Bacterial colonisation and degradation of geologically weathered and discard coal
- Authors: Olawale, Jacob Taiwo
- Date: 2018
- Subjects: Coal mine waste , Coal -- Biodegradation , Coal mines and mining -- Environmental aspects , Land degradation , Electron microscopy , Extracellular polymeric substances , Flagella (Microbiology) , Fourier transform infrared spectroscopy , Microbiologically influenced corrosion
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/61625 , vital:28043
- Description: Bacterial beneficiation of low-grade coal, coal discard, and waste has the potential to mitigate land degradation, water and soil pollution and, be a strategy for mining companies to responsibly extract and process coal with environmental sustainability. This study investigated the colonisation and biodegradation or depolymerisation of coal discard and geologically weathered coal by selected strains of bacteria, and an attempt has been made to describe the mechanisms associated with colonisation and biodegradation of this carbonaceous material. Ten bacterial strains, Bacillus strain ECCN 18b, Citrobacter strain ECCN 19b, Proteus strain ECCN 20b, Exiguobacterium strain ECCN 21b, Microbacterium strain ECCN 22b, Proteus strain ECCN 23b, Serratia strain ECCN 24b, Escherichia strain ECCN 25b, Bacillus strain ECCN 26b and Bacillus strain ECCN 41b, isolated from diesel-contaminated soil and coal slurry and identified using DNA sequencing, were rescreened and their coal biodegradation potential ranked. The ranking of the bacterial strains was undertaken using several indicators including; formation of brown halos on the plate culture (solid), change in colour intensity of the medium in liquid culture, change in culture media pH, and an increase in absorbance at 280nm and 450nm. Although, all the ten strains showed evidence of biodegradation of coal discard and geologically weathered coal based on the ranking employed, and the three strains considered the best candidates were Citrobacter strain ECCN 19b, Exiguobacterium strain ECCN 21b and Serratia strain ECCN 24b. The actions of the three bacterial strains were further studied and characterised in relation to coal degradation. Electron microscopy revealed that Citrobacter strain ECCN 19b, Exiguobacterium strain ECCN 21b and Serratia strain ECCN 24b attached to the surface of coal discard and geologically weathered coal by a process that appeared to involve extracellular polymeric substances (EPS), and flagella. The presence of flagella for Citrobacter strain ECCN 19b and Serratia strain ECCN 24b was confirmed by transmission electron microscopy. Bacterial degradation of coal discard and geologically weathered coal by these selected strains resulted in the release of soluble and insoluble products. Ultraviolet/ visible spectrophotometric (UV/VIS) analysis revealed that the soluble products resembled humic acid-like substances, which was confirmed following Fourier Transform Infrared (FTIR) spectroscopy. Analysis revealed that the coal-derived humic acid-like substances were similar to commercial humic acid extracted from bituminous coal. Elemental analysis of the insoluble product residue after bacterial biodegradation revealed the modification of the chemical compositions of the coal discard and geologically weathered coal substrates. Characterisation of the functional groups of the insoluble product using FTIR spectroscopy indicated changes, with the appearance of new peaks at 1737cm-1, 1366cm-1, 1228cm-1, and 1216cm-1 characteristic of aldehyde, ketones, carboxylic acids, esters, amines, and alkanes. Broad spectra regions of 3500 -3200cm-1, characteristic of alcohol and phenol, were also observed. Together, these results were taken as evidence for increased oxidation of the coal substrates, presumably as a consequence of bacterial catalysed biodegradation of coal discard and geologically weathered coal. During bacterial degradation of coal discard and geologically weathered coal, strains produced extracellular protein, which was detected and further investigated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE). At least three protein bands with molecular mass 53 kDa, 72 kDa, and 82 kDa were common to the three bacterial strains. Following ammonium sulphate precipitation and gel filtration chromatography, additional bands with molecular mass 16 kDa, 33 kDa, 37 kDa, and 43 kDa were detected. An extracellular laccase activity was detected in cultures of Exiguobacterium strain ECCN 21b and Serratia strain ECCN 24b. Cytochrome P450 activity was detected in all the bacterial strains in the presence of both coal discard and geologically weathered coal. This is the first time that cytochrome P450 activity has been reported following exposure of these three bacterial strains to a coal substrate. Overall, this research has successfully demonstrated the partial degradation of coal discard and geologically weathered coal by Citrobacter strain ECCN 19b, Exiguobacterium strain ECCN 21b and Serratia strain ECCN 24b and the release of humic acid-like substances. Thus, the biodegradation process involved adherence to and growth of the bacteria on the surface of coal substrate and appeared to require the formation of alkaline substances and the combined activities of extracellular LAC and cytochrome P450. Since bacterial degradation of low-grade coal and discard appears to be viable, the bacteria isolated in this study can potentially be used either for conversion of discard into valuable chemicals or to mitigate the deleterious effects of stockpiled coal discard on the environment.
