Water Research 159 (2019) 406e413

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Water Research
journal homepage: www.elsevier.com/locate/watres

An examination of the microbial community and occurrence of

potential human pathogens in rainwater harvested from different
roofing materials
Sungwoo Bae a, *, Juan P. Maestre b, Kerry A. Kinney b, Mary Jo Kirisits b
a
Department of Civil and Environmental Engineering, National University of Singapore, 1 Engineering Drive 2, E1A 07-03, 11576, Singapore
b
Department of Civil, Architectural, and Environmental Engineering, The University of Texas at Austin, Austin, TX, 78712, USA

a r t i c l e i n f o a b s t r a c t

Article history: While harvested rainwater can serve as an alternative water supply, microbial contaminants within the
Received 20 October 2018 collection system can negatively affect water quality. Here, we investigated the impact of roofing material
Received in revised form on the microbial quality of rainwater freshly harvested from pilot-scale roofs (concrete tile, cool, green,
29 April 2019
Galvalume® metal, and asphalt fiberglass shingle). The microbial quality of freshly harvested rainwater
Accepted 9 May 2019
Available online 14 May 2019
from six rain events over two years was analyzed by high-throughput sequencing and culture-dependent
and -independent techniques. The concentrations of total coliform were significantly different among
rainwaters harvested from the various roofing materials (p-value >0.05). However, the fecal coliform
Keywords:
Rainwater harvesting
concentrations and the copy numbers of Enterococcus 23S rRNA genes and total Bacteria 16S rRNA genes
Microbial community did not vary by type of roofing material in a statistically significant way. Potential human pathogens such
Microbial source tracking as Legionella, Escherichia coli O157:H7, Shiga-toxin-producing Escherichia coli, and adenovirus were
Waterborne pathogen detected at least once in rainwater harvested from the different roofing materials, even though the
PCR lowest occurrence of those potential human pathogens was noted from the metal roof. Also, substantial
High-throughput sequencing variation in the microbial communities from the different roofing materials was observed at the family
and genus levels. These results demonstrate that the type of roofing material affects the microbial quality
of freshly harvested rainwater, indicating that the choice of roofing material could shape the microbial
community structure entering a rainwater storage tank. Given that detection of potential pathogens in
the freshly harvested rainwater also differed between roofing materials, the type of roofing used to
capture rainwater needs to be considered in rainwater harvesting system design, particularly if the water
is intended for potable use.
© 2019 Published by Elsevier Ltd.

1. Introduction materials in RWH applications are composite asphalt shingle and

Rainwater harvesting (RWH) has received significant attention
as a supplement to traditional freshwater supplies for potable and
non-potable uses (Kim et al., 2016; Lye, 2009; Rozos and
Makropoulos, 2012). The suitability of harvested rainwater for a
particular application depends on its physical, chemical, and
microbiological qualities, which can be impacted by the type of
roofing material used in the catchment (Lee et al., 2012; Mendez
et al., 2011; van der Sterren et al., 2013). A recent survey of rain-
water harvesters in the U.S. showed that the most common roofing
* Corresponding author.
E-mail address: ceebsw@nus.edu.sg (S. Bae).
metal (Thomas et al., 2014). Some studies have shown that rain-
water harvested from metal roofs contains lower concentrations of
fecal indicator bacteria (FIB) as compared to rainwater harvested
from other roofing materials (Lee et al., 2012; Mendez et al., 2011).
While this and other basic differences in the microbiological quality
of rainwater harvested from various roofing materials have been
noted (Mendez et al., 2011), the literature does not contain a
detailed study of the impact of roofing material on the microbial
community of harvested rainwater.
Catchment surfaces can be directly exposed to sources of fecal
contamination, such as birds and small mammals; therefore, the
concentrations of FIB often have been used to assess the microbi-
ological quality of harvested rainwater (Ahmed et al., 2012; Despins

