doi: 10.2965/jwet.23-149 Journal of Water and Environment Technology, Vol.22, No.

3: 133–143, 2024

Original Article
Influence of Legionella pneumophila Viability States on Colonization
in Granular Activated Carbon Filters
Tomohiro Nakanishi a*, Masataka Kimura a, Yasuhiro Asada b, c, Sadahiko Itoh a

aDepartment of Environmental Engineering, Graduate School of Engineering, Kyoto University, Kyoto, Japan
b Department of Environmental Health, National Institute of Public Health, Wako, Japan

c Research Center for Environmental Quality Management, Graduate School of Engineering, Kyoto University, Otsu, Japan

ABSTRACT
Legionella is an important opportunistic pathogen in drinking water distribution and premise plumb-
ing systems. This study investigated the potential growth of Legionella pneumophila during granular
activated carbon (GAC) filtration, considering their viability states in the filter beds. Laboratory-scale
biologically active GAC columns were used, and L. pneumophila cells were spiked with different
viability states (culturable, viable but non-culturable (VBNC), and dead cells). The gene concentra-
tions in the effluents were monitored for 70 days. In columns spiked with the culturable cells, high
levels of L. pneumophila were detected in the effluents even after 70 days of operation, suggesting
that the GAC filter bed could serve as an ecological niche for L. pneumophila colonization. However,
when VBNC-cells were introduced, the levels of L. pneumophila in the effluents were significantly
lower, though still higher than in the column spiked with dead cells. This suggested that the growth
potential of L. pneumophila was influenced by their viability states in the influent water. These find-
ings underscored the ecological potential for Legionella regrowth and emphasized the necessity of
monitoring their behavior during GAC treatment, particularly when incomplete inactivation during
ozonation is concerned.

Keywords: Legionella, granular activated carbon, viable but non-culturable, biofilm, spiking
experiment

INTRODUCTION are frequently employed in drinking water treatment to ef-

Legionella spp. represents a notably significant oppor-
tunistic pathogen within drinking water distribution and
premise (i.e., building) water plumbing systems [1,2]. Due
to its opportunistic nature, Legionella contamination in
buildings housing high-risk populations (e.g., hospitals and
elderly care facilities) can result in outbreaks of Legion-
naires’ disease [3–5]. Controlling Legionella solely through
water treatment would be challenging, given its capacity to
regrow in distribution and plumbing systems [1]. Nonethe-
less, understanding the behavior of this organism in the
water treatment process remains crucial.
Ozonation and granular activated carbon (GAC) filtration
fectively remove dissolved organic carbon, micropollutants,
and compounds responsible for taste and odor [6,7]. As the
GAC filter bed operates without residual disinfectant, the
surface of the GAC is prone to colonization by microorgan-
isms. While these microbes can be beneficial due to their
biodegradation capabilities, caution should be exercised
regarding microbial leakage. Given that Legionella has
been reported to reside in biofilms and can infect free-living
amoeba (FLA) within these biofilms [8], a biologically active
GAC filter is considered a preferable environment for their
proliferation. Consequently, the GAC filter may serve as a
potential source of Legionella in the water treatment process.
Although disinfection is conducted as a final barrier, bacteria

Corresponding author: Tomohiro Nakanishi, E-mail: nakanishi.tomohiro.8r@kyoto-u.ac.jp

Received: December 28, 2023, Accepted: April 9, 2024, Published online: June 10, 2024
Open Access This is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY) 4.0 License. http://
creativecommons.org/licenses/by/4.0/

