Available online at www.sciencedirect.
com
ScienceDirect
Drinking water microbiology — from measurement
to management
Caitlin R Proctor and Frederik Hammes
New microbial tools enable detailed quantification and
characterization of complex drinking water (DW) microbiomes.
Many opportunities exist from source to tap to apply this
knowledge toward management of the microbiology. This
requires consideration of the microbiome continuum across all
phases harboring microbes (planktonic cells, biofilms, and cells
attached to loose deposits) and across all stages (source,
treatment, distribution, and premise). Biofilters can be
optimized toward specific compound removal and can seed
the distribution network (DN) with beneficial bacteria.
Disinfection aggressively controls the microbiome, but may
select for unwanted bacteria. Within premise plumbing,
dramatic changes occur with unavoidable stagnation and pipe
material influence. To supply safe DW sustainably, it is
imperative that the field progress from characterization toward
management of the DW microbiome.
Addresses
Eawag, Swiss Federal Institute of Aquatic Science and Technology,
Überlandstr. 133, CH-8600 Dübendorf, Switzerland
Corresponding author: Hammes, Frederik (frederik.hammes@eawag.ch)
Current Opinion in Biotechnology 2015, 33:87–94
This review comes from a themed issue on Environmental
biotechnology
Edited by Spiros N Agathos and Nico Boon
http://dx.doi.org/10.1016/j.copbio.2014.12.014
0958-1669/# 2014 Elsevier Ltd. All rights reserved.
Introduction
The presence of bacteria in drinking water (DW) has been
recognized since the earliest microbial studies. Although
research initially focused on fecal-associated pathogens, it
has broadened considerably in recent years toward includ-
ing nonfecal opportunistic pathogens (e.g. Legionella,
Mycobacteria), process-related microbial problems (e.g. bio-
fouling, biocorrosion), and microbes that are functionally
relevant for DW treatment (e.g. nutrient and micropollu-
tant removal) [1–3]. The fact is, from source to tap, different
stages of DW systems offer unique habitats where microbes
develop thriving complex communities that in turn influ-
ence downstream microbiomes (Figure 1). However, there
are still broad knowledge gaps with respect to how these
www.sciencedirect.com
microbiomes are shaped and in turn the consequences for
both the DW system and consumers. In order to treat and
distribute water sustainably, it is imperative to accept the
omnipresence of microbes in DW, understand their behav-
ior, and apply that knowledge to manage them [4]. This
review focuses on the microbiology of centralized treat-
ment and distribution of DW within the context of indus-
trialized countries. It examines potential avenues for
microbial management throughout such systems and high-
lights critical research areas and opportunities.
Exciting new methods reveal a complex DW
microbiome
Next-generation sequencing (NGS) technologies (i.e.
454 pyrosequencing, Illumina, and Ion Torrent) enable
high-throughput, high-resolution characterization of the
microbiome. This includes amplicon sequencing of the
16S rRNA gene for identifying bacterial community
members [2,5–8,9,10], analysis of RNA for describing
the active microbiome [11], DNA-based metagenomic
analysis for discerning functional capacity [12,13], and
potential applications for metatranscriptomic analysis of
gene expression [5,6]. With the exception of straightfor-
ward bacterial community analysis, which captures a
taxonomic snapshot with resolution ranging from major
phyla down to rare taxa, application of these methods in
DW research has so far been rather limited.
All community composition studies concur that DW
microbiomes are complex, comprising up to 48 phyla
and in excess of 4 000 unique operational taxonomical
units (OTUs) [7,10,14,15,16], with many defined OTUs
presently unmatched to known organisms (i.e. ‘unclassi-
fied’). DW microbiomes tend to be dominated by Pro-
teobacteria and share most other phyla, even with
considerable differences in geography and treatment
processes (Figure 2). Figure 2 shows a broad comparison
of phyla from similar effluent points of three treatment
plants, where 15 out of 25 total phyla were shared by at
least two samples. With the exception of one candidate
phylum (G02), the phyla unique to any one DW system
represented 5% or less of sequences from that specific
system (Figure 2). While some of these similarities are
potentially driven by the similar low-nutrient profile
across all DW systems, it is erroneous to label DW
microbiomes as similar based solely on phyla-level data.
Substantial differences occur in the distribution of
lower classifications and the occurrence of rare taxa
[17]. The relative abundance of Alpha-proteobacteria,
Beta-proteobacteria, or Gamma-proteobacteria is inconsistent
Current Opinion in Biotechnology 2015, 33:87–94
88 Environmental biotechnology