https://doi.org/10.1016/j.watres.2019.05.029
0043-1354/© 2019 Published by Elsevier Ltd.
 S. Bae et al. / Water Research 159 (2019) 406e413
et al., 2009; Kim and Han, 2011; Lee et al., 2012). It also has been
shown that harvested rainwater contains potential human patho-
gens such as Campylobacter spp., Campylobacter jejuni, Legionella
pneumophila, and Salmonella spp. (Ahmed et al. 2008, 2011a, 2012).
However, the concentration of FIB and the occurrence of potential
human pathogens generally do not correlate in harvested rainwater
(Ahmed et al., 2008; Kim et al., 2016). Thus, the utility of traditional
FIB as indicators of microbial water quality in RWH systems has
been questioned, just as FIB use has been questioned for the eval-
uation of recreational water quality (Savichtcheva and Okabe,
2006). Bacteroides has been suggested as an alternative indicator
of fecal contamination from warm-blooded animals for harvested
rainwater (Ahmed et al., 2008). Recently, microbial source tracking
(MST) has been applied to rainwater harvesting systems and
showed that the human-specific Bacteroides HF183 marker
commonly occurs in gutter debris (Waso et al., 2016) and in tank
water (Waso et al. 2016, 2018); these same studies also demon-
strated the presence of adenovirus (Waso et al. 2016, 2018) in gutter
debris and tank water.
A few studies have employed traditional tools such as culturing
and denaturing gradient gel electrophoresis (DGGE) to examine the
bacterial community of harvested rainwater and have found
phylogenetically diverse microbial communities in rainwater stor-
age tanks (Evans et al., 2007, 2009; Kim and Han, 2011). Broad
sequencing of the bacterial community in rainwater has been per-
formed in only a few studies (Ahmed et al., 2017; Chidamba and
Korsten, 2015). The microbial diversity and richness observed in
rainwater tanks were not significantly different between urban
areas and peri-urban areas (Ahmed et al., 2017), but high-
throughput sequencing revealed the presence of diverse potential
pathogenic genera in rainwater tanks (Ahmed et al., 2017;
Chidamba and Korsten, 2015).
To our knowledge, the effect of the type of roofing material on
microbiological water quality and community composition in
freshly harvested rainwater has not been thoroughly examined. In
the current study, rainwaters were freshly harvested from five
different types of roofing material and were interrogated for
microbiological water quality and community composition. The
objectives of this study were as follows: (i) to investigate the
occurrence of selected bacteria, amoebae, and viruses - some of
which are potential human pathogens - from rainwater freshly
harvested from different roofing materials; (ii) to describe bacterial
community structure using 16S rRNA gene pyrosequencing, and
(iii) to examine potential sources of fecal contamination in har-
vested rainwater using host-associated genetic markers. While the
current study is focused on the quality of freshly harvested rain-
water, it should be noted that many rainwater harvesters utilize
first-flush diversion, storage, filtration, and/or disinfection to
render the rainwater suitable for a particular use, including potable
use (Thomas et al., 2014).
2. Materials and methods
Study site and pilot-scale roofs. Rainwater was harvested from
five pilot-scale roofs (concrete tile [concrete], an acrylic-surfaced 2-
ply atactic-polypropylene-modified bituminous membrane [cool],
unfertilized green [green], Galvalume®-coated steel [metal], and
asphalt fiberglass shingle [shingle]) at the Lady Bird Johnson
Wildflower Center in Austin, Texas. Detailed descriptions of these
pilot-scale roofs have been published previously (Mendez et al.,
2011).
Rainwater sample collection. A passive sampling device was
installed at each pilot-scale roof as described previously (Mendez
407
et al., 2011). Briefly, a PVC-insert was placed in the gutter of each
roof, which directed the first-flush rainwater to a sterile 2-l glass
bottle and subsequent rainwater to a sterile 10-l polypropylene
container. A sterile polyethylene funnel (46-cm inner-diameter)
was used to collect ambient rainwater (i.e., rainwater that had no
roofing contact) in a sterile 10-l polypropylene tank. Rainwater
samples were collected during six rain events.
Culture-based enumeration of FIB. Total and fecal coliform in
ambient and harvested rainwater were measured using standard
membrane-filter procedures. Total coliform were enumerated ac-
cording to Standard Method 9222B with m-Endo broth, and fecal
coliform were enumerated according to Standard Method 9222D
with m-FC agar (APHA, 1998).
DNA extraction. To examine the microbial community in har-
vested rainwater, DNA was extracted from bulk rainwater samples.
