Towards a bacterial biofertiliser for the rehabilitation of disturbed and degraded land
- Authors: Masudi, Wiya Leon
- Date: 2024-10-11
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/466826 , vital:76790 , DOI https://doi.org/10.21504/10962/466826
- Description: Agriculture, mining, industry, and human activity disturb, degrade, and pollute pristine environments and particularly the soil environment. Excessive land exploitation slows or disrupts the soil potential, rendering it incapable of playing its role leading to land degradation. In the mining sector, specifically coal mining, rehabilitation of disturbed and degraded land involves strategies that include importing topsoil and using fossil fuel-derived fertilisers. Both practices are unsustainable. To address the unsustainability, a myco-phytoremediation technology known as Fungcoal was developed to facilitate successful revegetation of mining-disturbed and degraded land following the bioconversion of waste coal into a soil-like humic-rich substrate. To offset the dependence on chemical-based fertilisers, efforts were/are focussed on finding mutualistic and cost-effective microbial resources with plant growth-promoting (PGP) activity as a bacterial biofertiliser. This study made use of 22 isolated bacteria and the three Fungcoal coal-degrading fungi viz., Aspergillus sp. ECCN 84, Aspergillus sp. ECCN 225 and Penicillium sp. ECCN 243 as the microbial resource. Initially, characterisation of the substrate waste coal and molecular identification of the selected bacterial isolates were carried out. Physicochemical analysis of the low-rank coal (LRC) substrate revealed a pH of 3.60 with background S content equivalent to 7.13 g L-1, N at 20 mg L-1, P at 7.8 mg L-1 and K at 3.3 mg L-1. Energy-dispersive X-ray spectroscopy (EDX) analysis revealed a C and O content of 23.09 and 69.03 wt%, respectively. Metagenomic analysis of the microbial population associated with the LRC substrate showed that among the 96.32% of bacteria, 59.46 to 62.18% belonged to Bacillota (also called Firmicutes), a phylum of largely Gram-positive bacteria, and 33.01 to 35.74% to Pseudomonadota (synonymous with Proteobacteria), a phylum of mostly Gram-negative bacteria. Following purification of the selected bacterial isolates and molecular characterisation by PCR, phylogenetic relatedness to known plant growth-promoting bacteria (PGPB) contained in the GenBank database showed that these bacterial isolates clustered with high bootstrap values to the reference PGPB strains. Only Pseudomonas sp. ECCN 10b (MW672582) was outside of the tree and shared significant similarity (100%) with Pseudomonas fluorescens (CP015638). A biochemical study revealed that the two Proteus sp. strains, Exiguobacterium sp., Enterobacter sp., and Ancylobacter, tolerated high salt and a wide range of temperatures. Bacterial isolates showed a high pH tolerance between 3 and 11, with the best growth at pH around 7. Nine of the identified strains, four Bacillus sp., Exiguobacterium sp., Enterobacter sp., Pseudomonas sp., Arthrobacter sp., and Aeromonas sp., were able to grow and increase in a medium containing either glucose, mannitol, sodium L-glutamate, sucrose, or fructose. Growth was highest in media containing either sodium L-glutamate, sucrose, or fructose. All the coal degrading strains and 83% of those isolated from municipal wastewater used more complex carbon sources such as high and LRC. The potential for PGP activity was quantified spectrophotometrically by measuring the production of auxins, as indole-3-acetic acid (IAA) equivalents; gibberellins, as gibberellic acid (GA3) equivalents, along with 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and siderophore activity. Additionally, nutrient mobilisation was evaluated by monitoring an ability to mineralise NH4+, PO43−, and K+. Competent PGP strains for the coal degrading isolates included Proteus strain ECCN 20b, Proteus strain ECCN 23b, and Serratia strain ECCN 24b. In response to L-trp supplementation, the concentration of indolic compounds (measured as indole-3-acetic acid) increased. Production of ammonium and solubilisation of insoluble P by these strains was also apparent. Only Serratia strain ECCN 24b could solubilise insoluble K. Production of indoles increased following exposure to increasing aliquots of LRC, suggesting no negative effect of this material on indole production and that these bacteria may possess PGP potential. Of the twelve bacterial strains isolated from wastewater MaB-flocs, three produced indoles, nine mineralised NH4+, seven solubilised P, and one K. Potential of isolated strains for PGP activity according to a one-way ANOVA on ranks was: ECCN 7b > ECCN 4b > ECCN 6b > ECCN 3b = ECCN 10b > ECCN 1b = ECCN 5b > ECCN 8b > ECCN 2b > ECCN 12b > ECCN 9b = ECCN 11b. Further study revealed that cell-free filtrate from indole-producing cultures of Aeromonas strain ECCN 4b, Enterobacter strain ECCN 7b, and Arthrobacter strain ECCN 6b promoted mung bean adventitious root formation. Based on a biochemical study and the outcome of the ranking of bacterial strains according to PGP-like activities, three bacteria, Enterobacter sp., strain ECCN 7b, Proteus sp., strain ECCN 20b and Serratia sp., strain ECCN 24b that showed great mutualistic relationship with the most effective Fungcoal biocatalyst, A. fischeri ECCN 84, were used to prepare a bacterial bio-fertiliser. This consortium grew well in NB supplemented with L-tryptophan and produced indole compounds that could activate the adventitious rooting of mung bean (Vigna radiata L.) hypocotyls. Finally, the consortium showed no antibiotic resistance activity; however, they produced better biofertiliser with good responses to root/plant biomass production of the same Fabaceae, mung bean (Vigna radiata L.). The further development of this consortium into a cost-effective, environmentally friendly biofertiliser may help reduce dependence on chemical-based fertilisers and improve the sustainability of Fungcoal and other land rehabilitation strategies. Further studies are therefore underway to investigate in greater detail the PGP activity of these isolates individually and in consortium under field conditions to support the Fungcoal myco-phytoremediation strategy. , Thesis (PhD) -- Faculty of Science, Institute for Environmental Biotechnology, 2024
