- Title
- Evaluation of bacteria laccase hybrid biosensor and application in the detection of phenolic contaminants in water
- Creator
- Edoamodu, Chiedu Epiphany https://orcid.org/0000-0002-9254-3955
- Subject
- Laccase
- Subject
- Water -- Purification
- Date
- 2021-09
- Type
- Doctoral theses
- Type
- text
- Identifier
- http://hdl.handle.net/10353/22820
- Identifier
- 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.
- Description
- Thesis (PhD) -- Faculty of Science and Agriculture, 2021
- Format
- computer
- Format
- online resource
- Format
- application/pdf
- Format
- 1 online resource (308 leaves)
- Format
- Publisher
- University of Fort Hare
- Publisher
- Faculty of Science and Agriculture
- Language
- English
- Rights
- University of Fort Hare
- Rights
- All Rights Reserved
- Rights
- Open Access
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UFH Department of Biochemistry & Microbiology
| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | SOURCE1 | PhD Thesis.pdf | 7 MB | Adobe Acrobat PDF | View Details Download |