- Full Text:
- Authors: Olawale, Jacob Taiwo
- Date: 2018
- Subjects: Coal mine waste , Coal -- Biodegradation , Coal mines and mining -- Environmental aspects , Land degradation , Electron microscopy , Extracellular polymeric substances , Flagella (Microbiology) , Fourier transform infrared spectroscopy , Microbiologically influenced corrosion
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/61625 , vital:28043
- Description: Bacterial beneficiation of low-grade coal, coal discard, and waste has the potential to mitigate land degradation, water and soil pollution and, be a strategy for mining companies to responsibly extract and process coal with environmental sustainability. This study investigated the colonisation and biodegradation or depolymerisation of coal discard and geologically weathered coal by selected strains of bacteria, and an attempt has been made to describe the mechanisms associated with colonisation and biodegradation of this carbonaceous material. Ten bacterial strains, Bacillus strain ECCN 18b, Citrobacter strain ECCN 19b, Proteus strain ECCN 20b, Exiguobacterium strain ECCN 21b, Microbacterium strain ECCN 22b, Proteus strain ECCN 23b, Serratia strain ECCN 24b, Escherichia strain ECCN 25b, Bacillus strain ECCN 26b and Bacillus strain ECCN 41b, isolated from diesel-contaminated soil and coal slurry and identified using DNA sequencing, were rescreened and their coal biodegradation potential ranked. The ranking of the bacterial strains was undertaken using several indicators including; formation of brown halos on the plate culture (solid), change in colour intensity of the medium in liquid culture, change in culture media pH, and an increase in absorbance at 280nm and 450nm. Although, all the ten strains showed evidence of biodegradation of coal discard and geologically weathered coal based on the ranking employed, and the three strains considered the best candidates were Citrobacter strain ECCN 19b, Exiguobacterium strain ECCN 21b and Serratia strain ECCN 24b. The actions of the three bacterial strains were further studied and characterised in relation to coal degradation. Electron microscopy revealed that Citrobacter strain ECCN 19b, Exiguobacterium strain ECCN 21b and Serratia strain ECCN 24b attached to the surface of coal discard and geologically weathered coal by a process that appeared to involve extracellular polymeric substances (EPS), and flagella. The presence of flagella for Citrobacter strain ECCN 19b and Serratia strain ECCN 24b was confirmed by transmission electron microscopy. Bacterial degradation of coal discard and geologically weathered coal by these selected strains resulted in the release of soluble and insoluble products. Ultraviolet/ visible spectrophotometric (UV/VIS) analysis revealed that the soluble products resembled humic acid-like substances, which was confirmed following Fourier Transform Infrared (FTIR) spectroscopy. Analysis revealed that the coal-derived humic acid-like substances were similar to commercial humic acid extracted from bituminous coal. Elemental analysis of the insoluble product residue after bacterial biodegradation revealed the modification of the chemical compositions of the coal discard and geologically weathered coal substrates. Characterisation of the functional groups of the insoluble product using FTIR spectroscopy indicated changes, with the appearance of new peaks at 1737cm-1, 1366cm-1, 1228cm-1, and 1216cm-1 characteristic of aldehyde, ketones, carboxylic acids, esters, amines, and alkanes. Broad spectra regions of 3500 -3200cm-1, characteristic of alcohol and phenol, were also observed. Together, these results were taken as evidence for increased oxidation of the coal substrates, presumably as a consequence of bacterial catalysed biodegradation of coal discard and geologically weathered coal. During bacterial degradation of coal discard and geologically weathered coal, strains produced extracellular protein, which was detected and further investigated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE). At least three protein bands with molecular mass 53 kDa, 72 kDa, and 82 kDa were common to the three bacterial strains. Following ammonium sulphate precipitation and gel filtration chromatography, additional bands with molecular mass 16 kDa, 33 kDa, 37 kDa, and 43 kDa were detected. An extracellular laccase activity was detected in cultures of Exiguobacterium strain ECCN 21b and Serratia strain ECCN 24b. Cytochrome P450 activity was detected in all the bacterial strains in the presence of both coal discard and geologically weathered coal. This is the first time that cytochrome P450 activity has been reported following exposure of these three bacterial strains to a coal substrate. Overall, this research has successfully demonstrated the partial degradation of coal discard and geologically weathered coal by Citrobacter strain ECCN 19b, Exiguobacterium strain ECCN 21b and Serratia strain ECCN 24b and the release of humic acid-like substances. Thus, the biodegradation process involved adherence to and growth of the bacteria on the surface of coal substrate and appeared to require the formation of alkaline substances and the combined activities of extracellular LAC and cytochrome P450. Since bacterial degradation of low-grade coal and discard appears to be viable, the bacteria isolated in this study can potentially be used either for conversion of discard into valuable chemicals or to mitigate the deleterious effects of stockpiled coal discard on the environment.
