Joint Meeting Presentation

21 October 2019

Factors Influencing Microbial Regrowth and

Occurrence of Opportunistic Pathogen in
Premise Plumbing

Iftita Rahmatika (D3-W)

Water Environment Technology Lab
Department of Urban Engineering
The University of Tokyo
Challenge in premise plumbing 2

Premise plumbing: portion of water distribution system beyond property line,

includes showerheads, faucets, water heaters, etc.

• Long stagnation time Favorable

• Temperature fluctuation condition for
• Loss of disinfection microbial regrowth
• Large specific surface area1)

Microbial regrowth had been associated with the occurrence of opportunistic pathogen.
Source Microorganisms
Household tap water in US 2)
Legionella pneumophila
Mycobacterium avium
Pseudomonas aeruginosa
Washroom and kitchen tap water in China3) Legionella pneumophila
Pseudomonas aeruginosa
Biofilm shower hoses in Europe Legionella pneumophila
(Proctor et al., 2018) 4) Mycobacterium avium
Acanthamoeba spp
Number
9.8 copies/mL
1.1 copies/mL
1.8 copies/mL
3 log copies/mL
1.04 log copies/mL
6.6x105 copies/cm2
6.6x107 copies/cm2
2.5x104 copies/cm2

Microbial regrowth is serious issue and the factors influencing microbial

regrowth needs to be evaluated.
What caused microbial regrowth? 3

Polymeric material are widely used

in building plumbing. What
happened inside the pipes?

Image source: Eawag

Migration of organic Current method to evaluate the microbiological

Microbial matter from plastic effect of carbon migration from plastic pipes 5):
regrowth material of pipes to
water 5) Method Parameters to
Organic determine microbial
regrowth
matter
MDOD (UK) Oxygen consumption
Biodegradable
BOM organic matter W270 (Germany) Volume of surface
Biofilm supporting growth
regrowth BPP (Netherlands) Adenosine triphosphate
(ATP)
BioMig (EaWag, TCC, ATP
Additional organic matter released from pipes Switzerland)
could promote microbial regrowth.
Previous method to evaluate contaminant migration from pipes 4

The most used method for migration test: (1) static immersion testing, (2) static pipe segment,
and (3) water flowing intermittently or continuously through a pipe. 6)

Previous study evaluated the impact of pipe material to microbial regrowth:

1) Biomass production potential (BPP) test: A method to determine the concentration of
active biomass on the surface of piece of pipe material that is incubated in the inoculated
drinking water.7)
2) Biomig test: method to evaluate the potential release of compounds from polymeric
material in contact with drinking water.5)

After incubation Measurement

• Total organic carbon (TOC): to evaluate the amount
of organic carbon released from pipes.
• Assimilable organic carbon (AOC): to evaluate the
amount of organic carbon that is biodegradable for
Static immersion specific strain of bacteria.
testing method

Limitation:
Composition of biodegradable organic matter released from pipes and their impact on
microbial community had not been addressed yet.
Research framework 5

Research objective:
to reveal risks related to microbial regrowth in premise plumbing and to evaluate the
factors influencing microbial regrowth in premise plumbing.

Research framework:
Part 1: One – year monitoring of microbial regrowth and opportunistic
pathogen occurrence in premise plumbing.
1. To evaluate the effect of stagnation on microbial regrowth and opportunistic pathogen
occurrence in premise plumbing.
2. To evaluate seasonal variation on microbial regrowth and opportunistic pathogen
occurrence in premise plumbing.
3. To estimate the health risk of opportunistic pathogen in premise plumbing.
4. To evaluate the contribution of treatment plant in shaping microbial community in premise
plumbing.

Part 2: To evaluate the impact of organic matter to microbial regrowth.

1. To evaluate the impact of organic matter migrated from pipe material to microbial
community in drinking water.
2. To identify BOM composition released from pipe material that influences microbial
regrowth in premise plumbing.
Summary from previous experiment 6

One-year monitoring of microbial regrowth in premise plumbing

Sample: water from different faucets : (1) before stagnation, and (2) after 24 h of stagnation.

Sampling location: Engineering building 8 and 14, UTokyo.

Sampling time: May 2018 – April 2019.

