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 Number
Household tap water in US 2)
Legionella pneumophila 9.8 copies/mL
Mycobacterium avium 1.1 copies/mL
Pseudomonas aeruginosa 1.8 copies/mL
Washroom and kitchen tap water in China3) Legionella pneumophila 3 log copies/mL
Pseudomonas aeruginosa 1.04 log copies/mL
Biofilm shower hoses in Europe Legionella pneumophila 6.6x105 copies/cm2
(Proctor et al., 2018) 4) Mycobacterium avium 6.6x107 copies/cm2
Acanthamoeba spp 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

Limitation:
Composition of organic matter released from pipes and their impact on microbial
community had not been studied yet.
Research framework 4

Research objective:
to reveal risks related to microbial regrowth in premise plumbing and 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.
Results from previous experiment 5
Research Methods 6

Identification of BOM composition promoting microbial regrowth

1) Migration potential 1) 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 7

1) Migration potential
Orbitrap MS. analysis:
Sterilized tap water Sterilized tap water

Peak intensity
Before
with free Cl2 (0.2-0.3 mg/L)
migration

m/z Orbirap 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
• HPLC column separation with using
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 8

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 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 9

2) Regrowth potential

Post migration sample Sterilized tap water (reference) Control:

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

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:

Microbial

Peak intensity
Peak intensity

community
from tap * *

*
4 days
of incubation m/z
m/z
TCC is monitored
* BOM candidates
every day After incubation
Before incubation whose intensities
decreased by more
than 30%.
Results and Discussions 10

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, indicated that contact of drinking water with pipe
material accelerates the depletion of free chlorine, even after repeated migration
experiment.
Results and Discussions 11

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

• The release of DOC was higher in PECl, indicated that materials exposed to chlorine
accelerated the migration of organic carbon to water.
• However, in PE condition, bacterial contamination might occur, as seen in Mig3, Mig4,
and Mig7.
Results and Discussions 12

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

Sample Number of newly formed Newly-formed formulae is the

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

Number of newly-formed molecules Top individual assigned molecules

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 alter the composition of organic matter released from PE.
Results and Discussions 13

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

Mig1 Mig7
C18H36O2 (stearic acid)
C17H26O4 C18H36O2
C8H10N4 C30H36O3N2
C18H22N4 C16H32O2
C7H8O4 C12H26O4S
Used as coating agent in high-
density polyethylene pipe6)
C8H10O4 C4H6O5N2S

The top components released in Mig1 and Mig7 were different, indicated that
fresh and used pipes released different organic matter.
Common crosslinking by-product
of PEX, methyl tert-butyl ether
(C5H12O) were not detected. Analysis of size distribution
of DOM migrated from PE is
important.
Results and Discussions 14

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

Mig1
1E+06

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

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
Results and Discussions 15

2) Regrowth potential
Microbial regrowth during incubation (Mig7)
 6. Effects of stearic acid coating on zeolite in
LDPE, LLDPE, and HDPE composites

Production and Characterization of

Polyethylene/Calcium Carbonate Composite
Materials by Using Calcium Carbonate Dry and
Wet Coated With Different Fatty Acids

The effects of glutamine palmitic acid content

on properties of high density
polyethylene/silica composites