that GH5D protein structure is an (α/β)8 barrel with a tunnel-like active site. Enzymes with this type of protein structure are able to perform transglycosylation, a process in which GH5D produced methyl, ethyl and propyl cellobiosides. We concluded that the GH5D, GH5E, GH5H and GH45 enzymes possess novel biochemical properties and that they form synergy during the hydrolysis of complex substrates (SSB and CC). GH5D transglycosylation could be used to produce novel biodegradable chemicals with special properties (e.g. anti-microbial properties). In conclusion, our findings suggest that GH5D, GH5E, GH5H and GH45 can potentially be used to improve biorefinery processes. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2019
- Full Text:
- Date Issued: 2019
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