Towards a bacterial biofertiliser for the rehabilitation of disturbed and degraded land
- Authors: Masudi, Wiya Leon
- Date: 2024-10-11
- Subjects: Biofertilizers , Land degradation , Ecological disturbances , Plant-growth promoting rhizobacteria , Restoration ecology
- 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:
- Authors: Masudi, Wiya Leon
- Date: 2024-10-11
- Subjects: Biofertilizers , Land degradation , Ecological disturbances , Plant-growth promoting rhizobacteria , Restoration ecology
- 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:
Towards the development of a bio-fertiliser using mixed liquor from high rate algal oxidation ponds
- Authors: Masudi, Wiya Léon
- Date: 2020
- Subjects: Biofertilizers , Microalgae - Biotechnology , Algae -- Culture
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/142969 , vital:38181
- Description: Mixed liquor includes consortia of microalgae and bacteria produced in high rate algal oxidation ponds (HRAOPs). The consortia of microalgae and bacteria occur as flocs called microalgae-bacterial flocs (MaB-flocs). This study aimed to source bacteria from MaB-flocs generated in HRAOPs and, after isolation and identification, evaluate their potential as plant growth promoting (PGP) microorganisms. Twelve bacterial strains namely ECCN 1b, ECCN 2b, ECCN 3b, ECCN 4b, ECCN 5b, ECCN 6b, ECCN 7b, ECCN 8b, ECCN 9b, ECCN 10b, ECCN 11b, and ECCN 12b were successfully isolated and their molecular identity established using amplified 16S rRNA gene sequence analysis that was compared to sequences deposited in the NCBI gene database. Blast analysis identified these isolates at the genus level as Bacillus strain ECCN 1b, Fictibacillus strain ECCN 2b, Bacillus strain ECCN 3b, Aeromonas strain ECCN 4b, Exiguobacterium strain ECCN 5b, Arthrobacter strain ECCN 6b, Enterobacter strain ECCN 7b, Exiguobacterium strain ECCN 8b, Microbacterium strain ECCN 9b, Pseudomonas ECCN strain 10b, Ancylobacter strain ECCN 11b and Microbacterium strain ECCN 12b. These isolates were able to grow in nutrient broth in a pH range between 6 and 10, with the best growth achieved at pH 8 to 9. The results on the use of carbon substrate revealed that 5 strains including Arthrobacter strain ECCN 6b, Aeromonas strain ECCN 4b, Pseudomonas strain ECCN 10b, Enterobacter strain ECCN 7b and Bacillus strain ECCN 3b were capable of using glucose, sucrose and mannitol. No faecal coliforms were found. However, of the 12 isolates screened for bio-fertilisation potential, Bacillus strain ECCN 1b, Fictibacillus strain ECCN 2b, Bacillus strain ECCN 3b, Aeromonas strain ECCN 4b, Exiguobacterium strain ECCN 5b, Arthrobacter strain ECCN 6b, Enterobacter ECCN strain 7b, Exiguobacterium strain ECCN 8b and Pseudomonas strain ECCN 10b showed multifunctional plant growth promoting (PGP) potential. The potential for PGP included the production of ammonium-N, solubilisation of phosphate-P and potassium-K, oxidation of Mn and production of auxin, indole-3-acetic acid (IAA). Results are discussed in terms of the amount or concentration (mg L-1) of plant essential nutrient and growth regulator produced by these isolated bacteria. Even so, further studies are needed to test and confirm the bio-fertiliser and plant growth promoting activity of these strains in pot trials and field experiments, or both.