133
 134 Journal of Water and Environment Technology, Vol. 22, No. 3, 2024
in GAC-treated water have been reported to be associated
with fine carbon particles [9], which could potentially sur-
vive subsequent disinfection.
Several field observations have identified the GAC filter
bed as a potential source of Legionella. Li et al. [10] directly
observed a high abundance of Legionella genes on GAC
filters in an operational treatment plant, and increases in
Legionella gene concentration through GAC filtration were
observed [10,11]. Sharma [12] conducted a laboratory-scale
experiment in which L. pneumophila was introduced into
GAC columns, and notable Legionella colonization on the
GAC particles was observed concurrently with the formation
of biofilms. Molloy et al. [13] also observed the long-term
persistence of spiked Legionella in the effluents of point-of-
use GAC filters. On the other hand, Liu et al. [14] have sug-
gested that the GAC filter may function as a less preferable
environment for opportunistic pathogens due to potential
competition and/or antagonism with indigenous bacteria.
In the aquatic environment, gram-negative bacteria like
Legionella can enter the viable but non-culturable (VBNC)
state when exposed to stressors such as starvation and
chemical oxidation [15,16]. Legionella in the VBNC state is
of potential concern since they retain the capability to infect
and replicate within FLA present in biofilms [17]. Con-
sequently, they can make an ecological balance with FLA
within the biofilm environment, leading to their long-term
persistence [8]. In the ozonation − GAC filtration process,
the presence of VBNC pathogenic bacteria, such as Pseudo-
monas aeruginosa and Salmonella spp., have been observed
in ozonated water [18]. Therefore, it is highly probable that
VBNC-Legionella in ozonated water continuously flows into
GAC filter beds. However, the direct investigation of the im-
pact of Legionella viability states on their growth potential
within the filter is yet to be conducted.
The objective of this study is to obtain fundamental insights
into the ecology and growth potential of L. pneumophila in
the GAC filter bed, taking into consideration their viability
states in the influent water. To this end, culturable, VBNC,
and dead L. pneumophila were prepared and introduced into
lab-scale biologically active GAC columns. Subsequently,
the trends in L. pneumophila gene concentrations in the ef-
fluents were monitored over 60 days.
MATERIALS AND METHODS
Column setup and stabilization
GAC filter and ozonated water
GAC was sampled from a drinking water treatment plant
that operates in the Kansai region of Japan, sourcing water
from the Yodo River. The plant utilizes the following pro-
cess trains: coagulation/sedimentation, rapid sand filtration,
ozonation, GAC filtration, and chlorination. During the sam-
pling event, the GAC had been in operation for more than
five years, thereby indicating its biological maturity. The
GAC sample was obtained from the uppermost layer of the
bed because it was technically challenging to take samples
from the lower layer while the filter bed remained in opera-
tion. The GAC sample was stored in 2-litter sterile plastic
containers. Additionally, ozonated water was weekly col-
lected to serve as the feed water for laboratory-scale columns
(see below). The basic water quality (turbidity, pH, TOC, and
UV260) of the ozonated water is presented in Table S1 in
Supplementary Materials. The collected GAC samples and
ozonated water were transported to Kyoto University and
kept at 4°C for 24 h (GAC) or maximum period of 1 week
(ozonated water) before use.
GAC column setup and operation
A total of nine laboratory-scale GAC columns were pre-
pared. Approximately 100 mL of the collected GAC samples
were aseptically deposited into glass columns, each with
a length of 300 mm, an inner diameter of 2.5 cm, and a
volume of 150 mL. These columns were sealed with rubber
lids. The height of the filter beds within the columns was
set at 200 mm. Since the GAC sample was originated solely
from the uppermost layer of the operational filter bed, the
microbial abundance along the filter depth could be po-
tentially different from the real situation. To mitigate this,
each column underwent a 70-day period of being supplied
with ozonated water (i.e., stabilization period) to develop
and stabilize mature biofilms throughout the filter depth.