For the first three rain events, DNA was extracted from ambient
and harvested rainwater using the UltraClean® Soil DNA kit (MO
BIO Laboratories, Carlsbad, CA). For the final three rain events,
DNA was extracted from ambient and harvested rainwater using
the Ultraclean® Water Isolation kit (MO BIO Laboratories, Carls-
bad, CA), which streamlined the isolation of DNA from the water
samples. Both kits yielded similar DNA concentration and quality.
Two liters of ambient rainwater and 0.5e4 l of harvested rainwater
(depending on how much rainwater could be filtered before
clogging the filter) were used for DNA extraction. The concentra-
tion and purity of DNA were measured with a Nanodrop® ND-
1000 Spectrophotometer (Nanodrop Technologies, Wilmington,
DE).
Quantitative, real-time PCR (qPCR) analysis. qPCR assays were
conducted for alternative FIB (host-associated Bacteroidales, uni-
versal Bacteroidales and Bacteroides spp. 16S rRNA gene) and
traditional FIB (Enterococcus 23S rRNA gene). qPCR assays also were
conducted for total Bacteria (16S rRNA gene), pathogenic E. coli
O157:H7 (rfBE), Shiga-toxigenic E. coli (stx1 and stx2), Legionella
(16S rRNA gene), five genera of free-living amoebae (Acanthamoeba,
Hartmannella, Echinamoeba, Vahlkampfia, and Naegleria), and all
known human adenovirus serotypes (hexon gene). Gene quantities
were normalized to the volume of rainwater from which DNA was
extracted so that gene concentrations could be compared among
the various rainwater samples. Amplification and detection were
carried out in 384-well plates in a ViiA™ 7 Real-Time PCR system
(Applied Biosystems, Foster City, CA). Each 10-ml PCR reaction
contained 5 ml of either qPCR MasterMix Plus (Eurogentec, San
Diego, CA) or Power SYBR® Green PCR Master Mix (Applied Bio-
systems, Foster City, CA), 2 ml of DNA template, and the optimal
primer/probe concentrations summarized in Table S1. Each run
included negative controls without DNA template. The thermal
cycler qPCR profile for host-associated Bacteroidales, universal
Bacteroidales, Bacteroides spp., and Enterococcus consisted of
2 min at 50
C, 10 min at 95
C, and 40 cycles of 15 s at 95
C and
60 s at 60
C. The thermal cycler qPCR conditions for the remaining
assays (total Bacteria, E. coli O157, E. coli - stx1 and stx2, Legionella,
free-living amoebae, and adenovirus) followed previously pub-
lished protocols (Table S1). For SYBR® Green assays, a melting curve
analysis for triplicate qPCR reactions was conducted with a serial
dilution approach to identify PCR inhibition. To confirm a positive
qPCR amplification of E. coli, Legionella, free-living amoebae, or
adenovirus, end-point PCR with the same primers (i.e., those
shown in Table S1) was applied. The purified amplicon was
sequenced at the Genomic Sequencing and Analysis Facility (GSAF)
at the University of Texas at Austin; those sequences were
compared to the National Center for Biotechnology Information
(NCBI) database using the Basic Local Alignment Search Tool
408 S. Bae et al. / Water Research 159 (2019) 406e413
(BLAST).
454 pyrosequencing analysis of the Bacterial community in
rainwater samples. Twenty microliters of extracted DNA from
ambient and harvested rainwater were sent to the Research and
Testing Laboratory (Lubbock, TX) for pyrosequencing using a 454
FLX þ Titanium Genome Sequencer (454 Life Sciences, Branford,
CT). PCR amplification was performed using primers 28F (50 - GAG
TTT GAT CNT GGC TCA G -30 ) and 519R (5’ eGTN TTA CNG CGG CKG
CTG-30 ) to amplify ~500-bp fragments of the Bacterial 16S rRNA
gene (Fan et al., 2012). Sequences generated from pyrosequencing
analysis of the amplicon were processed using the Quantitative
Insight into Microbial Ecology (QIIME) pipeline (Caporaso et al.,
2010) with the default settings. Briefly, we used the USEARCH
script built in QIIME on a dataset of de-multiplexed sequences to
filter noise, remove chimeras, and pick operational taxonomic units
(OTUs). Taxonomy assignments were performed using the Ribo-
somal Database Project (RDP) classifier with clustering at 97%
sequence identity. The alignment was performed with the PyNAST
algorithm against the Greengenes reference dataset. After rarefying
the number of reads to that of the lowest sample, alpha- and beta-
diversity analyses were performed using the default recommen-
dations for QIIME parameters.
Statistical analysis. The nonparametric Spearman rank corre-
lation was used to examine the correlation between the concen-
trations of culturable FIB and specific genetic marker
concentrations (e.g., Enterococcus, Bacteroidales, Bacteroides, and
total Bacterial 16S rRNA genes). Additionally, binary and ordered
logistic regressions were applied to examine the correlation be-
