Grantee Research Project Results
Final Report: Rapid Detection of Sewer Pipeline Problems Using Bacterial DNA Markers and Q-PCR Technology
EPA Grant Number: R834871Title: 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 Amount: $299,956
RFA: Advancing Public Health Protection through Water Infrastructure Sustainability (2009) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
The project focused on identifying bacterial and DNA markers that are indicative of two major sewer pipeline problems, including microbiologically induced crown corrosion (MICC) and fat-oil-grease (FOG) deposition. The working hypothesis is that these pipeline problems are associated with specific microbial processes and populations and hence the problem severity levels are quantitatively related to the concentrations of the specific bacterial DNA markers present in the sewage water. In the first 2 years (2011-2013), the project focused on identifying microbial populations associated with MICC, and considerable efforts were made to elucidate the functions of Mycobacterium in the MICC process. From 2014, the project started focusing on the investigation of microbial processes that relate to the formation and biodegradation of FOG deposits in sewer collection systems. Overall, the project resulted in publication of three peer-reviewed journal papers and four conference presentations.
Summary/Accomplishments (Outputs/Outcomes):
Summary of Yearly Research Progress
2011-2012. In this year, the project focused on conducting laboratory microcosm experiments to investigate corrosion of concrete coupons under simulated sewer crown conditions. The objectives were to create different concrete crown corrosion conditions using different levels of H2S and then to characterize microbial communities on the corroded concrete coupons and to quantify the abundance of Acidithiobacillus in the samples. However, after more than 1 year of running laboratory microcosms, corrosion of the concrete coupons in the microcosms was very limited, as indicated by both pH measurement and mechanical strength of the coupon surface. This progress of corrosion of concrete coupons was significantly slower than those previously reported in literature when concrete coupons were placed in real sewer systems. This indicated limitations of the laboratory experimental conditions and prompted subsequent change of approach from laboratory experiments to field sampling.
2012-2013: In this year, field sampling was started as an alternative approach to overcome the technical challenges of simulating MICC in laboratory microcosms. Samples were collected from multiple locations in Honolulu where MICC was previously reported, which identified the Ala Moana Beach Park as a hot spot of H2S generation and MICC occurrence. Subsequently, extensive sampling was conducted in the gravity sewer pipes immediately downstream of a force main in the Ala Moana Beach Park. Pyrosequencing of 16S rRNA gene amplicons identified Mycobacterium spp., instead of Acidithiobacillus, as the most dominant microbial component in the MICC communities. Because this significantly differed from our working assumption that Acidithiobacillus spp. were the dominant MICC populations, additional experiments were conducted to verify that Mycobacterium spp. are metabolically active under the MICC conditions and are indeed sulfur-oxidizing. Direct RNA sequencing of the MICC samples was attempted to identify the mRNAs expressed in the samples, which would determine the metabolic activities of the Mycobacterium populations identified by pyrosequencing. However, RNA extraction from these MICC samples proved to be extremely difficult, presumably due to the high acidity and the complex biofilm compositions in the samples. Alternatively, we focused on isolating Mycobacterium to verify their ability to use H2S, elemental sulfur and/or thiosulfate as electron donors.
2013-2014: In this year, the project obtained numerous acidophilic bacterial isolates from the corroded sewer crown samples, including several Acidithiobacillus isolates and one Alicyclobacillus isolate but no Mycobacterium populations. This partially explains why previous cultivation-based approaches almost always have recovered Acidithiobacillus as the dominant populations in MICC, even though cultivation-independent analysis of the MICC microbial communities showed Mycobacterium populations to be numerically dominant. Additional efforts using nutrient rich clinical media resulted in the isolation of several Mycobacterium populations form the MICC samples. Efforts to verify these Mycobacterium isolates as acidophilic sulfur oxidizer, however, have failed. Because Mycobacterium spp. are important opportunistic pathogens, experiments also were conducted to determine whether the Mycobacterium populations can use volatile fatty acids from sewage and use sewer pipelines as habitats.