Throughout the stabilization phase, the columns experienced
daily water replacements, where 100–150 mL of water was
carefully decanted from the bottom, with minimal disrup-
tion to the filter beds, and simultaneously replenished with
an equal volume of fresh ozonated water. All columns were
operated in the dark at a room temperature of 20°C.
Preparation of spiking microorganisms in different
viable states
Culturable L. pneumophila and amoeba
Legionella pneumophila (ATCC 33152; obtained from the
American Type Culture Collection [ATCC], Rockvile, MD,
USA) was cultivated on BCYE (Buffered Charcoal Yeast
Extract) agar plates (Nissui Pharmaceutical, Tokyo, Japan)
at 35°C for 24 to 48 hours. Several colonies were suspended
Journal of Water and Environment Technology, Vol. 22, No. 3, 2024
in autoclaved PBS. These suspended colonies underwent a
washing process, involving three rounds of centrifugation at
8000 g for 10 minutes, discarding the supernatant each time,
and resuspending the cells in fresh PBS through vortexing.
Subsequently, the aliquots were immediately spiked to the
GAC columns after dilution.
Acanthamoeba castellanii (ATCC 30234), an amoeba
host of L. pneumophila commonly found in anthropogenic
aquatic environments and water supply systems [19,20], was
also co-spiked with L. pneumophila in specific columns.
A. castellanii was axenically cultivated in PYG (proteose
peptone-yeast extract-glucose) medium (ATCC Medium
712) within 25 cm 2 cell-culture flasks at 30°C. Once the tro-
phozoites reached confluence, they were rinsed by replacing
the medium with PAGE’s saline (composed of NaCl: 120 mg,
MgSO4: 4 mg, Na2HPO4: 142 mg, KH2PO4: 136 mg, CaCl2:
4 mg, in 1000 mL MQW, prepared according to the instruc-
tions of ATCC Medium 1323). The trophozoites were then
suspended in the same saline solution and utilized as the
spike to the GAC columns.
L. pneumophila in VBNC state
L. pneumophila was transformed to a VBNC state under
oligotrophic conditions, as oligotrophs are widely recognized
as primary inducers of the VBNC state in natural water
environments [15]. Culturable L. pneumophila, as described
earlier, were suspended in 500 mL of autoclaved PBS, result-
ing in a viable cell concentration of 2.3 × 105 cells/mL (quan-
tified via carboxyfluorescein diacetate (CFDA) staining).
Subsequently, the cell suspension was incubated without
agitation at a temperature of 20°C in the dark for up to 143
days. Throughout this period, the viability of the bacteria
was assessed using two distinct indicators: culturability and
esterase activity. Cells that displayed ongoing intracellular
enzymatic activity but lacked culturability were categorized
as VBNC-state cells [15,16]. These analyses were regularly
conducted for 120 days during the starvation period. Since
the majority of the cells transitioned into the VBNC state by
day 80, the suspension obtained on day 83 was used as the
spike into the GAC columns.
Dead L. pneumophila and amoeba
The suspensions containing culturable L. pneumophila or
A. castellanii, prepared as previously described, were sub-
jected to inactivation by autoclaving at 121°C for 20 minutes.
Subsequently, these inactivated suspensions were introduced
into the columns.
135
Spike of microbes in GAC columns
Following the stabilization period, GAC columns were
spiked with L. pneumophila and Acanthamoeba cells, exhib-
iting various viable states as outlined in Table 1. The objec-
tive was to investigate the behavior of L. pneumophila within
the GAC, considering whether the spiked cells were cultur-
able (columns No. 1, 2, 3, 4) or in a VBNC state (columns
No. 5, 6, 7, 8). Additionally, A. castellanii was co-spiked in
columns No. 3, 4, 7, 8 to examine the potential effects of
coexisting FLA. Each spiking condition was replicated in
duplicate columns, as shown in Table 1. For comparison
purposes, autoclaved L. pneumophila and amoeba were
spiked as dead cells in column No. 9.
The spiked concentrations of L. pneumophila and amoeba
were set as approximately 104 cells/mL and 102 trophozoites/
mL, respectively. One hundred milliliter suspension contain-
ing L. pneumophila and amoeba (if necessary) were poured
into the GAC columns, while simultaneously decanting the
old ozonated water. The suspension was allowed to contact
the GAC bed for 1 day to facilitate the colonization of the
spiked microorganisms. Subsequently, the column water was