tween the frequency of occurrence of genetic markers for E. coli,
Legionella, free-living amoebae, and adenovirus and the concen-
tration of FIB (culturable or measured with genetic markers). The
Kruskal-Wallis one-way analysis of variance was used to compare
concentrations of culturable FIB and genetic markers for FIB
among rainwaters harvested from different roofing materials. For
samples with non-detects of culturable FIB, the limit of detection
(1 colony/100 ml) was substituted (Antweiler and Taylor, 2008) to
perform the correlation analysis; for samples with non-detects of
genetic markers, one-half of the detection limit (1.25  102 gene
copies (gc)/100 ml for the Enterococcus assay and 1.13  102 gc/
100 ml for the Bacteroides [AllBac] assay) was substituted
(Antweiler and Taylor, 2008). Statistical analyses were performed
using Minitab 16 statistical software (Minitab Inc., State College,
PA). Rarefaction curves for 16S rRNA genes were generated from
QIIME (Caporaso et al., 2010) and were used to compare the
richness observed in different samples at the same sequence
depth of 4000 reads.
3. Results
3.1. Concentration of FIB in harvested rainwater and ambient
rainwater
The concentrations of total coliform (Fig. 1A) and fecal coliform
(Fig. 1B) in harvested and ambient (no direct contact with the roof)
rainwater were assessed using culture-based methods. Of the 36
samples (30 harvested rainwater and 6 ambient rainwater samples)
collected over six rain events, 92% were positive for total coliform
and 89% were positive for fecal coliform, respectively. The con-
centrations of total coliform and fecal coliform varied by orders of
magnitude in the ambient rainwater and harvested rainwater
across the six events. We performed Kruskal-Wallis one-way
analysis of variance to evaluate the concentration of total and fecal
coliform in harvested rainwater samples from different roofing
Fig. 1. The concentrations of (A) total coliform and (B) fecal coliform in harvested
rainwater and ambient rainwater samples collected from six rain events.
ND indicates non-detect. For instance, 1 ND means no coliform were detected in one
sample out of the six samples collected at that location. In the plot, the upper and
lower lines of the box indicate the first and third quartiles, the central horizontal line
represents the median, the white square represents the mean, and the black triangles
indicate the minimum and maximum values.
materials. The total coliform concentrations in rainwater were
significantly different among the roofing materials (p-value<0.05).
The median concentration of total coliform was highest in rain-
water collected from the shingle roof (3.6 x 103 colony-forming
units [CFU]/100 ml) and lowest in rainwater collected from the
green roof (3.3 x102 CFU/100 ml) followed by the metal roof (1.1 x
103 CFU/100 ml). Although the Kruskal-Wallis analysis showed that
the fecal coliform concentrations in the harvested rainwater were
not significantly different among the roofing materials (p-
value>0.05), the fecal coliform in the rainwater harvested from the
green roof had the lowest median concentration.
The concentrations of genetic markers for FIB, including
Enterococcus, host-associated Bacteroidales, universal Bacter-
oidales (BacUni-UCD), the Bacteroides spp. (AllBac), and for total
Bacteria (16S rRNA gene) were determined by qPCR in harvested
rainwater. Enterococcus was detected in 61% of the rainwater
samples (Fig. 2). The qPCR assays for universal Bacteroidales
(BacUni-UCD, which targets all Bacteroidales regardless of the
fecal source) and cow-, dog- and human-associated Bacteroidales
were non-detects in all harvested rainwater samples (data not
shown), but the other qPCR assay for the Bacteroides spp. (AllBac)
showed detections in 63% of the harvested rainwater samples
(Fig. 2). The Kruskal-Wallis one-way analysis of variance test
showed no significant difference among the rainwaters harvested
from the various roofing materials with respect to their concen-
trations of genetic markers for Enterococcus and Bacteroides. In
Fig. 2, the average total Bacteria 16S rRNA gene concentrations
were 5.26  109 gene copy number/100 ml in harvested rainwater,
indicating the high abundance of bacteria in harvested rainwater.
Nonparametric Spearman rank correlations were conducted to
examine the correlation between the concentrations of culturable
FIB and genetic markers for Enterococcus and Bacteroides. Total
coliform concentrations were positively correlated with the con-
centrations of both Bacteroides and Enterococcus in harvested
rainwater (Spearman's rank correlation coefficient r ¼ 0.64, p-
value < 0.05) (see Table 1).
 S. Bae et al. / Water Research 159 (2019) 406e413 409