- Full Text:
Evaluation of Fungcoal as a bioprocess technology for self-cladding of waste coal dumps
- Authors: Sekhohola, Lerato M
- Date: 2016
- Subjects: Coal mine waste , Fungi -- Biotechnology , Coal -- Biodegradation , Bermuda grass -- Biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:5959 , http://hdl.handle.net/10962/d1019992
- Description: Low-grade coal, a poor source of energy, has long been regarded as waste material by the coal mining industry. Biological degradation of this coal material by ligninolytic fungal strains presents a viable strategy towards eliminating this unusable fossil fuel. To this end, a novel and patented bioprocess termed Fungcoal was developed. Fungcoal is a biological process utilised in the in situ treatment of waste coal and is based on the mutualistic relationship between the fungus Neosartorya fischeri and the graminaceous species Cynodon dactylon. The process facilitates the rapid conversion of waste coal into soil-like material that stimulates establishment of vegetation for eventual coal dump rehabilitation. While a number of in vitro studies have identified various fungal strains as efficient coal degraders, the mechanisms involved in the Fungcoal-stimulated degradation process have not been fully elucidated. Furthermore, implementation of Fungcoal at both pilot and commercial scale has not been achieved. Thus the objective of this work was to investigate Fungcoal as a bioprocess via examining the role of coal degrading fungi (CDF) and grasses as biocatalysts in coal biodegradation and for the self-cladding of waste coal dumps. Initially, waste coal degradation by N. fischeri, strain ECCN 84, was investigated, specifically focusing on the mechanisms underpinning the process. In vitro studies showed the addition of waste coal induced active fungal colonisation resulting in increased fungal biomass. Increased extracellular laccase (LAC) activity, occuring concomitantly with an increase in hyphal peroxisome proliferation, was also observed in the coal supplied fungal cultures. Analysis of the colonised waste coal revealed a time dependent reduction in the percentage weight of elemental carbon coupled with an increase in elemental oxygen. The results supported metabolism and degradation of waste coal by N. fischeri strain ECCN 84 and involvement of fungal extracellular laccase. The contribution of C. dactylon, a C4 grass species to in situ biodegradation of waste coal in the presence of coal degrading and mycorrhizal fungi (MF) was also investigated. Enhanced degradation of the waste coal into a humic soil-like material was observed within the rhizosphere. Analysis of the resultant substrate revealed an increased concentration of highly oxidised humic-like substances (HS). Fungi remained viable in the rhizosphere up to 47 weeks post-inoculation and cultivation of C. dactylon, indicating the resultant humic substance-rich rhizosphere provided an environment conducive for microbial proliferation and activity. Furthermore, humic substance enrichment of waste coal substrates supported germination and seedling emergence of several agronomic species including Zea mays (corn), Phaseolus vulgaris (bean), Pisum sativum (pea), and Spinacia oleracea (spinach). Use of various cladding materials to support coal biodegradation, by fungus-grass mutualism and rehabilitation of waste dumps was evaluated at commercial scale. While substantial physico-chemical changes were not evident in the absence of cladding or where waste coal was used as cladding material, successful establishment of grass cover and diversity was achieved within three hydrological cycles on dumps cladded with weathered coal. Work presented in this thesis successfully demonstrates the degradation of waste coal by N. fischeri. The biodegradation process included enhanced extracellular LAC activity coupled with increased 3 waste coal oxidation. Increased HS concentration of waste coal substrate supported germination and early seedling establishment of several agronomic species. At commercial scale a co-substrate in the form of carbon-rich weathered coal was essential to support fungus-grass mutualism and Fungcoal-induced rehabilitation. These findings support the developed Fungcoal concept and the underpinning rationale that the phyto-biodegradation of waste coal indeed depends on the mutualistic interactions between grass root exudates and the ligninolytic and mycorrhizal fungi. Taken together, these findings provide practical evidence of the contribution of fungi and grasses as mutualists in the biodegradation of waste coal and sustainable rehabilitation of waste coal dumps
