Today, more than eight million tons of biosolids are generated annually for land application nationwide. These biosolids contain beneficial plant nutrients, soil conditioners, and may also contain pathogenic bacteria, viruses, protozoa and parasites. The fate of pathogens is a concern for biosolids generators, applicators, and the general public. The ability to detect the presence of microbial pathogens and the resulting health risks in biosolids is a significant issue confronting the wastewater industry. Ideally, wastewater treatment plants should be able to monitor for specific pathogens in biosolids. Since it is almost impossible to detect and quantify the presence of all possible pathogens in waste matrices, there is a compelling need to identify a suite of indicators that can be used to predict the presence of pathogenic microorganisms in biosolids. The overall objective of this project was to identify those pathogens and surrogate indicator organisms that are at the highest density in raw sludge and determine their time-temperature-pH relationships in the laboratory under controlled conditions.
There were surprisingly low numbers of culturable enteric viruses (median values shown) (< 1 PFU/g), Salmonella spp ( < 8 MPN/g), and helminth ova ( < 1 ova/g) in the untreated sewage samples. Other pathogens, such as Shigella spp (25 MPN/g), Legionella spp (108 CFU/g), Aeromonas spp (10
Chapter
1.2 Time-Temperature Studies
2.1 Sampling and Sample Handling Procedures
2.2 Experimental Approach – Traditional Methods
2.2.1 Anaerobic Heterotrophs
2.2.2 Aerobic Spore-Formers
2.2.3 Clostridium Perfringens Spores
2.2.7 Generic Escherichia coli
2.2.13 Coliform and Generic E. coli Estimation by Colilert™
2.2.14 Male-Specific and Somatic Coliphages
2.2.15 Total Culturable Enteric Virus
2.2.16 Total Community DNA Extraction
2.2.17 Adenovirus Sequences by Real-Time PCR
2.2.18 Detection of Cryptosporidium spp. Oocysts by Microscopy
2.2.19 Detection of Giardia spp. DNA Sequences by Real-Time PCR
2.2.20 Detection of Helminth Eggs by Microscopy
2.3 Experimental Approach – Deep Sequencing
2.3.1 Community DNA Extraction
2.3.2 Massively Parallel FLX-Titanium Pyrosequencing
2.3.3 Bacterial Diversity Data Analysis
2.3.4 Bacterial Identification
3.1 Experimental Approach
3.2 Experimental Protocols
3.2.1 Bacterial Sample Processing
3.2.2 Virus Sample Processing
4.1 Traditional Methods Results
4.2 Deep Sequencing Results
4.2.1 Phylogenetic Profiles of Bacterial Communities Within Sewage Sludge
4.2.2 Comparison of Microflora of Sewage Sludge Samples
4.2.3 Detection of Pathogens within Sewage Sludge Samples
6.1 Microbial Survey of Raw Sewage Samples
6.1.3 Comparison of Microbial Indicator Organisms and Specific Pathogens
6.2 Deep-Sequencing Evaluation of Microbial Diversity
6.2.2 Microbial Diversity and Conserved Organisms
6.2.3 Comparison of Samples
6.3 Time-Temperature Relationships
6.3.1 Response of Microorganisms to Temperature Stress
7.2 Significance and Recommendations for Wastewater Industry
Developing Better Indicators for Pathogen Presence in Sewage Sludge
( WERF Research Report Series )
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