Fig. 2. The concentration of (A) total Bacteria, (B) total Bacteroides (AllBac), and (C) Enterococcus measured by qPCR in harvested rainwater and ambient rainwater from six rain
events. ND indicates non-detection. The upper and lower lines indicate the first and third quartiles, the central horizontal line represents the median, the black square represents
the mean, and the upper/under whiskers indicate the minimum and maximum values.

Table 1
Occurrence of selected viruses, bacteria, and amoebae as shown by qPCR of ambient rainwater or freshly harvested rainwater from different roofing materials.

Source Sample Type Incidence of target gene per number of events sampled

E. coli O157 E. coli stx1 E. coli stx2 Adenovirus Legionella Amoebae Totals

Harvested rainwater Ambient rainwater 0/6 0/6 0/6 0/6 1/6 1/6 2/36
From green roof 1/6 0/6 0/6 0/6 3/6 4/6 8/36
From metal roof 0/6 0/6 1/6 0/6 0/6 2/6 3/36
From shingle roof 0/6 0/6 2/6 0/6 0/6 4/6 6/36
From concrete roof 0/6 0/6 2/6 1/6 2/6 5/6 10/36
From cool roof 0/6 1/6 1/6 0/6 1/6 4/6 7/36
Totals 1/36 1/36 6/36 1/36 7/36 20/36

3.2. Genetic markers for potential pathogens, free-living amoebae,
and viruses in rainwater harvested from different roofing materials
Genetic markers for selected potential pathogens, free-living
amoebae, and viruses were assayed by qPCR; positive results
from qPCR were confirmed by end-point PCR and sequencing to
verify the target gene identity. E. coli O157, E. coli stx1, and adeno-
virus were detected only once in the harvested rainwater samples,
while E. coli stx2 and Legionella occurred more frequently in the
harvested rainwater samples (Table 2). E. coli stx2 was detected in
rainwater harvested from the metal and shingle roofs, and Legion-
ella was detected in rainwater harvested from the green, concrete,
and cool roofs. As compared to potential pathogens and viruses,
free-living amoebae occurred more frequently in the harvested
rainwater samples. The collective occurrence of potential bacterial
pathogens, free-living amoebae, and viruses in harvested rainwater
follow in order from highest to lowest: concrete, green, cool,
shingle, and metal roofs, followed by ambient rainwater (Table 2).
Binary logistic regressions were utilized to examine the correlation
between the concentration of FIB (by culture- or molecular-based
methods) and the presence of selected organisms in harvested
rainwater. None of the FIB were significantly correlated with all of
the selected organisms; however, a few individual correlations
were noted. Table 2 shows evidence of a correlation between the
410 S. Bae et al. / Water Research 159 (2019) 406e413

Table 2
Binary logistic regression analysis of FIB concentrations and the presence of selected microorganisms.

p-value for correlation and the goodness-of-fit testa

Fecal Legionella E. coli O157 E. coli stx1 E. coli stx2 Adenovirus Amoebae
indicator
Correlation Fit of Correlation Fit of Correlation Fit of Correlation Fit of Correlation Fit of Correlation Fit of
(p-value) model (r2) (p-value) model (r2) (p-value) model (r2) (p-value) model (r2) (p-value) model (r2) (p-value) model (r2)

Total 0.148 0.55 0.757 1.00 0.499 1.00 0.039 0.66 0.885 0.69 0.92 0.45
coliform
Fecal 0.850 0.74 0.791 1.00 0.558 1.00 0.024 0.60 0.529 0.79 0.36 0.81
coliform
Enterococcus 0.204 0.61 0.704 0.99 0.978 0.98 0.848 0.30 0.664 0.78 0.138 0.65
Bacteroides 0.030 0.49 0.925 0.99 0.046 1.00 0.563 0.25 0.928 0.99 0.353 0.48
a
p-values were calculated from binary logistic analysis. p-values indicate whether the model fit the data adequately. Significant correlations (p-value < 0.05) are shown in
bold.