- Full Text:
- Date Issued: 2024-10-11
- Authors: Masudi, Wiya Leon
- Date: 2024-10-11
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/466826 , vital:76790 , DOI https://doi.org/10.21504/10962/466826
- Description: Agriculture, mining, industry, and human activity disturb, degrade, and pollute pristine environments and particularly the soil environment. Excessive land exploitation slows or disrupts the soil potential, rendering it incapable of playing its role leading to land degradation. In the mining sector, specifically coal mining, rehabilitation of disturbed and degraded land involves strategies that include importing topsoil and using fossil fuel-derived fertilisers. Both practices are unsustainable. To address the unsustainability, a myco-phytoremediation technology known as Fungcoal was developed to facilitate successful revegetation of mining-disturbed and degraded land following the bioconversion of waste coal into a soil-like humic-rich substrate. To offset the dependence on chemical-based fertilisers, efforts were/are focussed on finding mutualistic and cost-effective microbial resources with plant growth-promoting (PGP) activity as a bacterial biofertiliser. This study made use of 22 isolated bacteria and the three Fungcoal coal-degrading fungi viz., Aspergillus sp. ECCN 84, Aspergillus sp. ECCN 225 and Penicillium sp. ECCN 243 as the microbial resource. Initially, characterisation of the substrate waste coal and molecular identification of the selected bacterial isolates were carried out. Physicochemical analysis of the low-rank coal (LRC) substrate revealed a pH of 3.60 with background S content equivalent to 7.13 g L-1, N at 20 mg L-1, P at 7.8 mg L-1 and K at 3.3 mg L-1. Energy-dispersive X-ray spectroscopy (EDX) analysis revealed a C and O content of 23.09 and 69.03 wt%, respectively. Metagenomic analysis of the microbial population associated with the LRC substrate showed that among the 96.32% of bacteria, 59.46 to 62.18% belonged to Bacillota (also called Firmicutes), a phylum of largely Gram-positive bacteria, and 33.01 to 35.74% to Pseudomonadota (synonymous with Proteobacteria), a phylum of mostly Gram-negative bacteria. Following purification of the selected bacterial isolates and molecular characterisation by PCR, phylogenetic relatedness to known plant growth-promoting bacteria (PGPB) contained in the GenBank database showed that these bacterial isolates clustered with high bootstrap values to the reference PGPB strains. Only Pseudomonas sp. ECCN 10b (MW672582) was outside of the tree and shared significant similarity (100%) with Pseudomonas fluorescens (CP015638). A biochemical study revealed that the two Proteus sp. strains, Exiguobacterium sp., Enterobacter sp., and Ancylobacter, tolerated high salt and a wide range of temperatures. Bacterial isolates showed a high pH tolerance between 3 and 11, with the best growth at pH around 7. Nine of the identified strains, four Bacillus sp., Exiguobacterium sp., Enterobacter sp., Pseudomonas sp., Arthrobacter sp., and Aeromonas sp., were able to grow and increase in a medium containing either glucose, mannitol, sodium L-glutamate, sucrose, or fructose. Growth was highest in media containing either sodium L-glutamate, sucrose, or fructose. All the coal degrading strains and 83% of those isolated from municipal wastewater used more complex carbon sources such as high and LRC. The potential for PGP activity was quantified spectrophotometrically by measuring the production of auxins, as indole-3-acetic acid (IAA) equivalents; gibberellins, as gibberellic acid (GA3) equivalents, along with 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and siderophore activity. Additionally, nutrient mobilisation was evaluated by monitoring an ability to mineralise NH4+, PO43−, and K+. Competent PGP strains for the coal degrading isolates included Proteus strain ECCN 20b, Proteus strain ECCN 23b, and Serratia strain ECCN 24b. In response to L-trp supplementation, the concentration of indolic compounds (measured as indole-3-acetic acid) increased. Production of ammonium and solubilisation of insoluble P by these strains was also apparent. Only Serratia strain ECCN 24b could solubilise insoluble K. Production of indoles increased following exposure to increasing aliquots of LRC, suggesting no negative effect of this material on indole production and that these bacteria may possess PGP potential. Of the twelve bacterial strains isolated from wastewater MaB-flocs, three produced indoles, nine mineralised NH4+, seven solubilised P, and one K. Potential of isolated strains for PGP activity according to a one-way ANOVA on ranks was: ECCN 7b > ECCN 4b > ECCN 6b > ECCN 3b = ECCN 10b > ECCN 1b = ECCN 5b > ECCN 8b > ECCN 2b > ECCN 12b > ECCN 9b = ECCN 11b. Further study revealed that cell-free filtrate from indole-producing cultures of Aeromonas strain ECCN 4b, Enterobacter strain ECCN 7b, and Arthrobacter strain ECCN 6b promoted mung bean adventitious root formation. Based on a biochemical study and the outcome of the ranking of bacterial strains according to PGP-like activities, three bacteria, Enterobacter sp., strain ECCN 7b, Proteus sp., strain ECCN 20b and Serratia sp., strain ECCN 24b that showed great mutualistic relationship with the most effective Fungcoal biocatalyst, A. fischeri ECCN 84, were used to prepare a bacterial bio-fertiliser. This consortium grew well in NB supplemented with L-tryptophan and produced indole compounds that could activate the adventitious rooting of mung bean (Vigna radiata L.) hypocotyls. Finally, the consortium showed no antibiotic resistance activity; however, they produced better biofertiliser with good responses to root/plant biomass production of the same Fabaceae, mung bean (Vigna radiata L.). The further development of this consortium into a cost-effective, environmentally friendly biofertiliser may help reduce dependence on chemical-based fertilisers and improve the sustainability of Fungcoal and other land rehabilitation strategies. Further studies are therefore underway to investigate in greater detail the PGP activity of these isolates individually and in consortium under field conditions to support the Fungcoal myco-phytoremediation strategy. , Thesis (PhD) -- Faculty of Science, Institute for Environmental Biotechnology, 2024
