Water Research 130 (2018) 47e57

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Droplet distribution and airborne bacteria in an experimental shower

unit
C.E. Estrada-Perez b, K.A. Kinney c, J.P. Maestre c, Y.A. Hassan b, M.D. King a, *
a
Department of Biological and Agricultural Engineering, Texas A and M University College Station, TX, USA
b
Department of Nuclear Engineering, Texas A and M University College Station, TX, USA
c
Department of Civil, Architectural and Environmental Engineering, The University of Texas at Austin, USA

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

Article history: Although human exposure to water aerosols is common in residential showers, the droplet distribution
Received 31 May 2017 patterns generated in showers are not well understood nor is the bacteria released during shower
Received in revised form operation. In this study, a two-phase flow Particle Tracking Velocimetry (PTV) algorithm was successfully
10 November 2017
used to characterize the spatial spray pattern and velocity field in two experimental showers (one low-
Accepted 15 November 2017
Available online 16 November 2017
flow and one high-flow). In addition, the airborne bacteria present in the shower over nearly 5 months of
controlled operation was determined for both showers. The results indicate that the droplet velocity out
of the low-flow showerhead (which had fewer orifices) was significantly higher than that out of the high-
Keywords:
Shower
flow showerhead resulting in a higher aerosol number concentration in the low-flow shower and more
Particle tracking velocimetry (PTV) consistent wetting of the shower wall. Both showerheads generated droplets in the respirable range and
Wetted wall cyclone (WWC) genera of potential health concern were observed in the shower aerosols measured both prior to and
Bacterial aerosols following shower operation. The study provides one of the first visualizations of droplet spray patterns in
Microbiome residential showers and provides insight into the airborne bacteria present in showers.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction WaterSense, a voluntary partnership program sponsored by the U.S.

Water sprays are widely used for applications that range from
dispersing herbicides and washing down equipment in agricultural
or industrial facilities to running showers in residential settings
(Creech et al., 2015; Zhou et al., 2007). Water sprays in both occu-
pational and domestic settings may contain harmful chemicals or
bioparticles that, once aerosolized, pose an inhalation risk to hu-
man health (de Man et al., 2014; Barker et al., 2017; Angenent et al.,
2005). The extent to which potentially harmful particles can
penetrate the human respiratory system is strongly influenced by
the characteristics of the microbial or chemical agents present in
the aerosolized water and the size distribution of the aerosolized
droplets generated in the spray system (Thomas, 2013; Kleinstreuer
et al., 2008; Steiling et al., 2014). Spray nozzles are often designed to
achieve flow patterns needed for a specific industrial application
(Issa, 2009; Castillo et al., 2001) and/or to meet water conservation
goals via, for instance, the distribution of low-flow showerheads to
residents in water-stressed municipalities (Wong et al., 2016).
* Corresponding author.
E-mail address: mdking@tamu.edu (M.D. King).
Environmental Protection Agency (EPA), is both a label for water-
efficient products and a resource for saving water. Nationwide, if
WaterSense labeled showerheads with a flow rate of 2.0 gpm or less
were purchased for all normal showerhead replacements and
installed in all new construction, the estimated water savings could
reach 30.3 billion gallons per year. Estimated national energy sav-
ings could exceed 1.6 billion kWh of electricity and 10.9 million
cubic feet (Mcf) of natural gas each year (WaterSense® Specification
for Showerheads; www.epa.gov). Several studies have been con-
ducted to characterize the droplet size and velocity distributions in
spray systems (Ferreira et al., 2010) including a few that have
focused on aerosols generated during residential shower operation
(Zhou et al., 2007). However, more advanced techniques now
available for visualizing flow patterns (Kim et al., 2011; Cloeter
et al., 2010) have not yet been applied to residential showers. In
addition, the links between showerhead design, droplet spray
pattern and the bacteria aerosolized in residential showers are not
well understood.
Aerosolization of tap water from residential showerheads can
generate bioaerosols that contribute to the indoor microbiome
(Prussin and Marr, 2015; Perkins et al., 2009). Even with

