Removal of Fecal Indicators, Pathogenic Bacteria, Adenovirus, Cryptosporidium and Giardia (oo)cysts in Waste Stabilization Ponds in Northern and Eastern Australia
Abstract
:1. Introduction
2. Experimental Section
2.1. Sampling Sites
2.2. Physico-Chemical Measurements
Ponds | Zone | Year | T °C | pH | DO, mg/L | Turbidity, NTU | Conductivity, µS/cm |
---|---|---|---|---|---|---|---|
WSP1 | subtropical | 2014 | 17–22 | 8.8–9.4 | 8–23.7 | 26–72 | 842–1041 |
WSP2 | subtropical | 2014 | 13.6–17.5 | 10.2–10.6 | 17.4–24.4 | 34–66 | 929–1002 |
WSP3 | tropical | 2013 | 28.7–38.0 | 6.08–10.45 | 0–34.26 | 76–136 | 467–766 |
WSP4 | tropical | 2014 | 25–26.4 | 7.8–9.2 | 9.9–11.4 | 27–65 | 1750–1876 |
2.3. Culture Based Bacterial Quantification
2.4. F+ RNA Coliphage Quantification
2.5. Viral Precipitation and Infectivity Assay
2.6. Cryptosporidium and Giardia Enumeration
2.7. DNA Extraction and Recovery
2.8. Preparation of Stocks for qPCR Calibration Curves
2.9. PCR Primers and TaqMan Probes
2.10. Real-Time qPCR
Microorganisms | Gene | Function | Sequences (5′-3′) | Primer Concentration, µM | Reference |
---|---|---|---|---|---|
E. coli | uidA | glucuronidase | F: GTGTGATATCTACCCGCTTCGC | 0.7 | [14] |
R: AGAACGGTTTGTGGTTAATCAGGA | 0.7 | ||||
P: TCGGCATCCGGTCAGTGGCAGT | 0.2 | ||||
Enterococcus faecalis | 23s RNA | Ribosomal gene | F: GAGAAATTCCAAACGAACTTG | 0.5 | [15] |
R: CAGTGCTCTACCTCCATCATT | 0.5 | ||||
P: TGGTTCTCTCCGAAATAGCTTTAGGGCTA | 0.08 | ||||
DNA IAC pM13mp18 | NA | F: AAGATTTGAATCGGTTGCTTGG | 0.4 | [13] | |
R: GCCACTGCTCCATTATCTGG | 0.4 | ||||
P: CCGATTGTTAGCCAGCCCATGCCA | 0.2 | ||||
Adenovirus (all types) | Hexon gene | F: GCCACGGTGGGGTTTCTAAACTT | 0.5 | [16] | |
R: GCCCCAGTGGTCTTACATGCA | 0.5 | ||||
P: TGCACCAGACCCGGGCTCAGGAGGTACTCCGA | 0.2 | ||||
Campylobacter jejuni | mapA | Mucus adhesion-promoting protein | F: GGTTTTGAAGCAAAGATTAAAGG | 0.5 | [17] |
R: AAGCAATACCAGTGTCTAAAGTGC | 0.5 | [17] | |||
P: TGGCACAACATTGAATTCCAACATCGCTA | 0.3 | [18] | |||
VS1 | n/a | F: GAATGAAATTTTAGAATGGGG | 0.4 | [19] | |
R: GATATGTATGATTTTATCCTGC | 0.4 | ||||
P: TTTAACTTGGCTAAAGGCTAAGGCT | 0.1 | ||||
Salmonella enterica | gyrB | gyrase protein | F: CGTGGGCGTCTCGGTAGTY | 0.5 | [20] |
R: CTCATATTCAAATTCAGTGACG | 0.5 | [20] | |||
P: AAACCGGCACGATGGTACGTTTCT | 0.25 | This study | |||
ttrRSBCA | tetrathionate respiration | F: CTCACCAGGAGATTACAACATGG | 0.4 | [21] | |
