Evaluation of bacteria laccase hybrid biosensor and application in the detection of phenolic contaminants in water
- Edoamodu, Chiedu Epiphany https://orcid.org/0000-0002-9254-3955
- Authors: Edoamodu, Chiedu Epiphany https://orcid.org/0000-0002-9254-3955
- Date: 2021-09
- Subjects: Laccase , Water -- Purification
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/22820 , vital:52802
- Description: The continuous outpour of aromatic pollutants in diverse groups, emanating from the industrial and domestic system due to population density, industrialisation and technological advancement is alarming. The increasing strength in wastewater discharge constitutes the main cause of the natural water pollution load, causing scarcity of potable water for consumption with an increasing health challenge. The physiochemical treatment approach has faced a series of limitations with little or no success. Hindrance to wastewater management can cause a point source contamination problem that might increase treatment cost and release a broad range of chemical contaminants in the environment. Hence, green, eco-friendly and cost-effective tools are imperative. The application of laccase has received much attention in bioremediation and bioprocessing matters owing to the oxidising capacity of a wide range of substrates. The process requires available molecular oxygen for its activation, releasing water as a by-product thus, establishing this research. This study was devised to examine the bioprocessing potentials of hybrid and amalgamated laccases extracted from the diverse environmental milieu of the Eastern Cape Province, South Africa. Bacteria producing laccase were isolated from marine sediment, cow dung, and wastewater samples via selective enrichment with some aromatic compounds. The axenic cultures were screened for laccase activity on various phenolic and non-phenolic substrates. The isolates were identified via molecular techniques and they belonged to the gammaproteobacteria and Bacilli classes under the following genera, Enterobacter and Bacillus. They were deposited in the NCBI database as Enterobacter asburiae ES1, Enterobacter sp. Kamsi, Enterobacter sp. AI1, and Bacillus sp. NU2 with the assigned accession number MN686602, MN686603, MN686605, MN686607, respectively. Optimisation of the laccase production via one factor at a time technique (OFAT) from the four bacteria species showed more enzyme yield in all lignocellulosic agro-waste media. However, wheat bran and mandarin peel maximally enhanced laccase production. In addition, xylose, galactose, fructose, and sorbitol were the best carbon sources utilised while (NH₄)₂SO8, KNO3 and NaNO3 were noted as the best nitrogen sources employed. Laccase yields were increased at pH 4 and 5, at temperatures 45 and 55 o C, and at 50 and 100 rpm, and precisely, at day eight of the incubation period. Further purification of the crude laccase yielded a purification fold of 4.18, 4.39, 2.78, 8.11, and the SDS-PAGE analysis showed a molecular size of 90, 55, 75 and 50 kDa for ES1, Kamsi, AI1, and NU2 laccases, respectively. The characterised purified laccase demonstrated polyextremotolerant potentials. The laccases were active through a wide temperature regime (30-90 o C) with maximum activity at 50 o C (ES1/AI1 and Kamsi/NU2) 60 o C (AI1), 70 o C (ES1, Kamsi, NU2); and were stable at 60 o C (ES1, AI1, NU2), 70 o C (ES1/AI1), 80 o C (Kamsi and Kamsi/NU2). Also, the laccases remained active through pH 3 - 8 and optimal at pH 4 (AI1, NU2), pH 5 (Kamsi, ES1/AI1), pH 7 (ES1), pH 8 (Kamsi/NU2), and the individual stability was measured at pH 4 (Kamsi, NU2), pH 5 (AI1), pH 6 (ES1), pH 7 (Kamsi/NU2), pH 10 (ES1/AI1). The purified laccases were either enhanced or left unchanged by a variable concentration of metallic salts, inhibitors, chelating agents and organic solvents. Clearly, the activities of the laccase were enhanced when pre-incubated with 1, 3, and 6 mm of CuCl2, FeCl3, MgCl2, ZnCl2 and AgCl, and 1, 2, 3 mm of Triton x-100, PMSF, EDTA, Tween 20, and NaCl. Additionally, 20, 30, and 10 percent v/v of acetone and DMSO were prominent organic solvents that also stimulated both the hybrid and amalgamated laccase activity. The gene of the purified laccases targeted showed a clear band size of 690 bp for the Enterobacter species laccases and 775 bp for the laccase from Bacillus sp. The protein sequence was deposited in NCBI database with the assigned accession numbers, MW251989, MW25990, MW251992, and MW251994 for ES1, Kamsi, AI1, and NU2 laccases, respectively. The optimised pH and temperature parameter examined on the decolourising potential of the bacteria laccases showed an effective dye removal on the five synthetic dyes (Congo Red (CR), Methyl Orange (MO), Malachite Green (MG), Reactive Blue 4 (RB4), Ramazol Brilliant Blue R (RBBR)) applied. The purified laccases were successfully immobilised in Na-alginate with cca. 88.49, 70.91, 76.04, 76.13, 90.07, and 91.99 laccase yield for the hybrid (ES1, ES1, Kamsi, AI1, NU2) and amalgamated (ES1/AI1 and Kamsi/NU2) laccases. The immobilised laccases were able to retain an average activity of 32 – 52 percent after eight dye decolourising cycles, exhibiting strong catalytic activity than the free laccases. Nonetheless, no significant difference was examined between the hybrid and amalgamated laccase activity. Also, the immobilised laccases were shown to be more efficient in biotechnological application than the free laccases. The result suggests that immobilising an enzyme in a carrier matrix served effectively as the remediation approach than the hybrid and the amalgamation of the free enzymes. Also, the application of lignocellulosic waste served as a cheaper substrate for commercial production of laccase and could help s in promoting es the biotechnology application and the bioeconomy. , Thesis (PhD) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-09
- Authors: Edoamodu, Chiedu Epiphany https://orcid.org/0000-0002-9254-3955
- Date: 2021-09
- Subjects: Laccase , Water -- Purification
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/22820 , vital:52802
