Microorganisms have been suggested to be good indicators of ecosystem stability as they are sensitive to changes in the environment. In the DWDS, microbial community fingerprints have also been suggested for use as indicators of stability in the drinking water microflora (6
). In the last several years, many water utilities have started to switch from using chlorine for disinfection to chloramines due to the rising concern of DBP formed during chlorination. In the Urbana distribution system, the disinfectant residual is maintained by periodic use of chlorination and chloramination. During chlorination, breakpoint addition is conducted, and free chlorine is produced to control biofilm growth. While studies have examined the impact of chlorination and chloramination on microbial communities, to our knowledge, the present study is the first to use molecular methods for community composition analysis and to determine the influence of transitory switches between these two disinfection methods on drinking water microbial communities in a full-scale DWDS.
This study used two different approaches, T-RFLP and 454 pyrosequencing, to characterize the drinking water microflora. The results suggested that the free chlorine concentration was an obvious factor in explaining variations in drinking water microflora. NMDS analysis indicated that there was a temporal change in the microbial community, and further analysis revealed that the change was a result of the different disinfectant strategy conducted by the water utility during each season, which influenced the free chlorine concentration in the DWDS. Similar results were obtained by CCA, with some degree of effects due to seasonal or temporal change. It is known that chlorination and chloramination exhibited different degrees of effectiveness in shaping the microbial composition through chemically oxidizing bacterial cells and suppressing bacterial growth (12
). In our study, 49 or 69 OTUs were mainly detected in chloraminated or chlorinated water, respectively. The different dominant bacterial groups detected in each cluster could represent differences in planktonic cells that were damaged but not lysed, survived and possibly proliferate, or dislodged from distribution systems surfaces during the disinfection treatments.
Previous studies have reported the predominance of Alphaproteobacteria
or both in the drinking water (40
). In contrast, our results showed the predominance of the Gammaproteobacteria
population over the Alpha
- and Betaproteobacteria
populations in most of the samples. This could be attributed to the differences in the source water quality (groundwater versus surface water), physicochemical variables of the water, and efficiency of disinfection treatment (28
). Studies have also demonstrated the influence of disinfectants on the Proteobacteria
populations where Alphaproteobacteria
predominated in both chloraminated and chlorinated water (40
), whereas Betaproteobacteria
in drinking water biofilms were favored with increased chlorination (24
Although we did not observe the same trend on the overall proteobacterial populations (see Fig. S4 in the supplemental material), our results did show the influence of chlorination and chloramination on certain major bacterial groups (≥5% abundance) or core microbial populations at the family level of classification. Under chloramination, Methylophilaceae
, and Pseudomonadaceae
were the dominant families detected, whereas, under chlorination, Cyanobacteria
, and Xanthomonadaceae
were dominant. The presence of Methylophilaceae
, and Methylobacteriaceae
were possibly influenced by dissolved methane, which has been detected in the groundwater that serves as the source water in Urbana and Champaign area (18
). However, we do not have sufficient information to explain the presence of other bacterial families detected, their resistance to chlorine and chloramine and the microbial ecology roles they played. For example, Blastomonas
sp. was the main population detected in Sphingomonadaceae
, which comprise of a group of bacteria that have a versatile metabolism and are ubiquitous in soil and water habitats. Although the close relatives of Blastomonas
sp. are common inhabitants of chlorinated water (11
), its resistance to chlorination remains unclear. Lysobacter
species, a common soil and freshwater bacterium linked to the production of the musty-earthy odor detected in water (8
), were the most abundant members found in Xanthomonadaceae
and were also detected from a biofilm microbial community a water meter collected from the same distribution system (15
). The possibility for Lysobacter
spp. to be sloughed from the biofilms should be further confirmed. Other studies have demonstrated that not only are Cyanobacteria
present in chlorinated DWDS but some are also potentially active members of the DWDS microbial community (17
), suggesting that they could survive from different physical and chemical treatments and enter the DWDS (13
). We think the use of cultivation-based methods together with metagenomics approaches should be pursued to further understand the ecological roles of predominant populations in DWDS.