replaced every day with fresh ozonated water using the same
procedure as during the stabilization period. This replace-
ment process was repeated for 70 days. Throughout this pe-
riod, the genes of L. pneumophila and the heterotrophic plate
count (HPC) bacteria in the effluent water were regularly
analyzed using qPCR and culture methods, respectively.
Following the operation, core samples were collected from
the upper surfaces of the GAC beds for the analysis of L.
pneumophila.
Analytical methods
Culturability and enzymatic activity of L. pneumophila
cells
The culturability of starving L. pneumophila cells was
assessed by spreading 100 µL of the undiluted sample onto
duplicate BCYE-agar plates, followed by incubation at 35°C
Table 1 Spiked microorganisms and their viability states in
GAC columns.
Spiked microorganism
Column ID
L. pneumophila A. castellanii
No.1, 2 Culturable NA
No.3, 4 Culturable Culturable
No.5, 6 VBNC NA
No.7, 8 VBNC Culturable
No.9 Dead Dead
NA: not added
 136 Journal of Water and Environment Technology, Vol. 22, No. 3, 2024
under humidified conditions. The plates were monitored for
a minimum of 10 days, checking for the presence of Legio-
nella colonies. Loss of culturability was confirmed when no
colonies were observed on the both plates.
Enzymatic activity was examined microscopically us-
ing CFDA as an esterase substrate, following established
staining procedures [21,22]. In brief, 965 µL of starving L.
pneumophila suspension was spiked with 20 µL of 25 mM
EDTA and 15 µL of 10 mg/mL CFDA (Sigma-Aldrich, St.
Louis, USA) in a microcentrifuge tube, and then incubated at
35°C for 60 minutes. The suspension was filtered through a
polycarbonate membrane with 0.22 µm pores (K020N025A,
Advantec, Tokyo, Japan). The retained green cells on the
membrane were counted using an epifluorescent microscope
(Eclipse 80i, Nikon, Tokyo, Japan) equipped with an excita-
tion/emission filter set at 488 nm and 515 nm, respectively.
Microscopic observation was conducted at a magnification
of x400 or x200 in at least ten fields.
DNA extraction from water and GAC core samples
DNA extraction was carried out on the column effluents
and core GAC samples using the DNeasy PowerWater Kit
(Qiagen, Tokyo, Japan). For the column effluent, 100 mL was
filtered through polycarbonate filters with 0.22 µm pores
(K020N047A, Advantec). As for the core GAC samples, 0.14
to 0.31 grams (wet weight) of GAC were suspended in 20 mL
of sterile PBS, vortexed for 1 minute to detach the adhered
biomass, and then the supernatant was filtered through the
same filters. The treatment condition for detaching biomass
was determined in a preliminary experiment (see Text S1
and Table S2). The DNA was extracted and eluted to 100 µL
following the manufacturer’s protocol.
Quantification of L. pneumophila and HPC
Enumeration of L. pneumophila was performed using
qPCR assays targeting the mip gene [23]. The qPCR assays
were conducted on a StepOnePlus™ platform (Thermo
Fisher Scientific, Waltham, USA) in 20 µL reaction mix-
tures, consisting of TaqPath™ qPCR Master Mix (Applied
Biosystems, Tokyo, Japan), 400 nM of each primer, 100 nM
of the probe, and 2 µL of DNA template. Primer sequences,
target regions, and PCR conditions are provided in Table S3
in Supplementary Materials. Each qPCR run included DNA
extracts, negative controls, and 10-fold serial dilutions of
the standard, all in triplicate. Oligo DNAs containing the
qPCR target sequences (synthesized by Thermo Fisher
Scientific, Tokyo, Japan) were used as the DNA standards.
The amplification efficiency ranged from 96% to 105%. The
limit of quantification (LOQ) for all qPCR assays was set at
a concentration of 10 copies/µL. Samples in which all three
replicates exhibited values above the LOQ were considered
quantifiable. Samples that showed significant amplification
in only one or two wells were considered to be below the
LOQ but still positive for the presence of the target gene.
The total number of HPC bacteria in the column effluent
was determined by culturing on R2A agar (Daigo, Nihon
Pharmaceutical, Tokyo, Japan) at 20°C in the dark for one
week.
Data analysis
The HPC in effluents from each column were analyzed by
R (version 3.5.1) within the RStudio environment. Paramet-
ric one-way analysis of variance (ANOVA) was used and fol-
lowed by the Bonferroni p-value corrected t-test. Statistical