Figure 1

source-to-network seeding (= no treatment or treatment breakthrough)

microbiome
continuum source-to-biofilter seeding biofilter-to-network seeding network-to-household seeding
source water treatment distribution household

no treatment reservoirs
Ground water
Surface water
Sea water
Reclaimed water
oxidation biofiltration
(= disinfection) (= growth)
• planktonic, biofilm, &
chlorination disinfection particle-associated cells
• indigenous communities • filter-specific microbiome residuals • widely varied pipe
• anthropogenic • functionally relevant materials
contamination • pollutant biodegradation • planktonic, biofilm and particle- • warm temperatures
• seasonal variation • bio-augmentation membrane associated cells • hot & cold water
• sudden events potential filtration • pipes materials: cast iron, cement, distribution
PVC, etc... • disinfection residual loss
• seasonal temperature fluctuations • long stagnation times
• varied residence times • potential for growth and
final treatment step: UV • contamination through pipe community structural
elimination of viable microbes disinfection breakage changes

Current Opinion in Biotechnology

Schematic overview of drinking water systems, highlighting major influences on microbial quantity and composition across the four stages of the
microbiome continuum - source, treatment, distribution and household. The figure is theoretical; numerous variations in system configuration
occur.

across different DW microbiomes, as well as over time
and between stages within one system [9,15,18]. The
dynamics of individual and rare OTUs can also differ
completely from that of the overall phyla [15,17].
This raises the key question of whether a universal DW
microbiome can actually be defined. One much-needed
step forward would be a meta-study of all existing DW
microbiome data, if provision can be made for differ-
ences in sampling (i.e. stages, phases, and temporal
variation discussed below), biases (e.g. DNA extraction
[19], reagent contamination [20], and PCR [21]), and
analytical methods (i.e. sequencing platform differences
[22,23]). Moreover, there is clear potential for applica-
tion of more advanced statistical methods to all studies.
For example, Pinto and colleagues [15] used such tools
to select 66 ‘key’ OTUs from thousands and map the
interactions that were responsible for most of the dy-
namics seen in their DW system, perhaps setting a
precedent for a more functional definition of the ‘core’
microbiome.
Research opportunities:
 Establish a comprehensive definition of the ‘core’
microbiome and test for the existence of a general core
DW microbiome (versus system-specific ones) [15].
 Implement NGS applications beyond ‘taxonomic
snapshots’, with further consideration for metagenome,
transcriptome, viruses, and eukaryotic organisms,
including free-living amoebae, fungi, and invertebrates
[24,25] (Figure 3).
 Fundamental studies to identify and annotate unclas-
sified OTUs in DW.
Considering stages, phases, and temporal
variations
A major challenge with respect to defining a DW micro-
biome is that it changes dynamically in both absolute
abundance and community structure through stages of
DW treatment and distribution (Figure 1), with each
stage potentially seeding the downstream system
[9,14,16,18,26]. In this manner, a microbiome continu-
um develops, necessitating consideration across all stages
for accurate interpretation of the causes of change.
Moreover, the DW microbiome varies considerably over
the phases of planktonic cells, attached biofilm cells, and
bacteria attached to particles or loose deposits [27]
(Figure 3). The planktonic phase is arguably most rele-
vant to the consumer, with total cell concentrations
typically ranging from 103 to 105 cells/mL [16,28,29],
but representing less than 2% of bacteria in a distribution
network (DN) [30]. Biofilms offer an environment