Among minor core populations (<0.1% of total pyrosequences), correlation between their abundances and disinfection practice, and sampling time and location was not obvious. These included, for example, metal reducers (e.g., Gallionellaceae, Geobacteraceae, and Shewanellaceae), methanogens (e.g., Methanobacteriaceae, Methanosaetaceae, and Methanosarcinaceae), anaerobic syntrophs (e.g., Syntrophaceae and Syntrophobacteraceae), and nitrifying populations (e.g., Nitrosomonadaceae, Nitrospinaceae, Nitrospirales, and Nitrosopumilus). Most of these populations were likely those present in source water and have survived through the water treatment processes or perhaps were those that have established themselves in the biofilm and were detected in the planktonic phase upon biofilm detachment. Legionella spp. (<0.006 of total sequences obtained for a given sample) and Mycobacterium spp. (<0.008) were also detectable in some of the samples. However, due to the short 16S pyrosequence reads obtained, we could not precisely determine the identity and confirm the presence of potential pathogenic organisms. It can be anticipated that some of these populations could serve as inocula and proliferate in the DWDS if optimal growth conditions are provided.
We were also able to identify microbial populations that exhibited transient responses. Flavobacteriaceae and Clostridiaceae were two major populations observed during winter 2010 and fall 2011 (i.e., season 6), respectively, and Methylobacteriaceae was only detected in sample F10_S2. In particular, most of the sequences in Clostridiaceae were associated with Clostridium species, which are known spore-forming organisms and have strong resistance to disinfection treatments. Their appearances could likely be due to compromises or perturbations in treatment barriers or to changes in source water microbial composition. Thus, it will be useful to find out the correlation between the transient microbial populations and causes and use the information to monitor the integrity of system operation in the future.
No spatial patterns in the microbial communities were observed during most sampling time points ( and ), suggesting that the water age at the sampling sites might not be long enough to allow the development of distinct local drinking water microflora. This finding is good for water utilities, since almost the same water quality produced at the water treatment plant can be provided to individual households. It was further observed that the microbial community clustering between the sites was less pronounced in samples collected during chlorination than chloramination. One possible explanation is that free chlorine is a stronger disinfectant than chloramines and can rapidly react with microbial cells. As a result, the cells could be lysed, and microbial community structures in the water phase and biofilms could be altered to an extent greater than that observed between two given sampling points. Alternately, chlorine can cause an increase in assimilable organic carbon (AOC) due to the reaction of free chlorine with the dissolved organic carbon present in natural water (10
). If the AOC generated could serve as substrates for growth of heterotrophic bacteria, although unlikely under a short water age, shifts in the microbial communities could be expected.
Biodiversity is a subject of interest in ecological studies and is usually used as an indicator of ecosystem stability to cope with perturbations. With this notion, biodiversity could be used as an indicator of distribution system compromise (e.g., inefficient treatment, pipe leak, contamination, corrosion, etc.). However, in our study, the bacterial diversity estimations based on 16S pyrosequencing were insensitive for detecting the influences of disinfectant strategies. It is possible that diversity measurements may exhibit less variability than species composition in response to environmental factors because changes in some taxonomic groups can be compensated for by others, and thus the changes observed in microbial communities are not necessarily reflected in microbial diversity (9
). This is in agreement with previous soil microbial ecological studies reporting that microbial community composition and structures were important for environmental monitoring rather than microbial diversity measurement (2
). Overall, we characterized here the drinking water microflora of a DWDS that receives both chlorination and chloramination treatments over a 2-year period. Although our study is based on DNA-based analysis that is useful for identifying present members, unlike RNA-based analysis that could provide information to active members, our study nonetheless demonstrated that chlorine and chloramines caused microbial community shifts and that certain populations are particularly affected by the different treatments (i.e., a reduction in cell numbers or reduced sensitivity to the treatments). Chlorination or chloramination treatments are reported to exert a strong selection process in the microflora in the drinking water that is consumed at the tap (6
). Our findings revealed that the reversible shifts in microbial communities were especially pronounced with chloramination, which perhaps suggested a stronger selection pressure of microbial populations exerted from chloramines compared to chlorine. This impact of different disinfection methods on the overall drinking water microflora and detailed analysis of microbial communities in the distribution system can provide insight to the selection of disinfection strategies used by water utilities that provide potable water to the end users.