https://doi.org/10.1016/j.watres.2017.11.039
0043-1354/© 2017 Elsevier Ltd. All rights reserved.
48 C.E. Estrada-Perez et al. / Water Research 130 (2018) 47e57
disinfection of the water leaving municipal treatment plants, tap
water used in residential showers contains a diverse microbial
community that contributes to the airborne microbial community
as well as to the development of biofilms, some of which can harbor
potential pathogens that are resistant to biocidal treatment (Taylor
et al., 2000; Soto-Giron et al., 2016). Although many microorgan-
isms are not of concern, potentially pathogenic bacteria identified
in tap water include Legionella pneumophila, Mycobacteria, Bur-
kholderia, Streptococcus, Anaerococcus and Corynebacterium (Bauer
et al., 2008; Falkinham et al., 2008; Krojgaard et al., 2011;
Herna
ndez-Gardun ~ o and Elwood, 2012; Diaz-Flores et al., 2015).
Similarly, potentially pathogenic bacteria have been detected on
shower curtains (Kelley et al., 2004), in biofilms within shower-
heads (Feazel et al., 2009) as well as in the bioaerosols generated
during showering (Perkins et al., 2009).
An important consideration when evaluating potential human
exposures during showering is the size distribution of the aerosols
generated. In an experimental shower stall fitted with a manikin to
generate a realistic spray pattern, Zhou et al. (2007) confirmed that
respirable droplets are generated during showering but the effect of
showerhead design was not considered. Quantitative microbial risk
assessment models rely on this type of data as well as other pa-
rameters to assess the risk posed by specific pathogens such as
Legionella spp. (Hamilton and Haas, 2016). Another important
feature of shower systems is the velocity of the spray generated and
the effect of showerhead design on the distribution of droplets
within the shower stall (Pana ~o and Delgado (2014). There has been
a concerted effort in many municipalities to replace standard, high-
flow (2.5 gpm, 9.46 lpm) showerheads with water efficient, low-
flow (<2 gpm ¼ <7.57 lpm) showerheads. The effect of shower-
head design on the spray pattern generated has not been examined
yet although recent advances in two phase flow visualization make
such investigations possible. Particle tracking velocimetry (PTV) is
one such technique that is typically used for visualizations but
which can also be used as a tool to measure the motion of indi-
vidual objects (including droplets) in experimental images. The
object tracking capability of the PTV algorithm utilized in the cur-
rent study has previously been validated for two-phase flow
measurements (Kataoka et al., 1986; Estrada-Perez, 2004; Estrada-
Perez and Hassan, 2010; Estrada Perez et al., 2015). Using object
characteristics such as size and shape enables the easy discrimi-
nation of phases, allowing the independent study of liquid and gas
velocities within a single image, and providing a series of images for
statistical analysis of the results.
The goal of the current study was to assess the effect of two
different showerhead designs on the liquid droplet distribution and
bacterial release in two full-scale experimental showers. The
experimental shower stalls were identical except one was equipped
with a low-flow showerhead and the other with a high-flow
showerhead. Particle Tracking Velocimetry experiments were
conducted to assess the water droplet size, direction and speed in
each shower. In addition, the quantity and composition of the
bacterial aerosols released over five months of daily operation were
determined in each shower.
2. Materials and methods
2.1. Experimental shower unit
A 9 m  6 m x 3 m tent within a temperature-controlled
building was constructed to accommodate a full-scale, residential
dual shower unit (Sterling by Kohler Advantage White Shower Kit
(Model # 62040100-0), Fig. 1). The two shower stalls have a total
volume of 2.5 m3 (1.52 m width (0.76 m for each stall)  0.86 m
depth  1.93 m height) with a 0.75-m gap between the top of each
shower wall and the tent ceiling. The two showers were separated
in the middle with a vinyl shower curtain. Also the front opening of
each shower unit was covered by a vinyl shower curtain. A Heimlich
maneuver training manikin/torso (Model #1602; Simulaids Inc.,
Saugerties, NY) was mounted below the showerhead in each
shower to create realistic splash effects and droplet flow pattern
conditions similar to those found in occupied residential units. The
showers were equipped with two different showerheads. In the left
shower was a low-flow showerhead (Delta 1-Spray 1.5 gallons per
minute (gpm) Water-Efficient Shower Head in Chrome with 12
nozzles; Model # 52653-PK; Table 1, Fig. 1); in the right shower was
a high-flow showerhead (MOEN 2.5 gpm Banbury 5-Spray 4 in.
Shower Head in Spot Resist Brushed Nickel with 69 nozzles, Model
# 23045SRN; Table 1, Fig. 1). The showerheads were positioned at
approximately a 30-degree angle above the manikin heads. The
water supply to the shower consisted of chlorine-disinfected
groundwater supplied by the City of Bryan. Regarding the physi-
cochemical and microbiological characteristics of the Bryan
municipal water that was used to supply the experimental shower
units, the water had a chlorine residual that ranged between 0.42
and 3.10 ppm, with the pH at about 8.5. The detected level for ni-
trates was 0.41 ppm. (City of Bryan, 2014). The temperature of the
water in the pipes between individual shower events was
measured to be between 23  Ce25  C.
An electric water heater (Whirlpool E1F20US015V 120 V) with a
capacity of 20 gallons was used to supply hot water (45e46  C) for
the shower testing. During shower operation, the hot water was
mixed continuously with cold water for a realistic shower event
and to maintain the same water temperature for both sides of the
shower.
2.2. Shower droplet sizing and particle tracking velocimetry (PTV)
An Aerodynamic Particle Sizer (APS 3321, TSI Inc., Burnsville,
MN) was used to determine the particle size distribution of the
shower aerosols generated during shower operation. The APS has
an upper size limit of 20 mm and thus was not used to measure the
large shower droplets but rather the aerosols in the respirable
range (less than 10 mm), as shown in the studies by Zhou et al.
(2007), Cowen and Ollison (2006) and Xu and Weisel (2003). The
particle number and mass size distributions of the aerosols
generated during shower operation were measured by the APS
instrument via 10 replicate measurements, 20 s each, at two loca-
tions (one upper and one lower level, at about 1.2 m and 0.6 m
height) in each shower stall as indicated in Fig. 1.
PTV is a full-field, non-intrusive flow visualization technique
that can capture information on flow characteristics with high
spatial and temporal resolution. Instantaneous fluid velocities can
be determined using the PTV technique by recording the position of
images at successive time instants via detection of fluorescent
materials suspended in the fluid or by distinguishing the optical
characteristics of the fluid (e.g, water) droplets. The underlying
assumption is that these tracers closely follow the fluid motion
with minimal lag (Estrada-Perez and Hassan, 2010). However, in
this study, no tracers were required because the droplets could be
analyzed based on their reflection of light. The accuracy of the PTV
technique has been tested against international benchmarks
(Stanislas et al., 2005), where the algorithm uncertainties yielded
subpixel accuracy of about 0.1056 pixel/Dt, (with Dt ¼ time be-
tween frames).
In each experimental shower stall, the water droplet flow from
each showerhead was recorded with a high-speed high-resolution
camera (Photron USA Inc., San Diego, CA). A total of five trials were
completed for each showerhead with two different camera views,
namely top and bottom views, illuminated by a continuous halogen
 C.E. Estrada-Perez et al. / Water Research 130 (2018) 47e57 49

Fig. 1. (a) Experimental shower unit with two separate but identical shower stalls each equipped with identical manikins but different showerheads (low-flow (1.5 gpm; 12
nozzles), left; high-flow (2.5 gpm; 69 nozzles), right). White and black X symbols indicate the location for the particle size measurements in the upper and lower levels of each
shower stall, respectively. Black arrows and dotted line indicate the wall seam where the upper and lower shower stall parts meet. (b) Measurement of the different nozzle di-
ameters on the low-flow (1.5 gpm) and high-flow (2.5 gpm) showerhead.