presence of E. coli stx2 and the concentration of total coliform (p-
value ¼ 0.039) and fecal coliform (p-value ¼ 0.024). The concen-
tration of Bacteroides shows correlation with the presence of
Legionella (p-value ¼ 0.03).
3.3. Microbial community in rainwater harvested from different
roofing materials
We investigated the microbial community in ambient rainwater
and rainwater harvested from different roofing materials. Quality
filtering recovered an average number of 5139 reads per sample,
with an average read length of 420 bp. Thirteen phyla were
recovered from the rainwater samples, with Proteobacteria being
the dominant phylum for rainwater harvested from all roofing
materials, accounting for 61e97% of the OTUs detected in Fig. 3A.
The second most common phylum was Bacteroidetes in the ambient
rainwater and rainwater harvested from the green and shingle
roofs. The microbial communities also were examined at the family
and genus levels, and substantial variation among the microbial
communities was observed (Fig. S1). The microbial communities of
rainwater harvested from the cool and green roofs were dominated
by Betaproteobacteria, with a majority belonging to Oxalobacter-
aceae and Comamonadaceae at the family level (Fig. S1); Methyl-
obacteriaceae of the Alphaproteobacteria dominated the microbial
community in rainwater harvested from the shingle roof; and
Enterobacteriaceae and Pseudomonadaceae dominated the micro-
bial community in rainwater harvested from the metal roof. At the
genus level, Methylobacterium and Pseudomonas were most
frequently detected in rainwater harvested from the cool and
shingle roofs, whereas Janthinobacterium and Spirosoma, both
genera of Gram-negative soil bacteria, were the dominant genera in
rainwater harvested from the green roof. Also, the nitrite-oxidizing
genus, Nitrospira (1.25% relative abundance), was detected in
rainwater harvested from the metal roof, which contained a higher
nitrite concentration in its harvested rainwater as compared to
rainwater harvested from other roofing materials in our previous
study (Mendez et al., 2011). The genera present only in harvested
rainwater (i.e., not found in ambient rainwater) include Williamsia,
Nocardia, Granulicatella and Methylosinus for the concrete roof,
Pantoea for the green and shingle roofs, and Gallionella for the
metal roof.
The OTUs and diversity indices for the microbial communities in
ambient and harvested rainwater are shown in Fig. 3B. The highest
number of unique OTUs was observed in ambient rainwater, and
the lowest number was found in the rainwater harvested from
shingle roof (Fig. 3B). The Shannon diversity index indicates the
highest diversity in the ambient rainwater sample followed by (in
descending order) rainwater harvested from the green, concrete,
metal, shingle and cool roofs. Chao1 and abundance-based
coverage estimators (ACE) also indicate that the ambient rain-
water sample showed the highest diversity followed by the rain-
water harvested from the green roof. The inverse of Simpson's
original index (InvSimpson) showed that the ambient rainwater,
the concrete and green roofs had diverse microbial communities.
The shared OTUs between the ambient rainwater and the rainwater
harvested from the different roofing materials was calculated,
suggesting that a major fraction of the microbial community in
harvested rainwater originates outside of the ambient rainwater
itself (Table S2).
4. Discussion
Microbial water quality and microbial communities in rainwater
harvested from five different roofing materials were examined by
culture-based and molecular methods for six rain events over two
years. Most previous studies have investigated microbial water
quality in rainwater storage tanks, where the sampled water has an
unknown residence time; however, only a few studies have
examined the microbial quality of ambient rainwater and rainwater
freshly harvested from roofs (Dobrowsky et al., 2017; Gikas and
Tsihrintzis, 2017; Mendez et al., 2011). In this study, we examined
the concentration of FIB and the occurrence of free-living amoebae
and potential human pathogens (viruses and bacteria) in ambient
rainwater and rainwater freshly harvested from co-located pilot
roofs constructed with five different roofing materials.
The occurrence of potential pathogens was different among
rainwaters harvested from different roofing materials, suggesting
that the type of roofing material exerts a selective pressure on the
microbial community. Additionally, the abundance of certain mi-
croorganisms in the harvested rainwaters was higher than that in
ambient rainwater (i.e., no direct contact with the roof). For
instance, the concentration of Bacteroides and Enterococcus were
lower and often non-detected in ambient water. Shiga-toxin genes
(stx1 and stx2) were detected in 19% of the harvested rainwater
samples. E. coli producing a Shiga-like toxin, especially serotype
O157, is an important emerging human pathogen in water (Heijnen
and Medema, 2006). Interestingly, we detected adenovirus DNA
once and only in rainwater harvested from the concrete roof. Ad-
enoviruses are important human pathogens that are responsible for
enteric illnesses as well as for respiratory and eye infections (Jiang,
2006). It has been reported that human adenovirus has been
detected up to 52% of harvested rainwater tanks (Waso et al. 2016,
2018). In contrast to previous studies which frequently detected
adenovirus in rainwater storage tanks (Waso et al. 2016, 2018),
adenovirus was detected rarely in our freshly harvested rainwater
samples suggesting that sediments in rainwater tanks may harbor
adenovirus as virus adsorption to sediment has been shown to
greatly increase survival time.
 S. Bae et al. / Water Research 159 (2019) 406e413 411