- Description: The continuous outpour of aromatic pollutants in diverse groups, emanating from the industrial and domestic system due to population density, industrialisation and technological advancement is alarming. The increasing strength in wastewater discharge constitutes the main cause of the natural water pollution load, causing scarcity of potable water for consumption with an increasing health challenge. The physiochemical treatment approach has faced a series of limitations with little or no success. Hindrance to wastewater management can cause a point source contamination problem that might increase treatment cost and release a broad range of chemical contaminants in the environment. Hence, green, eco-friendly and cost-effective tools are imperative. The application of laccase has received much attention in bioremediation and bioprocessing matters owing to the oxidising capacity of a wide range of substrates. The process requires available molecular oxygen for its activation, releasing water as a by-product thus, establishing this research. This study was devised to examine the bioprocessing potentials of hybrid and amalgamated laccases extracted from the diverse environmental milieu of the Eastern Cape Province, South Africa. Bacteria producing laccase were isolated from marine sediment, cow dung, and wastewater samples via selective enrichment with some aromatic compounds. The axenic cultures were screened for laccase activity on various phenolic and non-phenolic substrates. The isolates were identified via molecular techniques and they belonged to the gammaproteobacteria and Bacilli classes under the following genera, Enterobacter and Bacillus. They were deposited in the NCBI database as Enterobacter asburiae ES1, Enterobacter sp. Kamsi, Enterobacter sp. AI1, and Bacillus sp. NU2 with the assigned accession number MN686602, MN686603, MN686605, MN686607, respectively. Optimisation of the laccase production via one factor at a time technique (OFAT) from the four bacteria species showed more enzyme yield in all lignocellulosic agro-waste media. However, wheat bran and mandarin peel maximally enhanced laccase production. In addition, xylose, galactose, fructose, and sorbitol were the best carbon sources utilised while (NH₄)₂SO8, KNO3 and NaNO3 were noted as the best nitrogen sources employed. Laccase yields were increased at pH 4 and 5, at temperatures 45 and 55 o C, and at 50 and 100 rpm, and precisely, at day eight of the incubation period. Further purification of the crude laccase yielded a purification fold of 4.18, 4.39, 2.78, 8.11, and the SDS-PAGE analysis showed a molecular size of 90, 55, 75 and 50 kDa for ES1, Kamsi, AI1, and NU2 laccases, respectively. The characterised purified laccase demonstrated polyextremotolerant potentials. The laccases were active through a wide temperature regime (30-90 o C) with maximum activity at 50 o C (ES1/AI1 and Kamsi/NU2) 60 o C (AI1), 70 o C (ES1, Kamsi, NU2); and were stable at 60 o C (ES1, AI1, NU2), 70 o C (ES1/AI1), 80 o C (Kamsi and Kamsi/NU2). Also, the laccases remained active through pH 3 - 8 and optimal at pH 4 (AI1, NU2), pH 5 (Kamsi, ES1/AI1), pH 7 (ES1), pH 8 (Kamsi/NU2), and the individual stability was measured at pH 4 (Kamsi, NU2), pH 5 (AI1), pH 6 (ES1), pH 7 (Kamsi/NU2), pH 10 (ES1/AI1). The purified laccases were either enhanced or left unchanged by a variable concentration of metallic salts, inhibitors, chelating agents and organic solvents. Clearly, the activities of the laccase were enhanced when pre-incubated with 1, 3, and 6 mm of CuCl2, FeCl3, MgCl2, ZnCl2 and AgCl, and 1, 2, 3 mm of Triton x-100, PMSF, EDTA, Tween 20, and NaCl. Additionally, 20, 30, and 10 percent v/v of acetone and DMSO were prominent organic solvents that also stimulated both the hybrid and amalgamated laccase activity. The gene of the purified laccases targeted showed a clear band size of 690 bp for the Enterobacter species laccases and 775 bp for the laccase from Bacillus sp. The protein sequence was deposited in NCBI database with the assigned accession numbers, MW251989, MW25990, MW251992, and MW251994 for ES1, Kamsi, AI1, and NU2 laccases, respectively. The optimised pH and temperature parameter examined on the decolourising potential of the bacteria laccases showed an effective dye removal on the five synthetic dyes (Congo Red (CR), Methyl Orange (MO), Malachite Green (MG), Reactive Blue 4 (RB4), Ramazol Brilliant Blue R (RBBR)) applied. The purified laccases were successfully immobilised in Na-alginate with cca. 88.49, 70.91, 76.04, 76.13, 90.07, and 91.99 laccase yield for the hybrid (ES1, ES1, Kamsi, AI1, NU2) and amalgamated (ES1/AI1 and Kamsi/NU2) laccases. The immobilised laccases were able to retain an average activity of 32 – 52 percent after eight dye decolourising cycles, exhibiting strong catalytic activity than the free laccases. Nonetheless, no significant difference was examined between the hybrid and amalgamated laccase activity. Also, the immobilised laccases were shown to be more efficient in biotechnological application than the free laccases. The result suggests that immobilising an enzyme in a carrier matrix served effectively as the remediation approach than the hybrid and the amalgamation of the free enzymes. Also, the application of lignocellulosic waste served as a cheaper substrate for commercial production of laccase and could help s in promoting es the biotechnology application and the bioeconomy. , Thesis (PhD) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-09
Performance of an integrated algal pond for treatment of domestic sewage: a process audit
- Authors: Dube, Anele
- Date: 2020
- Subjects: Water -- Purification , Sewage -- Purification -- Anaerobic treatment , Algae -- Biotechnology , Waste disposal -- South Africa , Integrated algae pond systems (IAPS)
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167043 , vital:41432