lamp (Fig. 1). The top view covered the upper shower region which Table 1
included the showerhead and most of the manikin's torso, while Flow calculations based on the diameters for the different types of nozzles on the
showerheads.
the bottom view covered the lower part of the manikins and was
intended to capture the behavior of the bouncing droplets before QShowerhead Nozzle diameter QNozzle Nozzle QTotal Nozzle
hitting the wall or ground. The high-speed camera was operated at gpm (lpm) mm gpm (lpm) # gpm (lpm)

1000 frames per second. For each trial, 5000 images were captured Low-flow
and over a period of 4 s 4000 frames were selected. 1.5 (5.678) 1.3 0.125 (0.473) 12 1.5 (5.678)
High-flow
2.5 (9.463) 0.97 0.011 (0.042) 48 0.538 (2.038)
2.3. PTV algorithm 1.68 0.034 (0.127) 9 0.303 (1.146)
2.34 0.065 (0.247) 6 0.391 (1.482)
4.21 0.211 (0.799) 6 1.267 (4.798)
To determine the droplet velocity fields from the experimental
images, a particle tracking velocimetry (PTV) algorithm originally
developed by Hassan et al. (1992a, 1992b) and updated as needed
for different applications (Estrada-Perez and Hassan, 2010) was some cases a local gray-scale threshold is suggested. In the present
used. Sensitivity studies performed previously with experimental study, the illumination distribution was fairly constant throughout
and synthetic images (Estrada-Perez, 2004; Stanislas et al., 2005) the entire image; therefore, there was no need to implement a local
provide confirmation that PTV can be used successfully for study- gray-scale threshold. The mask correlation threshold allows
ing the velocity of particles non-intrusively in multiphase flows. assignment of a cut-off value for the mask correlation coefficient.
The main processes of the PTV algorithms are: (1) particle The cut-off depends on the signal to noise ratio within the exper-
detection, (2) particle centroid location estimation and (3) particle imental images and is a user input threshold to discriminate pixels
matching in between consecutive frames (particle tracking). A representing a droplet from those representing the background.
detailed description of the PTV algorithm can be obtained from Generally, it is obtained with a trial and error process until the al-
Estrada-Perez (2004). The components of the algorithm that are gorithm selects only the objects to be tracked, in this case, until all
new or relevant for the present study are described below. the droplets are appropriately discriminated from the background.
Pixels with coefficients larger than the cut-off value are considered
2.4. Procedure for particle detection part of a particle. The coefficient value depends on the likelihood of
a pixel to represent the central pixel of an ideal particle template.
The object identification procedure classifies every pixel of the This ideal template is generated by assuming that the light reflected
image into two parts: pixels part of a particle (in this study, a from an ideal particle will follow a 2D Gaussian distribution as
droplet) and pixels part of the background. To this end, two follows:
discriminatory thresholds are considered: The gray-scale threshold
. . !
and the mask correlation threshold. The gray-scale threshold is
ðx  x0 Þ2 a2 þ ðy  y0 Þ2 b2
used to discern whether or not a pixel is part of a particle based on Iðx; yÞ ¼ I0 exp
its gray-scale intensity, and the mask correlation threshold which is 2d20
based on the approach of Takehara (1998). For this approach, it is
assumed that the gray-scale intensity distribution of an ideal par- where I is the image pixel intensity, I0 is the maximum gray-scale
ticle has a Gaussian distribution with the peak on the center of the intensity, (x0, y0) is the centroid location of the ideal particle, a
particle, i.e., brighter pixels are more likely to be part of a particle and b are shape modification constants to make the ideal particle
and to be located near the center of such particle. The gray-scale into ellipsoidal shapes, and d0 is the particle diameter. The range of
threshold is sensitive to non-uniform illumination; therefore in x and y will determine how large the template is. Typically x and y
50 C.E. Estrada-Perez et al. / Water Research 130 (2018) 47e57
are larger than d0.
Once a correlation value is assigned to each pixel, they are
combined together in groups of pixels that will form an object (part
of a particle or part of the background). The centroids of the
identified particles are calculated using sub-pixel interpolation. If
the interpolation fails, the center of gravity method is used to locate
the center with sub-pixel accuracy.
2.5. Procedure for particle centroid estimation
Once the pixel representing the droplet centroid is located,
Gaussian three-point sub-pixel interpolation and center of gravity
methods are used to determine the droplet centroid location plus
or minus a sub-pixel correction with subpixel accuracy. The sub-
pixel corrections εx for the x coordinate and εy for the y coordinate
are expressed as:
logðIðx0  1; y0 ÞÞ  logðIðx0 þ 1; y0 ÞÞ
εx ¼
2 logðIðx0  1; y0 ÞÞ þ logðIðx0 þ 1; y0 ÞÞ  2 logðIðx0 ; y0 ÞÞ
and
logðIðx0 ; y0  1ÞÞ  logðIðx0 ; y0 þ 1ÞÞ
εy ¼
2 logðIðx0 ; y0  1ÞÞ þ logðIðx0 ; y0 þ 1ÞÞ  2 logðIðx0 ; y0 ÞÞ
The particle centroid position with sub-pixel accuracy (xc, yc) is
calculated using:
xc ¼ x0 þ εX
and
yc ¼ y0 þ εy
2.6. Procedure for particle matching in between consecutive frames
The particle matching algorithm is based on direct spatial cor-
relation. This algorithm computes a correlation coefficient between
two consecutives PTV sub-images separated in time by Dt. The
correlation coefficient between the two sub-images IA and IB with
a  b dimensions is computed using the following relation:
X b h
a X ih i WWC was operated at an air sampling flow rate of 100 L/min and a
CIA IB ¼ IA ði; jÞ  IA IB ði; jÞ  IB
0
i¼1 j¼1
11=2
X b h
a X i in sterile, DNA-free Milli-Q water. The collector inlet was positioned
@ IA ði; jÞ  IA A
i¼1 j¼1
0 11=2
X b h
a X i locations denoted on Fig. 1.
@ IB ði; jÞ  IB A
i¼1 j¼1
where IA and IB are the average intensities of sub-images A and B,
respectively. The correlation coefficient will determine which par-
ticle in picture B is the best match of a particle in picture A. Since
particle locations are estimated to sub-pixel accuracy, and the in-
terval between pictures is known, an accurate particle velocity
estimation is achieved. For instance, a droplet to be matched on
image frame j will have multiple correlation coefficient values
assigned. Each of these values corresponds to candidate droplets in
frame j þ1. Based on these correlation values, the droplet pair that
shows the highest correlation is estimated to represent the same
droplet at different instance of time and at a different location.
Following the successful pairing of the droplets, and recalling that
the position in frame j and frame jþ1 are known together with the
time intervals between the frames, the velocity components can be
calculated with:
dxi xijþ1  xij
ui ¼ ¼
dt DPIV
dyi yijþ1  yij
vi ¼ ¼
dt DPIV
where ui and vi represent the velocities in the stream-wise and
normal flow directions of droplet i, xij and xijþ1 represent the x
coordinate position in frame j and jþ1 respectively, and yij and yijþ1
represent the y coordinate position of droplet i in frame j and jþ1.
2.7. Shower operation and microbiome sampling
Each of the shower units was operated daily for 20 min to mimic
routine shower operation for a period of five months. The tem-
perature of the water exiting the showers was controlled at
45e46  C and the water temperature measured at the manikins
was between 35 and 37  C. To model routine occupant activity in
residential showers, the surface of the manikins in each of the two
units was wiped thoroughly with wet soap (Irish Spring®) prior to
each daily shower actuation. Bioaerosol samples for microbial
analysis were collected on the first day of operation as well as
periodically throughout the five-month operating period. The re-
sults presented herein focus primarily on the concentration of
bacterial aerosol concentrations measured during the study as well
as identification of potentially pathogenic bacterial taxa that
developed in each shower.
Portable Wetted Wall Cyclone (WWC) collectors designed in our
laboratory and manufactured at TSI Inc. (Shoreview, MN)
(McFarland et al., 2010; King and McFarland, 2012) were used to
collect aerosol samples in each experimental shower. For the 100 L/
min cyclone, the cut point (the particle size where the collection
efficiency is 50%) of the aerosol-to-hydrosol efficiency curve is
1.2 mm AD (Aerodynamic Diameter), and the average collection
efficiency for single cells and clusters of bacterial particles is 86%
over a size range of 1e8.6 mm (McFarland et al., 2010), while
maintaining the culturability of the collected bacteria (King and
McFarland, 2012). For each 20-min bioaerosol sampling event, a
collection liquid inflow rate of 100 mL/min to concentrate the
airborne particles present in the shower air by a factor of up to 106
near the center point of each shower unit (at approximately 0.4 m
width  0.4 m depth  1 m height), in between the APS sampling
Aerosol samples from each shower unit were collected consec-
utively, starting in the low-flow unit and repeating the sampling in
the high-flow unit. Aerosol samples were collected with the WWC
in each shower unit for a 20 min period before the water in the
shower was turned on and for a 20-min period during shower
operation.
To clean the WWC units between sample collections, the col-
lector was turned on and the inlet covered with a 102 mm A/D filter
(Pall, MA) for particle free airflow. Using a 1 mL pipette, 100 mL of
fresh 10% bleach was skirted into the WWC inlet three times within
a 2 min period. The bleach was removed by squirting 100 mL of
isopropanol five times during a 5 min period into the collector.
