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
Waste keratinous biomass valorization and characterization of keratinases produced by exiguobacteria species
- Authors: Dlume, Tutuka
- Date: 2021-02
- Subjects: Factory and trade waste -- Biodegradation , Bioremediation
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/20695 , vital:46438
- Description: Keratinous wastes are emanating in a million tons, as by-products, from various agro-industrial processing plants. Consequently, they create a serious solid waste problem in the environment due to poor handling. Microbial keratinases are proteolytic enzymes that effectively participate in keratin-rich biomass hydrolyses such as feathers, nail, hair, hooves, and horns. Therefore, proper management of these wastes via recycling into useful products is ecologically imperative. Biodegradation of keratin-rich biomass has been identified as an economical and environmentally friendly way of transforming these recalcitrant agro wastes into useful products, hence the motivation for this study. Feather degrading bacterial strains previously isolated from a municipal dumpsite and coded as SSB-02 and SSB-03 was identified through 16S rDNA sequencing and phylogenetic analysis. The fermentation conditions for keratinase production were optimized. The protein and amino acids constituents of the hydrolyzed chicken feather were analyzed. The biochemical properties of the keratinase produced were determined. Also, the effect of laundry detergents on the stability of the keratinase was studied. The isolates coded as SSB-02 and SSB-03 showed a high percentage of sequence homology with Exguobacterium spp., hence they were identified as Exiguobacterium sp. FBH5 and Exiguobacterium acetylicum FHBD, respectively. Exiguobacterium sp. FBH5 showed the highest extracellular keratinase production of 934.58 ± 27.27 U/mL at 72 h of incubation; in optimized fermentation conditions that included pH (5.0), temperature (30 oC), and chicken feather (0.5percent, w/v). Similarly, E. acetylicum FHBD displayed optimal keratinase production of 1023.64 ± 25.71 U/mL at 120 h of fermentation and improved fermentation conditions that involved pH (3.0), temperature (35 oC) and chicken feathers (0.5-1.5percent; w/v). The amino acid analysis showed that arginine, aspartic acid and glutamic acid were the most abundant amino acids cleaved from the degradation of chicken feathers by Exiguobacterium sp. FBH5 at a respective concentration of 1.16, 1.28 and 1.45 (g/100g sample). Additionally, hydrolysate that emanated from E. indicum FHBD degradation of feather showed high concentrations of arginine, serine, aspartic acid, glutamic acid, and glycine at the respective concentration (g/100g sample) of 1.2, 1.12, 1.34, 1.58 and 1.29. The keratinases were optimally active at pH and temperature of 8.0 and 50 oC, respectively. EDTA and PMSF did not highly repress keratinolytic proteases' activity, and this inhibitory pattern suggests that they may belong to a mixed protease family. Keratinase from E. acetylicum FHBD was highly stable in the presence of SDS, with 99percent residual activity and displayed variable stability in other chemical agents tested. A similar stability pattern was observed with keratinase from Exiguobacterium sp. FBH5, although the enzyme lost about 40percent of its original activity in the presence of SDS. Evaluation of metal ion stability indicated that E. acetylicum FHBD keratinase was remarkably stable in the presence of Fe3+, Mg2+, Cu2+, Zn2+, and Ba2+, with residual activity of 94percent, 88percent, 89percent, 90percent, and 97percent, respectively. Similarly, Exiguobacterium sp. FBH5 keratinase was considerably stable after treatment with Co2+, K+, and Zn2+ as it displayed a residual activity of 85percent, 84percent and 93percent, respectively. The study of the keratinases stability in laundry detergents showed that E. acetylicum FHBD keratinolytic proteases was activated in the presence of Omo, Surf, Sunlight, and Pro wash after 60 min of pre-incubation compared to 30 min, with residual activity of 94 ± 2.94percent, 91 ± 2.53percent, 95 ± 2.89percent and 87 ± 2.89percent respectively. Likewise, Exiguobacterium sp. FBH5 keratinase activity was promoted after 60 min of incubation compared to 30 min, with a residual enzyme activity of 79percent, 84percent, 101percent, 103percent and 105percent and 106percent for Ariel, Surf, Prowash, Freewave, Sky and Evaklin, respectively. Therefore Exiguobacterium spp., demonstrated excellent keratinolytic potentials that could be exploited for sustainable development of bio-innovative products. The study keratinases' properties suggest their industrial and biotechnological application potentials, especially as bio-additive in the formulation of laundry detergents. , Thesis (MSc) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-02
