EPA
EPA/600/R-17/284 I August 2017
www.epa.gov/homeland-security-research
United States
Environmental Protection
Agency
Persistence of Non-pathozabogenic
Bacillus Spores on Sewer Infrastructure
Surfaces and Assessment of
Decontamination Using Chlorine


altft
Office of Research and Development
Homeland Security Research Program

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EPA/600/R-17/284
August 2017
Persistence of Non-pathogenic Bacillus
Spores on Sewer Infrastructure and
Assessment of Decontamination Using
Chlorine
by
Jeffrey Szabo
U.S. Environmental Protection Agency
Cincinnati, OH 45268
Gune Silva and Greg Meiners
CB&I Federal Services, LLC
Cincinnati, OH 45204
Contract EP-C-12-014
U.S. Environmental Protection Agency Contracting Officer's Representative: Ruth Corn
Office of Research and Development
Homeland Security Research Program
Cincinnati, OH 45268

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Disclaimer
The U.S. Environmental Protection Agency (EPA) through its Office of Research and
Development funded and managed the research described herein under contract EP-C-12-014
with CB&I Federal Services, LLC. It has been subjected to the Agency's review and has been
approved for publication. Note that approval does not signify that the contents necessarily reflect
the views of the Agency. Any mention of trade names, products, or services does not imply an
endorsement by the U.S. Government or EPA. The EPA does not endorse any commercial
products, services, or enterprises.
The contractor role did not include establishing Agency policy.
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Table of Contents
Disclaimer	ii
1.0 Introduction	1
1.1	Background and Proj ect Description	1
1.2	Project Objective	2
2.0 Description of Experimental Protocol	2
2.1	Description of the SETBC Apparatus	2
2.2	Description of Bacillus globigii Preparation Procedure	7
2.3	Determination of Bacillus globigii Adhered to Infrastructure Materials	9
2.4	Preparation of Chlorine Solutions Using Sodium Hypochlorite	10
3.0 Analysis of Test Results	11
3.1	Evaluation of B. globigii Adhesion to Infrastructure Materials	11
3.2	Impact of Chlorine on Infrastructure Material-Adhered B. globigii	13
4.0 Conclusions	15
5.0 References	17
List of Figures
Figure 1. Schematic overview of the Secondary Effluent Test Bed Channel setup	4
Figure 2. View of the Secondary Effluent Test Bed Channel system	5
Figure 3. Sewer infrastructure coupons	6
Figure 4. Bacillus globigii removal from infrastructure materials overtime	13
Figure 5. Impact of chlorine on Bacillus globigii adhered to infrastructure materials	15
List of Tables
Table 1. List of Experimental Activities	7
Table 2. Randomized Coupons Sample Pattern	9
Table 3. Concentrations and Volumes of Stock Chlorine Concentrations	10
Table 4. Average Bacillus globigii Concentrations Adhered to Infrastructure Materials	12
Table 5. Water Quality Measured via Online Sensors	12
Table 6. Average Bacillus globigii Concentrations Before and After Chlorination	14
in