Finally, 100 mL aliquots of sterile collection liquid (0.01% Tween-20)
were sprayed five times into the WWC inlet over a 5 min period.
The collector was then operated with sterile collection liquid for
 C.E. Estrada-Perez et al. / Water Research 130 (2018) 47e57
20 min prior to each sample collection to prevent carryover of
collected particles.
2.8. Analysis of microbial samples
The concentration of viable bacteria present in the bioaerosol
collection fluid samples was determined by plating 100 mL volumes
in appropriate dilutions on Difco Tryptic Soy Agar (TSA) Petri dishes
(Becton Dickinson Co., Sparks, MD), incubating the plates overnight
for 24 h at 37  C, and counting the resulting colony forming units
(CFU) on the plates. Although water microbiology practices
commonly use minimal medium and room temperature for long
term growth of bacteria in water samples, our goal was to enhance
the culturability of bacteria that may have been affected during the
aerosol collection process and also to stimulate the growth of
bacteria that may cause waterborne diseases by incubating the cells
on rich medium (TSA), at 37  C for 24 h (Liao and Shollenberger,
2003). In addition, the aerosol samples were analyzed by whole
cell qPCR (An et al., 2006; Han et al., 2010). Briefly, the bacteria were
lysed by heating 1 mL aliquots of the diluted samples in 1.5 mL
Eppendorf tubes for 15 min at 95  C. The tubes were placed on ice
for 5 min to quickly chill the suspensions, and the cell debris was
sedimented by microcentrifuging at 117 g's for 2 min. Aliquots
(3 mL) of the supernatants in appropriate dilutions were added as
the DNA template to the PCR reaction mixture. The reaction
mixture contained 5 mL of 2X Power SYBR Green PCR Master Mix
(Applied Biosystems, Warrington, UK), 3 mL of the hydrosol sample
supernatant, 1 mL of the universal 16 S forward 1369 (50 - CGG TGA
ATA CGT TCY CGG), and 1 mL of the reverse 1492 (50 - GGT TAC CTT
GTT ACG ACT T) primers to amplify a 123 bp fragment. The ther-
mocycling program of the AB StepOne RT-PCR System (AB, Foster
City, CA) consisted of an initial cycle of 95  C for 10 min followed by
40 cycles at 95  C for 15 s and 60  C for 60 s. Standard curves were
generated via 10-fold dilutions of genomic DNA extracted from
mid-log phase E. coli K-12 MG1655 (The Coli Genetic Stock Center,
New Haven, NE). The average bacterial genome copy number (GCN)
for each PCR reaction was based on an estimated seven 16 S rRNA
copies per E. coli and assuming four 16 S rRNA copies per genome
on average for bacteria. (Hospodsky et al., 2012; Lee et al., 2009;
Stoddard et al., 2015). Also, a melting curve was constructed at
the end of each qPCR run to ensure specificity in each sample's
amplification.
2.9. Illumina sequencing
The alkaline lysis method was used to extract bacterial DNA
from all bioaerosol samples (Zhou et al., 1990) for Illumina
sequencing. Briefly, the cells were pelleted and then lysed in fresh
TENS lysis buffer containing 0.1 M NaOH, 1x TE buffer and 0.5% SDS.
The proteins were precipitated by adding 3 N sodium acetate at pH
5.2. The proteins were removed by centrifugation and the super-
natant was transferred to a sterile 1.5 mL micro centrifuge tube.
After the addition of 10 mL of Polyacryl Carrier (PC 152, Molecular
Research Center Inc., Cincinnati, OH), the DNA was precipitated by
isopropanol. The pellet was then washed with cold, 100% ethanol
and dissolved in DNA hydration (filter sterilized DNAse free Milli-Q
water) solution. The DNA concentration was determined by
measuring optical density at OD260 using a spectrophotometer
(NanoDrop Technology, Wilmington, ED).
Bacterial DNA recovered from the bioaerosol samples were
analyzed at the Genomic Sequencing and Analysis Facility (GSAF) at
the University of Texas at Austin (Austin, TX, USA) for Illumina®
paired-end (2  250) sequencing on the MiSeq platform (Maestre
et al., 2016). First-round PCR was used to amplify the V4/V5 re-
gions of the 16 S rRNA gene using the primers 515F (50 -
51
GTGYCAGCMGCCGCGGTA-30 ) (Baker et al., 2003) and 909R (50 -
CCCCGYCAATTCMTTTRAGT-30 ) (Wang and Qian, 2009). Bacterial
DNA sequences were processed and analyzed in QIIME v.1.8
(Caporaso et al., 2010). Sequences were demultiplexed and forward
and reverse reads were merged using FLASH v.1.2.11 (Magoc and
Salzberg, 2011) with maximum overlap of 250 bp. Sequences
were quality-filtered (-q 19), and chimeras were removed via QIIME
and USEARCH (Edgar, 2010). High-quality sequences were clustered
into operational taxonomic units (OTUs) at 97% similarity using
QIIME's USEARCH-based open-reference OTU clustering workflow
(pick_open_reference_otus.py). Global singleton OTUs were
removed. All samples were rarefied to the least number of se-
quences (2365) present in any individual sample as is commonly
done in microbiome studies (Table S1). The effect of rarifying to
10,000 sequences was also investigated but it was determined that
it did not significantly change the relative abundances of the taxa
identified in the bacterial community. Taxonomy was assigned
using the Ribosomal Database Project classifier (Wang et al., 2007)
with the reference database Greengenes13_8 16s rRNA (McDonald
et al., 2012; Klappenbach et al., 2001) for bacteria.
3. Results and discussion
3.1. Aerosol particle sizing
Both the low-flow and high-flow showerheads produced aero-
sols in the respirable range with mass median diameters (MMDs)
ranging from 2.7 to 3.7 mm (see Table 2 and Fig. 2). For both the
upper and lower sampling locations within each shower, a slightly
lower MMD was measured in the low-flow shower as compared to
the high-flow shower. The range of droplet sizes was similar in both
showers with the measured aerosols ranging mostly between
0.5 mme10 mm (Fig. 2). As noted earlier, the APS unit used to
measure the aerosol size distribution has an upper cut off of 20 mm
and thus does not measure the larger droplet sizes.
A comparison of the calculated aerosol concentrations from
both the lower and upper regions of each shower unit indicates that
the shower with the low-flow showerhead had significantly higher
aerosol number concentrations as compared to the high-flow
shower. In both showers, higher droplet concentrations were
detected in the lower parts of both shower units likely as a result of
the reflected spray off of the manikin obstacle in each shower. The
increased concentration of particles in the lower portion of the
shower stall is likely most relevant for children in showers and
should be considered when developing quantitative microbial risk
assessments (QMRA) for children. Although the current study
mimicked adult occupants in showers, we hypothesize that a child
or a shorter object standing in the shower would result in the
droplets bouncing off from the top of the object and hitting the
opposite wall, resulting in a similar, albeit lower bounce pattern. In
the case of an empty shower stall, without any object present, a
gravity driven straight flow could be expected.
The average aerosol mass concentration for the upper and lower
aerosol samples collected in the low-flow shower was 5 mg/m3; the
average for the high-flow shower was 3.5 mg/m3. These levels are
generally lower but on the same order of magnitude as the levels
(5e14 mg/m3) reported by Zhou et al. (2007) during hot water
operation at similar flow rates in a full-scale experimental shower
system. Although the water temperature and shower flow rates in
the previous study were similar to the current study, the aerosol
concentrations were measured at a greater height (1.5 m) and the
shower was located in a much smaller room (16.7 m3) than that
employed in the current study. Also, the mass concentrations
measured by Zhou et al. (2007) were significantly lower (e.g., on the
order of 0.1e1 mg/m3) when the relative humidity conditions in the
52 C.E. Estrada-Perez et al. / Water Research 130 (2018) 47e57
Table 2
Particle MMD diameter and concentration in the low-flow and high-flow experi-
mental shower units.
Shower Unit Mass Median Concentration,
Diameter, mm Number/cm3
Low-flow, Upper Part 2.74 147
Low-flow, Lower Part 3.05 234
High-flow, Upper Part 3.16 56
High-flow, Lower Part 3.65 101
room were below 70%. Nevertheless, our results confirm that
showers generate aerosols in the respirable range although the size
of the aerosol generated is a function of several factors including
the configuration of the showers within the room and the sampling
location.
3.2. PTV measurements
Results from the PTV algorithm consist of full-field velocity
fields and corresponding velocity profiles. The velocity profiles
were extracted from regions of interest by means of virtual line
probes. This approach provides qualitative and quantitative com-
parisons between the two showerheads. Moreover, the PTV tech-
nique provides insight into the influence of showerhead design on
the induced flow patterns, and ultimately it allows investigation of
the relationship between these flow patterns and the microbiome
that develops in each shower.
Fig. 3 shows the instantaneous fields of the droplet velocity
estimated using the PTV algorithm. An animated rendering of the
image is provided in Supplementary Information. Each vector on
these fields represents an individual droplet velocity which can be
tracked over time. The vectors are color coded to show the
magnitude of the droplet velocity. These fields are plotted on top of
the images representing the manikin's location. This helps to
interpret the effect that the different shower nozzle characteristics
Fig. 2. Mass concentration of the aerosolized particles in the experimental shower
units with the low-flow and high-flow showerheads; for the upper and lower stall
areas as indicated on Fig. 1. In addition, an outside sample was also collected 3 m
outside of the shower, at a height of 0.9 m.
have on the flow structure and distribution of the droplets in the