Sampling date
Faucet Sampling
Building
code location Summer 2018 Autumn 2018 Winter 2019 Spring 2019
(S) (A) (W) (SP)
Eng. 14-A Room No. 309 May, 24 – 25 Nov, 15 – 16 Jan, 21 – 22 April, 19 – 20
Building 14 14-B Room No. 309 June, 22 – 23 - - -
14-C Room No. 309 June, 21 – 22 Dec, 6 – 7 Jan, 23 – 24 April, 13 – 14
14-D Room No. 301 May, 26 – 27 Nov, 15 – 16 Jan, 24 – 25 April, 14 – 15
14-E Room No. 301 June, 24 – 25 Nov, 27 – 28 Jan, 25 – 26 April, 16 – 17
14-F Room No. 421 June, 23 – 24 - - -
14-G Room No. 409 June, 30 – July, 1 Nov, 23 – 24 Jan, 26 – 27 April, 16 – 17
14-H Room No. 401 July 1 – 2 Nov, 25 – 26 Jan, 26 – 27 April, 20– 21
Eng. 8-A Room No. 401 May 9 – 10 Nov, 21 – 22 Jan, 24 – 25 April, 17 – 18
Building 8
Summary from previous experiment 7

One-year monitoring of microbial regrowth in premise plumbing

T o t a l c e ll c o u n t s (c e lls /m L )

TCC in several faucets during one-year monitoring Microbial composition in faucet S-14A
100%

80%
Before stagnation First 100 mL after stagnation Others (<5%)
1E+06
Novosphingobium
60%
Blastomonas
1E+05
Sphingomonas
40% Effusibacillus
1E+04
Rhizobium
Cyanobacteria
1E+03
20%
Phreatobacter

1E+02 0%
Before After 24h
stagnation stagnation
S=summer, A=autumn, W= winter, SP= spring

Increase of bacterial abundance and shift of microbial community composition after

water stagnation was observed in faucets during one year monitoring.

What caused their regrowth? Organic matter from pipes?

Summary from previous experiment 8

The impact of organic matter from pipes to microbial regrowth

Migration test: static pipe segment method Dissolved organic carbon

6.0
Tap water in UT (Oct 2018) Pre-incubation
5.0 Post-incubation

4.0
DOC

DOC (mg /L)

3.0 migration
New cross-linked 2.0
polyethylene pipe (N-PEX) Carbon-free
glass bottle as 1.0
control (REF)
0.0
REF N-PEX N-SPE O-SPE
New steel pipe lined with
polyethylene powder • DOC released after incubation in
(N-SPE) pipes and PEX pipes released the
largest DOC.

Understanding the composition

Old SPE used for 24 years and biodegradability of DOM
(O-SPE) migrated from pipes is important.
Incubation at 25oC for 7 days
Research Methods 9

Identification of BOM composition promoting microbial regrowth

1) Migration potential 2) Regrowth potential

To evaluate the amount and
composition of organic matter
released from pipes by incubating
sterilized drinking water in pipes.
To evaluate composition of organic
matter released from pipes that
promotes microbial regrowth by
inoculating water contains organic
matter released from pipes with
microbes.

Pipes used in this experiment:

New cross-linked polyethylene pipe (PE):
Diameter: 2 cm
Length of pipe: 3 m
S/V: 2 cm-1
Prior to the experiment, pipes were washed with
sodium hypochlorite 0.3 mg/L for 2 h.
Research Methods 10

1) Migration potential: static pipe Orbitrap MS. analysis:

segment method

Peak intensity
Sterilized tap water Sterilized tap water Before
(Cl2= 0 mg/L) with free (Cl2= 0.2-0.3 mg/L) migration

m/z Orbitrap MS.

* DOM molecules
n=3 n=3 After newly released

Peak intensity
*
migration * * from PE
Incubation in room
temp. for 24 h

Post migration sample

• PE-1 m/z
• PECl-1
• PE-2 • PECl-2 1. SPE (BondElut PPL)
• PE-3 • PECl-3 2. Orbitrap MS
• Column separation with
Analysis:
1. Free chlorine depletion. InertSustain® AQ-C18 column.
• ESI negative ionization.
2. Migration of DOC.
• Molecular formulae assignment
3. DOM composition with Orbitrap MS.
4. Regrowth potential. using Compound Discoverer.
Research Methods 11

1) Migration potential
Migration potential was conducted in 7 sequential cycles

Sterilized tap water Mig1

with and without free chlorine Room temp. 24 h
Mig1 sample for DOC, free
chlorine, Orbitrap MS. and
regrowth potential analysis
Refilled with new sterilized Mig2
tap water Room temp. 24 h
Mig2 sample

Refilled with new sterilized Mig3

tap water Room temp. 24 h

Mig3- Mig6 sample

Refilled with new sterilized Mig7

tap water Room temp. 24 h
Mig7 sample
Research Methods 12

2) Regrowth potential (Mig1)

Post migration sample Sterilized tap water (reference) Control:

• PE-1 • PECl-1 • REF-1 • PE-3 • REF-3
• PE-2 • PECl-2 • REF-2 • PECl-3

Pasteurized (60oC) Pasteurized (60oC)

Inoculate with microbes from tap (initial TCC: 5×10 3 cells/mL) inoculate without microbes
25oC for 4 days 25oC for 4 days

Orbitrap MS. analysis to analyze DOM supporting regrowth:

Microbial

Peak intensity
Peak intensity

community
from tap * *

m/z 4 days
m/z
of incubation
TCC is monitored * BOM candidates
Before incubation every day After incubation whose intensities
decreased by more
than 30%.
Results and Discussions 13

1) Migration potential
Depletion of free chlorine

0.4
Before migration After 24 h migration

0.3
Free chlorine (mg/L)

0.2

0.1

0
Mig1 Mig2 Mig3 Mig4 Mig5 Mig6 Mig7

Free chlorine decreased after 24 h in all migration cycles, due to the reaction of
chlorine with variety compound of in the bulk and material surfaces.
Results and Discussions 14

1) Migration potential
Release of organic carbon

0.6 0.6
Before migration After 24 h of migration PECl Before migration After 24 h migration PE
DOC (mg/L)

0.5 0.5
DOC increased after migration.
What is their composition?
0.4 Are they biodegradable? 0.4

DOC (mg/L)
0.3 0.3
DOC decreased
0.2
Contaminated with bacteria?
0.2

0.1 0.1

0 0
Mig1 Mig2 Mig3 Mig4 Mig5 Mig6 Mig7 Mig1 Mig2 Mig3 Mig4 Mig5 Mig6 Mig7
-0.1 -0.1

• DOC migrated from material into the water was higher in the presence of chlorine,
indicated that materials exposed to chlorine accelerated the migration of organic carbon to
water. These are consistent with previous Biomig test. 8)
• However, in the absence of chlorine, bacterial contamination occurred, as seen in Mig3,
Mig4, and Mig6.
Results and Discussions 15

1) Migration potential
Composition of DOM released from PE pipes (Mig1)

Sample Number of newly released Newly-released molecules are the

molecules (CV≤20%, n=3) molecules that were not present in
PE 270 original drinking water sample but
were detected in the samples after
PECl 341
migration experiment.

Number of newly-released molecules Top assigned molecular formulae

PE PECl
PE (n=3) PECl (n=3)
C16H24O2 C18H22O4

145 C10H12O4 C17H27O4Cl

74 196
 
 
C11H16S2 C17H26O4
C7H16O5N6S C12H26O54S
C8H10O4 C12H16O5

• Some molecules in PE and PECl were different between each other.

• Chlorine might also alter the composition of organic matter released from PE.
Results and Discussions 16

1) Migration potential
Composition of DOM released from PE pipes (Mig1 and Mig7)
Top assigned molecular formulae of PECl

Mig1 Mig7 C18H36O2 (stearic acid)

C18H22O4 C18H36O2
C17H27O4Cl C30H36O3N2
C17H26O4 C16H32O2
Used as coating agent in high-
C12H26O54S C12H26O4S density polyethylene pipe9)
C12H16O5 C4H6O5N2S
The top components released in Mig1 and Mig7 were different, indicated that fresh and used
pipes might release different organic matter.
Common crosslinking by-product of PEX,
methyl tert-butyl ether (C5H12O)10) were not
Analysis of size distribution of
detected.
DOM migrated from PE is
important.
Results and Discussions 17

2) Regrowth potential
Microbial regrowth during incubation (Mig1)
TC C (cells/m L)

Mig1
1E+06
Before regrowth After regrowth (4 d)
1E+06
Bacterial
8E+05
contamination
6E+05

4E+05

2E+05
Contro Contro Contro
0E+00 l l l
PE-1 PE-2 PE-3 PECl-1 PECl-2 PECl-3 REF-1 REF-2 REF-3

BOM candidates supporting regrowth

• Increase of TCC in PECl was higher than that specifically released from pipes:
PE, indicated that chlorine might affect Sample Number of BOM
the release of DOM supporting candidates (CV≤20%,
microbial regrowth. n=3)
• The difference of TCC in REF with PECl
PE 24
was not significant.
PECl 117

DOM released from pipes contributes to microbial regrowth.

Results and Discussions 18

Migration potential: static immersion method

In previous migration experiment, bacterial contamination occurred. To limit the contamination,
static immersion method was applied.