Current Opinion in Biotechnology 2015, 33:87–94 www.sciencedirect.com

 Drinking water microbiology Proctor and Hammes 89

Figure 2 represent up to 80% of active biomass, but are often not

examined due to sampling difficulties [27,30]. This
Phylum WTP 1 WTP 2 WTP 3 latter phase is highly relevant since it can be mobilized
and transported in the network during hydraulic distur-
Proteobacteria
bances and has been shown to harbor hygienically rele-
Unclassified vant microbes, including mycobacteria [27,33,34]. Several
Bacteroidetes authors demonstrated convincingly that the phases differ
Planctomycetes in both absolute abundance and community composition
Nitrospira
[11,14,30,33,35]. It was demonstrated that combining
relative abundance measurements (i.e. NGS) with abso-
Acidobacteria
lute abundance measurements (i.e. flow cytometry
Actinobacteria (FCM)) and viability assessment (i.e. adenosine tri-phos-
Cyanobacteria phate) provides complimentary information for micro-
Chloroflexi biome characterization throughout DW treatment and
TM7 distribution [9,26].
Chlamydiae
In this regard, cultivation-independent methods that
Firmicutes allow accurate quantification of bacterial concentrations
Verrucomicrobia (i.e. FCM) have become invaluable as an alternative to
OP11 cultivation, keeping pace with NGS developments
Gemmatimonadetes [5,9,36]. FCM can also be extremely sensitive to tem-
GN02
poral variations in aquatic systems [36]. These variations,
including a daily pattern in premise plumbing bacterial
OD1
concentrations [36,37], add yet another dimension to
Elusimicrobia consider when designing sampling/monitoring schemes.
OP3
ZB2 Research opportunities:
TM6
 Determine the relevance of each phase to the DW
SM2F11
system and the consumer.
ABY1_OD1  Establish consensus of systematic and comparable
WS3 sampling and analytical methods to include all phases
Chlorobi in microbiome descriptions [27,28].
 Develop and apply ecological theories and models to
0 Lowest Relative Abundance Highest the DW microbiome continuum [15].
= reported in all WTP = reported in 2 WTP
= unique to 1 WTP Managing microbes from catchment to
Chlorination Final Filter consumer
WTP 1 [18] 0.5 mg/L w/NH3 Dual Media A sobering question is how the wealth of microbial data
WTP 2 [9] 0.1 mg/L Granular activated carbon acquired with state-of-the-art methods actually benefits
WTP 3 [26] none Slow sand filtration DW management and whether microbial resource man-
agement concepts [4] are applicable to DW systems. The
Current Opinion in Biotechnology
fact is that decisions at all stages of DW treatment and
distribution profoundly shape the microbiome. While this
Relative abundance of bacteria at the phylum level from the effluent of
three drinking water treatment plants [WTP] as reported: WTP 1 [18],
provides ample opportunities to purposefully manage
WTP 2 [9], and WTP 3 [26]. Studies were chosen for illustrative DW microbiology, it also highlights the need for a multi-
purposes based on use of similar sampling points and methods, and dimensional approach toward studying these systems and
to capture a wide variation in geography and treatment schemes. proposing management solutions.

protected from oxidative and hydraulic stress, while pipe
material can affect biofilms both positively and negative-
ly. Pipe-associated biofilm cell concentrations range from
104 to 107 cells/cm2 [31,32], comprise 20–60% of the
bacteria in a DW system [30], and are associated with
many unwanted reactions (e.g. biocorrosion). Bacteria
associated with loose deposits, sediments, and particles
Biofilter bacteria are useful for DW treatment
A primary goal of DW treatment is the production of safe
DW, and thus treatment plants apply multiple hygienic
barriers (e.g. UV, membrane filtration, ozonation)
(Figure 1). In this respect, quantitative microbial risk
assessment (QMRA) is a well-developed concept for
managing DW safety, with specific emphasis on source

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90 Environmental biotechnology

Figure 3

pipe material influences

biofilm cells community shifts, growth substrate leaching biofilm inhibition
(104 – 108 cells/cm2) (e.g. flexible tubing) (e.g., copper pipes)

attachment detachment
protozoa
transport

neglected biome
planktonic cells
(103 – 105 cells/mL)
hydraulic influences invertebrates
predation, protection, viruses
re-suspension
disinfectant residuals DNA exchange

free DNA

biofouling sediments / loose deposits bio-corrosion biodegradation

(excessive EPS formation) (altered environment) (stainless steel; concrete) (synthetic polymer pipes)

unwanted consequences of biofilm deposition

Current Opinion in Biotechnology

Distribution and interactions of the microbiome across various phases (planktonic, biofilm and particle-associated) in a drinking water system.
Adapted from Liu et al. and Nescerecka et al. [27,29,30].

