Grantee Research Project Results

2012 Progress Report: Rapid Detection of Sewer Pipeline Problems Using Bacterial DNA Markers and Q-PCR Technology

EPA Grant Number: R834871
Title: Rapid Detection of Sewer Pipeline Problems Using Bacterial DNA Markers and Q-PCR Technology
Investigators: Yan, Tao
Institution: University of Hawaii at Manoa
EPA Project Officer: Page, Angela
Project Period: February 1, 2011 through January 31, 2016
Project Period Covered by this Report: February 1, 2012 through January 31,2013
Project Amount: $299,956
RFA: Advancing Public Health Protection through Water Infrastructure Sustainability (2009) RFA Text | Recipients Lists
Research Category: Drinking Water , Water

Objective:

Sewer pipeline deterioration is the primary cause of sewer accidents that threaten the public health safety. This enormous water infrastructure problem requires a technical solution that can rapidly identify deterioration in a large number of pipelines with ease. The overall goal of this project is to develop a pipeline inspection technology that is based on Q-PCR quantification of bacterial DNA markers in sewage water, which indicate sewer pipe problems and is rapid and high throughput in order to be implemented at sewershed or sewer system level. In this project, the proposed qPCR-based technologies will address two specific sewer pipeline problems: FOG deposition and concrete crown corrosion. The working hypothesis is that these pipeline problems are associated with specific microbial processes and the problem severity levels are quantitatively related to the concentrations of the specific bacterial DNA markers present in the sewage water. The study will be performed through field monitoring and laboratory simulation. Bacterial DNA markers indicative of the different sewer pipeline problems will be first identified, and subsequently Q-PCR methods for the DNA markers will be developed and evaluated under actual field conditions.

Progress Summary:

The project has made significant progress on developing qPCR technology to detect sewer crown corrosion (Objective 2), which is the focus of this progress report. Initial work on detecting FOG deposition using qPCR (Objective 1) has just started in summer 2013. The first task in Objective 2 is to identify the microbial populations that can be used as biomarkers for different levels of sewer crown corrosion. The first experiment was conducted in microcosms to investigate how H₂S concentration affects concrete corrosion microbial communities, which unfortunately was not successful. Alternatively, field sampling at actual corroded gravity sewers was performed and microbial communities at the sewer crown were determined by using both culture-dependent and culture independent approaches. The overall microbial communities from concrete crown samples were assessed to determine the heterotrophic bacterial cell density (by MPN) and the total bacterial cell number was quantified (by qPCR), as well the presence of eukaryotic and archaeal cells. Then the bacterial biodiversity and its spatial and temporal variability were determined by 16S rRNA gene pyrosequencing and comparison amongst sewer crown samples collected from different locations and at different dates. One particularly interesting finding is the lack of dominance of Acidithiobacillus in some of the extremely acidic sewer crown samples, and the detection of Mycobacterium as the dominant populations. Since Mycobacterium, in addition to the widely acknowledged Acidithiobacillus populations, could be another biomarker indicative of sewer crown corrosion, extensive efforts have been make and are ongoing to isolate these Mycobacterium species. To date, several Mycobacterium isolates have been obtained from the sewer crown samples using the Middlebrook 7H9 broth that is commonly used to cultivate clinical samples of Mycobacterium populations.

Experiments have also been started to develop qPCR assays for the detection of Mycobacterium populations from sewage samples. The pan-mycrobial primers commonly used could not be used in qPCR since the amplicon is too big, and new qPCR primers were designed, which are shown to be capable of detecting Mycobacterium species. Additionally, as genes involved in sulfur-oxidation pathway are also potential good biomarkers for sewer crown corrosion, various sulfur oxidizing genes were PCR amplified from genomic DNA from the corrosion products. Tetrathionate hydrolase gene was not detected, and soxB gene was detected, which will be further tested for its suitability in detecting sewer crown corrosion. Interestingly, archaeal SOR genes were detected through PCR, which suggests potential importance of archaea in the MICC process. The possible involvement of archaea in sulfur oxidation under extreme acidic conditons is currently being investigated.

Future Activities:

Currently, we are testing whether the mycobacterial isolates can oxidise elemental sulfur. Also obtain longer length 16S rRNA gene sequences for identification. We have selected/developed several Mycobacteirum-specific and Acidithiobacillus-specific qPCR primers. Once we have proven that Mycobacterium can indeed carry out sulfur oxidation, we will start to test the qPCR assays in municipal wastewaters for the detection of concrete corrosion. We will also test if the soxB qPCR protocol is appropriate for screening wastewater for indication of concrete corrosion. Since our qPCR results indicated the presence of archaeal SOR genes, we will clone and sequence the full length archaeal SOR genes. If a diverse archaeal population is found, then pyrosequencing of the archaeal population in the corrosion products will be conducted, which could add to our understanding of MICC microbial communities as previous works have not yet identified the role of archarea in MICC. To further elucidate the role of eukarya in MICC, we are planning to place concrete coupons in a manhole known to have experienced severe corrosion to test for the microbial succession of eukarya (and perhaps archaea) as well as bacteria. Work for Objective 1 has just started. We will construct a lab-scale sewer system identify proper biomarkers for detecting FOG deposition. In the same time, we will also conduct field sampling of FOG deposition to determine the microbial communities associated.

Journal Articles:

No journal articles submitted with this report: View all 7 publications for this project

Progress and Final Reports:

Original Abstract

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.