Risk Assessment of Viral Pathogens in Land Applied Biosolids Using 454 Pyrosequencing

EPA Grant Number: FP917115
Title: Risk Assessment of Viral Pathogens in Land Applied Biosolids Using 454 Pyrosequencing
Investigators: Bibby, Kyle James
Institution: Yale University
EPA Project Officer: Michaud, Jayne
Project Period: September 1, 2010 through August 31, 2013
Project Amount: $111,000
RFA: STAR Graduate Fellowships (2010) RFA Text |  Recipients Lists
Research Category: Academic Fellowships , Fellowship - Human Health: Risk Assessment and Decision Making


To gain a better understanding of the viral content and diversity in biosolids, I propose the following three objectives: Objective 1. Perform massively parallel next-generation sequencing on viral DNA and cDNA from the influent and effluent of a mesophilic anaerobic digester. Objective 2. Develop approaches to increase the certainty of virus identification from sequence information. Objective 3. Use sequencing data to develop pathogen concentrations in biosolids and produce an aerosol risk analysis for individual and total virus content.


Biosolids are stabilized sewage sludges that are applied to agricultural lands to take advantage of their high nutrient content. Due to their source, biosolids have the potential to contain pathogens, and the risks posed to the public by their land application are poorly understood. This research will sequence the viral DNA in a biosolids sample, for the first time identifying the full diversity of viruses present in biosolids, and apply this measure to existing risk assessments.


This research will utilize next-generation DNA sequencing to describe the viral metagenome of biosolids from a mesophilic anaerobic digester. Virus-sized particles will be isolated from the biosolids, nucleic acids extracted and sequenced. These sequences will be identified, and the updated viral concentrations applied to existing risk assessments. This work will develop the tools necessary to properly produce and classify these sequences as well as to apply this sequence information to risk assessments.

Expected Results:

There are two primary expected results from this research. The first is the development of next-generation DNA sequencing-enabled tools and methodologies for risk assessment. The study of biosolids provides an excellent platform for the development of improved, next-generation sequencing-enabled risk assessment methods. In order for these methods to be properly applied, bioinformatics methods must be further developed to ensure the identity of pathogenic sequences. The completion of an in silico study will resolve remaining questions about the importance of BLAST search algorithm, selection of database, and read length. The development of the idea of “gene certainty” will be an important advancement in assuring the identity of sequences as pathogens. The second expected result of this research is an updated viral risk assessment of biosolids land application. A primary barrier to risk-based regulation of pathogens in biosolids has been the lack of reliable pathogen concentrations. Through the incorporation of infectious virus concentrations and qPCR measurements, virome data may be made quantitative. By including all highly enriched viruses in the sample and updating existing exposure and risk assessments, the risk estimates generated will be the most comprehensive to date. Using multiple aerosol transport models for risk assessment will facilitate a consensus view on the risk posed by biosolids land application. These results will promote the movement towards risk-based regulation of biosolids land application practices. Ensuring public safety during land application will encourage sustainable reuse of nutrient rich biosolids and the further development of renewable energy anaerobic digestion technologies. These methods may then be applied in the future to a myriad of quantitative microbial risk assessment cases.

Potential to Further Environmental/Human Health Protection:

This work will serve to encourage the sustainable practice of biosolids land application while ensuring the protection of public health. In addition, the use of next-generation DNA sequencing-enabled risk assessment tools will facilitate the move from indicator organism-based risk assessments to risk assessments based on the complete diversity of pathogens present.

Supplemental Keywords:

biosolids, sewage sludge, anaerobic digestion, compost, DNA sequencing, bioinformatics, pathogen, risk assessment,

Progress and Final Reports:

  • 2011
  • 2012
  • Final