vicinity of the manikins. From these fields, it is apparent that for
both nozzles, the velocity at the exit of the nozzles reaches a
maximum value. Thereafter, the droplets are directed towards the
manikin's body hitting first the manikin's head. Interestingly, the
water droplets exit the low-flow showerhead at a significantly
higher speed compared to the high-flow showerhead where the
droplet speed is about 50% lower. After hitting the manikin, mul-
tiple droplet trajectories are observed. There are elastic scattering
collisions between the droplets and the top of the manikin head.
The droplets gain elevation to later start a free-falling descent.
Other droplets tend to adhere to the manikin's surface. These
slowly slide down the manikin's body which effectively delays their
free-falling descent until much later when they detach from the
manikin. The velocity fields show the droplets transitioning from
the initial pressure driven velocity, just after exiting the shower
nozzles, to their final velocity induced by gravitational acceleration.
The maximum droplet velocities observed in Fig. 3 are within the
range of previously measured free falling droplets (up to 9 m/s) as
reported by Gunn and Kinzer (1949). Changes in the position of the
shower occupants would be expected to change the spray pattern,
in addition, the spray off a manikin could be different than off a
human.
To better understand the general behavior of the shower drop-
lets, the droplet streamlines were added to Fig. 3 (see left and right
images). These streamlines represent the most probable paths that
the droplets may follow after being expulsed from the nozzles. Han
and Moss (1997) described streamlines during a flow visualization
experiment in virtual impactor models. Similar vortices and
streamlines have been generated for the shower droplet pattern
(Fig. 3). One interesting finding from these streamline images is the
fact that the droplets' horizontal displacements on the left shower
are consistently larger than those on the right shower. Droplets in
the low-flow shower have a higher probability to reach the wall
opposite the manikin's location. Hence, different periods of active
wetting are expected for each shower wall depending on the type
of showerhead used. The PTV results indicate that the low-flow
shower wall is subjected to a greater number of high velocity
droplets than the high-flow shower wall which is less disturbed
during operation. These results suggest that the wall biofilm in the
low-flow shower may be continuously interrupted and broken up
by the shower droplets which may yield a less diverse biofilm
containing taxa that could tolerate these conditions (Cense et al.,
2006; Epstein et al., 2011).
The streamlines also show dramatic differences between the
flow patterns in the two units. The water droplets exit the 12
nozzles on the low-flow showerhead with sufficiently high velocity
to bounce off the manikin's body and reach the opposite wall. The
PTV images show higher droplet concentrations in the low-flow
unit, in agreement with the APS analysis (Fig. 2), indicating that
the streams break up earlier, resulting in a more turbulent pattern.
In contrast, while the water passing through the perimeter nozzles
of the high-flow showerhead seem to have higher speed compared
to the rest of the droplets exiting the head, the particles display a
turbulent pattern after bouncing off the manikin and they gravitate
towards the shower base before reaching the wall.
To achieve a better understanding of the droplets in-flight
behavior, we classify the droplets as experiencing three different
stages: a) pressure driven stage, b) scattering stage, and c) gravity
driven stage. The pressure driven stage happens near the droplet
generation, just after exiting the nozzle. At this stage the droplets
velocity is high reaching a maximum velocity magnitude of
approximately 4e5 m/s depending on the showerhead. In this
stage, the droplets' horizontal velocity component is significantly
larger than the vertical component. If the droplet is left
 C.E. Estrada-Perez et al. / Water Research 130 (2018) 47e57
Fig. 3. The instantaneous velocity magnitude (m/s) for the upper and lower view of the left (low-flow) and right (high-flow) shower units in the experimental shower. Streamlines
and vortices generated by the shower droplets in the low-flow (a) and high-flow (b) units are shown on the two sides of the PTV image. The manikins are shown in gray scale in the
background. Although the positions of the manikins are identical in both shower units, a different camera angle was selected for the right unit to optimize droplet visualization.
undisturbed, it will gain vertical acceleration due to gravity, how-
ever this is not the case in this experiment because of the presence
of the manikin in the droplets path. When the droplets reach the
manikin body, the second droplet stage begins, namely, the scat-
tering stage. In the scattering stage, the droplets are decelerated
through elastic collisions with the manikin. As noted above, the
droplets in this stage may go through different paths, for instance,
the droplets may bounce off the manikin gaining elevation prior to
free falling. Droplets in this path experience a sudden change of
direction towards the wall opposite the manikin's location. Also,
the droplets experience vertical acceleration due to gravity. If
droplets do not interact with each other, the droplets may undergo
a parabolic trajectory starting from the bouncing point and ending
either on the shower wall or on the shower floor. If droplets in this
path interact with each other, they may coalesce forming a larger
droplet which will change direction and vertical speed due to the
added mass. Another observed droplet path after the droplets
interact with the manikin surface is the sliding path. In this path,
droplets do not bounce off the manikin, but rather adhere to it as
they form a small water layer that covers the surface. Thereafter,
droplets of water begin to start falling off the manikin towards the
shower floor. Finally, the last droplet stage is the gravity driven
stage. This stage covers all droplets before they hit the shower wall
or floor. In the gravity driven stage, there is a vertical preferential
direction and a continued acceleration due to gravity. The velocity
at a given height in this stage will depend on the droplet size (mass)
and whether or not the droplets interact with each other during
flight.
Due to the complexity of droplet behavior, much of the indi-
vidual droplet interactions are difficult to quantify. To simplify
interpretation of the velocity field results, virtual line probes
located at five different positions in each shower (Fig. 4) were used
calculate the average magnitude of the droplets' velocity
1=2
ðv ¼ ðv2x þ v2y Þ Þ passing through each line. On the images, xn
denotes the length (in cm) along the virtual line profile where the
velocity measurements were recorded; thus, each profile starts at
point zero at the beginning of each arrow and extends to a
maximum of 60 cm along the length of the probe. Line probe 1 was
selected to analyze the water droplet velocities on their path from
each nozzle outlet to the region of first interaction with the
53
manikin. Line probe 2 is a cross section of the spray exiting the
shower nozzle and accounts for droplets traveling down to the
region where the shower stall base meets the shower wall, forming
a lip that retains moisture and supports the formation of a biofilm.
Line probe 3 captures close-to-body droplet interactions, droplets
bouncing off the body, and dispersing droplets from the shower-
head, passing the manikin's upper torso. Line probe 4 captures the
droplets dispersing directly from the initial showerhead spray, far-
from-body droplet interaction, and other droplet patterns. Finally,
line probe 5 captures the droplets passing at about the level of a
showering occupant's knee prior to reaching the stall base where it
impacts and re-aerosolizes from the shower floor surface.
The line probe 1 velocity profiles along the spray centerline and
the line probe 2 velocity profiles transverse to the showerhead
spray confirm that the velocity of water droplets exiting the low-
flow showerhead are higher than those exiting the high-flow
showerhead. The velocity of the spray droplets increases rapidly
as the spray exits each showerhead as evident in the step function
shape of the velocities from position 8e35 cm along line probe 1 in
both showers. However, in both showers, the magnitude of the
droplet velocities fluctuates after 35 cm along line probe 1, likely
due to the interference of water droplets bouncing off of the
manikins (Fig. 3). The higher droplet velocities in the low-flow
shower versus the high-flow shower persist as the spray reaches
the shower occupant (manikin) as evident by the velocity profiles
along line probe 3. However, the step velocity profiles for each
shower are slightly shifted along profile 3 suggesting that the
droplets are impacting the manikin's heads at slightly different
locations. Along line probe 4, the magnitude of the droplet veloc-
ities are similar near the manikin in each shower but ultimately the
magnitude of droplet velocities is higher in the low-flow shower as
the droplets approach the wall opposite the manikin. This result is
consistent with the observation evident in Fig. 3 that more spray
reaches the opposite wall in the low-flow shower as compared to
the high-flow shower. Finally, the droplet velocities in both
showers are similar along horizontal line probe 5 near the bottom
of the showers where re-aerosolization of droplets from the shower
floor is likely occurring.
The virtual line probes capture the magnitude of the droplets’
1=2
velocity crossing them ðv ¼ ðv2x þ v2y Þ Þ. Hence a profile of the
54 C.E. Estrada-Perez et al. / Water Research 130 (2018) 47e57