water contaminants [38]. However, DW treatment plants
are not solely antimicrobial environments. Biofiltration
(e.g. rapid sand, granular activated carbon, and slow sand
filtration) is one of the oldest treatment methods and is
operated with encouragement of bacterial growth in bio-
films supported on granular material. A basic function of
biofiltration is the removal of growth supporting nutrients,
essential for producing biologically stable DW [16]. This
function is common for a wide variety of heterotrophic
bacteria as demonstrated in studies of various biofilter
communities [26,39,40]. More specialized biofiltration
functions include the bacterial-mediated removal of un-
wanted compounds such as ammonium, arsenic, manga-
nese, iron, and a variety of micropollutants [1,41–46].
New microbiology methods benefit the understanding
and optimization of biofiltration processes by identifying
and quantifying major contributors to pollutant removal
[41,44,45]. For example, arsenic removal was linked to
the location of active communities within the biofilter and
optimized with filter operational parameters [44]. In
another example, iron oxidation, previously viewed as a
purely chemical process, was linked to iron oxidizing
bacteria [43]. While progress in this field has been made,
there is still ample opportunity to further develop bior-
emediated removal of micropollutants as an alternative to
expensive abiotic processes [1].
Biofilters harbor specific communities and consequential-
ly cause significant shifts in the microbiome, accounting
for 50–60% change with different filter types in series,
based on b-diversity metrics [26]. There is growing evi-
dence that biofilters at the treatment plant may direct the
entire downstream DN microbiome [14,16,18,26], poten-
tially providing operators the opportunity to control and
manage the DN microbiome from this easily observed
treatment stage.
Research opportunities:
 Combine metabolic potential analysis (metagenomics)
with optimization of biofilters.
 Determine feasibility of bioaugmentation of biofilters
with assembled communities [1].
 Understand further the role and risks of biofilters in
seeding and shaping the DN microbiology [18].
Disinfection before and during distribution
The final step of DW treatment is critical with respect to
the microbiome released into the DN and the tools with
which this is studied. Final disinfection eliminates most
bacteria and is a key component in risk management [38].
Often coupled to final disinfection is the use of residual
disinfectants (i.e. chlorine, chloramine) with the purpose