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Acronyms
B. globigii Bacillus atrophaeus subspecies globigii
CFU
colony forming units
cm
centimeter
DPD
N,N-di ethyl -p-phenyl enedi amine
EPA
U.S. Environmental Protection Agency
gal
gallon
gpm
gallons per minute
HDPE
high-density polyethylene
hr
hour
L
liter
L/m
liter per minute
min
minute
mL
milliliter
MSDGC
Metropolitan Sewer District of Greater Cincinnati
PVC
polyvinyl chloride
rpm
revolutions per minute
sec
second
SETBC
Secondary Effluent Test Bed Channels
T&E
Test and Evaluation
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Executive Summary
The objective of this study was to examine the persistence of Bacillus atrophaeus subsp. globigii
(B. globigii) spores (a surrogate for pathogenies, anthracis spores) on the surface materials that
make up common sewer systems. To achieve this goal, the U.S. Environmental Protection
Agency (EPA) has built a set of six identical pilot-scale Secondary Effluent Test Bed Channels
(SETBC) at the EPA Test and Evaluation (T&E) Facility in Cincinnati, Ohio. The SETBC
system consists of six 6-inch diameter polyvinyl chloride (PVC) pipes. Each pipe has been
fabricated with two open grids to mount and hold infrastructure test material coupons in the
effluent flow. The SETBC system has service connections that deliver a total flow of 280
gallons per minute (gpm) of unfiltered secondary effluent from the Metropolitan Sewer District
of Greater Cincinnati (MSDGC) (a maximum of approximately 47 gpm per pipe).
Coupons (excised samples) of various sewer infrastructure materials were randomly placed in the
SETBC and conditioned to grow biofilms for two months by exposing them to MSDGC
unfiltered secondary effluent before introduction of the B. globigii spores. The persistence of the
spores on the various infrastructure materials (high density polyethylene [HDPE], brick, rubber,
concrete, iron, clay, PVC) was examined for up to 42 days. In a subsequent experiment, the
efficacy of chlorine disinfection on infrastructure-adhered B. globigii was examined by
introducing chlorine bleach into the flow. The results of the pilot-scale study are presented in
this report along with SETBC structure, system operating conditions, and possible future
research.
A summary of the results from the experiments conducted in the six channel SETBC system are
as follows:
•	The data suggest that shear forces from water flow only (no disinfection) are capable of
achieving 2 to 4 log spore removal on all materials tested except for iron. Spore removal
of 3.2 and 3.4 log were observed from shear forces at 14 days for HDPE and concrete.
Shear forces from normal flow removed 1.9, 2.3 and 2.7 log of the adhered spores from
PVC, rubber and vitrified clay at 14 days of exposure.
•	Few spores adhered to brick above the background levels, and adhered spore levels
dropped below the background concentration by 4 hours after spore injection
•	At 42 days of exposure to secondary effluent flow, log removals of 3.7, 3.8 and >4.0
were observed for PVC, clay and rubber.
•	For PVC, rubber, clay, HDPE and concrete, the number of spores detected at the end of
the experiment were below the background levels. This may be due to variations in spore
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adherence to coupons due to different levels of biofilm and organic matter accumulation
on the coupons, variations in the secondary effluent concentrations, or variations in flow
patterns in the test bed channels.
•	Spores were observed on iron above background levels up to 42 days after injection. Log
removal due to flow was 1.2 at day 14 and 1.5 at day 42. Persistence on iron might be
due to corrosion products on the surface of the iron.
•	Secondary effluent may represent a dilute raw wastewater, but it still exerted a large free
chlorine demand. When enough free chlorine was added to initially achieve 10 and 25
mg/L in the secondary effluent, no chlorine was detected in the flow. When enough
chlorine was added to achieve 50 mg/L in the secondary effluent, 27 mg/L was detected.
This demand is due to the organic compounds in the secondary effluent and will likely be
more pronounced in raw wastewater.
•	Adding chlorine into the flowing wastewater to disinfect B. globigii spores adhered to
wastewater infrastructure coupons was ineffective. Log removals of 1.5 were observed
on both brick and clay after the 50 mg/L chlorine injection compared to 0.7 and 1.0 log
removal for brick and clay, respectively, after 4 hours in secondary flow only (in Phase
1). All other materials had more spore removal after 4 hours in secondary effluent flow
only compared to chlorination. However, it is unknown if the spores removed during
chlorination were the same fraction of spores removed due to shear forces from the flow.
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1.0	Introduction
1.1	Background and Project Description
The U.S. Environmental Protection Agency's (EPA's) Homeland Security Research Program
(HSRP) conducts research to protect infrastructure, and to detect, respond to, and recover from
terrorist attacks on the nation's water and wastewater infrastructure. The potential for biological
contamination of sewer system infrastructure is one area of concern. In the event of a drinking
water distribution system contamination incident involving a biological agent, contamination of
the sewer system infrastructure could result from flushing of the drinking water distribution
system to remove the contaminant. In addition, in the event of a biological contamination
incident over a wide outdoor area or a building exterior, contamination of the sewer system could
result from wash down activities or rain releasing biological agents into a sewer system. An
open question is whether biological agents like pathogenic Bacillus spores, which are hardy and
resistant to inactivation in the environment, will persist on wastewater infrastructure and if they
can be removed via flushing or disinfection.
Information on adhesion of microorganisms to drinking water infrastructure and their survival in
the presence of chlorine is abundant (LeChevallier et al. 1988; De Beer et al. 1994; Chu et al.