- Description: Integrated algae pond systems (IAPS) are energy efficient, robust, passive systems that use the principles of fermentation, photosynthesis and microbial metabolism to remediate wastewater, producing a good quality effluent with reuse potential. In addition to the treatment of wastewater, IAPS have the ability to generate two additional product streams viz. biogas and biomass. The latter adds to the attractiveness of the system. However, the implementation of this technology, like many passive systems, has remained limited at a commercial scale, and the inclination is still towards grey technologies. The aim of this research was to investigate the capabilities and potential of a demonstration-scale IAPS and use results obtained to establish a process audit framework. The aspects considered for the audit included performance efficiency, effluent water quality, biomass composition, quantity and productivity within the ponds, and cost analysis of operation and maintenance over a 9-year period. Plant performance was closely monitored during the course of the study and this led to a review of previously adopted plant management strategies. Troubleshooting exercises were also carried out when plant performance declined. Results showed that IAPS efficiently reduced standard water parameters with the exception of pH, dissolved oxygen, and nitrate whose values increased from raw influent to final effluent. The following water quality parameters were established for the final effluent: total suspended solids 55 ± 7.1 mg. L-1 (n = 28); chemical oxygen demand 94.1 ± 10.6 mg. L-1 (n = 28) (after removal of algae); pH 9.9 ± 0.01 (n = 26); ammonium nitrogen 1.7 ± 0.3 mg. L-1 (n = 25); nitrate 3.3 ± 0.6 mg. L-1 (n = 25); ortho-phosphate 1.6 ± 0.2 mg. L-1 (n = 25); electrical conductivity 98.7 ± 2.0 mS m-1 (n = 26) and faecal coliforms (per 100 mL) 1482.6 ± 636.0 (n = 24). The final effluent measured consistently high chemical oxygen demand and total suspended solids, however close analysis showed that total suspended solids could be controlled by increasing the frequency of removal of settled biomass within the settling ponds. Biomass produced contained microalgae, bacteria, metazoa, and protozoa. The biomass productivity achieved was as high as 130.6 kg ha-1 d-1; however, about 33% was lost to the final effluent due to inadequate settling. Results obtained during the course of this study and outcomes of earlier work on IAPS are taken as the baseline to determine parameters needed for the development of the process audit framework. Techniques utilised to derive the blue print process audit protocol for IAPS included a turtle diagram, a flow diagram and a checklist. Attention to plant management proved vital in determining overall performance. Cost, including operating and maintenance, of treating water using the demonstration scale system on a per person equivalent per year basis was determined as ZAR 123.87 (where, ZAR to USD = 0.07).
- Full Text:
- Date Issued: 2020
- Authors: Dube, Anele
- Date: 2020
- Subjects: Water -- Purification , Sewage -- Purification -- Anaerobic treatment , Algae -- Biotechnology , Waste disposal -- South Africa , Integrated algae pond systems (IAPS)
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167043 , vital:41432
- Description: Integrated algae pond systems (IAPS) are energy efficient, robust, passive systems that use the principles of fermentation, photosynthesis and microbial metabolism to remediate wastewater, producing a good quality effluent with reuse potential. In addition to the treatment of wastewater, IAPS have the ability to generate two additional product streams viz. biogas and biomass. The latter adds to the attractiveness of the system. However, the implementation of this technology, like many passive systems, has remained limited at a commercial scale, and the inclination is still towards grey technologies. The aim of this research was to investigate the capabilities and potential of a demonstration-scale IAPS and use results obtained to establish a process audit framework. The aspects considered for the audit included performance efficiency, effluent water quality, biomass composition, quantity and productivity within the ponds, and cost analysis of operation and maintenance over a 9-year period. Plant performance was closely monitored during the course of the study and this led to a review of previously adopted plant management strategies. Troubleshooting exercises were also carried out when plant performance declined. Results showed that IAPS efficiently reduced standard water parameters with the exception of pH, dissolved oxygen, and nitrate whose values increased from raw influent to final effluent. The following water quality parameters were established for the final effluent: total suspended solids 55 ± 7.1 mg. L-1 (n = 28); chemical oxygen demand 94.1 ± 10.6 mg. L-1 (n = 28) (after removal of algae); pH 9.9 ± 0.01 (n = 26); ammonium nitrogen 1.7 ± 0.3 mg. L-1 (n = 25); nitrate 3.3 ± 0.6 mg. L-1 (n = 25); ortho-phosphate 1.6 ± 0.2 mg. L-1 (n = 25); electrical conductivity 98.7 ± 2.0 mS m-1 (n = 26) and faecal coliforms (per 100 mL) 1482.6 ± 636.0 (n = 24). The final effluent measured consistently high chemical oxygen demand and total suspended solids, however close analysis showed that total suspended solids could be controlled by increasing the frequency of removal of settled biomass within the settling ponds. Biomass produced contained microalgae, bacteria, metazoa, and protozoa. The biomass productivity achieved was as high as 130.6 kg ha-1 d-1; however, about 33% was lost to the final effluent due to inadequate settling. Results obtained during the course of this study and outcomes of earlier work on IAPS are taken as the baseline to determine parameters needed for the development of the process audit framework. Techniques utilised to derive the blue print process audit protocol for IAPS included a turtle diagram, a flow diagram and a checklist. Attention to plant management proved vital in determining overall performance. Cost, including operating and maintenance, of treating water using the demonstration scale system on a per person equivalent per year basis was determined as ZAR 123.87 (where, ZAR to USD = 0.07).