Fig. 4. Schematic of the manikin's location and orientation relative to the low-flow (top image) and high-flow (bottom image) showerheads. Five virtual line probes are also shown
for each shower.

average velocity magnitude is provided for each virtual line. The
average velocity magnitude and the length along each line profile
are described with vn [m/s] and xn [cm] respectively with n ¼ 1, 2, 3,
4, 5 representing the number of the line probe used.
3.3. Microbial analysis of the collected aerosols
The total concentration of bacteria in CFU/m3 and in bacterial
genome copy number (GCN)/m3 present in the shower air prior to
shower operation and during shower operation were determined
for Days 0, 6, 56 and 146 of shower operation. To assess the effect of
shower operation on total bacteria aerosolized, the log of the ratio
of airborne bacterial concentrations (GCN/m3) measured during
and immediately prior to shower operation was calculated for each
shower. A similar process was used to calculate the log of the
airborne concentration ratio based on viable bacterial concentra-
tions (CFU/m3). Thus, positive concentration ratios indicate that
shower operation increased the estimated concentration of bacte-
ria in the shower air whereas negative values indicate that turning
on the shower reduced the airborne bacterial concentration. As
evident in Fig. 5, the log of the concentration ratios based on GCN
varied from approximately 1.0 (indicating substantial enrichment
of total bacterial concentrations with shower operation) to 1.0
(indicating a substantial decrease in bacterial concentrations with
shower operation). Removal of some fraction of the aerosol as a
result of the shower spray is certainly possible as this is a common
approach employed in wet scrubber technologies (Kim et al., 2001).
In general, however, an increase in airborne bacterial concentration
 C.E. Estrada-Perez et al. / Water Research 130 (2018) 47e57 55