R: AGCTCAGACCAAAAGTGACCATC | 0.4 | ||||
P: CG +ACG +GCG +AG+ACCG | 0.25 |
2.11. Identification of Campylobacter spp.
2.12. Statistical Analysis
3. Results
3.1. Concentrations of Microorganisms in Maturation Ponds
Microorganism | Year | Method | WSP1, QLD (n = 8) | WSP2 *, QLD (n = 8) | WSP3, NT (n = 8) | WSP4, NT (n = 4) | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Influent | Effluent | LRV | Influent | Effluent | LRV | Influent | Effluent | LRV | Influent | Effluent | LRV | |||
E. coli | 2013 | culture | 5.6 ± 0.6 × 104 | 2.6 ± 0.7 × 101 | 3.3 | 2.8 ± 0.07 × 102 | 2.9 ± 0.4 × 102 | 0.0 | 1.9 ± 0.01 × 104 | 2.30 ± 0.01 × 104 | −0.1 | NT | NT | - |
qPCR | 6.6 ± 3 × 104 | 2.2 ± 3.2 × 102 | 2.5 | 7.7 ± 3.3 × 103 | 3.64 ± 0.7 × 103 | 0.3 | 7.6 ± 3.3 × 105 | 4.1 ±1.12 × 105 | 0.3 | |||||
2014 | culture | 1.5 ± 0.2 × 105 | 3.5 ± 0.8 × 102 | 2.6 | 2 × 102 | ND | 2.3 | 1.3 ± 0.01 × 105 | 1.4 ± 0.1 × 105 | 0.0 | 1.7 ± 0.7 × 105 | 4.4 ± 0.5 × 104 | 0.6 | |
qPCR | 1.5 ± 0.1 × 106 | 1.2 ± 6.6 × 103 | 3.1 | 3.2 ± 0.8 × 104 | ND | - | 1.7 ± 0.2 × 106 | 2.07 ± 0.1 × 106 | −0.1 | 3.1 ± 0.0 × 106 | 8.97 ± 0.06 × 105 | 0.5 | ||
Enterococcus spp. | 2013 | culture | 1.9 ± 0.05 × 104 | 7.0 ± 4.2 × 102 | 1.4 | 7.2 ± 0.2 × 102 | 8.8 ± 1.6 × 102 | −0.1 | 4.7 × 103 | 4.3 × 103 | 0.0 | NT | NT | - |
qPCR | 2.7 ± 1.2 × 106 | 1.9 ± 1.7 × 103 | 3.2 | 1.6 ± 0.04 × 106 | 3.4 ± 0.1 × 105 | 0.7 | 3.2 ± 1.05 × 106 | 2.16 ± 1.32 × 106 | 0.2 | - | ||||
2014 | culture | 4.4± 0.07 × 104 | 4.3 ± 0.6 × 104 | 0.0 | 3.0 × 102 | ND | 2.4 | 1.1 ± 0.6 × 103 | 1.1 ± 0.3 × 103 | ND | 2.8 ± 0.6 × 104 | 9.1 ± 5.3 × 103 | 0.5 | |
qPCR | 1.5 × 107 | 3 × 106 | 0.7 | 4.4 ± 0.6 × 106 | 9.1 ± 2.2 × 102 | 3.7 | 1.9 ± 1.01 × 104 | 1.5 ± 0.21 × 104 | 0.1 | 2.5 ± 0.01 × 105 | 4.9 ± 0.7 × 104 | 0.7 | ||
Salmonella spp. | 2013 | culture | 3.0 ± 0.4 ×103 | 9.5 ± 7.8 × 101 | 1.5 | 3.2 ± 0.2 × 102 | 4.1 ± 0.8 × 102 | −0.1 | NT | NT | - | NT | NT | - |
qPCR | ND | ND | - | ND | ND | - | ND | ND | - | |||||
2014 | culture | 5.5 ± 1.5 × 104 | 3.4 ± 1.1 × 103 | 1.2 | ND | ND | 0 | NT | NT | - | NT | NT | - | |
qPCR | ND | ND | - | ND | ND | - | - | |||||||
Campylobacter spp. | 2013 | qPCR | ND | ND | - | ND | ND | - | ND | ND | - | NT | NT | - |
2014 | culture | 7.0 × 101 | 6.6 ± 5.7 | 1.0 | ND | ND | - | 9.6 ± 0.1 × 103 | 8.0 ± 1.3 × 103 | 0.1 | NT | NT | - | |
qPCR | ND | ND | - | ND | ND | - | ND | ND | - | ND | ND | |||
adenovirus | 2014 | qPCR | 4.8 ± 1.5 × 103 | 3.03 ± 1.3 × 103 | 0.2 | ND | ND | - | 2.0 × 105 | 2.4 × 105 | −0.1 | NT | 12.04 MPNIU | - |