- Full Text:
- Date Issued: 2024-10-11
An investigation into water and sanitation in the Eastern Cape Province and potential for implementation of biotechnology platforms
- Authors: Gumunyu, Wilbert
- Date: 2022-10-14
- Subjects: Sewage South Africa Eastern Cape , Sanitation South Africa Eastern Cape , Algae Biotechnology , Biotechnology , Sewage Purification Biological treatment , Algal biofuels
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362830 , vital:65366
- Description: In contemporary South Africa, a country in transition, destruction rather than reconstruction seems commonplace. Electricity supply is at an all-time low and ‘load shedding’ is an almost daily occurrence. Similarly, more fragility of water delivery and sanitation service is being reported with the likelihood of ‘water shedding’ a soon-to-be reality. In view of the ever-increasing reported mismanagement of South Africa’s water and sanitation infrastructure, which is likely nearing the point of collapse if not already collapsed, this thesis set out to interrogate at a provincial and municipal level the status of water and sanitation in Chris Hani District Municipality (CHDM), Eastern Cape Province. One major driver behind this study was to evaluate provincial and municipal water/wastewater infrastructure as a platform to support implementation of locally developed bioprocess technologies. First, publicly available historical data was used to derive a baseline or benchmark from which to determine gains/losses in compliance, water quality and efficiency. The period 2009-2013/4 during which Blue and Green Drop reporting was routine, was used as an appropriate start point. Data thus derived indicated that, at provincial and district municipality (DM) level, potable water supply was by bulk water schemes and, sanitation was typically by waste stabilization pond (WSP) treatment of municipal sewage. The derived benchmark for the period 2009-2013/14 indicated that most plants were not compliant (~75% of the Eastern Cape Province water treatment plants (WTP) operating between average performance to critical state), operated either at- or above design capacity (Eastern Cape Province, 16%; CHDM, 19%). A large number of plants for the province (62.4%) and district (81.25%) had hydraulic design capacity or average daily flows that were unknown (information not provided in available literature or plant reports) and final effluent did not always meet the general standard (70.3% of the CHDM wastewater treatment works (WWTW)). To determine the contemporary state (2020 - 2022) of water and sanitation within CHDM, a scoping exercise of WTP and WWTW in selected local municipalities was carried out. This was done along a west-east transect by appraising WTP and WWTW in the towns of Cradock, Tarkastad, Komani, Tsomo, Cacadu, and Ngcobo. Furthermore, targeted assessment of the Queenstown WTP and the Cradock WWTW was used to derive real-time data on the status of water and sanitation infrastructure. Results showed that water and sanitation services within CHDM had, in the years between 2009-2013/14 to date, deteriorated. Freshwater demand significantly exceeded capability of water supply schemes, where demand was as much as three times greater than reported available supply. For most WWTW, operation was in excess of capacity and between 1.52 and 12 times installed hydraulic loading. The targeted scoping exercise revealed that Queenstown WTP is a moderate risk plant (Cumulative risk rating, CRR = 11 and maximum risk rating, MRR = 47.8%), whereas Cradock WWTW was in working condition but with challenges and some infrastructural dysfunction. Assessment of water/effluent quality revealed that turbidity and TOC were above SANS 241 general limit. Non-compliance in terms of nitrate/nitrite-N, ammonium-N, phosphate-P, TSS, total coliforms, E. Coli, and free chlorine was evident at Cradock WWTW. Unstructured interviews with plant operators corroborated these findings. The overwhelming support for bulk schemes for potable water provision and WSP for sanitation indicated a partially water secure municipal district but derelict in terms of its sanitation services. The later, it was concluded, in particular provides an ideal opportunity for implementation of platform technologies to support bioprocesses for entrepreneurship, employment, economic benefit and to secure a closed circular economy for regional water and sanitation through valorisation of co-product streams. Among the co-product streams considered in this thesis are biomass, biogas, biofertilizers, biofuels and several high value chemical products. , Thesis (MSc) -- Faculty of Science, Institute for Environmental Biotechnology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Gumunyu, Wilbert
- Date: 2022-10-14
- Subjects: Sewage South Africa Eastern Cape , Sanitation South Africa Eastern Cape , Algae Biotechnology , Biotechnology , Sewage Purification Biological treatment , Algal biofuels
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362830 , vital:65366