- Full Text:
- Date Issued: 2020
Synthesis of indium phthalocyanines for photodynamic antimicrobial chemotherapy and photo-oxidation of pollutants
- Authors: Sindelo, Azole
- Date: 2019
- Subjects: Phthalocyanines , Azo dyes , Indium compounds , Photochemotherapy , Nanoparticles , Photodegradation , Pollutants , Water -- Purification
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/67581 , vital:29116
- Description: Indium (III) octacarboxyl phthalocyanine (ClInOCPc) alone and when conjugated to magnetic nanoparticles (MNP-ClInOCPc), 2(3),9(10),16(17),23(24)-octapyridylsulfanyl phthalocyaninato chloroindium (III) (ClInOPyPc) and its quaternized derivative 2(3),9(10),16(17),23(24)-octamethylpyridylsulfanyl phthalocyaninato chloroindium (III) (ClInOMePyPc) were synthesized. All Pcs were tested for both photodynamic antimicrobial chemotherapy (PACT) of an unknown water sample and photo-degradation of methyl red (MR). The singlet quantum yield (ΦΔ) for the ClInOCPc and MNP-ClInOCPc in PAN polymer fibers were 0.36 and 0.20 respectively using ADMA as a quencher in water. The photo-inactivation of bacteria in a water sample with unknown microbes was tested, with the MNP-ClInOCPc inactivating 90.6 % of the microbes and the ClInOCPc with 84.8 %. When embedded to the polymer, there was 48% bacterial clearance for ClInOCPc and 64% clearance for the MNP-ClInOCPc. The rate of degradation of MR increased with decrease of the MR concentration, with the MNP-ClInOCPc having the fastest rate. For ClInOPyPc and ClInOMePyPc, the singlet quantum yields were 0.46 and 0.33 in dimethylformamide (DMF), respectively. The PACT activity of ClInOMePyPc (containing 8 positive charges) was compared to those of 9(10),16(17),23(24)-tri-N-methyl-4-pyridylsulfanyl-2(3)-(4-aminophenoxy) phthalocyaninato chloro indium (III) triiodide (1) (containing 3 positive charges) and 2-[4-(N-methylpyridyloxy) phthalocyaninato] chloroindium (III) iodide (2) (containing 4 positive charges). When comparing ClInOMePyPc, 1 and 2, the largest log reduction for E. coli were obtained for complex 2 containing four positive charges hence showing it is not always the charge that determines the PACT activity, but the bridging atom in the phthalocyanine plays a role.
- Full Text:
- Date Issued: 2019
- Authors: Sindelo, Azole
- Date: 2019
- Subjects: Phthalocyanines , Azo dyes , Indium compounds , Photochemotherapy , Nanoparticles , Photodegradation , Pollutants , Water -- Purification
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/67581 , vital:29116
- Description: Indium (III) octacarboxyl phthalocyanine (ClInOCPc) alone and when conjugated to magnetic nanoparticles (MNP-ClInOCPc), 2(3),9(10),16(17),23(24)-octapyridylsulfanyl phthalocyaninato chloroindium (III) (ClInOPyPc) and its quaternized derivative 2(3),9(10),16(17),23(24)-octamethylpyridylsulfanyl phthalocyaninato chloroindium (III) (ClInOMePyPc) were synthesized. All Pcs were tested for both photodynamic antimicrobial chemotherapy (PACT) of an unknown water sample and photo-degradation of methyl red (MR). The singlet quantum yield (ΦΔ) for the ClInOCPc and MNP-ClInOCPc in PAN polymer fibers were 0.36 and 0.20 respectively using ADMA as a quencher in water. The photo-inactivation of bacteria in a water sample with unknown microbes was tested, with the MNP-ClInOCPc inactivating 90.6 % of the microbes and the ClInOCPc with 84.8 %. When embedded to the polymer, there was 48% bacterial clearance for ClInOCPc and 64% clearance for the MNP-ClInOCPc. The rate of degradation of MR increased with decrease of the MR concentration, with the MNP-ClInOCPc having the fastest rate. For ClInOPyPc and ClInOMePyPc, the singlet quantum yields were 0.46 and 0.33 in dimethylformamide (DMF), respectively. The PACT activity of ClInOMePyPc (containing 8 positive charges) was compared to those of 9(10),16(17),23(24)-tri-N-methyl-4-pyridylsulfanyl-2(3)-(4-aminophenoxy) phthalocyaninato chloro indium (III) triiodide (1) (containing 3 positive charges) and 2-[4-(N-methylpyridyloxy) phthalocyaninato] chloroindium (III) iodide (2) (containing 4 positive charges). When comparing ClInOMePyPc, 1 and 2, the largest log reduction for E. coli were obtained for complex 2 containing four positive charges hence showing it is not always the charge that determines the PACT activity, but the bridging atom in the phthalocyanine plays a role.
- Full Text:
- Date Issued: 2019
Water quality, biomass and extracellular polymeric substances in an integrated algae pond system
- Authors: Jimoh, Taobat Adekilekun
- Date: 2018
- Subjects: Water -- Purification , Sewage -- Purification -- Anaerobic treatment , Sewage lagoons , Sewage disposal plants , ASPAM model (Acid mine drainage) , Integrated algae pond systems (IAPS)
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/57307 , vital:26871
- Description: Integrated algae pond systems (IAPS) combine the use of anaerobic and aerobic bioprocesses to effect wastewater treatment. Although, IAPS as a technology process offers many advantages including efficient and simultaneous N and P removal, no requirement for additional chemicals, O2 generation, CO2 mitigation, and a biomass with potential for valorization, a lack of technological advancement and the need for large land area, has limited the reach of this technology at industrial scale. In mitigation, peroxonation was introduced as a tertiary treatment unit and its effect on COD and TSS of IAPS treated water investigated. An effort was made to characterize the soluble but persistent COD in IAPS treated water and, productivity of the HRAOP mixed liquor was investigated to gain insight into the potential use of this biomass. Results show that peroxone treatment effectively reduced COD, TSS, and nutrient load of IAPS water without any significant impact on land area requirement. Indeed, summary data describing the effect of peroxone on quality of IAPS-treated water confirmed that it complies with the general limit values for either irrigation or discharge into a water resource that is not a listed water resource for volumes up to 2 ML of treated wastewater on any given day. Extraction followed by FT-IR spectroscopy was used to confirm albeit tentatively, the identity of the soluble but persistent COD in IAPS treated water as MaB-floc EPS. Results show that MaB-flocs from HRAOPs are assemblages of microorganisms produced as discrete aggregates as a result of microbial EPS production. A relationship between photosynthesis and EPS production was established by quantification of the EPS following exposure of MaB-flocs to either continuous light or darkness. Several novel strains of bacteria were isolated from HRAOP mixed liquor and 16S ribosomal genomic sequence analysis resulted in the molecular characterization of Planococcus maitriensis strain ECCN 45b. This is the first report of Planococcus maitriensis from a wastewater treatment process. Productivity and change in MaB-flocs concentration, measured as mixed liquor suspended solids (MLSS) between morning and evening were monitored and revealed that MLSS is composed of microalgae and bacteria but not fungi. Concentration varied from 77 mg L-1 in September (winter) to 285 mg L-1 in November (spring); pond productivity increased from 5.8 g m-2 d-1 (winter) to 21.5 g m-2 d-1 (spring); and, irrespective of MLSS concentration in late afternoon, approximately 39% was lost overnight, which presumably occurred due to passive removal by the algae settling pond. The outcomes of this research are discussed in terms of the quality of treated water, and the further development of IAPS as a platform technology for establishing a biorefinery within the wastewater treatment sector.