significance was set at p < 0.05.
RESULTS
Viability states of L. pneumophila during the starva-
tion period
Figure 1 presents the concentrations of L. pneumophila
throughout the starvation period, considering both culturable
cells and enzymatically active cells. The culturable cell con-
centrations started at 3.5 × 103 CFU/mL on day 0. However,
they exhibited a rapid decline after one month and became
undetectable after day 80. On the other hand, the concentra-
tions of enzymatically active cells remained relatively stable
until day 123. From these results, we can conclude that the
majority of the spiked L. pneumophila cells at day 83 of the
starvation period were in the VBNC state.
Fluctuations of influent water quality
During the GAC column operation, ozonated water ob-
tained from the actual treatment plant was utilized as the in-
fluent water. Throughout the experimental period, basic water
quality parameters displayed small fluctuations (Table S1),
and no notable impact of such variations on the abundance of
L. pneumophila genes in the column effluent was observed.
Thus, it was inferred that fluctuations in influent water qual-
ity did not have a major effect on L. pneumophila growth in
this experiment.
HPC bacteria in effluent water
HPC bacteria in the effluent water from each GAC column
were enumerated during the spiking experiment, as depicted
in Fig. 2. The HPC counts exhibited a typical range from 103
Journal of Water and Environment Technology, Vol. 22, No. 3, 2024
Fig. 1 Change of culturable and viable L. pneumophila concentration during the
starvation period.
to 105 CFU/mL in each column effluent, and no statistically
significant differences were observed among the spiking
conditions (Bonferroni-adjusted p > 0.05). These HPC bac-
teria were higher than those observed in ozonated water (101
to 102 CFU/mL, data not shown) during preliminary mea-
surements. This marked difference in HPC counts strongly
indicates the extensive bacterial colonization within all the
GAC columns.
L. pneumophila concentration in effluent water from
GAC columns
The concentrations of L. pneumophila (mip gene) in the
column effluents are presented in Fig. 3. During the initial
phase up to day 21, the gene abundance showed a declin-
ing trend in all nine columns. The subsequent trends after
day 21 varied depending on the viability states of the spiked
L. pneumophila. In columns No. 1, 2, 3, and 4, which were
spiked with culturable cells, the concentrations after day 21
remained relatively high, ranging from 2 to 3 log copies/mL.
On the other hand, in columns No. 5, 6, 7, and 8, which were
spiked with the VBNC-cells, the gene levels were less stable.
The concentration in columns No. 5 and 7 was below the
limit of quantification but still detectable after day 21, while
sporadic high concentrations were observed in columns No.
6 and 8 after day 42. In column No.9, which was spiked
with autoclaved microbes, L. pneumophila was mostly not
detected after day 21. The spiking of A. castellani (columns
No.3, 4, 7, and 8) did not show clear impact on the L. pneu-
137
mophila concentrations.
Gene abundance of L. pneumophila in GAC core
samples
Figure 4 displays the gene abundance of L. pneumophila
in the GAC filters before spiking microbes and after the
monitoring period. Prior to spiking, L. pneumophila was not
detected in the GAC filters. Following the 70-day operation,
L. pneumophila was detected in all nine columns, with mip
gene abundances ranging from 3.6 to 6.3 log10 copies/g-wet.
Although no clear trends were observed among the different
spiking conditions, the results demonstrate the long-term
persistence of the spiked L. pneumophila in the filter bed.
DISCUSSION
This study investigated the possibility of L. pneumophila
for long-term persistence or even growth within the GAC
filter bed using lab-scale columns. L. pneumophila cells
were spiked in different viability states, with consideration
for their VBNC state, which is relevant to the actual presence
in aquatic environment and water treatment processes [15].
Induction of L. pneumophila to VBNC state
In this study, VBNC cells were induced through incuba-
tion under an oligotrophic condition. During the starvation
periods, complete loss of culturability was observed within
80 days, while enzymatically active cell number did not
 138 Journal of Water and Environment Technology, Vol. 22, No. 3, 2024