calculated estimate of the concentration of any given genera in the

Fig. 5. Log of the airborne bacterial concentration ratios based on viable CFU (left axis)
and on bacterial genome copy number (GCN), right axis, for the aerosol samples
collected before and during shower operation in the low-flow and high-flow showers.
shower air varied substantially (Table S3) and no consistent pattern
is evident with respect to the effect of shower operation. The
calculated concentrations of each genera in the experimental
shower units before shower operation were not statistically
different than those measured after shower operation (paired t-test
of square root transformed data, p < 0.05), indicating that, at least
at the genus level, multiple sources other than just the water spray
contribute to the composition of the airborne community. The
frequency of detection also varied substantially with some genera
such as Pseudomonas and Sphingomonas present in all samples and
others such as Mycobacterium only detected sporadically (e.g., in six
of 16 samples). Nevertheless, the estimated maximum concentra-
tions of potentially pathogenic bacteria detected over the course of
the study (Day 0 e Day 146) indicates that pathogenic genera of
potential concern were present in abundant quantities (Table 3).
While this data provides only an estimated concentration of genera
of potential concern (as compared to qPCR techniques that quantify
specific pathogenic taxa at the species level), the results never-
theless provide insight into the aerosols of potential concern pre-
sent in showers (Perkins et al., 2009; Bernard, 2012).