- Full Text:
- Date Issued: 2018
- Authors: Jimoh, Taobat Adekilekun
- Date: 2018
- Subjects: Water -- Purification , Sewage -- Purification -- Anaerobic treatment , Sewage lagoons , Sewage disposal plants , ASPAM model (Acid mine drainage) , Integrated algae pond systems (IAPS)
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/57307 , vital:26871
- Description: Integrated algae pond systems (IAPS) combine the use of anaerobic and aerobic bioprocesses to effect wastewater treatment. Although, IAPS as a technology process offers many advantages including efficient and simultaneous N and P removal, no requirement for additional chemicals, O2 generation, CO2 mitigation, and a biomass with potential for valorization, a lack of technological advancement and the need for large land area, has limited the reach of this technology at industrial scale. In mitigation, peroxonation was introduced as a tertiary treatment unit and its effect on COD and TSS of IAPS treated water investigated. An effort was made to characterize the soluble but persistent COD in IAPS treated water and, productivity of the HRAOP mixed liquor was investigated to gain insight into the potential use of this biomass. Results show that peroxone treatment effectively reduced COD, TSS, and nutrient load of IAPS water without any significant impact on land area requirement. Indeed, summary data describing the effect of peroxone on quality of IAPS-treated water confirmed that it complies with the general limit values for either irrigation or discharge into a water resource that is not a listed water resource for volumes up to 2 ML of treated wastewater on any given day. Extraction followed by FT-IR spectroscopy was used to confirm albeit tentatively, the identity of the soluble but persistent COD in IAPS treated water as MaB-floc EPS. Results show that MaB-flocs from HRAOPs are assemblages of microorganisms produced as discrete aggregates as a result of microbial EPS production. A relationship between photosynthesis and EPS production was established by quantification of the EPS following exposure of MaB-flocs to either continuous light or darkness. Several novel strains of bacteria were isolated from HRAOP mixed liquor and 16S ribosomal genomic sequence analysis resulted in the molecular characterization of Planococcus maitriensis strain ECCN 45b. This is the first report of Planococcus maitriensis from a wastewater treatment process. Productivity and change in MaB-flocs concentration, measured as mixed liquor suspended solids (MLSS) between morning and evening were monitored and revealed that MLSS is composed of microalgae and bacteria but not fungi. Concentration varied from 77 mg L-1 in September (winter) to 285 mg L-1 in November (spring); pond productivity increased from 5.8 g m-2 d-1 (winter) to 21.5 g m-2 d-1 (spring); and, irrespective of MLSS concentration in late afternoon, approximately 39% was lost overnight, which presumably occurred due to passive removal by the algae settling pond. The outcomes of this research are discussed in terms of the quality of treated water, and the further development of IAPS as a platform technology for establishing a biorefinery within the wastewater treatment sector.
- Full Text:
- Date Issued: 2018
Assessment of the physicochemical and microbiological qualities of Tyume River in Amathole District in the Eastern Cape Province, South Africa
- Sibanda, Timothy https://orcid.org/0000-0002-6864-3796
- Authors: Sibanda, Timothy https://orcid.org/0000-0002-6864-3796
- Date: 2013-05
- Subjects: Water -- Purification , Sewage -- Purification
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/24465 , vital:62812
- Description: The bioflocculant-producing potentials of three marine bacteria isolated from the sediment samples of Algoa Bay in the Eastern Cape Province of South Africa were assessed. Analysis of the partial nucleotide sequence of the 16S rDNA of the bacteria revealed 99 percent, 99 percent, and 98 percent similarity to Cobetia sp. L222, Bacillus sp. A-5A, and Bacillus sp. HXG-C1 respectively and the sequence was deposited in GenBank as Cobetia sp. OAUIFE, Bacillus sp. MAYA and Bacillus sp. Gilbert (accession number JF799092, JF799093, and HQ537128 respectively). Cultivation condition studies for Cobetia sp. OAUIFE revealed that bioflocculant production was optimal with an inoculum size of 2 percent (v/v), initial pH of 6.0, Mn2+ as the metal ion, and glucose as the carbon source. Metal ions, including Na+, K+, Li+, Ca2+and Mg2+ stimulated bioflocculant production resulting in flocculating activity of above 90 percent. This crude bioflocculant is thermally stable, with about 78 percent of its flocculating activity remaining after heating at 100 oC for 25 min. Analysis of the purified bioflocculant revealed it to be an acidic extracellular polysaccharide. FTIR analysis revealed the presence of methoxyl, hydroxyl, and carboxyl - groups in the compound bioflocculant and SEM micrograph of the bioflocculant revealed a crystal-linear structure. On the other hand, bioflocculant production by Bacillus sp. MAYA was optimal when glucose (95.6 percent flocculating activity) and ammonium nitrate (83.3 percent flocculating activity) were used as carbon and nitrogen sources respectively; inoculum size was 2 percent (v/v); initial pH 6; and Ca2+ as coagulant aid. Chemical analysis of the purified bioflocculant shows that it is composed of uronic acid, neutral sugar and protein. FTIR analysis also revealed the presence of methoxyl, hydroxyl, carboxyl and amino- groups in this bioflocculant. The bioflocculant is thermostable with about 65.6 percent residual flocculating activity retained after heating the bioflocculant at 100 oC for 25 min. However bioflocculant production by Bacillus sp. Gilbert was optimal when sodium carbonate (95.2 percent flocculating activity) and potassium nitrate (76.6 percent flocculating activity) were used as carbon and nitrogen sources respectively; inoculum size was 3 percent (v/v); initial pH 9; and Al3+ as cation. The crude bioflocculant retained 44.2 percent residual flocculating activity after heating at 