- Description: In contemporary South Africa, a country in transition, destruction rather than reconstruction seems commonplace. Electricity supply is at an all-time low and ‘load shedding’ is an almost daily occurrence. Similarly, more fragility of water delivery and sanitation service is being reported with the likelihood of ‘water shedding’ a soon-to-be reality. In view of the ever-increasing reported mismanagement of South Africa’s water and sanitation infrastructure, which is likely nearing the point of collapse if not already collapsed, this thesis set out to interrogate at a provincial and municipal level the status of water and sanitation in Chris Hani District Municipality (CHDM), Eastern Cape Province. One major driver behind this study was to evaluate provincial and municipal water/wastewater infrastructure as a platform to support implementation of locally developed bioprocess technologies. First, publicly available historical data was used to derive a baseline or benchmark from which to determine gains/losses in compliance, water quality and efficiency. The period 2009-2013/4 during which Blue and Green Drop reporting was routine, was used as an appropriate start point. Data thus derived indicated that, at provincial and district municipality (DM) level, potable water supply was by bulk water schemes and, sanitation was typically by waste stabilization pond (WSP) treatment of municipal sewage. The derived benchmark for the period 2009-2013/14 indicated that most plants were not compliant (~75% of the Eastern Cape Province water treatment plants (WTP) operating between average performance to critical state), operated either at- or above design capacity (Eastern Cape Province, 16%; CHDM, 19%). A large number of plants for the province (62.4%) and district (81.25%) had hydraulic design capacity or average daily flows that were unknown (information not provided in available literature or plant reports) and final effluent did not always meet the general standard (70.3% of the CHDM wastewater treatment works (WWTW)). To determine the contemporary state (2020 - 2022) of water and sanitation within CHDM, a scoping exercise of WTP and WWTW in selected local municipalities was carried out. This was done along a west-east transect by appraising WTP and WWTW in the towns of Cradock, Tarkastad, Komani, Tsomo, Cacadu, and Ngcobo. Furthermore, targeted assessment of the Queenstown WTP and the Cradock WWTW was used to derive real-time data on the status of water and sanitation infrastructure. Results showed that water and sanitation services within CHDM had, in the years between 2009-2013/14 to date, deteriorated. Freshwater demand significantly exceeded capability of water supply schemes, where demand was as much as three times greater than reported available supply. For most WWTW, operation was in excess of capacity and between 1.52 and 12 times installed hydraulic loading. The targeted scoping exercise revealed that Queenstown WTP is a moderate risk plant (Cumulative risk rating, CRR = 11 and maximum risk rating, MRR = 47.8%), whereas Cradock WWTW was in working condition but with challenges and some infrastructural dysfunction. Assessment of water/effluent quality revealed that turbidity and TOC were above SANS 241 general limit. Non-compliance in terms of nitrate/nitrite-N, ammonium-N, phosphate-P, TSS, total coliforms, E. Coli, and free chlorine was evident at Cradock WWTW. Unstructured interviews with plant operators corroborated these findings. The overwhelming support for bulk schemes for potable water provision and WSP for sanitation indicated a partially water secure municipal district but derelict in terms of its sanitation services. The later, it was concluded, in particular provides an ideal opportunity for implementation of platform technologies to support bioprocesses for entrepreneurship, employment, economic benefit and to secure a closed circular economy for regional water and sanitation through valorisation of co-product streams. Among the co-product streams considered in this thesis are biomass, biogas, biofertilizers, biofuels and several high value chemical products. , Thesis (MSc) -- Faculty of Science, Institute for Environmental Biotechnology, 2022
- Full Text:
- Date Issued: 2022-10-14
Microalgal-bacterial flocs and extracellular polymeric substances for optimum function of integrated algal pond systems
- Authors: Jimoh, Taobat Adekilekun
- Date: 2021-10-29
- Subjects: Flocculation , Extracellular polymeric substances , Water Purification , Sewage Purification Anaerobic treatment , Integrated algae pond systems (IAPS) , Microalgal-bacterial flocs
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/191214 , vital:45071 , 10.21504/10962/191214
- Description: Despite the dire state of sanitation infrastructures, water scarcity, and the dwindling reserve of natural resources due to ever-increasing population growth, implementation of a suitable technology that can provide a solution to all these issues continues to be ignored. The integrated algal pond system (IAPS) is a wastewater treatment technology that combines the processes of anaerobic digestion and photosynthetic oxygenation to achieve wastewater treatment and facilitate the recovery of treated water and resources in the form of biogas and microalgal-bacterial biomass. The natural process of bioflocculation through microalgal-bacterial mutualism and production of extracellular polymeric substances (EPS) in high rate algal oxidation ponds (HRAOPs) of an IAPS increases efficiency of wastewater treatment and potentially enhances harvestability and biomass recovery, which could contribute significantly to the successful establishment of a biorefinery. Using a 500 PE pilot-scale IAPS supplied domestic sewage coupled with laboratory experiments, this study investigated the importance and function of in situ EPS production and MaB-floc formation in HRAOP. A metagenomic study revealed the biological components of the biomass or mixed liquor suspended solids (MLSS) produced in HRAOP and showed that the suspended biomass is composed largely of eukaryotes that were