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INNOVATIVE RESEARCH FOR A SUSTAINABLE FUTURE
Effect of Changing Treatment Disinfectants on the Microbiology of Distributed Water
and Pipe Biofilm Communities using Conventional and Metagenomic Approaches
Risk Management Research Project - Addressing Drinking Water Challenges through Science and Innovation
Background & Problem
Drinking water distribution systems are a potential source of waterborne illnesses from the growth of specific pathogens
associated with larger microbial communities, known as biofilms, which occur on drinking water pipe surfaces.
Mycobacterium avium, Legionella pneumophila, and Naegleria fowleri are examples of these pathogens that are currently
listed as priority contaminants on the EPA's Contaminant Candidate List 3. Previous studies have demonstrated that the
viability and virulence of these pathogens is influenced by other microbes in the biofilm communities; therefore, a better
understanding of the entire range of microbes in the biofilms is needed to develop pathogen control strategies. Current
treatment includes the use of halogen disinfectants, such as chlorine and monochloramine. Chlorine is the most
commonly used drinking water disinfectant in the U.S., but the sustainability of this treatment has been questioned due to
the potential for carcinogenic chlorinated byproducts. Because of this, many utilities are now using monochloramine as
both the primary and residual disinfectant. Previous studies have documented the differences between the effectiveness
of chlorine and monochloramine at killing specific types of pathogens and the ability to penetrate into biofilms; however,
there is limited data on their effectiveness at controlling the occurrence and growth of pathogens in distribution systems.
In addition, the impact to the overall microbial community composition by these disinfectants is also unknown.
Purpose of the Studies
The purpose of this research was to add to our knowledge of chlorine and monochloramine disinfectants, with regards to
effects on the microbial communities in drinking water distribution systems. A whole metagenome-based approach using
sophisticated molecular tools (e.g., next generation sequencing) was used to assess the microbial composition and the
metabolic potential in these communities. In addition, this research produced insights on the importance of certain
operational parameters in biofilm-related research.
Results and Conclusions to Date
| The implementation of metagenomic tools has provided a robust dataset on the diverse and highly complex
nature of drinking water pipe biofilm communities. Metabolic pathways1 and sequences assigned to a variety of
pathogen-related functions (e.g., virulence factors and antibiotic resistance) were identified in both chlorine and
monochloramine treated drinking water samples. The results showed differences in community structure and function
among communities exposed to the disinfectants:
*J* Genes indicative of Legionella were more abundant in the free chlorine treated samples.
*J* Genes indicative of Mycobacterium were more abundant in monochloramine treated samples.
*J* Although genes that code for proteins needed to survive/avoid/repair oxidative damage were identified in both the
chlorine and monochloramine treated communities, exopolysaccharide (EPS)2 genes of Mycobacterium were more
abundant in communities from monochloramine treated water, which likely translates to more biofilm material.
*J* Occurrence of antibiotic resistance was not preferential to either treatment; however, protein groups associated
with specific oxidant defense mechanisms were more abundant in one or the other treated systems3.
1 As evidenced by specific gene sequences that potentially code for the enzymes needed to complete complex biological transformations.
2 Mycobacterium is one group of bacteria that produce a significant proportion of the EPS in biofilms. EPS serves to protect biofilm organ isms from direct exposure to
disinfectants and to promote microbial adhesion to surfaces and overall biofilm formation.
3 Example: glutathione reductase and thioredoxin reductase, which collectively code for proteins needed to protect other proteins from oxygen radicals, were more
abundant in the chlorine samples. In contrast, the dps gene, needed to protect DNAfrom oxidation damage, was more abundant in monochloramine samples.
U.S. Environmental Protection Agency, Office of Research and Development
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Electron microscope images of biofilm growth on copper (images 1 & 2)
and PVC (images 3 & 4) drinking water pipe material from the studies.
The data from Study 1 suggest that disinfection
treatments can influence the occurrence of
specific types of gene systems in pipe biofilm
communities. The impact of these differences on
the occurrence and virulence of waterborne
pathogens needs to be the subject of continuing
future research.
Publication Available:
Gomez-Alvarez V., Revetta R.P., Santo Domingo
J.W. (2012). Metagenomic analyses of drinking
water receiving different disinfection treatments.
Applied Environmental Microbiology,
7S(17):6095-6102. (DOI:10.1128/AEM.01018-12)
A molecular approach was used to
characterize biofilm communities formed in
monochloramine treated drinking water.
Samples of communities that formed on various
types of biofilm capture devices were evaluated
to determine if they differ in their particular
community structure and if the compositions
vary over time as the biofilms form. Overall the results supported one of the observations of Study 1: Mycobacterium was
a dominant bacterial group in biofilms from the monochloramine treated waters. However, this study expanded on the
observation by showing that these slow-growing Mycobacterium are not the main initial biofilm colonizers. Their
predominance after two months suggests that they become integrated into the communities as persistent secondary
colonizers of biofilms. In general, the most significant variable that influenced the composition of biofilm communities
was the age of the biofilm and not the type of surface material. This observation has important practical implications for
future biofilm sampling schemes and for identifying potential management strategies.
Publication Available:
Revetta R.P., Gomez-Alvarez V., et al. (2013). Establishment and early succession of bacterial communities in
monochloramine-treated drinking water biofilms. FEMS Microbiology Ecology, S6(3):404-414. (DOl: 10.1111/1574-6941.12170)
A current study of the operational parameters of a simulated chloraminated drinking water distribution system
was designed to reflect three distinct operational conditions of a typical distribution system: (1) normal, (2) failure leading
to nitrification, and (3) restoration via a chlorine burn. Preliminary results show little variation in biomass in bulk water
and biofilm samples during the normal operation. However, an increase in biofilms was detected when the concentration
of chlorine residual was decreased in the system, which lead to nitrification conditions and failure of the system. Biomass
levels decreased after a chlorine burn. Analysis of the biofilms showed clustering of samples and significant differences in
functional and community structures during these different operational conditions. For example, environmental
Mycobacterium-\\ke sequences peaked during normal operations but decreased after failure of the system, while the
abundance of nitrifying bacteria increased as nitrification increased. Data on the
impact of the chlorine burn has yet to be analyzed. The results of this study will be
submitted for publication in 2015.
Principal Investigators
Expected Outcome
The metagenomic approaches used in these studies will help to develop a more
comprehensive understanding of the microbial ecology of drinking water
distribution systems relative to what disinfectants are used. Such information is
critical to the design of effective management practices and ultimately helps to
prevent waterborne disease and safeguard human health.
Randy Revetta
(513)569-7129
revetta.randy@epa.gov
Vicente Gomez-Alvarez
(513) 569-7362
gomez-alvarez.vicente@epa.gov
U.S. Environmental Protection Agency, Office of Research and Development
EPA/600/F-14/327 | September 2014
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