100 oC for 15 min. FTIR analysis reveals the presence of hydroxyl, carboxyl and methylene - groups in the compound bioflocculant. SEM micrograph of the bioflocculant revealed an amorphous compound. The consortia of these bacteria strains also produced bioflocculants with high flocculating activities which were highly efficient in removing turbidity and chemical oxygen demand (COD) from brewery wastewater, diary wastewater and river water. The bioflocculants from the consortia seemed better than traditional flocculants such as alum . The characteristics of the bioflocculant produced by the consortium of Cobetia sp. OAUIFE and Bacillus sp. MAYA showed that this extracellular bioflocculant, composed of 66percent uronic acid and 31percent protein and an optimum flocculation (90 percent) of kaolin suspension, when the dosage concentration was 0.8 mg/ml, under weak alkaline pH of 8, and Ca2+ as a coagulant aid. The bioflocculant is thermally stable, with a high residual flocculating activity of 86.7 percent, 89.3 percent and 87.0 percent after heating at 50 oC, 80 oC and 100 oC for 25 min respectively. The FTIR analysis of the bioflocculant indicated the presence of hydroxyl, amino, carbonyl and carboxyl functional groups. Scanning electron microscopy (SEM) image revealed a crystal-linear spongy-like bioflocculant structure and EDX analysis of the purified bioflocculant showed that the elemental composition in mass proportion of C,N,O,S and P was 6.67:6.23:37.55:0.38:4.42 (percent w/w). However, the characteristics of the bioflocculant produced by the consortium of Cobetia sp OAUIFE and Bacillus sp. Gilbert showed an optimum flocculation (90 percent) of kaolin suspension when the dosage concentration was 0.2 mg/ml, under neutral pH of 7, and Ca2+ as a coagulant aid. The FTIR analysis of the bioflocculant Tyume River water samples were collected monthly, over a 12-month period starting from August 2010 and ending in July 2011, and transported on ice to the Applied and Environmental Microbiology Research Group (AEMREG) Laboratory at the University of Fort Hare, Alice for analyses within 6 h of collection. Electrical conductivity (EC), total dissolved solids (TDS), temperature, pH and dissolved oxygen (DO) of water samples were determined in situ using a multi-parameter ion-specific meter. Concentrations of orthophosphate and total nitrogen (nitrate + nitrite) were determined by standard photometric methods. Total coliforms (TC), faecal coliforms (FC) and enterococci were determined by the membrane filtration method. Viruses in water samples were concentrated using the adsorption-elution method, followed by extraction of viral nucleic acids and purification done using commercially available kits. The concentrations of human enteric viruses in the river-water samples were estimated using quantitative PCR. RNA viruses were quantified in a two-step protocol where RNA was first transcribed into cDNA in a separate reverse-transcription step. Adenovirus species and serotypes were simultaneously detected using serotype-specific multiplex PCR. Norovirus genogroups GI and GII were detected by semi-nested PCR. The risk of infection associated with recreational and domestic use of the water was also estimated. Biochemical oxygen demand (BOD) levels fell within the stipulated BOD guideline of 10 mg/ℓ for surface waters where full contact use is allowed and ≤ 30 mg/ℓ where public access is prohibited, restricted, or infrequent. DO concentrations generally ranged between 7.47 mg/ℓ and 10.42 mg/ℓ, well within the target water quality requirements. The temperature regime ranged between 6°C and 28°C and for most sampling sites, the temperature regimes were within the acceptable limit of no risk (≤ 25°C) for domestic water uses in South Africa. EC ranged between 47 μS/cm and 408 μS/cm well within the South African target water quality EC guideline of 700 μS/cm though it was observed to increase as the river flowed through settlements. The pH in the period beginning September 2010 through to January 2011 was consistently below pH 9, but from February 2011 to June 2011 the pH significantly increased to between pH 10 and pH 11 at most sampling sites. Unpolluted waters normally show a pH of between 6.5 and 8.5. Most of the pH values observed in this study lie between pH 8.5 and pH 10.8 levels which are not far off from the upper level guideline of pH 9.0 for domestic use. Turbidity ranged between 6 NTU and 281 and fell short of the target water quality range (0 NTU to 1 NTU) of no risk for domestic water uses in South Africa. Monthly TDS values and EC values showed direct proportionality. TDS concentrations at all sites fell within the acceptable guideline of 0 mg/ℓ to 450 mg/ℓ of TDS for domestic use. Nutrient profiles were as follows: nitrate (0.18 mg/ℓ to 4.21 mg/ℓ); nitrite (0.02 mg/ℓ to 2.35 mg/ℓ); and orthophosphate (0.06 mg/ℓ to 2.72 mg/ℓ). The bacteriological qualities of the water were poor, exceeding the guideline of 200 CFU/100 mℓ and 33 CFU/100 mℓ for FC and enterococci respectively, for recreational water. FC counts also exceeded the 1 000 CFU/100 mℓ guideline for water used in fresh produce irrigation. Generally, higher counts of TC, FC and enterococci were recorded at the sampling sites located at the lower reaches of the river compared to the upper reaches. Adenovirus was detected in 31percent of the river samples in concentrations ranging between 1.0×100 genome copies/ℓ and 8.49×104 genome copies/ℓ. Serotyping showed the presence of species C adenovirus serotypes 1, 2, 6 and 7, and species F adenovirus serotype 41. The prevalence of norovirus was 4percent while rotavirus was detected in 4percent of river samples in concentrations ranging between 9×100 genome copies/ℓ and 5.64×103 genome copies/ℓ. Hepatitis A virus was detected in 13percent of river samples in concentrations ranging between1.67×103 and 1.64×104 genome copies/ℓ while enteroviruses were not detected. Detection of enteric viruses was inversely correlated to temperature. Risk analysis showed that both hepatitis A virus and adenovirus presented significantly higher risk of infection