dominated by the colonial microalgae Pseudopediastrum sp. and Desmodesmus sp., and a diverse range of prokaryotes including bacteria and cyanobacteria. Dominance, within the bacterial population, by a sulphur-oxidizing bacterium, Thiothrix which comprised up to 80% of the prokaryotes, coincided with a period of poor flocculation and was therefore rationalized to have contributed to bulking and poor biomass settleability. Otherwise, good flocs were formed in the MLSS with settleability up to 95% and, within 1 h. The formation of MaB-flocs appeared to be dependent on EPS concentration of the mixed liquor due to the observed positive correlation between soluble EPS (S-EPS), biomass concentration, and settleability. The contribution and role of MLSS components towards the formation and sustenance of MaB-flocs were further demonstrated in laboratory experiments using pure strains of microalgae, cyanobacteria, and bacteria. Results showed that pure cultures of dominant microalgae in MLSS, Pseudopediastrum sp. and Desmodesmus sp. achieved a rapid 92 and 75% settleability within 3 h. A self-flocculating filamentous cyanobacterium, Leptolyngbya strain ECCN 20BG was isolated, characterized, and shown to achieve 99% settleability within 5 min by forming large tightly aggregated flocs. In further experiments, this strain was found to improve the settleability of MLSS by an average of 20%. Bacterial strains identified as Bacillus strain ECCN 40b, Bacillus strain ECCN 41b, Planococcus strain ECCN 45b, and Exiguobacterium strain ECCN 46b were also observed to produce sticky EPS-like materials in pure cultures that could also contribute to the aggregation of cells in a mixed environment. Given these results, various factors and/or mechanisms that might enhance microbial aggregation and biomass recovery from HRAOP MLSS were identified in this study and include; (1) dominance by larger colonial microalgae prevents disintegration of MaB-flocs and enhances recovery of biomass from MLSS by gravity sedimentation, (2) presence of filamentous cyanobacteria species that can self-flocculate to form an interwoven network of filaments may play an important role in the structural stability and settleability of MaB-flocs in MLSS, and (3) production of EPS to form the matrix or scaffold whereon all microbial components aggregate to develop a microenvironment. Indeed, all forms of EPS, except for that produced by Bacillus strain ECCN 41b, showed bioflocculating property and were able to serve as flocculants for the recovery of Chlorella, an alga known for its poor settleability. A combination of biochemical analyses and FTIR spectroscopy revealed the importance of carbohydrate enrichment of these biopolymers. Carbohydrate concentration in all forms of EPS was between 12 and 41% suggesting that production of these compounds by microbes within the MLSS contributed to MaB-floc formation. EPS extracted from bulk MLSS and EPS produced by Bacillus strains possessed some surface-active properties that were comparable to Triton X-100, indicating potential application in bioremediation and recovery of oil from contaminated soil and water. In particular, EPS generated from Bacillus strain ECCN 41b displayed relatively distinct properties including the quantity produced (> 500 mg/L), increased viscosity, inability to flocculate microalgal cells, a rhamnolipid content of 32%, and a higher surface-activity. Based on these results, Bacillus strain ECCN 41b was rationalized to produce anionic EPS with potential application in metal or oil recovery. In addition to EPS production, the bacteria Planococcus strain ECCN 45b and Exiguobacterium strain ECCN 46b appeared pigmented. Based on partial characterization using UV/Vis spectrophotometry, thin-layer chromatography, FTIR, and NMR, the pigments produced by these two strains appeared to be identical and were tentatively identified as ketocarotenoids. This study successfully demonstrated the importance of EPS production and formation of MaB-flocs in the MLSS from HRAOP of an IAPS treating domestic sewage. It is evident that increased settleability of the biomass does contribute to the reported efficiency of wastewater treatment by IAPS and would reduce both total suspended solids (TSS) and chemical oxygen demand (COD). In addition, demonstration that this biomass contains products of value such as carotenoids and EPS with potential for commercial use strengthens the idea of using IAPS as a platform technology for innovation of the wastewater treatment process to a biorefinery. , Thesis (PhD) -- Faculty of Science, Institute for Environmental Biotechnology, 2021
- Full Text:
- Date Issued: 2021-10-29
- Authors: Jimoh, Taobat Adekilekun
- Date: 2021-10-29
- Subjects: Flocculation , Extracellular polymeric substances , Water Purification , Sewage Purification Anaerobic treatment , Integrated algae pond systems (IAPS) , Microalgal-bacterial flocs
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/191214 , vital:45071 , 10.21504/10962/191214
- Description: Despite the dire state of sanitation infrastructures, water scarcity, and the dwindling reserve of natural resources due to ever-increasing population growth, implementation of a suitable technology that can provide a solution to all these issues continues to be ignored. The integrated algal pond system (IAPS) is a wastewater treatment technology that combines the processes of anaerobic digestion and photosynthetic oxygenation to achieve wastewater treatment and facilitate the recovery of treated water and resources in the form of biogas and microalgal-bacterial biomass. The natural process of bioflocculation through microalgal-bacterial mutualism and production of extracellular polymeric substances (EPS) in high rate algal oxidation ponds (HRAOPs) of an IAPS increases efficiency of wastewater treatment and potentially enhances harvestability and biomass recovery, which could contribute significantly to the successful establishment