Current Opinion in Biotechnology 2015, 33:87–94 www.sciencedirect.com


of inhibiting microbial growth during distribution. Final
and/or residual disinfection has three major implications.
Firstly, as it partially or completely destroys the treatment
plant microbiome, it effectively eliminates any meaning-
ful seeding of the DN with biofilter communities. Sec-
ondly, it means that interpretation of microbiome data is
incomplete without viability considerations. Many popu-
lar methods (e.g. FCM total cell counts and all DNA-
based methods) include effectively dead, and thus po-
tentially irrelevant, cells. In this case, it is imperative to
use viability-targeting methodology for quantification and
characterization [9,11,29,47,48]. Henne and colleagues
showed clear differences between RNA and DNA popu-
lations [11], while the work of Chiao and colleagues
clearly demonstrated a completely different DNA-based
microbiome after chlorination when applying propidium
monoazide to exclude DNA from membrane-compro-
mised cells [47]. Finally, some viable bacteria are still
detected in systems with disinfection, and thus disinfec-
tants potentially act as a stress-based selective pressure.
Uncontrolled growth-related problems arise when disin-
fectant residual is lost [29]. Furthermore, there is growing
concern that use of disinfection residuals selects for a
microbiome that is resistant to oxidants and to other
stressors such as antibiotics [13,49]. There is also evi-
dence that chloramine versus chlorine use selects for
different microbiome composition [7,10,12,39,50,51].
Research opportunities:
 Validate methodologies to assess bacterial viability in
conjunction with community analysis of disinfected
DW [47,48].
 Establish the importance of extracellular DNA and
DNA from dead cells in DW.
 Establish relationship between disinfection and selec-
tion of multiresistant microorganisms and determine
consequences for the consumer [13,49].
The alternative: distributing a viable microbiome
DN microbiology is essentially dictated by opposing
philosophies regarding the use or nonuse of disinfectants.
The alternative approach to both final disinfection and
use of residual disinfectants is the continuous release of a
viable microbiome during treatment (Figure 1), with
control of subsequent regrowth through nutrient limita-
tion [16]. Several large European treatment plants use
biofiltration as a final treatment step, thus releasing
benign communities that remain stable during distribu-
tion, with concentrations in the order of 104 to 105 cells/
mL [37]. Wang and colleagues [3] recently proposed
the provocative idea to use benign bacteria, either from
a designed inoculum or from indigenous bacteria, as
‘probiotics’ in premise plumbing to competitively ex-
clude those that are detrimental to the system or con-
sumers. It can be argued that European systems without
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Drinking water microbiology Proctor and Hammes 91
disinfectants have unintentionally been employing this
strategy with indigenous bacteria at full-scale for decades
and are therefore particularly interesting models to study
the concept (Box 1).
Challenges along the last meters
Within buildings, water effectively stagnates for up to
23 hours per day [52]. Stagnation allows water tempera-
ture to increase to household levels [37] and leads to
depletion of disinfectant residuals [29,51]. In addition,
stagnation exposes the pipe biofilm to bioavailable nutri-
ents in the water phase and additionally allows exchanges
between the suspended cells and pipe biofilm micro-
biomes. These factors favor microbial growth and consid-
erable changes in both concentration (up to 320%
increase) and composition (up to 100% shift) of the
DW microbiome have been observed after stagnation
[37].
Pipe material is critical to DW microbiology, especially in
premise plumbing where pipe diameters are considerably
smaller than in the DN, resulting in a surface-area-to-
volume ratio increase. For a typical house, 150 m of 2 cm
diameter pipe provides a 94 000 cm2 surface for biofilm
development. With DW biofilm concentrations of up to
107 cells/cm2 [32], this implies up to 1012 DW biofilm
bacteria in a single household. It has been demonstrated
that pipe diameter [34] and material affect the biofilm
microbiome [51,53,54,55]. Metal-based materials (e.g.
copper pipes) may be effective at inhibiting short-term
biofilm formation compared to synthetic materials [54],
but this growth limitation may result in unwanted selec-
tion. For example, it was suggested that copper selects for
certain opportunistic pathogens (i.e. Legionella pneumo-
phila) and more resistant free-living amoeba [55,56].
Alternatively, polymeric substrates, shown to support
Box 1 Indigenous communities as drinking water ‘probiotics’
Concept
Altering system conditions across the entire microbiome continuum
to promote establishment of preferred indigenous communities [3].
Considerations for full-scale implementation
(1) Use biofilter seeding to continuously provide a controllable
indigenous inoculum to the distribution network [18,26].
(2) Stop final/residual disinfection as this is counterproductive to
point 1.
(3) Apply nutrient limitations to encourage biological stability [37]
and reduce risk.
(4) Smartly design system to encourage/discourage desirable/
undesirable microorganisms (e.g. hot water configuration [60],
pipe material [56]).
(5) Incorporate ‘extreme’ selective antimicrobial measures tar-
geting specific unwanted bacteria (e.g. bacteriophage or
competitor introduction) [3].
(6) Promote consumer acceptance with proof of concept research
(i.e. European systems) including detailed risk assessment.
Current Opinion in Biotechnology 2015, 33:87–94
92 Environmental biotechnology
extensive bacterial growth [31,54,57], leach from synthet-
ic pipe material long after installation [31], although the
composition and quantity of these leached polymeric
substances differ considerably between different materi-
als [57,58]. Even small amounts of synthetic material, as
added with automatic versus standard faucets, may in-
duce community shifts [8]. Pipe material selection pre-
sents one of the clearest opportunities to influence the
household microbiome, given high surface-area-to-vol-
ume ratios.
The hot water distribution system is a distinct part of the
premise plumbing system, often with a distinct micro-
biome [24,31] that presents a different risk to the con-
sumer, including the risk for Legionella infection
[3,56,59,60]. At high boiler temperatures (ca. 608C), dis-
infection may occur, but lower water heater temperatures
(i.e. 488C), and/or the combination with cold water, and/or
stagnation between uses means fluctuating lower tem-
peratures in relevant pipes [60]. Thus, initial high tem-
peratures may provide a selection mechanism followed by
more ideal temperatures for regrowth [60]. In one ex-
treme example, biofilms exposed to warm water, as in
showerheads, were shown to enrich opportunistic patho-
gens [61]. Design of the hot water distribution system,
including instantaneous hot water heaters and recirculat-
ing lines, may limit this risk [60].
Designing household solutions is difficult, as each prem-
ise varies greatly from the next. Certain prescribed mea-
sures of regrowth control may also inadvertently
encourage unwanted microbial regrowth and fail without
proper upkeep [8,39,61]. Thus, legislation with respect to
microbiology in premise plumbing is disturbingly limited.
Research opportunities:
 Establish effective premise plumbing sampling strate-
gies which balance with practicality and privacy issues.
 Provide guidance on pipe material selection consider-
ing supportive and inhibitory microbiological effects
[51,53,54,55,56,57].
 Demonstrate proposed pathogen mitigation strategies
[37,60,61] at full-scale.
Conclusions
 The DW microbiome develops along a dynamic
continuum from source to tap wherein engineered
strategies have profound microbiological impact.
 Full understanding of the DW microbiome requires a
broader and deeper assessment using advanced tools
(both quantitative and qualitative) to evaluate stage,
phase, and temporal variations.
 The challenge is to evolve from characterization toward
understanding, in order to benefit consumer and
practitioner acceptance and to drive the knowledge
Current Opinion in Biotechnology 2015, 33:87–94
toward sustainable management of the DW micro-
biome.
Acknowledgement
The authors acknowledge financial support from MERMAID, a Marie
Sklodowska-Curie Initial Training Network, under grant number 607492.
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