Destruction of parasites in biosolids from wastewater treatment lagoons

Khallaf, B 2019, Destruction of parasites in biosolids from wastewater treatment lagoons, Doctor of Philosophy (PhD), Science, RMIT University.

Document type: Thesis
Collection: Theses

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Title Destruction of parasites in biosolids from wastewater treatment lagoons
Author(s) Khallaf, B
Year 2019
Abstract Wastewater from domestic and industrial activities contains harmful compounds and pathogens which constitute significant health risks if released into the environment without adequate treatment. Wastewater treatment results in the generation of biosolids which after post-treatment measures can be used as soil fertilizers, as road base or sent to landfills. However, biosolids, especially those from developing countries, may contain substantial levels of Ascaris spp. (helminths) eggs which survive most post-treatment measures. These can cause human infections when these biosolids are used in agriculture. Helminths such as Ascaris spp. infect about 1.2 billion people in the developing world causing morbidity and mortality and are of public health concern. Therefore, the aims of this study are to determine the prevalence of Ascaris spp. eggs in biosolids from waste treatment plants in Victoria and their decay rates following pan drying and stockpiling of biosolids. The microbial communities in biosolids are characterized using PCR-DGGE and metagenomics-based approaches, and their roles in the decay of Ascaris spp. evaluated with MT2 Biolog-plate based assays.

Investigations of the prevalence of Ascaris spp. eggs were carried out on samples obtained from three wastewater treatment plants (WWTP) in Victoria (Heyfield, Rochester and Cobden WWTP). No eggs of Ascaris spp. were detected in these samples evaluated with the Tulane method indicating that helminths were either not present or that the Tulane method was not sensitive enough to detect them. Thereafter, biosolid samples were spiked with Ascaris suum eggs to assess the suitability of the Tulane method for egg detection. Ascaris suum eggs were detected (recovered) from spiked samples; the recovery efficiencies ranged from 64% in Cobden to 70% in Heyfield biosolid samples. The efficiency of egg recovery was inversely correlated with the concentration of spiked eggs. The Tulane method was found to be suitable for egg recovery from biosolids confirming that helminths was largely absent in the wastewater samples examined.

The next experimental investigation was to determine the decay rates of Ascaris suum eggs in spiked biosolid samples (Cobden and Rochester) subject to pan-handling and stockpiling at a specific temperature (20 oC) in laboratory-based assays over 17 weeks. While the Tulane method was suitable for egg recovery, its use for determining the viability of eggs can be time-consuming. Therefore, a Live/Dead Baclight staining procedure was used for determining the viability of recovered eggs in this study. Results showed that the viability of Ascaris suum eggs was reduced by 22% in Cobden samples and 31% in Rochester samples. Consequently, if the die-off (decay rates) were constant, storing Cobden biosolids for 13 months and Rochester biosolids for 18 months would lead to complete elimination of viable Ascaris suum eggs and render the biosolid samples safe for use in agriculture-related applications. The Live/Dead Baclight staining procedure was also successfully used to discriminate between viable and non-viable eggs, making it an ideal additional technique to accompany the Tulane's method.

Most research activities on the microbial composition of biosolids have assessed the prevalence of pathogens for public health safety. However, microorganisms naturally present in biosolids produce an array of enzymes, some of which may play important roles in the decay of Ascaris suum eggs alongside other factors. Therefore, two culture-independent approaches (PCR-DGGE and metagenomics) were used to characterize the microbial communities in selected biosolid samples.

Cluster analysis of the DGGE profiles of Cobden, Rochester and Heyfield samples indicated substantial differences (70-80%) between their microbial communities. Bacterial diversity (PCR-DGGE) assessed with Shannon diversity (H') was highest in Rochester samples (3.5), followed by Cobden (2.3) and Heyfield (1.8) samples. The microbial community from these samples was used to inoculate MT2 Biolog plates containing either chitin, lipid or protein substrates and incubated for 168 hours. Both Cobden and Rochester samples showed substantial utilization of protein substrates; Cobden samples also substantially degraded chitin and all the three samples showed some degree of lipid utilization. Key microbial groups from the bacterial community in the Biolog plates were identified and some putatively assigned to Flavobacteria, Cytophaga, Alpha-, Beta- and Gamma-proteobacteria groups. These groups produce enzymes such as lipases, proteinases and chitinases which can degrade the outer coating of Ascaris suum eggs rendering them non-viable.

Metagenomic analyses were used to evaluate the bacterial communities in the biosolid samples. The data indicated substantial differences in the biosolid communities. About 70% of the bacterial population belonged to the Gammaproteobacteria in Heyfield samples compared to ~25% in Rochester and ~12% in Cobden. In Rochester, both Gamma- and Beta-proteobacteria were dominant and Cytophagia, Betaproteobacteria and Epsilon bacteria were the key groups in the Cobden samples.

The final investigation involved the use of isolates biosolid samples to degrade the eggs of Ascaris suum. Pure bacterial isolates obtained from Heyfield, Rochester and Cobden samples, grown on chitin, lipid or protein substrates were screened based on growth and protein production characteristics in nutrient broth. Three isolates showing relatively high levels of activity, identified as Pedobacter sp., Acidovorax sp. and Brevundimonas sp were applied (as either cell-free, pellet-based, or uncentrifuged culture broth inocula) to Ascaris eggs to assess their effects on the rate of decay of Ascaris eggs compared to commercial enzymes. Individually, the egg decay efficiencies between the three isolates and the commercial enzymes were similar. However, when used as a mixture of the three isolates (uncentrifuged culture broth), there was a higher decay of Ascaris eggs (~23%) compared to the commercial enzyme mixture (~19%) and individual isolates and cell-free samples (up to 19%). This indicated that microbial synergy was important in the decay of eggs in biosolids.

This study has successfully shown that the Tulane method was suitable for Ascaris eggs recovery while the Live/Dead Baclight staining procedure was excellent at discriminating between viable and non-viable eggs. Bacterial community composition was dependent on the biosolid source and specific bacterial species such as Pedobacter sp., Acidovorax sp. and Brevundimonas sp. working in synergy do play a role in the decay of Ascaris eggs in controlled conditions. This opens the possibility of a microbial (biological) approach to the decay of helminths eggs, used independently or as adjuncts to existing sludge treatment processes. Future investigations should evaluate the impact of physicochemical and environmental factors on the synergistic decay of eggs in biosolid samples.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Science
Subjects Environmental Science and Management not elsewhere classified
Keyword(s) Wastewater treatment
Lagoon treatment
Lagoon simulation
Parasite destruction
Ascaris destruction
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Created: Wed, 11 Sep 2019, 08:33:25 EST by Adam Rivett
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