of a biorefinery. Using a 500 PE pilot-scale IAPS supplied domestic sewage coupled with laboratory experiments, this study investigated the importance and function of in situ EPS production and MaB-floc formation in HRAOP. A metagenomic study revealed the biological components of the biomass or mixed liquor suspended solids (MLSS) produced in HRAOP and showed that the suspended biomass is composed largely of eukaryotes that were dominated by the colonial microalgae Pseudopediastrum sp. and Desmodesmus sp., and a diverse range of prokaryotes including bacteria and cyanobacteria. Dominance, within the bacterial population, by a sulphur-oxidizing bacterium, Thiothrix which comprised up to 80% of the prokaryotes, coincided with a period of poor flocculation and was therefore rationalized to have contributed to bulking and poor biomass settleability. Otherwise, good flocs were formed in the MLSS with settleability up to 95% and, within 1 h. The formation of MaB-flocs appeared to be dependent on EPS concentration of the mixed liquor due to the observed positive correlation between soluble EPS (S-EPS), biomass concentration, and settleability. The contribution and role of MLSS components towards the formation and sustenance of MaB-flocs were further demonstrated in laboratory experiments using pure strains of microalgae, cyanobacteria, and bacteria. Results showed that pure cultures of dominant microalgae in MLSS, Pseudopediastrum sp. and Desmodesmus sp. achieved a rapid 92 and 75% settleability within 3 h. A self-flocculating filamentous cyanobacterium, Leptolyngbya strain ECCN 20BG was isolated, characterized, and shown to achieve 99% settleability within 5 min by forming large tightly aggregated flocs. In further experiments, this strain was found to improve the settleability of MLSS by an average of 20%. Bacterial strains identified as Bacillus strain ECCN 40b, Bacillus strain ECCN 41b, Planococcus strain ECCN 45b, and Exiguobacterium strain ECCN 46b were also observed to produce sticky EPS-like materials in pure cultures that could also contribute to the aggregation of cells in a mixed environment. Given these results, various factors and/or mechanisms that might enhance microbial aggregation and biomass recovery from HRAOP MLSS were identified in this study and include; (1) dominance by larger colonial microalgae prevents disintegration of MaB-flocs and enhances recovery of biomass from MLSS by gravity sedimentation, (2) presence of filamentous cyanobacteria species that can self-flocculate to form an interwoven network of filaments may play an important role in the structural stability and settleability of MaB-flocs in MLSS, and (3) production of EPS to form the matrix or scaffold whereon all microbial components aggregate to develop a microenvironment. Indeed, all forms of EPS, except for that produced by Bacillus strain ECCN 41b, showed bioflocculating property and were able to serve as flocculants for the recovery of Chlorella, an alga known for its poor settleability. A combination of biochemical analyses and FTIR spectroscopy revealed the importance of carbohydrate enrichment of these biopolymers. Carbohydrate concentration in all forms of EPS was between 12 and 41% suggesting that production of these compounds by microbes within the MLSS contributed to MaB-floc formation. EPS extracted from bulk MLSS and EPS produced by Bacillus strains possessed some surface-active properties that were comparable to Triton X-100, indicating potential application in bioremediation and recovery of oil from contaminated soil and water. In particular, EPS generated from Bacillus strain ECCN 41b displayed relatively distinct properties including the quantity produced (> 500 mg/L), increased viscosity, inability to flocculate microalgal cells, a rhamnolipid content of 32%, and a higher surface-activity. Based on these results, Bacillus strain ECCN 41b was rationalized to produce anionic EPS with potential application in metal or oil recovery. In addition to EPS production, the bacteria Planococcus strain ECCN 45b and Exiguobacterium strain ECCN 46b appeared pigmented. Based on partial characterization using UV/Vis spectrophotometry, thin-layer chromatography, FTIR, and NMR, the pigments produced by these two strains appeared to be identical and were tentatively identified as ketocarotenoids. This study successfully demonstrated the importance of EPS production and formation of MaB-flocs in the MLSS from HRAOP of an IAPS treating domestic sewage. It is evident that increased settleability of the biomass does contribute to the reported efficiency of wastewater treatment by IAPS and would reduce both total suspended solids (TSS) and chemical oxygen demand (COD). In addition, demonstration that this biomass contains products of value such as carotenoids and EPS with potential for commercial use strengthens the idea of using IAPS as a platform technology for innovation of the wastewater treatment process to a biorefinery. , Thesis (PhD) -- Faculty of Science, Institute for Environmental Biotechnology, 2021
- Full Text:
- Date Issued: 2021-10-29
Plant-fungal mutualism as a strategy for the bioremediation of hydrocarbon polluted soils
- Authors: Keshinro, Olajide Muritala
- Date: 2021-10-29
- Subjects: Mutualism (Biology) , Plant-fungus relationships , Bioremediation , Mucilage , Plant exudates , Extracellular polymeric substances , Laccase , Peroxidase , Phytoremediation , Ligninolytic enzymes
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/190918 , vital:45041 , 10.21504/10962/190918