Fig. 2 Change in HPC bacteria in the effluents of columns No. 1–4 (upper half) and
No.5–9 (lower half) during the experiments. L. pneumophila was spiked at day 0.

decrease significantly even after 125 days. This trend is
in agreement with the previous observation [24], in which
they observed long persistence of esterase activity of L.
pneumophila serogroup-6 strain for more than 200 days.
Therefore, the long persistence of L. pneumophila in oligo-
trophic condition was again confirmed in this study. It should
be noted that the actual inducer during ozonation would be
oxidative stress, and the oligotrophic stressor may elicit
different physiological and regulatory responses from the
VBNC cells compared to those induced by oxidation [16].
Nevertheless, it is highly probable that starved VBNC cells
originating prior to the ozonation steps remain abundant
in the ozonated water. Nonideal flow conditions such as
short-circuiting and internal recirculation in ozone contac-
tors can result in insufficient mixing and reduced ozone
exposure [25]. Consequently, significant concentrations of
viable bacteria within ozone contact chambers are actually
reported in full-scale plants [26]. Thus, not all the VBNC
cells in ozonated water may be the result of oxidative stress.
Therefore, the starved VBNC cells prepared in this study can
be considered as initial proxies for VBNC L. pneumophila in
ozonated water, particularly after incomplete ozone contact.
Persistence of L. pneumophila in GAC filter bed as
assessed by qPCR
In the spiking experiments, we utilized qPCR method to
monitor L. pneumophila concentrations in the column ef-
fluent water, aiming to evaluate the potential growth of the
spiked L. pneumophila. It is important to acknowledge that
qPCR analysis is not able to differentiate between culturable,
VBNC, and dead cells. However, assessing culturability or
enzymatic activity using cultivation or microscopy-based
methods, respectively, has limited selectivity for detecting
L. pneumophila species. Therefore, qPCR analysis, being a
sensitive and selective method, was deemed appropriate as
the primary approach. Thus, long-term monitoring of gene
concentrations in each column effluent is expected to enable
a qualitative assessment of the influence of the viability
states of spiked L. pneumophila.
During the initial 21 days after the spiking, L. pneumophila
Journal of Water and Environment Technology, Vol. 22, No. 3, 2024 139

