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
Cell-free biosynthesis of abscisic acid (ABA) in extracts of flavedo from Citrus sinensis (L.) osbeck
- Richardson, Gaynor Rose-Marie
- Authors: Richardson, Gaynor Rose-Marie
- Date: 1996
- Subjects: Biosynthesis Abscisic acid Citrus fruits
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4221 , http://hdl.handle.net/10962/d1003790
- Description: The biosynthetic origin of the plant growth regulator abscisic acid remains equivocal and almost nothing is known about the enzymes involved in this process. The present research programme describes the development of a cell-free system, capable of synthesizing abscisic acid and attempts to provide further information about the biochemistry and enzymology of this important biosynthetic pathway. Cell-free extracts were prepared either directly from the flavedo (crude) or from an acetone powder derived from flavedo, of mature coloured fruits of Citrus sinensis L. cv. Midknight and incubated with mevalonic acid, isopentenyl pyrophosphate, famesylpyrophosphate, geranylgeranyl pyrophosphate, ß-carotene and 1',4'-trans-abscisic acid diol. The neutral and acidic products formed were purified by thin-layer chromatography and high performance liquid chromatography, and quantified by high performance liquid chromatography, gas chromatography-electron capture and unequivocally identified by combined gas chromatography-mass spectrometry. Abscisic acid, 1',4'-trans-abscisic acid diol and phaseic acid were unequivocally identified as the major acidic products formed in this cell-free system. The acid fraction also contained xanthoxin acid. Labelled and unlabelled ß-carotene was converted into the neutral compounds xanthoxin and xanthoxin alcohol. In addition. high performance liquid chromatography-photodiode array analYSis of the oxy-carotenoid fraction revealed the complete spectrum of ß, ß-carotenoids induding zeaxanthin, antheraxanthin and violaxanthin with accumulation of an oxygenated carotenoid tentatively identified as 9- cis-violaxanthin. Identification of putative C₁₅ intermediates was achieved by either UV spectrophotometry and combined capillary gas chromatography-mass spectrometry or microchemical analYSis and co-chromatography. Refeeding studies using (±)-[2-¹⁴C]_ abscisic acid diol as substrate revealed that abscisic acid was not metabolized to abscisic acid diol, suggesting that it was/is produced as an intermediate rather than as a catabolite of ABA in this system. Stigmasterol, and to a lesser extent cholesterol reduced conversion of ß-carotene to abscisic acid but did not influence transformation of 1',4'-trans-abscisic acid diol to abscisic acid. AM01618 stimulated fonnation of abscisic acid and appeared to exert its effect at the level of conversion of 1' ,4'-trans-abscisic acid diol. Zeatin and the cytokinin analogue, ancymidol inhibited the biosynthesis of abscisic acid whereas dithiothreitol increased incorporation of label from ß-carotene into abscisic acid suggesting involvement of a cytochrome P450-type mixed function oxidase in this reaction sequence. Sodium dodecylsulphate polyacrylamide gel electrophoresis of the enzyme extract derived from Citrus flavedo revealed the presence of a 53 kD protein with peroxidase activity characteristic of a cytochrome P-450. Abscisic acid biosynthesizing activity was always greater in extracts from acetone powder and abscisic acid biosynthesis was enhanced in the presence of AMO 1618, NAD+, NADH, NADPH, MgCI₂ and Molybdate but was inhibited by FAD. Activity was further enhanced by the addition of (R,S)-abscisic acid as a cold-pool trap and by induding 0.1% w/v of either Tween 20 or Triton X 100 in the extraction buffer. When cis-ß-carotene was used as substrate, no abscisic acid was produced. Conversely when either all-trans-ß-carotene or a mixture of the two isomers was used, incorporation into abscisic acid occurred. Upoxygenase activity in cell-free extracts of Citrus flavedo increased with increasing protein concentration. As the ability of lipoxygenase to make xanthoxin from violaxanthin, had been reported, increased activity in the cell-free system implied that carotenoid deavage was being brought about by a non-haem oxygenase with lipoxygenase-like properties. Reports had implicated phoshorylation in the activation of many catalytic enzymes (Hanks et aI., 1985). Phosphorylation of the enzymes in this cell-free system proved unsuccessful. Further, it had been reported that in vitro phosphorylation of several membrane polypeptides and soluble polypeptides from com, had been promoted by the addition of Ca²₊ In this cell-free system Ca + did not have a stimulatory effect on protein phosphorylation. Dioxygenases generally occur as soluble enzymes, where they catalyse many oxygenation reactions in metabolic pathways. The addition of 2-oxo-glutarate, a requirement of most soluble oxidases, did not affect the activity of the cell-free system.