values compared to rotavirus in the case of ingestion of 10 mℓ or 100 mℓ of water from Tyume River while enteroviruses did not present any significant risk of infection. Tyume River water samples also did not conform to the US Environmental Protection Agency (US EPA) bacterial criteria of 200 CFU/100 mℓ faecal coliforms and 33 CFU/100 mℓ enterococci for bathing waters. Whereas the physicochemical parameters showed that Tyume River water was relatively clean, the bacteriological water quality was poor. Most of the microbiological contamination observed in this study (especially FIBs) can be blamed on inadequate sanitary infrastructure as we observed that open defecation is commonplace in this catchment, which also serves as a conduit for effluent discharges from wastewater-treatment facilities. Enteric viruses were detected along the course of the river in a sporadic pattern, generally not related to natural hydrological cycles and so we conclude that the presence of enteric viruses in the river is suggestive of the dynamics of the same in the host population. Even though the proportion of infective viruses was estimated in this study, fact remains that there is considerable risk of infection posed by the use of raw surface water for either domestic or recreational use. This study further confirmed the lack of correlation between faecal indicator bacteria and enteric virus occurrence in environmental waters, showing that assaying for enteric viruses in environmental waters remains the best method for determining the health risks associated with the use of faecally contaminated water. , Thesis (PhD) -- Faculty of Science and Agriculture, 2013
- Full Text:
- Date Issued: 2013-05
- Authors: Sibanda, Timothy https://orcid.org/0000-0002-6864-3796
- Date: 2013-05
- Subjects: Water -- Purification , Sewage -- Purification
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/24465 , vital:62812
- Description: The bioflocculant-producing potentials of three marine bacteria isolated from the sediment samples of Algoa Bay in the Eastern Cape Province of South Africa were assessed. Analysis of the partial nucleotide sequence of the 16S rDNA of the bacteria revealed 99 percent, 99 percent, and 98 percent similarity to Cobetia sp. L222, Bacillus sp. A-5A, and Bacillus sp. HXG-C1 respectively and the sequence was deposited in GenBank as Cobetia sp. OAUIFE, Bacillus sp. MAYA and Bacillus sp. Gilbert (accession number JF799092, JF799093, and HQ537128 respectively). Cultivation condition studies for Cobetia sp. OAUIFE revealed that bioflocculant production was optimal with an inoculum size of 2 percent (v/v), initial pH of 6.0, Mn2+ as the metal ion, and glucose as the carbon source. Metal ions, including Na+, K+, Li+, Ca2+and Mg2+ stimulated bioflocculant production resulting in flocculating activity of above 90 percent. This crude bioflocculant is thermally stable, with about 78 percent of its flocculating activity remaining after heating at 100 oC for 25 min. Analysis of the purified bioflocculant revealed it to be an acidic extracellular polysaccharide. FTIR analysis revealed the presence of methoxyl, hydroxyl, and carboxyl - groups in the compound bioflocculant and SEM micrograph of the bioflocculant revealed a crystal-linear structure. On the other hand, bioflocculant production by Bacillus sp. MAYA was optimal when glucose (95.6 percent flocculating activity) and ammonium nitrate (83.3 percent flocculating activity) were used as carbon and nitrogen sources respectively; inoculum size was 2 percent (v/v); initial pH 6; and Ca2+ as coagulant aid. Chemical analysis of the purified bioflocculant shows that it is composed of uronic acid, neutral sugar and protein. FTIR analysis also revealed the presence of methoxyl, hydroxyl, carboxyl and amino- groups in this bioflocculant. The bioflocculant is thermostable with about 65.6 percent residual flocculating activity retained after heating the bioflocculant at 100 oC for 25 min. However bioflocculant production by Bacillus sp. Gilbert was optimal when sodium carbonate (95.2 percent flocculating activity) and potassium nitrate (76.6 percent flocculating activity) were used as carbon and nitrogen sources respectively; inoculum size was 3 percent (v/v); initial pH 9; and Al3+ as cation. The crude bioflocculant retained 44.2 percent residual flocculating activity after heating at 100 oC for 15 min. FTIR analysis reveals the presence of hydroxyl, carboxyl and methylene - groups in the compound bioflocculant. SEM micrograph of the bioflocculant revealed an amorphous compound. The consortia of these bacteria strains also produced bioflocculants with high flocculating activities which were highly efficient in removing turbidity and chemical oxygen demand (COD) from brewery wastewater, diary wastewater and river water. The bioflocculants from the consortia seemed better than traditional flocculants such as alum . The characteristics of the bioflocculant produced by the consortium of Cobetia sp. OAUIFE and Bacillus sp. MAYA showed that this extracellular bioflocculant, composed of 66percent uronic acid and 31percent protein and an optimum flocculation (90 percent) of kaolin suspension, when the dosage concentration was 0.8 mg/ml, under weak alkaline pH of 8, and Ca2+ as a coagulant aid. The bioflocculant is thermally stable, with a high residual flocculating activity of 86.7 percent, 89.3 percent and 87.0 percent after heating at 50 oC, 80 oC and 100 oC for 25 min respectively. The FTIR analysis of the bioflocculant indicated the presence of hydroxyl, amino, carbonyl and carboxyl functional groups. Scanning electron microscopy (SEM) image revealed a crystal-linear spongy-like bioflocculant structure and EDX analysis of the purified bioflocculant showed that the elemental composition in mass proportion of C,N,O,S and P was 6.67:6.23:37.55:0.38:4.42 (percent w/w). However, the characteristics of the bioflocculant produced by the consortium of Cobetia sp OAUIFE and Bacillus sp. Gilbert showed an optimum flocculation (90 percent) of kaolin suspension when the dosage concentration was 0.2 mg/ml, under neutral pH of 