Pipe pieces
(total length= Mig1
20 cm) Room temp. 24 h Mig1 sample for
DOC, free
chlorine and
regrowth
200 mL of sterilized 200 mL of sterilized Mig2
potential analysis
tap water tap water (with Cl2) Room temp. 24 h
n=3 n=3
Mig2 sample
Incubation
25OC for 24 h Mig3
Room temp. 24 h
Post migration sample
Mig3 sample
• PE-1 • PECl-1
• PE-2 • PECl-2 Mig4
• PE-3 • PECl-3 Room temp. 24 h
Mig4 sample

TCC was checked in every sample to ensure no

contamination occurred
Results and Discussions 19

Migration potential: static immersion method

Depletion of free chlorine and release of DOC
F re e c h lo rin e (m g /L )

Consistent with previous

method, chlorine depleted
0.5
Before migration After 24 h migration after 24 h of migration.
0.4

0.3

0.2

0.1

0
Mig1 Mig2 Mig3 Mig4
1.4 1.4 • DOC increased and
DOC (mg/L)

1.2
PE 1.2
PECl the increase in
1 1 PEClwas obviously
higher than in PE
DOC (mg/L)

0.8 0.8

0.6
• Chlorine
0.6
accelerated the
0.4 0.4
release of organic
0.2
0.2 carbon
0 0
Mig1 Mig2 Mig3 Mig1 Mig2
Results and Discussions 20

Migration potential: static immersion method

Regrowth potential (Mig1)
Post migration sample Sterilized tap water (reference) Control:
• PE-1 • PECl-1 • REF-1 • PE-3 • REF-3
• PE-2 • PECl-2 • REF-2 • PECl-3

Inoculate with microbes from tap (initial TCC: 5×10 3 cells/mL) inoculate without microbes
25oC for 4 days 25oC for 4 days
TCC (cells/mL)

1E+06
Before regrowth After regrowth (4 d)
1E+06

8E+05

6E+05

4E+05

2E+05
Control Control Control
0E+00
PE-1 PE-2 PE-3 PECl-1 PECl-2 PECl-3 REF-1 REF-2 REF-3

• No contamination occurred in the assay

• Consistent with previous experiment, increase of TCC in PECl was higher than in PE, which
indicated that chlorine released DOM that supports microbial regrowth.
Conclusion and Future Plan 21

Conclusions:
• Contact of water with pipe material influences the depletion of free chlorine.
• Pipe material had the potential to release organic matter, and material exposed
to chlorine could release higher DOC.
• Several DOM released from PE pipes were found to be biodegradable and could
support microbial regrowth.

Future plan:
• To estimate the size distribution of DOM released from pipe using HPLC-size
exclusion chromatography with UV and TOC (NDIR).
• To conduct more migration experiment using static immersion method to test the
reproducibility.
• To evaluate the effect of other factors (e.g. stagnation time, temperature) to the
release of organic matter and their impact to microbial regrowth.
• To conduct regrowth potential assay by using single or multiple colonies, instead of
microbial community, to evaluate the substrate utilization of specific bacteria.
References 22

1) Lautenschlager, K., Boon, N., Wang, Y., Egli, T. and Hammes, F. (2010). Overnight stagnation of
drinking water in household taps induces microbial growth and changes in community composition.
Water Research, 44, 4868–4877.
2) Wang, H., Edwards, M., Falkinham, J. O. and Pruden, A. (2012). Molecular survey of the occurrence of
legionella spp., mycobacterium spp., pseudomonas aeruginosa, and amoeba hosts in two
chloraminated drinking water distribution systems. Applied and Environmental Microbiology, 78(17),
6285–6294
3) Liu, L. Xing, X. Hu, C. and Wang, H. (2018). One-year survey of opportunistic premise plumbing
pathogens and free-living amoebae in the tap-water of one northern city of China. Journal of
Environmental Sciences, article in press.
4) Proctor, C.R. Reimann. M., Vriens, B. and Hammes, F. (2018). Biofilm in Shower Hoses. Water
Research, 131, 274-286.
5) Wen, G., Kötzsch, S., Vital, M., Egli, T. and Ma, J. (2015). BioMig-A Method to Evaluate the Potential
Release of Compounds from and the Formation of Biofilms on Polymeric Materials in Contact with
Drinking Water. Env. Science & Tech., 49, 11659–11699.
6) Zhang, L., and Liu, S. (2014). Investigation of organic compounds migration from polymeric pipes into
drinking water under long retention times. Procedia Engineering, 70: 1753.1761.
7) Van der Kooij, D. and Veenendaal, H, R. (2017). Biomass Production Potential of Materials in Contact
with Drinking Water: Method and Practical Importance. Water Science & Technology, 1(3): 39 – 45 .
8) Mao, G,. Wang, U. and Hammes, F. (2018). Short-term organic carbon migration from polymeric
materials in contact with chlorinated drinking water. Science of the Total Environment, 613-614, 1220-
1227.
9) Kim, H., Biswas, J. and Choe, S. (2006). Effects of stearic acid coating on zeolite in LDPE, LLDPE, and
HDPE composites. Polymer, 47, 3891-3992.
10) Lund V, Anderson-Glenna N, Skjevrak I, Steffensen IL: Long-term study of migration of volatile organic
compounds from cross-linked polyethylene (PEX) pipes and effects on drinking water quality. Journal of
Water and Health, 9(3), 483-497, 2011.
References 23

THANK YOU FOR YOUR KIND ATTENTION