- Full Text:
- Date Issued: 1996
- Authors: Richardson, Gaynor Rose-Marie
- Date: 1996
- Subjects: Biosynthesis Abscisic acid Citrus fruits
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4221 , http://hdl.handle.net/10962/d1003790
- Description: The biosynthetic origin of the plant growth regulator abscisic acid remains equivocal and almost nothing is known about the enzymes involved in this process. The present research programme describes the development of a cell-free system, capable of synthesizing abscisic acid and attempts to provide further information about the biochemistry and enzymology of this important biosynthetic pathway. Cell-free extracts were prepared either directly from the flavedo (crude) or from an acetone powder derived from flavedo, of mature coloured fruits of Citrus sinensis L. cv. Midknight and incubated with mevalonic acid, isopentenyl pyrophosphate, famesylpyrophosphate, geranylgeranyl pyrophosphate, ß-carotene and 1',4'-trans-abscisic acid diol. The neutral and acidic products formed were purified by thin-layer chromatography and high performance liquid chromatography, and quantified by high performance liquid chromatography, gas chromatography-electron capture and unequivocally identified by combined gas chromatography-mass spectrometry. Abscisic acid, 1',4'-trans-abscisic acid diol and phaseic acid were unequivocally identified as the major acidic products formed in this cell-free system. The acid fraction also contained xanthoxin acid. Labelled and unlabelled ß-carotene was converted into the neutral compounds xanthoxin and xanthoxin alcohol. In addition. high performance liquid chromatography-photodiode array analYSis of the oxy-carotenoid fraction revealed the complete spectrum of ß, ß-carotenoids induding zeaxanthin, antheraxanthin and violaxanthin with accumulation of an oxygenated carotenoid tentatively identified as 9- cis-violaxanthin. Identification of putative C₁₅ intermediates was achieved by either UV spectrophotometry and combined capillary gas chromatography-mass spectrometry or microchemical analYSis and co-chromatography. Refeeding studies using (±)-[2-¹⁴C]_ abscisic acid diol as substrate revealed that abscisic acid was not metabolized to abscisic acid diol, suggesting that it was/is produced as an intermediate rather than as a catabolite of ABA in this system. Stigmasterol, and to a lesser extent cholesterol reduced conversion of ß-carotene to abscisic acid but did not influence transformation of 1',4'-trans-abscisic acid diol to abscisic acid. AM01618 stimulated fonnation of abscisic acid and appeared to exert its effect at the level of conversion of 1' ,4'-trans-abscisic acid diol. Zeatin and the cytokinin analogue, ancymidol inhibited the biosynthesis of abscisic acid whereas dithiothreitol increased incorporation of label from ß-carotene into abscisic acid suggesting involvement of a cytochrome P450-type mixed function oxidase in this reaction sequence. Sodium dodecylsulphate polyacrylamide gel electrophoresis of the enzyme extract derived from Citrus flavedo revealed the presence of a 53 kD protein with peroxidase activity characteristic of a cytochrome P-450. Abscisic acid biosynthesizing activity was always greater in extracts from acetone powder and abscisic acid biosynthesis was enhanced in the presence of AMO 1618, NAD+, NADH, NADPH, MgCI₂ and Molybdate but was inhibited by FAD. Activity was further enhanced by the addition of (R,S)-abscisic acid as a cold-pool trap and by induding 0.1% w/v of either Tween 20 or Triton X 100 in the extraction buffer. When cis-ß-carotene was used as substrate, no abscisic acid was produced. Conversely when either all-trans-ß-carotene or a mixture of the two isomers was used, incorporation into abscisic acid occurred. Upoxygenase activity in cell-free extracts of Citrus flavedo increased with increasing protein concentration. As the ability of lipoxygenase to make xanthoxin from violaxanthin, had been reported, increased activity in the cell-free system implied that carotenoid deavage was being brought about by a non-haem oxygenase with lipoxygenase-like properties. Reports had implicated phoshorylation in the activation of many catalytic enzymes (Hanks et aI., 1985). Phosphorylation of the enzymes in this cell-free system proved unsuccessful. Further, it had been reported that in vitro phosphorylation of several membrane polypeptides and soluble polypeptides from com, had been promoted by the addition of Ca²₊ In this cell-free system Ca + did not have a stimulatory effect on protein phosphorylation. Dioxygenases generally occur as soluble enzymes, where they catalyse many oxygenation reactions in metabolic pathways. The addition of 2-oxo-glutarate, a requirement of most soluble oxidases, did not affect the activity of the cell-free system.