- Description: Inasmuch as coal remains the linchpin for the generation of electricity and liquid petroleum products in South Africa, hydrocarbon waste and coal discard will continue to pose a threat to the environment. Therefore, the onus is on the associated industries to develop and implement efficient and sustainable strategies to mitigate the negative impacts of energy generating activities on the environment. Most conventional efforts in this regard, although successful for soil repair and the initiation of vegetation, have been deemed unsustainable. In an effort to find a sustainable remediation strategy a novel technology termed “FungCoal” was conceptualized and patented as a strategy for the rehabilitation of open cast coal mines, carbonaceous-rich spoils and coal wastes. This biotechnology, which exploits plant-fungal mutualism to achieve effective biodegradation of coal on discard dumps and the breakdown of the carbonaceous component in spoils, promotes revegetation to facilitate rehabilitation of mining-disturbed land. However, one limiting factor of the FungCoal bioprocess is that it requires oxidized weathered coal, a highly complex and variable resource for use as a co-substrate, for growth and proliferation of the coal degrading microorganisms. To fully exploit the potential of plant-fungal mutualism and its interaction for use in the remediation of coal contaminated soils, this study investigated the proposed relationship between plant roots, root exudate and the coal degrading fungus “Aspergillus sp.” (previously Neosartorya fischeri) strain 84 in more detail, in an effort to gain further insight into the mechanisms underpinning plant-fungal mutualism as a strategy for re-vegetation of coal discard dumps and the rehabilitation of hydrocarbon-contaminated soil using the FungCoal approach. A pot-on-beaker (PoB) method was developed for the easy cultivation and collection of extracellular polymeric substance (EPS)-containing exudates from Zea mays L. (maize) and Abelmuschus esculentus (okra). Characterisation of the EPS material from these exudates was carried out using a combination of physicochemical and biochemical methods. The results from analysis of phenolics and indoles showed that exudates contain some form of indoles and phenolic compounds, although in little proportions, which may fulfil a signalling function, responsible for attracting soil microorganisms into the rhizosphere. Spectroscopic analysis of the exudates using FT-IR revealed vibrations corresponding to functional groups of alkanes, alkenes, alkynes, and carboxylic acids. These compounds likely provide an easily accessible source of carbon to soil microorganisms and are also a better alternative to the poly-aromatics which are an inherent component locked-up in the supposed recalcitrant coal material. The results from biochemical analyses also revealed the presence of carbohydrate, proteins, lipids, and low amounts of α-amino-nitrogen in the EPS of maize and okra. These components of EPS are all essential for the stimulation of enzymatic activities in soil microorganisms and, which may in turn aid biodegradation. The action of the root EPS from maize was further tested on three coal-degrading fungal isolates identified as Aspergillus strain ECCN 84, Aspergillus strain ECCN 225 and Penicillium strain ECCN 243 for manganese peroxidase (MnP) and laccase (LAC) activities. The results revealed that the Aspergillus species, strains ECCN 84 and ECCN 225, showed with or without EPS, observable black halos surrounding each of the colonies after 7d incubation indicative of positive MnP activity, while no activity was observed for the Penicillium sp. strain ECCN 243. Analysis for LAC revealed little or no activity in any of the coal degrading fungi following addition of pulverized coal to the growth medium. Interestingly, the addition of EPS-containing exudate to the coal-containing medium resulted in increased LAC activity for all fungal isolates. This finding affirmed the positive contribution of EPS to extracellular LAC activity, purported as an important enzyme in the coal biodegradation process. Finally, the impact of plant-derived exudate on the colonisation and biodegradation of coal was investigated in situ using rhizoboxes, to simulate a coal environment, and was carried out for 16 weeks. Microscopic examination of coal samples after termination of the experiment showed fungal proliferation and attachment to coal particles. All of the rhizoboxes that contained plants had higher medium pH and EC, and the concentration of phenolics, indoles and humic acids was greater than that of control treatments. These observations indicated better rhizosphere colonisation, substrate biodegradation and humification. Therefore, root exudate appears to play a significant role in coordination of soil microorganisms within the rhizosphere and likely serves both as a scaffold for rhizospheric interactions by providing microorganisms with accessible carbon and as a likely ‘trigger’ for induction of coal-degrading enzymes such as fungal LAC for mobilisation of recalcitrant carbon. This study has shown that EPS exuded from roots of Zea mays together with coal degrading fungus Aspergillus strain ECCN 84 can alkalinise the coal substrate and facilitate introduction of oxygen, possibly as a result of increased laccase activity, and increase availability of nutrients (as indicated by higher EC) in a coal-polluted rhizosphere, to provide plants and their associated mycorrhizae and presumably other beneficial microorganisms a more mesic environment for sustained phytoremediation with enhanced rehabilitation potential. In conclusion, this study confirms the positive role of root exudate in mediating a mutualistic rehabilitation strategy involving plants and fungi such as the FungCoal bioprocess. , Thesis (PhD) -- Faculty of Science, Institute for Environmental Biotechnology, 2021
- Full Text:
- Date Issued: 2021-10-29
- Authors: Keshinro, Olajide Muritala
- Date: 2021-10-29
- Subjects: Mutualism (Biology) , Plant-fungus relationships , Bioremediation , Mucilage , Plant exudates , Extracellular polymeric substances , Laccase , Peroxidase , Phytoremediation , Ligninolytic enzymes
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/190918 , vital:45041 , 10.21504/10962/190918