Fig. 3. (A) The relative abundance at the phylum level and (B) the alpha-diversity in the microbial communities of rainwater harvested from different roofing materials. In panel (B),
“observed” represents the number of unique OTUs found in the sample at 97% sequence identity; Chao, ACE (abundance-based coverage estimator), Shannon and InvSimpson
(Inverse Simpson) indices estimate the OTUs richness and diversity.

The presence of non-enteric potential human pathogens, such as
Legionella spp., in rainwater harvesting systems has been reported
in the United States, Australia, and New Zealand (Ahmed et al.
2010a, 2010b; Kim et al., 2016; Lye, 2002; Simmons et al., 2008). In
our study, seven out of thirty-six rainwater samples were positive
for Legionella. Free-living amoebae act as environmental reservoirs
for Legionella, aiding in the propagation and distribution of
Legionella. In our study, Legionella always co-occurred with free-
living amoebae, suggesting that amoebae could be acting as
Legionella reservoirs in these systems (Greub and Raoult, 2004).
Since most amoebae are known to be disinfection-resistant (Loret
et al., 2008), multiple treatment barriers (e.g., filtration and disin-
fection) are strongly recommended for treatment of harvested
rainwater for potable use. Thus, properly designed storage tanks
and treatment systems are necessary for harvested rainwater to be
used for potable purposes (Kim et al., 2016; Lye, 2009).
Our study aimed to identify potential human pathogens and to
examine microbial community structure in rainwaters freshly
harvested from different roofing materials. Understanding the
impact of roofing material on microbial community structure and
the presence of potential human pathogens in harvested rainwater
will provide insight into the design of rainwater harvesting catch-
ments as well as to guide disinfection strategies. The number of
OTUs observed (Sobs), the Chao estimator, and the Shannon index
for the microbial communities in ambient and harvested rainwater
demonstrated that the type of roofing material affected microbial
diversity (Fig. 3B). The microbial community of the ambient rain-
water is highly diverse and substantially different in composition as
compared to the microbial communities of the harvested rainwa-
ters. The major Bacterial phyla detected in the harvested rainwater
412 S. Bae et al. / Water Research 159 (2019) 406e413
in the current study were Proteobacteria, Bacteroidetes Actino-
bacteria and Firmicutes; this microbial distribution is similar to that
reported in previous studies of rainwater harvesting systems using
cultivated isolates, DGGE, and high-throughput sequencing
(Ahmed et al., 2017; Evans et al., 2009; Kim and Han, 2011).
An increase in the relative abundance of the Proteobacteria
phylum in harvested rainwater as compared to ambient rainwater
implies that the roofing materials could offer favorable niches for
Proteobacteria. The most abundant and frequently detected taxa
included Burkholderiales, Sphingobacteriales, and Pseudomonadales,
which are similar to the taxa reported previously from cultivated
isolates in rainwater storage tanks (Evans et al., 2009). Although
relative abundances at the phylum level were similar to those
found in a previous study where high-throughput amplicon
sequencing was applied (Ahmed et al., 2017), our study showed
differences at the family level as compared to Ahmed et al. (2017);
we found Methylobacteriaceae, Oxalobacteraceae, Pseudomonada-
ceae, and Comamonadaceae to be the most abundant families in
freshly harvested rainwater, while Comamonadaceae and Plancto-
mycetaceae were the most abundant families in aged rainwater
tank samples (Ahmed et al., 2017).
Several problems associated with using FIB to indicate human
health risk are known, including the weak correlation between FIB
and the presence of particular pathogens and the lack of indication
of the source of the fecal contamination (Savichtcheva and Okabe,
2006). Alternatively, the application of PCR-based methods has
been used to identify fecal pollution and specific pathogens in
surface water and rainwater (Ahmed et al. 2008, 2011b, 2012;