7, and Ca2+ as a coagulant aid. The FTIR analysis of the bioflocculant Tyume River water samples were collected monthly, over a 12-month period starting from August 2010 and ending in July 2011, and transported on ice to the Applied and Environmental Microbiology Research Group (AEMREG) Laboratory at the University of Fort Hare, Alice for analyses within 6 h of collection. Electrical conductivity (EC), total dissolved solids (TDS), temperature, pH and dissolved oxygen (DO) of water samples were determined in situ using a multi-parameter ion-specific meter. Concentrations of orthophosphate and total nitrogen (nitrate + nitrite) were determined by standard photometric methods. Total coliforms (TC), faecal coliforms (FC) and enterococci were determined by the membrane filtration method. Viruses in water samples were concentrated using the adsorption-elution method, followed by extraction of viral nucleic acids and purification done using commercially available kits. The concentrations of human enteric viruses in the river-water samples were estimated using quantitative PCR. RNA viruses were quantified in a two-step protocol where RNA was first transcribed into cDNA in a separate reverse-transcription step. Adenovirus species and serotypes were simultaneously detected using serotype-specific multiplex PCR. Norovirus genogroups GI and GII were detected by semi-nested PCR. The risk of infection associated with recreational and domestic use of the water was also estimated. Biochemical oxygen demand (BOD) levels fell within the stipulated BOD guideline of 10 mg/ℓ for surface waters where full contact use is allowed and ≤ 30 mg/ℓ where public access is prohibited, restricted, or infrequent. DO concentrations generally ranged between 7.47 mg/ℓ and 10.42 mg/ℓ, well within the target water quality requirements. The temperature regime ranged between 6°C and 28°C and for most sampling sites, the temperature regimes were within the acceptable limit of no risk (≤ 25°C) for domestic water uses in South Africa. EC ranged between 47 μS/cm and 408 μS/cm well within the South African target water quality EC guideline of 700 μS/cm though it was observed to increase as the river flowed through settlements. The pH in the period beginning September 2010 through to January 2011 was consistently below pH 9, but from February 2011 to June 2011 the pH significantly increased to between pH 10 and pH 11 at most sampling sites. Unpolluted waters normally show a pH of between 6.5 and 8.5. Most of the pH values observed in this study lie between pH 8.5 and pH 10.8 levels which are not far off from the upper level guideline of pH 9.0 for domestic use. Turbidity ranged between 6 NTU and 281 and fell short of the target water quality range (0 NTU to 1 NTU) of no risk for domestic water uses in South Africa. Monthly TDS values and EC values showed direct proportionality. TDS concentrations at all sites fell within the acceptable guideline of 0 mg/ℓ to 450 mg/ℓ of TDS for domestic use. Nutrient profiles were as follows: nitrate (0.18 mg/ℓ to 4.21 mg/ℓ); nitrite (0.02 mg/ℓ to 2.35 mg/ℓ); and orthophosphate (0.06 mg/ℓ to 2.72 mg/ℓ). The bacteriological qualities of the water were poor, exceeding the guideline of 200 CFU/100 mℓ and 33 CFU/100 mℓ for FC and enterococci respectively, for recreational water. FC counts also exceeded the 1 000 CFU/100 mℓ guideline for water used in fresh produce irrigation. Generally, higher counts of TC, FC and enterococci were recorded at the sampling sites located at the lower reaches of the river compared to the upper reaches. Adenovirus was detected in 31percent of the river samples in concentrations ranging between 1.0×100 genome copies/ℓ and 8.49×104 genome copies/ℓ. Serotyping showed the presence of species C adenovirus serotypes 1, 2, 6 and 7, and species F adenovirus serotype 41. The prevalence of norovirus was 4percent while rotavirus was detected in 4percent of river samples in concentrations ranging between 9×100 genome copies/ℓ and 5.64×103 genome copies/ℓ. Hepatitis A virus was detected in 13percent of river samples in concentrations ranging between1.67×103 and 1.64×104 genome copies/ℓ while enteroviruses were not detected. Detection of enteric viruses was inversely correlated to temperature. Risk analysis showed that both hepatitis A virus and adenovirus presented significantly higher risk of infection values compared to rotavirus in the case of ingestion of 10 mℓ or 100 mℓ of water from Tyume River while enteroviruses did not present any significant risk of infection. Tyume River water samples also did not conform to the US Environmental Protection Agency (US EPA) bacterial criteria of 200 CFU/100 mℓ faecal coliforms and 33 CFU/100 mℓ enterococci for bathing waters. Whereas the physicochemical parameters showed that Tyume River water was relatively clean, the bacteriological water quality was poor. Most of the microbiological contamination observed in this study (especially FIBs) can be blamed on inadequate sanitary infrastructure as we observed that open defecation is commonplace in this catchment, which also serves as a conduit for effluent discharges from wastewater-treatment facilities. Enteric viruses were detected along the course of the river in a sporadic pattern, generally not related to natural hydrological cycles and so we conclude that the presence of enteric viruses in the river is suggestive of the dynamics of the same in the host population. Even though the proportion of infective viruses was estimated in this study, fact remains that there is considerable risk of infection posed by the use of raw surface water for either domestic or recreational use. This study further confirmed the lack of correlation between faecal indicator bacteria and enteric virus occurrence in environmental waters, showing that assaying for enteric viruses in environmental waters remains the best method for determining the health risks associated with the use of faecally contaminated water. , Thesis (PhD) -- Faculty of Science and Agriculture, 2013
- Full Text:
- Date Issued: 2013-05
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