2003; Emtiazi et al. 2004; Szabo etal. 2007; Miller et al. 2015). Previous studies on drinking
water infrastructure materials have noted the increased resistance of Bacillus atrophaeus
subspecies globigii (B. globigii) to free chlorine disinfection while they are associated with
biofilms (Miller et al. 2015). (In infrastructure studies related to biological agents,
nonpathogenic Bacillus spp., such as B. globigii, are used a surrogate for pathogenic Bacillus
anthracis) However, information on persistence of microbiological agents and/or their survival
ability on sewer pipe materials is limited. Additionally, the literature cited above focuses mostly
on drinking water biofilms grown on limited numbers of materials with smooth surfaces, which
do not fully represent the composition of the infrastructure materials in sewer systems.
To address the absence of data on biological contaminant persistence on sewer infrastructure, the
EPA's HSRP has built six identical pilot-scale Secondary Effluent Test Bed Channels (SETBC)
at the EPA Test and Evaluation (T&E) Facility in Cincinnati, Ohio. The SETBC system consists
of six 6-inch* diameter polyvinyl chloride (PVC) pipes; each pipe has been fabricated with two
open grids to mount and hold wastewater infrastructure test material coupons (excised samples)
in the secondary effluent flow. The SETBC system is plumbed to deliver a total of 280 gallons
*Note that English units are used in this text when they are the industry standard in the U.S.
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per minute (gpm) of unfiltered secondary effluent from the Metropolitan Sewer District of
Greater Cincinnati (MSDGC). B. globigii spores were released into the flow and their
persistence on wastewater infrastructure coupons was examined over time. These studies also
examined the effect of adding chlorine to the wastewater flow on the persistence of B. globigii
spores.
1.2 Project Objective
The main purpose of this pilot scale study was to evaluate the persistence of B. globigii on
various conditioned (biofilm covered) sewer infrastructure materials in flowing, unfiltered
secondary effluent, which was used as a simulant for dilute raw wastewater. The secondary
objective of this study was to determine the effect of adding chlorine bleach on the persistence of
B. globigii adhered to various sewer infrastructure materials.
2.0	Description of Experimental Protocol
This experiment had two phases. Phase 1 focused on the persistence of B. globigii, a surrogate
for B. anthracis spores, on sewer infrastructure materials after conditioning (growing biofilm)
the infrastructure for two months using secondary effluent. The persistence of B. globigii was
conducted initially for three 1-inch diameter coupons representing sewer infrastructure materials
(brick, concrete and high density polyethylene [HDPE]) followed by testing on four additional
materials (vitrified clay [clay], iron, polyvinyl chloride [PVC] and rubber). Phase 2 focused on
persistence of B. globigii on sewer infrastructure materials after introduction of chlorine into the
wastewater flow. All seven types of infrastructure material coupons were tested together during
Phase 2.
At the conclusion of an experiment, the SETBC system was drained and its wetted surfaces were
disinfected with 70% methanol to eliminate residual B. globigii from the previous test.
Similarly, used coupons were decontaminated with undiluted regular bleach (Clorox) followed
by 70% methanol and tested for residual B. globigii prior to use in the next experiment.
Additional details on the test protocol and time-line are presented later in Table 1.
2.1	Description of the SETBC Apparatus
The Test and Evaluation (T&E) Facility SETBC system consists of six 6-inch diameter PVC
pipes, six secondary effluent flow control valves, six injection ports, one secondary effluent flow
open/close and flow control valve, one secondary effluent drain port, six sets of flow sensors
(Grevline AVFM 5.0 velocity area flow meters. Grevline Instruments Inc.. Ontario. Canada)
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(Greyline, 2017) connected to a flow rate display panel, two sets of pH and temperature
monitoring sensors (not shown) (Hach® GLI online temperature/ORP meter; Hach Company.
Loveland. CO) (Hach, 2005), a data logger (not shown) and two sections (per each pipe) of
fabricated coupon-holding grids (Figure 1). Temperature and pH sensors were calibrated and
maintained according to the manufacturer's instructions.
Figure 2 shows the SETBC apparatus. The top of the figure shows sections A and B where
coupon-holding grids were located (also, see the labeled grids in Figure 1). The bottom portion
of the figure shows the flow sensors (white sections of pipe with cords coming out). The SETBC
system was directly connected to the MSDGC secondary effluent service pipe line via an effluent
valve. This effluent valve allowed secondary effluent to flow into the SETBC setup.
Additionally, each of the six pipes has a globe valve that can be turned to refine the flow in each
pipe. These valves appear as circular handles on each pipe and are visible at the top of Figure 2.
One-inch diameter coupons, prepared from various infrastructure materials, were secured to a
metal rod which was mounted on the grid, allowing exposure of the coupon surfaces to flowing
secondary effluent (Figure 3). Each coupon was mounted on the grid ensuring that the surface of
the coupon was parallel with the water flow. During testing, the secondary effluent flow rates in
each of the six SETBC pipes was adjusted, via the control valve, to 45 gallons per minute (gpm)
(Figure 3).
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8'
Secondary Effluent from MDS
Flow Rate Display Panel
PI-43 P2-45 P3-42
P4-42 P5-43 P6-45
Coupon Holding Grid
Legend
• Injection ports
Secondary effluent flow controlling valves
o Secondary effluent drainage port
H Secondary effluent open/close and flow control
Flow sensors
P Pipes
Figure 1. Schematic overview of the Secondary Effluent Test Bed Channel setup.
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Figure 2. View of the Secondary Effluent Test Bed Channel system.