Fig. 3 Change in L. pneumophila (mip) gene levels in the effluents of GAC columns spiked with:
a) culturable L. pneumophila (Lp), b) culturable Lp + viable A. castellanii (Ac), (c) VBNC Lp, (d)
VBNC Lp + viable Ac, and e) dead Lp + dead Ac. Microbes were spiked at day 0. Data points are
obtained from single replicate trials. Detected data below the LOQ are presented as 1 log copies/
mL with a downward arrow. Undetected data is shown as 0 log copies/mL.

concentration in the effluents declined during all the GAC
columns. This indicated that the spiked L. pneumophila cells
physically entrapped in GAC filters were discharged during
daily water exchange.
In columns spiked with culturable L. pneumophila (No. 1,
2, 3, and 4), the concentration after day 21 remained at high
levels, clearly differing from the column No. 9. This result
strongly implied that a fraction of the spiked cells success-
fully established a stable colonization within the filter bed,
resulting in the continuous sloughing off of L. pneumophila-
harboring biofilms into the effluent. This scenario closely
aligns with earlier observations. Molloy et al. [13] observed
a continual release of Legionella from the carbon filter over
six weeks after spiking this organism. In the experiment
conducted by Sharma [12], the colonization of the spiked Le-
gionella in the GAC filter became evident after the matura-
tion of the biofilms within the filter bed. In the current study,
the GAC columns were heavily colonized by biofilms, as
evidenced by the rapid increase (> 2 log) in HPC between the
influent and effluent water. Moreover, although not explicitly
verified in this study, it is highly probable that amoeba hosts
colonized those biofilms, as observed in previous research
with Acanthamoeba spp. [27] or Echinamoeba spp. [20] in
operational GAC filters. Thus, the above results strongly
 140 Journal of Water and Environment Technology, Vol. 22, No. 3, 2024