- Full Text:
- Date Issued: 1996
Constituent processes of leaf senescence in Hordeum vulgare cv. Dyan
- Afitlhile, Meshack Mosimanegape
- Authors: Afitlhile, Meshack Mosimanegape
- Date: 1994
- Subjects: Leaves , Leaves -- Development
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4184 , http://hdl.handle.net/10962/d1003752
- Description: Changes in chlorophyll content, carotenoid content and composition, abscisic acid and phaseic acid levels, hydrolytic enzyme activity and polypeptide pattern were monitored during senescence of the primary attached leaves of Hordeum vulgare L. cv. Dyan. Senescence occurred due to the normal course of leaf development or was induced by incubation of leaves in darkness. Loss of chlorophyll and total leaf protein was retarded in light whereas it continued rapidly in leaves from dark-incubated seedlings. Chlorophyll alb ratio increased with the progression of senescence, suggesting that chlorophyll b was referentially degraded during this process. Loss of total protein coincided with enhanced activity of acid and neutral proteases. In contrast, loss of chlorophyll was not accompanied by an increase in· peroxidase activity, suggesting that this enzyme was not responsible for initiating chlorophyll breakdown. Carotenoid and abscisic acid levels were monitored in the same tissue extracts. The results obtained show that the increase in endogenous levels of abscisic acid, induced by senescence, correlated with enhanced epoxidation of the xanthophyll cycle, ie., increased conversion of zeaxanthin to antheraxanthin and all-trans-violaxanthin. In addition, an increase in abscisic acid levels occurred concomitant with a decrease in all-trans-violaxanthin and 9'-cis-neoxanthin, suggesting an apparent 1:1 relationship on a molar basis. It is therefore proposed that enhanced abscisic acid production, due to foliar senescence, arises from fluctuations in carotenoid turnover. Polypeptide patterns in isolated chloroplasts, purified thylakoid and stromal fractions were very similar for leaves incubated in either light or darkness. A decrease in intensity of bands was observed in isolated chloroplasts and stromal fractions. Intensity of bands in thylakoids remained unchanged with the progression of senescence. Protein standards of peroxidase and lipoxygenase co-migrated with proteins of the isolated chloroplast. Although tentative, some proteins of the chloroplast may be representative of precursors of hydrolytic enzymes which are known to increase during senescence.
- Full Text:
- Date Issued: 1994
- Authors: Afitlhile, Meshack Mosimanegape
- Date: 1994
- Subjects: Leaves , Leaves -- Development
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4184 , http://hdl.handle.net/10962/d1003752
- Description: Changes in chlorophyll content, carotenoid content and composition, abscisic acid and phaseic acid levels, hydrolytic enzyme activity and polypeptide pattern were monitored during senescence of the primary attached leaves of Hordeum vulgare L. cv. Dyan. Senescence occurred due to the normal course of leaf development or was induced by incubation of leaves in darkness. Loss of chlorophyll and total leaf protein was retarded in light whereas it continued rapidly in leaves from dark-incubated seedlings. Chlorophyll alb ratio increased with the progression of senescence, suggesting that chlorophyll b was referentially degraded during this process. Loss of total protein coincided with enhanced activity of acid and neutral proteases. In contrast, loss of chlorophyll was not accompanied by an increase in· peroxidase activity, suggesting that this enzyme was not responsible for initiating chlorophyll breakdown. Carotenoid and abscisic acid levels were monitored in the same tissue extracts. The results obtained show that the increase in endogenous levels of abscisic acid, induced by senescence, correlated with enhanced epoxidation of the xanthophyll cycle, ie., increased conversion of zeaxanthin to antheraxanthin and all-trans-violaxanthin. In addition, an increase in abscisic acid levels occurred concomitant with a decrease in all-trans-violaxanthin and 9'-cis-neoxanthin, suggesting an apparent 1:1 relationship on a molar basis. It is therefore proposed that enhanced abscisic acid production, due to foliar senescence, arises from fluctuations in carotenoid turnover. Polypeptide patterns in isolated chloroplasts, purified thylakoid and stromal fractions were very similar for leaves incubated in either light or darkness. A decrease in intensity of bands was observed in isolated chloroplasts and stromal fractions. Intensity of bands in thylakoids remained unchanged with the progression of senescence. Protein standards of peroxidase and lipoxygenase co-migrated with proteins of the isolated chloroplast. Although tentative, some proteins of the chloroplast may be representative of precursors of hydrolytic enzymes which are known to increase during senescence.
- Full Text:
- Date Issued: 1994
- «
- ‹
- 1
- ›
- »