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From top to bottom: high-density
polyethylene, brick, rubber,
concrete, iron (uncorroded), clay,
PVC
Figure 3. Sewer infrastructure coupons.
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2.2 Description of Bacillus globigii Preparation Procedure
A stock of B. globigii cells was grown in generic spore medium (8 g nutrient broth, 40 mg
MnSC>4 and 100 mg CaCh in 1 L deionized water) for 5 days at 35°C in a shaking (145 rpm)
incubator. The concentration of spores in the resulting suspension was determined by spread
plating. A sub-sample of the purified spores was heat-shocked at 80° C for 10 min and analyzed
to determine the stock spore concentration (Coroller et al. 2001; Szabo et al. 2007). The
concentration of B. globigii in the injection solution was approximately 108 colony forming units
(CFU)/mL. The B. globigii stock solution was injected into each SETBC pipe separately at 170
mL/min for one minute using a pre-calibrated peristaltic pump to achieve a target B. globigii
concentration of approximately 105 CFU/mL.
Table 1. List of Experimental Activities
Phase and
Number of
Tests
Testing Date
Sampling
Time
Activities
Phase 1
Test 1
May 24th 2016

The brick, concrete and HDPE infrastructure material coupons
were placed in the pilot SETBC system for conditioning after
setting the secondary effluent flow to each pipe of the pilot
unit at 45 ± 5 gpm for the duration of the experiment. The
system was operated 24 hours per day during the experiment.
Jul. 18th 2016
0 hour
A pair of coupons from each material was collected prior to
Bacillus globigii spiking to determine the background B.
globigii concentration.
Jul. 18th 2016

Bacillus globigii was injected for 1 minute
Jul. 18th 2016
1 hour
A pair of coupons from each material was collected
Jul. 18th 2016
4 hour
A pair of coupons from each material was collected
Jul. 19th 2016
Day 1
A pair of coupons from each material was collected
Jul. 20th 2016
Day 2
A pair of coupons from each material was collected
Jul. 21st 2016
Day 3
A pair of coupons from each material was collected
Jul. 22td 2016
Day 4
A pair of coupons from each material was collected
Jul. 25th 2016
Day 7
A pair of coupons from each material was collected
Jul. 28th 2016
Day 10
A pair of coupons from each material was collected
Aug. 1st 2016
Day 14
A pair of coupons from each material was collected
Phase 1
Test 2
Aug. 29th 2016

The clay, iron, PVC and rubber infrastructure material
coupons were placed in the pilot SETBC system for
conditioning after setting the secondary effluent flow to each
pipe of the pilot unit at 45 ± 5 gpm. The system was operated
24 hours per day during the experiment.
Oct. 24th 2016
0 hour
A pair of coupons from each material was collected prior to B.
globigii spiking to determine the background B. globigii
concentration.
Oct. 24th 2016

B. globigii was injected for 1 minute
Oct. 24th 2016
1 hour
A pair of coupons from each material was collected
Oct. 24th 2016
4 hour
A pair of coupons from each material was collected
Oct. 25th 2016
Day 1
A pair of coupons from each material was collected
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Phase and
Number of
Tests
Testing Date
Sampling
Time
Activities

Oct. 26th 2016
Day 2
A pair of coupons from each material was collected
Oct. 27th 2016
Day 3
A pair of coupons from each material was collected
Oct. 28th 2016
Day 4
A pair of coupons from each material was collected
Oct. 31st 2016
Day 7
A pair of coupons from each material was collected
Nov. 03rd 2016
Day 10
A pair of coupons from each material was collected
Nov. 07th 2016
Day 14
A pair of coupons from each material was collected
Nov. 14th 2016
Day 21
A pair of coupons from each material was collected
Nov. 21st 2016
Day 28
A pair of coupons from each material was collected
Nov. 28th 2016
Day 35
A pair of coupons from each material was collected
Dec.05th 2016
Day 42
A pair of coupons from each material was collected
Phase 2
Test 1
Feb. 1st 2017