- Description: Inasmuch as coal remains the linchpin for the generation of electricity and liquid petroleum products in South Africa, hydrocarbon waste and coal discard will continue to pose a threat to the environment. Therefore, the onus is on the associated industries to develop and implement efficient and sustainable strategies to mitigate the negative impacts of energy generating activities on the environment. Most conventional efforts in this regard, although successful for soil repair and the initiation of vegetation, have been deemed unsustainable. In an effort to find a sustainable remediation strategy a novel technology termed “FungCoal” was conceptualized and patented as a strategy for the rehabilitation of open cast coal mines, carbonaceous-rich spoils and coal wastes. This biotechnology, which exploits plant-fungal mutualism to achieve effective biodegradation of coal on discard dumps and the breakdown of the carbonaceous component in spoils, promotes revegetation to facilitate rehabilitation of mining-disturbed land. However, one limiting factor of the FungCoal bioprocess is that it requires oxidized weathered coal, a highly complex and variable resource for use as a co-substrate, for growth and proliferation of the coal degrading microorganisms. To fully exploit the potential of plant-fungal mutualism and its interaction for use in the remediation of coal contaminated soils, this study investigated the proposed relationship between plant roots, root exudate and the coal degrading fungus “Aspergillus sp.” (previously Neosartorya fischeri) strain 84 in more detail, in an effort to gain further insight into the mechanisms underpinning plant-fungal mutualism as a strategy for re-vegetation of coal discard dumps and the rehabilitation of hydrocarbon-contaminated soil using the FungCoal approach. A pot-on-beaker (PoB) method was developed for the easy cultivation and collection of extracellular polymeric substance (EPS)-containing exudates from Zea mays L. (maize) and Abelmuschus esculentus (okra). Characterisation of the EPS material from these exudates was carried out using a combination of physicochemical and biochemical methods. The results from analysis of phenolics and indoles showed that exudates contain some form of indoles and phenolic compounds, although in little proportions, which may fulfil a signalling function, responsible for attracting soil microorganisms into the rhizosphere. Spectroscopic analysis of the exudates using FT-IR revealed vibrations corresponding to functional groups of alkanes, alkenes, alkynes, and carboxylic acids. These compounds likely provide an easily accessible source of carbon to soil microorganisms and are also a better alternative to the poly-aromatics which are an inherent component locked-up in the supposed recalcitrant coal material. The results from biochemical analyses also revealed the presence of carbohydrate, proteins, lipids, and low amounts of α-amino-nitrogen in the EPS of maize and okra. These components of EPS are all essential for the stimulation of enzymatic activities in soil microorganisms and, which may in turn aid biodegradation. The action of the root EPS from maize was further tested on three coal-degrading fungal isolates identified as Aspergillus strain ECCN 84, Aspergillus strain ECCN 225 and Penicillium strain ECCN 243 for manganese peroxidase (MnP) and laccase (LAC) activities. The results revealed that the Aspergillus species, strains ECCN 84 and ECCN 225, showed with or without EPS, observable black halos surrounding each of the colonies after 7d incubation indicative of positive MnP activity, while no activity was observed for the Penicillium sp. strain ECCN 243. Analysis for LAC revealed little or no activity in any of the coal degrading fungi following addition of pulverized coal to the growth medium. Interestingly, the addition of EPS-containing exudate to the coal-containing medium resulted in increased LAC activity for all fungal isolates. This finding affirmed the positive contribution of EPS to extracellular LAC activity, purported as an important enzyme in the coal biodegradation process. Finally, the impact of plant-derived exudate on the colonisation and biodegradation of coal was investigated in situ using rhizoboxes, to simulate a coal environment, and was carried out for 16 weeks. Microscopic examination of coal samples after termination of the experiment showed fungal proliferation and attachment to coal particles. All of the rhizoboxes that contained plants had higher medium pH and EC, and the concentration of phenolics, indoles and humic acids was greater than that of control treatments. These observations indicated better rhizosphere colonisation, substrate biodegradation and humification. Therefore, root exudate appears to play a significant role in coordination of soil microorganisms within the rhizosphere and likely serves both as a scaffold for rhizospheric interactions by providing microorganisms with accessible carbon and as a likely ‘trigger’ for induction of coal-degrading enzymes such as fungal LAC for mobilisation of recalcitrant carbon. This study has shown that EPS exuded from roots of Zea mays together with coal degrading fungus Aspergillus strain ECCN 84 can alkalinise the coal substrate and facilitate introduction of oxygen, possibly as a result of increased laccase activity, and increase availability of nutrients (as indicated by higher EC) in a coal-polluted rhizosphere, to provide plants and their associated mycorrhizae and presumably other beneficial microorganisms a more mesic environment for sustained phytoremediation with enhanced rehabilitation potential. In conclusion, this study confirms the positive role of root exudate in mediating a mutualistic rehabilitation strategy involving plants and fungi such as the FungCoal bioprocess. , Thesis (PhD) -- Faculty of Science, Institute for Environmental Biotechnology, 2021
- Full Text:
- Date Issued: 2021-10-29
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