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Bio-delipidation of pre-treated poultry slaughterhouse wastewater by enzymes from the wastewater isolates
Author(s)
Mbulawa, Siyasanga
Date Issued
2017
Type
Thesis
Publisher
Cape Peninsula University of Technology
Abstract
Pre-treatment of wastewater such as that from poultry slaughterhouses, which contains fats, oil,and grease (FOG) is necessary prior to the primary biological treatment of the wastewater to meet legislated discharge standards and to prevent environmental pollution. Physico- chemical pre-treatment is often applied to remove FOG in poultry slaughterhouse wastewater (PSW) before biological treatment. These pre-treatment methods, in particular physical pre- treatment systems, use synthetic chemicals, known to cause environmental contamination challenges, with FOG being inefficiently removed in certain instances. Biological techniques such as bio-delipidation using enzymatic catalysis for the pre-treatment of FOG-laden PSW could enhance the efficiency of the downstream biological treatment processes. This research focused on further bio-delipidation of PSW pre-treated with a dissolved air flotation system (DAF) for FOG removal using microbial lipases from bacterial strains isolated from the PSW itself.
Bacterial strains (n = 2) isolated from the PSW and screened for their potential to produce lipases were found to have a higher bio-delipidation potential when compared to other isolates (n = 18). Both isolates were identified using 16s rRNA as Bacillus sp., i.e. both Bacillus cereus AB1 (BF3) and CC-1 (B3O). These isolates were used to produce lipases, whereby are sponse surface methodology (RSM) was used to optimise pH (4-8) and temperature (30-60°C) as critical production conditions. achieving an optimum lipase production was achieved, with activity of 11.25 U/mL at 60°C, a pH of8 for BF3, and 15.50U/mL at 45°C and pH of 8.8 for B3O respectively, after 72 hours of bioreactor operation. The enzymes produced from both isolates were partially purified using a Bio-Rad size exclusion chromatography column (Bio-Gel® P-60) prior to use in subsequent experiments. The presence and activity of lipase were further determined using p-nitrophenyl acetate (p- NPA) as a substrate with the functionality of the semi-purified enzymes being characterized by optimizing the conditions in which the enzymes were required to function. Lipase activity was enhanced by Mg2+ while Fe2+, Na+, K+, Ca2+ were observed to have an inhibitory effect on the enzymes from both strains. Similarly, reduced stability of the lipases in organic solvents, namely toluene, methanol, and isopropanol, was also established. Additionally, detergents, Triclosan (TCS) (5-chloro-2-(2,4-dichlorophenoxy-phenol) and trichlorocarbonilide (3,4,4- trichlorocarbonilide)(TCC), usually found in PSW as antimicrobial and disinfectant agents to sanitise poultry product processing facilities, were used assess the activity of the enzyme in their presence at a concentration of 30% (v/v) (although these anti- microbial agents are used in minute quantities in cleaning products). The lipases from isolate BF3 maintained an activity of 91.43% and 81.36% in the presence of TCS and TCC, while that of B3O enzyme had 85.32% and 73.91% acitivity, when compared to the reference (control) experiments. The bio-delipidation efficacy was studied under varying pH and temperature conditions using DAF pre-treated PSW, observing a further removal efficiency of fatty acids from the protein- laden PSW at different pH and temperature. Bio-delipidation was found to be largely influenced by pH, as a pH below 7 and above 10 at 40°-45°C, calculated in the bio- delipidation efficiency reduction to below 50%. The temperature range mentioned, i.e 40°- 45°C, had a positive effect on further deffating of the protein-rich DAF pre-treated PSW, as high removal efficiency was observed at this temperature range. This could be due to the characteristic of the enzymes used,or the formation of stable FOG agglomerates and/oremulsion. Overall, a DAF effluent containing residual FOG and proteins was bio-delipidated effectively using enzymes from the PSW isolates, achieving further removal of FOG and proteins by 64.35% to 80.42%, culminating in tCOD reduction and reduced PSW turbidity, further resulting in improved wastewater quality characteristics meeting disposal standards. This study demonstrated that sequential DAF pre-treated PSW bio-delipidation has the potential to enhance the efficiency of downstream biological anaerobic treatment processes for PSW by further reducing residual FOG from a DAF system.
Bacterial strains (n = 2) isolated from the PSW and screened for their potential to produce lipases were found to have a higher bio-delipidation potential when compared to other isolates (n = 18). Both isolates were identified using 16s rRNA as Bacillus sp., i.e. both Bacillus cereus AB1 (BF3) and CC-1 (B3O). These isolates were used to produce lipases, whereby are sponse surface methodology (RSM) was used to optimise pH (4-8) and temperature (30-60°C) as critical production conditions. achieving an optimum lipase production was achieved, with activity of 11.25 U/mL at 60°C, a pH of8 for BF3, and 15.50U/mL at 45°C and pH of 8.8 for B3O respectively, after 72 hours of bioreactor operation. The enzymes produced from both isolates were partially purified using a Bio-Rad size exclusion chromatography column (Bio-Gel® P-60) prior to use in subsequent experiments. The presence and activity of lipase were further determined using p-nitrophenyl acetate (p- NPA) as a substrate with the functionality of the semi-purified enzymes being characterized by optimizing the conditions in which the enzymes were required to function. Lipase activity was enhanced by Mg2+ while Fe2+, Na+, K+, Ca2+ were observed to have an inhibitory effect on the enzymes from both strains. Similarly, reduced stability of the lipases in organic solvents, namely toluene, methanol, and isopropanol, was also established. Additionally, detergents, Triclosan (TCS) (5-chloro-2-(2,4-dichlorophenoxy-phenol) and trichlorocarbonilide (3,4,4- trichlorocarbonilide)(TCC), usually found in PSW as antimicrobial and disinfectant agents to sanitise poultry product processing facilities, were used assess the activity of the enzyme in their presence at a concentration of 30% (v/v) (although these anti- microbial agents are used in minute quantities in cleaning products). The lipases from isolate BF3 maintained an activity of 91.43% and 81.36% in the presence of TCS and TCC, while that of B3O enzyme had 85.32% and 73.91% acitivity, when compared to the reference (control) experiments. The bio-delipidation efficacy was studied under varying pH and temperature conditions using DAF pre-treated PSW, observing a further removal efficiency of fatty acids from the protein- laden PSW at different pH and temperature. Bio-delipidation was found to be largely influenced by pH, as a pH below 7 and above 10 at 40°-45°C, calculated in the bio- delipidation efficiency reduction to below 50%. The temperature range mentioned, i.e 40°- 45°C, had a positive effect on further deffating of the protein-rich DAF pre-treated PSW, as high removal efficiency was observed at this temperature range. This could be due to the characteristic of the enzymes used,or the formation of stable FOG agglomerates and/oremulsion. Overall, a DAF effluent containing residual FOG and proteins was bio-delipidated effectively using enzymes from the PSW isolates, achieving further removal of FOG and proteins by 64.35% to 80.42%, culminating in tCOD reduction and reduced PSW turbidity, further resulting in improved wastewater quality characteristics meeting disposal standards. This study demonstrated that sequential DAF pre-treated PSW bio-delipidation has the potential to enhance the efficiency of downstream biological anaerobic treatment processes for PSW by further reducing residual FOG from a DAF system.
Additional information
Thesis (MEng (Chemical Engineering))--Cape Peninsula University of Technology, 2017.
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