The brick, clay, concrete, HDPE, iron, PVC and rubber
infrastructure material coupons were placed in the pilot
SETBC system for conditioning after setting the secondary
effluent flow to each pipe of the pilot unit at 45 ± 5 gpm for
the duration of the experiment. The system was operated 24
hours per day during the experiment.
Apr. 11th 2017
0 hour
A pair of coupons from each material was collected prior to B.
globigii spiking to determine the background B. globigii
concentration.
Apr. 11th 2017

B. globigii was injected for 1 minute
Apr. 11th 2017
1 hour
A pair of coupons from each material was collected
Apr. 11th 2017
1.5 hour
Chlorine 10 mg/L was injected for 5 minutes
Apr. 11th 2017
1.5 hour
A pair of coupons from each material was collected (after
chlorine exposure)
Apr. 11th 2017
2.0 hour
Chlorine 25 mg/L was injected for 5 minutes
Apr. 11th 2017
2.0 hour
A pair of coupons from each material was collected (after
chlorine exposure)
Apr. 11th 2017
2.5 hour
Chlorine 50 mg/L was injected for 5 minutes
Apr. 11th 2017
2.5 hour
A pair of coupons from each material was collected (after
chlorine exposure)
gpm, gallons per minute; HDPE, high-density polyethylene; SETBC, secondary effluent test bed channels
During the Phase 1 of the experiment, 1-inch diameter coupons made from seven different
infrastructure materials were placed in both Sections A and B. Coupon materials included brick,
clay, concrete, HDPE, iron, PVC and rubber. Due to the nature of the Phase 2 of the experiment
only Section A was used. Regardless of the phase, test material coupons were conditioned for
two months in flowing MSDGC unfiltered secondary effluent. Two months of conditioning
were chosen based on the time available for experimentation. The purpose of this two-month
conditioning period was to grow biofilm on the coupons. At the end of two-month conditioning
period, coupons from each infrastructure material were collected in pairs prior to and after B.
globigii injection (Table 1). The specific pair of coupon sampling was based on a complete
randomization technique as outlined in the Table 2.
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All experiments in Phase 1 were originally planned to span 42 days. However, based on the low
levels of B. globigii detected on the coupons, Test 1 of the Phase 1 experiments was terminated
after two weeks (Table 1).
Table 2. Randomized Coupons Sample Pattern
Phase 1 of the Experiment
Time of Coupon
Collection
Section A Coupon positions
Section B Coupon positions
0 hour
23
01
1 hour
07
29
4 hour
01
07
Day 1
29
03
Day 2
09
11
Day 3
27
25
Day 4
13
19
Day 7
03
27
Day 10
21
09
Day 14
11
05
Day 21
15
17
Day 28
25
21
Day 35
05
23
Day 42
19
13
Phase 2 of the Experiment
0 hour
04 and 07
Not required
1 hour
01 and 03
1.5 hour
08 and 13
2.0 hour
09 and 10
2.5 hour
05 and 11
2.3 Determination of Bacillus globigii Adhered to Infrastructure Materials
After coupons were removed from the SETBCs, their surfaces were sampled to determine the
density of surface-adhered B. globigii. The method used to sample coupon surfaces varied
depending upon the type of coupon material. Concrete, iron, brick and clay pipe coupons had
their surfaces scraped with sterile scalpels until a visible layer of the surface had been removed.
Surface deposits from those coupons were scraped into a sterile coliform sample bottle. The
coupon surface and scalpel were then rinsed with sterile dilution buffer (Hardy Diagnostics,
Springboro, OH) into the respective coliform sample bottle. Large pieces of coupon material
that peeled off during surface scraping were crushed using sterile metal rods and each rod was
rinsed into the respective sample bottle using sterile dilution buffer.
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Each of the other infrastructure material (HDPE, PVC and rubber) coupons were sampled using
sterile swabs stored in lOmL Butterfield's buffer (Copan Diagnostics, Murrieta, CA). After
swabbing, each swab was shaken for 30 sec in 10 mL of Butterfi eld's buffer. After shaking, the
content in Butterfi eld's buffer was added to 90mL of sterile dilution buffer in a coliform sample
bottle.
Samples were then heat-shocked to remove vegetative cells from the secondary effluent, and
analyzed using membrane filtration. Three different volumes (10 mL, 1.0 mL, and 0.1 mL) were
filtered, in duplicate, from each sample. Filters were placed on tryptic soy agar plates and
incubated at 35°C for 24 hours.
2.4 Preparation of Chlorine Solutions Using Sodium Hypochlorite
Chlorination was performed by adding three successive injections of free chlorine into the
secondary effluent flow for 5 minutes each. If no chlorine demand were present, the first
injection would have achieved 10 mg/L in the flow. The subsequent injections would have
produced 25 and 50 mg/L in the flow. Each injection was separated by 30 min. Stock solutions
of chlorine were prepared by diluting sodium hypochlorite (Clorox® bleach [The Chlorox
Company, Oakland, CA]) and chlorine-free granular activated carbon filtered water. Prior to
preparing the stock solutions, the bleach was diluted in granular activated carbon filtered water,
and the concentration of the bleach was determined by using colorimetric DPD (N,N-diethyl-p-
phenylenediamine) Hach method 8021 (Hach, 2014). In order to achieve the target in-pipe
chlorine concentrations (10, 25 and 50 mg/L), 10.2 L stock solutions were prepared with varying
amounts of bleach, and then metered into the flow. Table 3 shows how each chlorine solution
was prepared.
Table 3. Concentrations and Volumes of Stock Chlorine
Waste
Chlorine
Chlorine
Stock
Volume
Total time
~Total
water
Application
Concentration
Solution
of Source
of Stock
Stock
Flow
Rate
in Source
Chlorine
Bleach
application
Volume
Rate
(mL/min)
Bleach (mg/L)
Concentration
Required
to each
prepared
(L/min)


