The efficiency of lytic enzymes for Ascaris eggs inactivation

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.