was observed more frequently during shower operation than a 4. Conclusions

decrease in concentration, especially for the high- flow shower.
This result suggests that bacteria from the shower water itself (or
biofilms associated with the shower) are released during shower
operation as has been documented previously (Perkins et al., 2009).
During the initial period of operation, the log of the CFU and GCN-
based concentration ratios were in general agreement but more
significant differences between the two metrics were observed
over time, particularly on Day 56 and on Day 146 (low-flow shower
only).
An examination of the sequencing results indicates that several
genera associated with potential human pathogens were detected
in the air of the experimental shower units. Microorganisms in
these genera which included Burkholderia, Streptococcus, Ralstonia,
among others, have been shown to cause a range of adverse human
health effects ranging from pneumonia to skin infections (Table S2).
To calculate the estimated airborne concentration of each of these
genera in the showers, the relative abundance of each genera in the
rarified sequence dataset was multiplied by the total airborne
bacterial genome concentration in the air (GCN/m3) following the
method proposed previously by Dannemiller et al. (2014). The re-
sults summarized in Table 3 indicate that the peak concentrations
for the 14 genera examined varied from 102 to 105 GCN/m3. The
This study successfully demonstrated the adaptation of a par-
ticle tracking velocimetry (PTV) algorithm for multiphase flows to
provide qualitative (visualization) and quantitative data on the
spray patterns and velocity fields generated in two full-scale,
experimental showers. Since this process was possible without
the introduction of fluorescent particles into the shower water, it
greatly simplifies the application of this technology and suggests
that it may be applicable to other water spray systems. Specific
findings from the current study include:
 A complex droplet pattern is evident in showers. Nevertheless,
the droplets generated in each shower can conceptually be
divided into three stages: (1) a pressure driven stage, (2) a
scattering stage, and (3) a gravity driven stage.
 The spray flow pattern and velocity profiles in residential
showers are a function of showerhead design. The number and
size of the nozzles in the two showerheads investigated in this
study substantially affected the water velocity profiles and spray
patterns observed. The droplets exiting the low-flow shower-
head had higher velocities (up to 5 m/s) than the high-flow
showerhead (up to 3 m/s) and, as a result, generated a greater
number of small droplets, some of which bounced off the

Table 3
Maximum calculated concentrations of potentially pathogenic bacterial genera detected in the air samples collected from the low-flow and high-flow showers before and after
shower operation.

Day 0e146 GCN/m3 GCN/m3 GCN/m3 GCN/m3

Genus Low-flow, before Low-flow, during High-flow, before High-flow, during

Acinetobacter 3.72 Eþ03 7.31 Eþ03 1.79 Eþ03 9.74 Eþ03

Anaerococcus 6.95 Eþ04 1.43 Eþ04 1.06 Eþ04 2.25 Eþ04
Burkholderia 4.33 Eþ04 1.29 Eþ04 2.92 Eþ03 6.51 Eþ03
Klebsiella 7.38 Eþ02 8.52 Eþ02 1.21 Eþ02 3.42 Eþ03
Legionella 0.00 Eþ00 6.28 Eþ02 0.00 Eþ00 0.00 Eþ00
Methylobacterium 1.16 Eþ05 3.35 Eþ03 3.35 Eþ03 9.54 Eþ03
Mycobacterium 0.00 Eþ00 1.12 Eþ02 3.98 Eþ02 2.09 Eþ03
Nocardioides 0.00 Eþ00 2.65 Eþ02 0.00 Eþ00 7.52 Eþ02
Pseudomonas 1.13 Eþ04 1.45 Eþ04 4.18 Eþ03 1.19 Eþ04
Ralstonia 7.93 Eþ04 2.19 Eþ04 1.39 Eþ04 2.41 Eþ04
Sphingomonas 2.11 Eþ05 2.41 Eþ04 7.54 Eþ03 4.08 Eþ04
Staphylococcus 1.63 Eþ04 1.55 Eþ03 1.37 Eþ03 4.27 Eþ02
Streptococcus 1.44 Eþ04 1.08 Eþ04 2.59 Eþ03 7.27 Eþ03
56 C.E. Estrada-Perez et al. / Water Research 130 (2018) 47e57
shower occupant (e.g, manikin) to reach the opposite shower
wall.
 The particle size distribution data collected from the experi-
mental showers confirms that both showers generate aerosols
in the respirable range.
 The concentration ratio of total bacteria in the air during shower
operation relative to the baseline values before shower opera-
tion varied by day of shower operation indicating that while
turning on the shower spray could increase the bacterial load in
the shower air relative to that present prior to shower operation,
it could also decrease the total and viable bacterial concentra-
tions, presumably through scrubbing of the air.
 Genera of potential health concern including Burkholderia,
Staphylococcus and Streptococcus were observed frequently in
the shower air of both experimental showers. However, there
was substantial variability observed in the estimated airborne
concentrations of these genera and the concentrations in the
shower air during shower operation were not statistically
different than those present in the shower air prior to operation.
Additional research would be required to confirm the presence
of specific pathogenic species and to identify the primary
sources (e.g, water, air or biofilm) contributing to the calculated
airborne concentrations.
The current study highlights the potential for linking PTV
measurements with microbiome investigations to assess aerosols
that may be important to human health. However, additional
studies are required to elucidate how droplet distribution and ve-
locities affect the aerosolized bacterial communities. In addition,
for residential showers, other factors such as type of shower stall,
water temperature, piping materials, and human occupants should
be investigated further as these activities will impact the micro-
biome that develops.
Acknowledgements
The authors are grateful to the National Science Foundation
(Award Number: CBET-1236040) for supporting this study. We also
wish to thank N.D. Allinson for his help with the PTV algorithm
evaluations.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.watres.2017.11.039.
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