- Full Text:
- Authors: Sekhohola, Lerato M
- Date: 2016
- Subjects: Coal mine waste , Fungi -- Biotechnology , Coal -- Biodegradation , Bermuda grass -- Biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:5959 , http://hdl.handle.net/10962/d1019992
- Description: Low-grade coal, a poor source of energy, has long been regarded as waste material by the coal mining industry. Biological degradation of this coal material by ligninolytic fungal strains presents a viable strategy towards eliminating this unusable fossil fuel. To this end, a novel and patented bioprocess termed Fungcoal was developed. Fungcoal is a biological process utilised in the in situ treatment of waste coal and is based on the mutualistic relationship between the fungus Neosartorya fischeri and the graminaceous species Cynodon dactylon. The process facilitates the rapid conversion of waste coal into soil-like material that stimulates establishment of vegetation for eventual coal dump rehabilitation. While a number of in vitro studies have identified various fungal strains as efficient coal degraders, the mechanisms involved in the Fungcoal-stimulated degradation process have not been fully elucidated. Furthermore, implementation of Fungcoal at both pilot and commercial scale has not been achieved. Thus the objective of this work was to investigate Fungcoal as a bioprocess via examining the role of coal degrading fungi (CDF) and grasses as biocatalysts in coal biodegradation and for the self-cladding of waste coal dumps. Initially, waste coal degradation by N. fischeri, strain ECCN 84, was investigated, specifically focusing on the mechanisms underpinning the process. In vitro studies showed the addition of waste coal induced active fungal colonisation resulting in increased fungal biomass. Increased extracellular laccase (LAC) activity, occuring concomitantly with an increase in hyphal peroxisome proliferation, was also observed in the coal supplied fungal cultures. Analysis of the colonised waste coal revealed a time dependent reduction in the percentage weight of elemental carbon coupled with an increase in elemental oxygen. The results supported metabolism and degradation of waste coal by N. fischeri strain ECCN 84 and involvement of fungal extracellular laccase. The contribution of C. dactylon, a C4 grass species to in situ biodegradation of waste coal in the presence of coal degrading and mycorrhizal fungi (MF) was also investigated. Enhanced degradation of the waste coal into a humic soil-like material was observed within the rhizosphere. Analysis of the resultant substrate revealed an increased concentration of highly oxidised humic-like substances (HS). Fungi remained viable in the rhizosphere up to 47 weeks post-inoculation and cultivation of C. dactylon, indicating the resultant humic substance-rich rhizosphere provided an environment conducive for microbial proliferation and activity. Furthermore, humic substance enrichment of waste coal substrates supported germination and seedling emergence of several agronomic species including Zea mays (corn), Phaseolus vulgaris (bean), Pisum sativum (pea), and Spinacia oleracea (spinach). Use of various cladding materials to support coal biodegradation, by fungus-grass mutualism and rehabilitation of waste dumps was evaluated at commercial scale. While substantial physico-chemical changes were not evident in the absence of cladding or where waste coal was used as cladding material, successful establishment of grass cover and diversity was achieved within three hydrological cycles on dumps cladded with weathered coal. Work presented in this thesis successfully demonstrates the degradation of waste coal by N. fischeri. The biodegradation process included enhanced extracellular LAC activity coupled with increased 3 waste coal oxidation. Increased HS concentration of waste coal substrate supported germination and early seedling establishment of several agronomic species. At commercial scale a co-substrate in the form of carbon-rich weathered coal was essential to support fungus-grass mutualism and Fungcoal-induced rehabilitation. These findings support the developed Fungcoal concept and the underpinning rationale that the phyto-biodegradation of waste coal indeed depends on the mutualistic interactions between grass root exudates and the ligninolytic and mycorrhizal fungi. Taken together, these findings provide practical evidence of the contribution of fungi and grasses as mutualists in the biodegradation of waste coal and sustainable rehabilitation of waste coal dumps
- Full Text:
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