2015 | culture | 23 MPNIU | 6.6 MPNIU | 1.2 | ||||||||||
Cryptosporidium spp. | 2014 | microscopy | 0.5 ± 0.7 (31%) | ND (22 %) | - | ND (16%) | ND (30%) | - | NT | NT | - | 2 ± 1.7 (3%) | 2.5 ± 1.4 (20%) | −0.3 |
2015 | microscopy | 0.5 ± 0.7 (25%) | ND (24%) | - | NT | NT | ||||||||
Giardia spp. | 2014 | microscopy | 119 ± 130 (21%) | 1 ± 1.4 (11%) | 2.2 | 7.5 (18%) | ND (30%) | 1.6 | NT | NT | - | 329.9 ± 7.6 (3%) | 154.7 ± 30.7 (30%) | 1.3 |
2015 | microscopy | 76.5 ± 64.3 (26%) | ND (14%) | 1.8 | ||||||||||
F+ RNA coliphage | 2013 | culture | 1.0 × 107 ± 2.0 | 0 | n/a | ND | ND | - | NT | NT | - | NT | NT | - |
2014 | culture | <1.0 × 105 | 2.0 × 101 | 3.7 | ND | ND | - | NT | NT | - | 3.4 ± 4.0 × 103 | 3.6 ± 4.9 × 101 | 2.0 | |
2015 | culture | 9 × 103 | 13 | 3.0 | ||||||||||
Total coliforms | 2013 | culture | 3.4 ± 0.2 × 104 | 4.1 × 102 | 1.9 | 8.8 ± 1.3 × 102 | 5.9 ± 1.2 × 102 | 0.2 | NT | NT | - | NT | NT | - |
2014 | culture | 2.0 ± 0.1 × 105 | 2.4 ± 0.2 × 103 | 1.9 | 7.6 ± 0.2 × 103 | 1.5 ± 1.0 × 102 | 1.7 | NT | NT | - | NT | NT | - |
3.2. Further Analysis of Microbial Concentration of WSP1
4. Discussion
4.1. Faecal Indicators and Pathogen Concentrations
4.2. Comparison of qPCR Data and Culture Methods for Fecal Indicators
4.3. Salmonella spp. and Campylobacter spp. Enumeration
5. Conclusions
Supplementary Files
Supplementary File 1Acknowledgments
Author Contributions
Conflicts of Interest
References
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Sheludchenko, M.; Padovan, A.; Katouli, M.; Stratton, H. Removal of Fecal Indicators, Pathogenic Bacteria, Adenovirus, Cryptosporidium and Giardia (oo)cysts in Waste Stabilization Ponds in Northern and Eastern Australia. Int. J. Environ. Res. Public Health 2016, 13, 96. https://doi.org/10.3390/ijerph13010096
Sheludchenko M, Padovan A, Katouli M, Stratton H. Removal of Fecal Indicators, Pathogenic Bacteria, Adenovirus, Cryptosporidium and Giardia (oo)cysts in Waste Stabilization Ponds in Northern and Eastern Australia. International Journal of Environmental Research and Public Health. 2016; 13(1):96. https://doi.org/10.3390/ijerph13010096
Chicago/Turabian StyleSheludchenko, Maxim, Anna Padovan, Mohammad Katouli, and Helen Stratton. 2016. "Removal of Fecal Indicators, Pathogenic Bacteria, Adenovirus, Cryptosporidium and Giardia (oo)cysts in Waste Stabilization Ponds in Northern and Eastern Australia" International Journal of Environmental Research and Public Health 13, no. 1: 96. https://doi.org/10.3390/ijerph13010096