In parallel, the project started characterizing microbial communities associated with FOG deposits in sewer systems. Laboratory microcosm experiments were conducted to understand FOG biodegradation under different redox conditions and to characterize the microbial communities associated with FOG using next generation sequencing.
2014-2015: In this year, the project focused on studying FOG deposits in sewer systems, including the biodegradation of FOG deposits and the source of long chain fatty acids (LCFAs), which can react with calcium ion to form FOG deposits. First, we completed the study on the biodegradation of FOG in laboratory microcosm experiments that were aimed to determine FOG biodegradability under various redox conditions. Microbial communities in these microcosms were analyzed by Illumina sequencing of the 16S rRNA gene amplicons. The results showed that overall biodegradation of FOG deposits was very slow, and different rates of biodegradation occurred under different redox conditions. Considerable biodegradation occurred under the aerobic condition and the anaerobic nitrate-reducing condition, while very limited biodegradation occurred under the anaerobic sulfate-reducing condition and methanogenic condition. Microbial community analysis by Illumina sequencing of 16S rRNA gene amplicons identified corresponding microbial communities in the microcosms. However, no clear bacterial DNA markers associated with FOG biodegradation were identified, which makes it difficult to use qPCR to quantify FOG deposit levels in sewer pipes. Then, the project pivoted to understand the potential sources of FOG precursors, in particular LCFAs, in sewer environments. The first source of LCFAs to be investigated was grease interceptors (GIs), which are widely used to prevent fat, oil, and grease from entering sewer systems. Bench-scale GIs were established in the laboratory to determine the production of LCFAs with and without microbial activities as well as production of LCFAs under different hydraulic retention times (HRTs).
2015-2016: In this year, the experiments on the production of LCFAs in GIs were completed. Bench-scale GIs were established in the laboratory to determine the production of LCFAs with and without microbial activities as well as production of LCFAs under different HRTs. The results showed that although LCFAs could be produced from abiotic, chemical hydrolysis of FOGs in GIs, microbial activities contributed to the majority of LCFAs in GI effluent, which was five times higher than that under only chemical hydrolysis conditions. A similar LCFA profile was found between GI effluent under the impact of microbial activities and FOG deposits. Longer HRT resulted in higher concentration of LCFAs in GI effluent as well as higher percentage of unsaturated LCFAs in total LCFA production within 60 days. However, the largest discharge amount of LCFAs was obtained under short HRT (HRT = 0.5 d) while a higher portion of produced LCFAs was retained in GIs under HRT = 1 d and 2 d, suggesting that GIs under short HRT would be a bigger concern for downstream FOG deposit formation.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 7 publications | 3 publications in selected types | All 3 journal articles |
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He X, Zhang Q, Cooney MJ, Yan T. Biodegradation of fat, oil and grease (FOG) deposits under various redox conditions relevant to sewer environment. Applied Microbiology and Biotechnology 2015;99(14):6059-6068. |
R834871 (2014) R834871 (Final) |
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He X, Yan T. Impact of microbial activities and hydraulic retention time on the production and profile of long chain fatty acids in grease interceptors:a laboratory study. Environmental Science:Water Research & Technology 2016;2(3):474-482. |
R834871 (Final) |
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Pagaling E, Yang K, Yan T. Pyrosequencing reveals correlations between extremely acidophilic bacterial communities with hydrogen sulphide concentrations, pH and inert polymer coatings at concrete sewer crown surfaces. Journal of Applied Microbiology 2014;117(1):50-64. |
R834871 (2013) R834871 (2014) R834871 (Final) |
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Progress and Final Reports:
Original AbstractThe 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.
Project Research Results
- 2014 Progress Report
- 2013 Progress Report
- 2012 Progress Report
- 2011 Progress Report
- Original Abstract
3 journal articles for this project