Fig. 4 Gene abundance of L. pneumophila at the surface of the GAC core be-
fore and after the spiking experiments. Before spiking the microbes, L. pneu-
mophila were not detected from any columns.

suggest that GAC filters may serve as potential niches for
L. pneumophila colonization if culturable L. pneumophila is
introduced.
On the other hand, the columns spiked with VBNC cells
(No. 5–8) exhibited different trends after day 21, character-
ized by predominantly lower concentrations than those
spiked with culturable cells (No. 1–4). According to the
previous infectivity assay [28], starved VBNC Legionella
strains exhibited reduced efficacy in infecting FLA or hu-
man macrophages compared to their culturable state. Thus,
the less efficient growth of VBNC cells may explain the
observed low abundance of L. pneumophila in the effluents.
Upon closer examination of the gene levels of L. pneu-
mophila in each column effluent, the behavior under each
column was variable: No. 6 and 8 exhibited a rapid increase
after day 40, while No. 5 and 7 displayed mostly lower
concentrations than LOQ. Elevated L. pneumophila levels in
columns No. 6 and 8 might be attributed to the resuscita-
tion of VBNC cells after prolonged contact with biofilms,
subsequently leading to replication. Similar trends were also
reported in previous studies [17,28], in which starved VBNC
L. pneumophila maintained the capacity to invade FLA and
undergo replication. On the other hand, such observation
was not reproduced in columns No. 5 and 7. The factors
contributing to these fluctuations between column replicates
(i.e., No. 5 and 6, and No. 7 and 8) remain unknown, possibly
indicating the presence of specific conditions that triggered
the regrowth. Nevertheless, it is important to highlight that,
even in columns No. 5 and 7, L. pneumophila genes were
more frequently detected (though mostly below the LOQ)
than the column No. 9. This implies that VBNC cells still
retained the potential for longer persistence in the GAC filter
bed than the dead cells.
L. pneumophila abundance within GAC core sam-
ples
After the monitoring period, gene abundance of L. pneu-
mophila in the GAC core from the surface layer of the filter
bed was measured (Fig. 4). However, no distinct patterns
were evident among each column, which was inconsistent
with the above monitoring results of the column effluent.
In column No. 9, unexpectedly high gene abundance was
observed within the GAC core. It can be inferred that the
detected genes originated from extracellular DNA released
from lysed cells during the autoclaving process, and that
these DNA fragments were irreversibly adsorbed onto the
GAC when the autoclaved microbes were introduced into
the column. This aligns with the relatively low initial L.
pneumophila concentration observed in the effluent from
column No. 9 (Fig. 3), indicating that a significant portion
of extracellular L. pneumophila genes from dead cells re-
mained at the surface of the filter via adsorption during the
Journal of Water and Environment Technology, Vol. 22, No. 3, 2024
column operation.
Moreover, with respect to columns No. 1–4 (culturable Lp)
and No. 5–8 (VBNC Lp), no clear difference was observed
between them. One possible explanation is the heterogeneous
distribution of L. pneumophila within the filter, leading to
significant deviations in measured abundances, particularly
among columns No. 1–4. As demonstrated in a previous
study [29], GAC is characterized by a heterogeneous cov-
erage of biofilms, rather than forming a dense, cotton-like
biofilm. Thus, the collected quantity (0.14 to 0.31 grams) of
the GAC core in the current study may not have adequately
represented the true conditions.
Although it was difficult to provide quantitative discus-
sions about the impact of L. pneumophila viability states, the
analysis of GAC core could confirm the persistence of spiked
L. pneumophila for more than 70 days within the filter bed
(columns No. 1–8).
Effect of co-spiked amoebae
The co-spiking of A. castellanii (No. 3, 4, 7, and 8) had no
discernible impact on the concentration of L. pneumophila in
the effluents. Although we selected this specific FLA species
as it is known to be a host for Legionella [19], it is likely that
A. castellanii failed to establish colonization in the current
GAC filter. Further investigations using indigenous FLA
species are warranted to evaluate the potential effects of FLA
introduction on the behavior of Legionella in the system.
Implications to public health risk
This study was conducted to gather fundamental insights
into the ecology of L. pneumophila in GAC filter beds, with
a focus on the regrowth potential of various viability states.
In summary, two significant possibilities were highlighted
under the present experimental conditions. Firstly, culturable
L. pneumophila most likely had the capability to establish
a stable colonization within the GAC filter, which in turn
resulted in the release of this bacterium into the treated ef-
fluents. Secondly, despite their reduced growth potential,
VBNC L. pneumophila retained at least the potential for long
persistence within the filter bed. These findings underscore
the ecological significance of the GAC filter as a niche for
L. pneumophila proliferation, even when they are in starved
VBNC states. Moreover, these results emphasize the impor-
tance of closely monitoring Legionella behavior during GAC
treatment, especially when incomplete inactivation during
ozonation is a concern. As discussed in the “Induction of
L. pneumophila to VBNC state” section, full-scale ozone
contactors sometimes suffer from incomplete inactivation
141
efficacy due to inadequate mixing conditions [25,26]. In
such scenarios, our findings would be significant to consider
regarding the possible impact on public health risks.
It should be noted, however, that these findings do not im-
mediately imply a public health risk in operational drinking
water treatments. This study introduced excessively large
quantity of L. pneumophila (approx. 106 cells) into the small
GAC columns to ensure clear observations. According to
Camper et al. (1985), autochthonous microbial community
within the GAC filter could influence the survival of patho-
gens through competition for nutrients and space [30]. In
case of lower L. pneumophila concentration in the ozonated
water, more vigorous competition might occur, potentially
resulting in a lesser magnitude of growth within the GAC
filter. Such potential effect of L. pneumophila concentration
needs to be further investigated.
This study has other significant limitations. Firstly, due to
the utilization of the qPCR method to monitor L. pneumoph-
ila levels, precise differentiation among culturable, VBNC
(e.g., enzymatically active cells or those with intact mem-
branes), and dead cells was not feasible. Evaluating these
viability states would be valuable for elucidating the ability
of L. pneumophila growth in GAC filter beds. Secondly, our
observations are limited in lab-scale experiments. Further
investigation under realistic conditions is desirable to fully
understand the significance of this regrowth phenomenon on
public health.
CONCLUSIONS
This study investigated the possibility of L. pneumophila
growth within the biofilm environment of GAC filter bed,
focusing on the influence of their viability states through
lab-scale spiking experiments. Notably, when culturable
cells were spiked, the L. pneumophila concentration in the
effluents remained high for 70 days, suggesting the poten-
tial of the GAC filter bed as a niche for their colonization.
Conversely, when VBNC-cells were introduced, the levels of
L. pneumophila in the effluents exhibited significant reduc-
tions. This underscored the considerable impact that the vi-
ability states have on their growth potential. Simultaneously,
higher gene detection rates were observed in these columns
compared to the column spiked with autoclaved microbes,
implying that the VBNC L. pneumophila still had the po-
tential to persist for a longer time than the dead cells. These
findings underscored the ecological potential for L. pneu-
mophila regrowth and emphasized the necessity of closely
monitoring their behavior during GAC treatment, particu-
 142 Journal of Water and Environment Technology, Vol. 22, No. 3, 2024
larly when concerns arise regarding incomplete inactivation
during ozonation. Further research is essential to determine
the potential impacts of this regrowth on public health.
ACKNOWLEDGEMENTS
This work was supported by JSPS KAKENHI Grant
Number 17K06618. Additionally, we would like to thank the
waterworks bureau for providing the GAC samples.
SUPPLEMENTARY MATERIALS
Supplementary Materials file for this article is available at the
link below. https://www.jstage.jst.go.jp/article/jwet/22/3/22_23-
149/_supplement/_download/22_23-149_1.pdf
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