Kaushik and Balasubramanian, 2012; Layton et al., 2006). Our data
show a correlation between total or fecal coliform concentrations
and the presence of E. coli stx2 (p-value < 0.05; Table 2), which
makes sense because E. coli is a subset of the total and fecal coli-
form. More broadly, and consistent with previous reports (Ahmed
et al., 2008; Lye, 2002), our results demonstrate that culture-
based and (q)PCR methods for FIB provide an inadequate assess-
ment of the overall microbial quality of rainwater due to the poor
correlation between those FIB and a suite of potential human
pathogens (E. coli O157, E. coli stx1, and adenovirus). Therefore, a
suitable bacterial indicator assay still needs to be developed for
harvested rainwater.
Culturable FIB and MST markers have been applied to investi-
gate presence or quantity of pathogens and their fecal sources in
harvested rainwater (Ahmed et al. 2011b, 2014, 2017; Waso et al.
2016, 2018). For instance, some studies have shown the presence
of the human-associated Bacteroides HF183 marker and possum
fecal DNA in rainwater tanks (Ahmed et al., 2016; Dobrowsky et al.,
2017; Waso et al. 2016, 2018). In our study, only two of six ambient
rainwater samples (i.e., rainwater that had no contact with roofing
materials) were positive for the AllBac marker (targeting Bacter-
oides), while rainwaters harvested from all five roofing materials
were positive for this marker, suggesting that this contamination
originates from direct fecal deposition to the roof or dry atmo-
spheric deposition rather than from wet atmospheric deposition
(rainfall). The fraction of the genus Bacteroides present in the
overall bacterial community of harvested rainwater was low
(~0.0001%, with an average of 104 gc/100 mL for the genus Bacter-
oides and an average of 1010 gc/100 mL for Bacteria in our study
(Fig. 2)). Similarly, our 16S rRNA gene sequencing results demon-
strated that the order of Bacteroidales was rare in all harvested
rainwater samples (data not shown). Thus, our data suggest that the
use of the BacUni-Bacteroidales and AllBac markers might be
insufficient to discern fecal contamination in harvested rainwater
samples due to their low abundance relative to other bacteria
present in the harvested rainwater and poor correlations with po-
tential human pathogens (Table 2).
5. Conclusion
 The microbial communities in harvested rainwater were
different from that of ambient rainwater and varied as a function
of the type of roofing material, suggesting that choice of roofing
material could shape the microbial community structure
entering a rainwater storage tank.
 Given that rainwater harvested from the metal roof showed the
lowest occurrence of potential bacterial and viral pathogens
among the tested roofing materials, metal roofing materials
could be recommended as one way to positively affect the mi-
crobial community structure of freshly harvested rainwater.
 The concentration of culturable fecal indicator in freshly har-
vested rainwater was not clearly correlated with the presence of
potential pathogens such as Legionella spp., pathogenic E. coli,
and adenovirus suggesting that development of a more suitable
bacterial indicator is needed to monitor microbial water quality
in rainwater harvesting systems.
 The selection of an appropriate roofing material for rainwater
catchment is an important first step in the design of a roof-
harvested rainwater system. Given the microbial diversity pre-
sent in freshly harvested rainwater, a multi-barrier treatment
strategy, and maintenance practices appropriate for the inten-
ded use of the harvested rainwater are needed to ensure the
long-term success of harvested rainwater systems.
Acknowledgements
This research was supported by the Texas Water Development
Board (contract number 0804830855). We thank Carolina Mendez
for sample collection and DNA extraction, Sarah Keithley for criti-
cally reading the manuscript, and Taegyu Kim for editing assistance.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.watres.2019.05.029.
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