Please use this identifier to cite or link to this item: https://hdl.handle.net/10321/5451
Title: The efficiency of lytic enzymes for Ascaris eggs inactivation
Authors: Zikalala, Thabiso 
Keywords: Lytic enzymes;Ascaris eggs
Issue Date: May-2024
Abstract: 
Faecal sludge (FS) contains organic matter that can enhance soil quality if pathogenic
organisms, such as Ascaris lumbricoides, are reduced to safe levels. A. lumbricoides is a highly
resistant nematode used to assess the efficiency of FS and wastewater treatment. The standard
for reusing FS is to reduce Ascaris spp eggs to <1 egg/g for helminth reduction. However, in
many developing countries, untreated FS is used as a soil enhancer without following proper
guidelines. There are a number of FS treatment technologies in use, such as compositing,
drying and the use of calcium carbonate. These techniques are able to reduce the pathogen
concentration, especially Ascaris spp to accepted standard prior to application. Other
alternative FS treatment technologies are under assessment and development.
The objective of this study was to evaluate the use of lytic enzymes to inactivate Ascaris spp
eggs by targeting the egg shell of the parasite. Ascaris spp egg shells consist of protein, chitin,
and a lipid layer. Therefore, it may be possible to disintegrate Ascaris spp by targeting these
layers of the egg shell with enzymes such as protease, chitinase, and lysozyme, which are
commonly produced by indigenous soil bacteria.
Ascaris spp eggs were detected in all FS samples collected from urine dehydration diversion
toilets (UDDTs) in Durban, with an average concentration of 976-1118 helminth eggs/gram of
FS. However, the viability of recovered eggs from FS was low and eggs were in different stages
of development. This would negatively affect the experiments and produce inconsistent data.
Therefore, for the lytic enzyme inactivation experiments, commercially bought eggs were used
instead of the helminth eggs recovered from the UDDTs FS. Exposure of the eggs to
commercial lytic enzymes was done following three different approaches. Firstly, Ascaris spp
eggs were exposed to each of the enzymes individually, a second approach was employed
where the eggs were consecutively exposed to the eggs with a rinse in between, and lastly, the
eggs were exposed to a mixture of all three enzymes at once. Viability of the Ascaris spp eggs
was determined via incubation, followed by microscopic examination of the eggs for visible
motile larvae. For the single enzyme exposure, chitinase was the most detrimental enzyme
resulting in a reduction of viability by 34% at room temperature. Exposure of the Ascaris spp
eggs using the enzymes in series, achieved an egg viability reduction of up to 90 % at 37 °C,
after 5 days exposure. Exposing the eggs to the mixed enzymes gave a reduced egg viability of
75 % at 37 °C, after 5 days exposure. Furthermore, the detection of microorganisms in the FS
capable of producing lytic enzymes used was also confirmed. Enzymes were produced using
selective media that resulted in the production of enzymes where the concentration (mg/L) and
specific activity (U/mg) was determined to 0,68 mg/mL (0,08 U/mg) of protease from nutrient
broth enriched with 2.5% milk, 3,17 mg/mL (0,006 U/mg) of chitinase produced from 1%
colloidal chitin, and 7132 mg/mL (2600 U/mg) lysozyme from nutrient broth enriched with 1%
Micrococcus lysodeikticus culture.
The lytic enzymes showed to have an antagonistic effect on the Ascaris spp eggs. This therefore
serves as a proof of concept that lytic enzymes produced by microorganisms found in FS could
potentially be used for the inactivation of Ascaris spp eggs. However, further work is required
focusing on enhancing the enzyme production, testing of the inactivation potential of these
enzymes in the presence of solids and other materials present in FS, and finally the technique
for field application of such technology.
Description: 
Submitted in fulfilment for the Degree of Master of Applied Sciences in Biotechnology at Durban University of Technology, Durban, South Africa, 2024.
URI: https://hdl.handle.net/10321/5451
DOI: https://doi.org/10.51415/10321/5451
Appears in Collections:Theses and dissertations (Applied Sciences)

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