- Authors: Dlume, Tutuka
- Date: 2021-02
- Subjects: Factory and trade waste -- Biodegradation , Bioremediation
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/20695 , vital:46438
- Description: Keratinous wastes are emanating in a million tons, as by-products, from various agro-industrial processing plants. Consequently, they create a serious solid waste problem in the environment due to poor handling. Microbial keratinases are proteolytic enzymes that effectively participate in keratin-rich biomass hydrolyses such as feathers, nail, hair, hooves, and horns. Therefore, proper management of these wastes via recycling into useful products is ecologically imperative. Biodegradation of keratin-rich biomass has been identified as an economical and environmentally friendly way of transforming these recalcitrant agro wastes into useful products, hence the motivation for this study. Feather degrading bacterial strains previously isolated from a municipal dumpsite and coded as SSB-02 and SSB-03 was identified through 16S rDNA sequencing and phylogenetic analysis. The fermentation conditions for keratinase production were optimized. The protein and amino acids constituents of the hydrolyzed chicken feather were analyzed. The biochemical properties of the keratinase produced were determined. Also, the effect of laundry detergents on the stability of the keratinase was studied. The isolates coded as SSB-02 and SSB-03 showed a high percentage of sequence homology with Exguobacterium spp., hence they were identified as Exiguobacterium sp. FBH5 and Exiguobacterium acetylicum FHBD, respectively. Exiguobacterium sp. FBH5 showed the highest extracellular keratinase production of 934.58 ± 27.27 U/mL at 72 h of incubation; in optimized fermentation conditions that included pH (5.0), temperature (30 oC), and chicken feather (0.5percent, w/v). Similarly, E. acetylicum FHBD displayed optimal keratinase production of 1023.64 ± 25.71 U/mL at 120 h of fermentation and improved fermentation conditions that involved pH (3.0), temperature (35 oC) and chicken feathers (0.5-1.5percent; w/v). The amino acid analysis showed that arginine, aspartic acid and glutamic acid were the most abundant amino acids cleaved from the degradation of chicken feathers by Exiguobacterium sp. FBH5 at a respective concentration of 1.16, 1.28 and 1.45 (g/100g sample). Additionally, hydrolysate that emanated from E. indicum FHBD degradation of feather showed high concentrations of arginine, serine, aspartic acid, glutamic acid, and glycine at the respective concentration (g/100g sample) of 1.2, 1.12, 1.34, 1.58 and 1.29. The keratinases were optimally active at pH and temperature of 8.0 and 50 oC, respectively. EDTA and PMSF did not highly repress keratinolytic proteases' activity, and this inhibitory pattern suggests that they may belong to a mixed protease family. Keratinase from E. acetylicum FHBD was highly stable in the presence of SDS, with 99percent residual activity and displayed variable stability in other chemical agents tested. A similar stability pattern was observed with keratinase from Exiguobacterium sp. FBH5, although the enzyme lost about 40percent of its original activity in the presence of SDS. Evaluation of metal ion stability indicated that E. acetylicum FHBD keratinase was remarkably stable in the presence of Fe3+, Mg2+, Cu2+, Zn2+, and Ba2+, with residual activity of 94percent, 88percent, 89percent, 90percent, and 97percent, respectively. Similarly, Exiguobacterium sp. FBH5 keratinase was considerably stable after treatment with Co2+, K+, and Zn2+ as it displayed a residual activity of 85percent, 84percent and 93percent, respectively. The study of the keratinases stability in laundry detergents showed that E. acetylicum FHBD keratinolytic proteases was activated in the presence of Omo, Surf, Sunlight, and Pro wash after 60 min of pre-incubation compared to 30 min, with residual activity of 94 ± 2.94percent, 91 ± 2.53percent, 95 ± 2.89percent and 87 ± 2.89percent respectively. Likewise, Exiguobacterium sp. FBH5 keratinase activity was promoted after 60 min of incubation compared to 30 min, with a residual enzyme activity of 79percent, 84percent, 101percent, 103percent and 105percent and 106percent for Ariel, Surf, Prowash, Freewave, Sky and Evaklin, respectively. Therefore Exiguobacterium spp., demonstrated excellent keratinolytic potentials that could be exploited for sustainable development of bio-innovative products. The study keratinases' properties suggest their industrial and biotechnological application potentials, especially as bio-additive in the formulation of laundry detergents. , Thesis (MSc) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-02
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