- Full Text:
- Authors: Masudi, Wiya Léon
- Date: 2020
- Subjects: Biofertilizers , Microalgae - Biotechnology , Algae -- Culture
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/142969 , vital:38181
- Description: Mixed liquor includes consortia of microalgae and bacteria produced in high rate algal oxidation ponds (HRAOPs). The consortia of microalgae and bacteria occur as flocs called microalgae-bacterial flocs (MaB-flocs). This study aimed to source bacteria from MaB-flocs generated in HRAOPs and, after isolation and identification, evaluate their potential as plant growth promoting (PGP) microorganisms. Twelve bacterial strains namely ECCN 1b, ECCN 2b, ECCN 3b, ECCN 4b, ECCN 5b, ECCN 6b, ECCN 7b, ECCN 8b, ECCN 9b, ECCN 10b, ECCN 11b, and ECCN 12b were successfully isolated and their molecular identity established using amplified 16S rRNA gene sequence analysis that was compared to sequences deposited in the NCBI gene database. Blast analysis identified these isolates at the genus level as Bacillus strain ECCN 1b, Fictibacillus strain ECCN 2b, Bacillus strain ECCN 3b, Aeromonas strain ECCN 4b, Exiguobacterium strain ECCN 5b, Arthrobacter strain ECCN 6b, Enterobacter strain ECCN 7b, Exiguobacterium strain ECCN 8b, Microbacterium strain ECCN 9b, Pseudomonas ECCN strain 10b, Ancylobacter strain ECCN 11b and Microbacterium strain ECCN 12b. These isolates were able to grow in nutrient broth in a pH range between 6 and 10, with the best growth achieved at pH 8 to 9. The results on the use of carbon substrate revealed that 5 strains including Arthrobacter strain ECCN 6b, Aeromonas strain ECCN 4b, Pseudomonas strain ECCN 10b, Enterobacter strain ECCN 7b and Bacillus strain ECCN 3b were capable of using glucose, sucrose and mannitol. No faecal coliforms were found. However, of the 12 isolates screened for bio-fertilisation potential, Bacillus strain ECCN 1b, Fictibacillus strain ECCN 2b, Bacillus strain ECCN 3b, Aeromonas strain ECCN 4b, Exiguobacterium strain ECCN 5b, Arthrobacter strain ECCN 6b, Enterobacter ECCN strain 7b, Exiguobacterium strain ECCN 8b and Pseudomonas strain ECCN 10b showed multifunctional plant growth promoting (PGP) potential. The potential for PGP included the production of ammonium-N, solubilisation of phosphate-P and potassium-K, oxidation of Mn and production of auxin, indole-3-acetic acid (IAA). Results are discussed in terms of the amount or concentration (mg L-1) of plant essential nutrient and growth regulator produced by these isolated bacteria. Even so, further studies are needed to test and confirm the bio-fertiliser and plant growth promoting activity of these strains in pot trials and field experiments, or both.
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Exploring the fertiliser potential of biosolids from algae integrated wastewater treatment systems
- Authors: Mlambo, Patricia Zanele
- Date: 2014
- Subjects: Sewage disposal plants , Sewage sludge as fertilizer , Algae -- Biotechnology , Sewage -- Purification -- Anaerobic treatment , Plant regulators , Biofertilizers , Microalgae -- Biotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5957 , http://hdl.handle.net/10962/d1013342
- Description: High rate algae oxidation ponds (HRAOP) for domestic wastewater treatment generate biosolids that are predominantly microalgae. Consequently, HRAOP biosolids are enriched with minerals, amino acids, nutrients and possibly contain plant growth regulator (PGR)-like substances, which makes HRAOP biosolids attractive as fertiliser or PGR. This study investigated HRAOP biosolids as a starting material for a natural, cost-effective and readily-available eco-friendly organic fertiliser and/or PGRs. Various HRAOP extract formulations were prepared and their effect on plant growth and development was evaluated using selected bioassays. Initial screening included assessing the effect on change in specific leaf area, radish cotyledon expansion as an indicator of PGR-like activity, and seed germination index (GI). More detailed studies on fertiliser efficacy and PGR-like activity utilised bean (Phaseolus vulgaris) and tomato (Solanum lycopersicum) plants. Combined effects of sonicated (S) and 40% v/v methanol (M) extract (5:1 SM) had impressive plant responses, comparable to Hoagland solution (HS). Other potentially fertiliser formulations included 0.5% M, 1% M, 2.5% S and 5% S formulations. The 5:1 SM and 5% S showed greater PGR-like activity, promoting cotyledon expansion by 459 ± 0.02% and 362 ± 0.01%, respectively. GI data showed that none of the formulations negatively impacted germination. Further investigation showed that the 5% S formulation increased leaf length, width and area by 6.69 ± 0.24, 6.21 ± 0.2 mm and 41.55 ± 0.2 mm². All formulated fertiliser extracts had no adverse effect on chlorophyll content and plant nutrient balance as indicated by C:N (8-10:1) ratio. In addition, plants appeared to actively mobilise nutrients to regions where needed as evidenced by a shift in shoot: root ratio depending on C, N and water availability. Furthermore, 5% S caused a 75% increase in tomato productivity and had no effect on bean productivity. Whereas, 5:1 SM and 1% M formulation improved bean pod production by 33.3% and 11%, respectively but did not affect tomato production. Harvest index (HI) however indicated a 3% reduction in tomato productivity with 5:1 SM and little or no enhancement in bean productivity with both 5:1 SM and 5% S treatments. Bean plants treated with 5:1 SM and 5% S produced larger fruits, which could be an indication of the presence of a PGR effect. Overall, HRAOP biosolids extracts prepared and investigated in this study demonstrated both fertiliser characteristics and PGR-like activity with performances comparable and in some cases exceeding that of commercial products. However additional research is needed to confirm presence of PGR-like activities and fertiliser efficacy.
- Full Text:
- Authors: Mlambo, Patricia Zanele
- Date: 2014
- Subjects: Sewage disposal plants , Sewage sludge as fertilizer , Algae -- Biotechnology , Sewage -- Purification -- Anaerobic treatment , Plant regulators , Biofertilizers , Microalgae -- Biotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5957 , http://hdl.handle.net/10962/d1013342
- Description: High rate algae oxidation ponds (HRAOP) for domestic wastewater treatment generate biosolids that are predominantly microalgae. Consequently, HRAOP biosolids are enriched with minerals, amino acids, nutrients and possibly contain plant growth regulator (PGR)-like substances, which makes HRAOP biosolids attractive as fertiliser or PGR. This study investigated HRAOP biosolids as a starting material for a natural, cost-effective and readily-available eco-friendly organic fertiliser and/or PGRs. Various HRAOP extract formulations were prepared and their effect on plant growth and development was evaluated using selected bioassays. Initial screening included assessing the effect on change in specific leaf area, radish cotyledon expansion as an indicator of PGR-like activity, and seed germination index (GI). More detailed studies on fertiliser efficacy and PGR-like activity utilised bean (Phaseolus vulgaris) and tomato (Solanum lycopersicum) plants. Combined effects of sonicated (S) and 40% v/v methanol (M) extract (5:1 SM) had impressive plant responses, comparable to Hoagland solution (HS). Other potentially fertiliser formulations included 0.5% M, 1% M, 2.5% S and 5% S formulations. The 5:1 SM and 5% S showed greater PGR-like activity, promoting cotyledon expansion by 459 ± 0.02% and 362 ± 0.01%, respectively. GI data showed that none of the formulations negatively impacted germination. Further investigation showed that the 5% S formulation increased leaf length, width and area by 6.69 ± 0.24, 6.21 ± 0.2 mm and 41.55 ± 0.2 mm². All formulated fertiliser extracts had no adverse effect on chlorophyll content and plant nutrient balance as indicated by C:N (8-10:1) ratio. In addition, plants appeared to actively mobilise nutrients to regions where needed as evidenced by a shift in shoot: root ratio depending on C, N and water availability. Furthermore, 5% S caused a 75% increase in tomato productivity and had no effect on bean productivity. Whereas, 5:1 SM and 1% M formulation improved bean pod production by 33.3% and 11%, respectively but did not affect tomato production. Harvest index (HI) however indicated a 3% reduction in tomato productivity with 5:1 SM and little or no enhancement in bean productivity with both 5:1 SM and 5% S treatments. Bean plants treated with 5:1 SM and 5% S produced larger fruits, which could be an indication of the presence of a PGR effect. Overall, HRAOP biosolids extracts prepared and investigated in this study demonstrated both fertiliser characteristics and PGR-like activity with performances comparable and in some cases exceeding that of commercial products. However additional research is needed to confirm presence of PGR-like activities and fertiliser efficacy.
- Full Text:
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