(mg/L)
(mL)
SETBC
(L)





(min)

170.3
170.3
101,000
10,000
1,010
5
10.2
170.3
170.3
101,000
25,000
2,525
5
10.2
170.3
170.3
101,000
50,000
5,050
5
10.2
concentrations
*Total stock volume was calculated based on 10 minute application time for 6 pipes. SETBC, Secondary Effluent
Test Bed Channels
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In addition to measuring the chlorine concentration of the source bleach and stock solutions, grab
samples from the SETBC system were analyzed prior to and after spiking with chlorine. The
measured chlorine concentration in the SETBC system prior to chlorine spiking was non-
detectable. The average chlorine concentrations in the SETBC system after spiking at the 10
mg/L and 25 mg/L levels were also non-detectable. The average chlorine concentration in the
SETBC after spiking at the 50 mg/L level was 27 mg/L. All the grab samples were obtained 30-
45 cm from the injection port at 2.5 minutes after the chlorine injection.
3.0	Analysis of Test Results
3.1	Evaluation of B. globigii Adhesion to Infrastructure Materials
The data in Table 4 shows that, for each material tested, B. globigii spores were detected on the
coupons before the injection of B. globigii. The spores could be coming from the secondary
effluent, or from external contamination in the T&E facility. As this was an unexpected result,
analysis of B. globigii spores did not occur during the coupon conditioning stage. The data in
Table 4 also shows that B. globigii adhered to the coupons at concentrations ranging from
8.2x 103 to 1,9x 104 CFU/in2 on all the materials except brick at one hour after the injection.
There was no apparent persistence of B. globigii above the background levels on the brick
coupons at the 4-hours after injection sample point and beyond. For the other materials, the level
of B. globigii adhesion at 1 and 4 hours appears to be an increase beyond the background levels
of adhered spores.
Based on the one-hour B. globigii adhesion concentration, HDPE and concrete had the highest
log removal of B. globigii (3.2 to 3.4) within fourteen days, which is attributed to the shear force
of the flowing wastewater (Table 4 and Figure 4). At day 14, the water shear force resulted in
1.2, 1.9, 2.3 and 2.7 log removal of B. globigii for iron, PVC, rubber and clay material coupons,
respectively (Table 4 and Figure 4). The highest overall log removal of B. globigii was observed
for clay (3.8), PVC (3.7) and rubber (>4.0) at forty-two days (Table 4 and Figure 4). However,
the amount of adhered B. globigii changed little on the iron coupons after the first two days.
Through day 42, the level of B. globigii detected on the iron coupons was above the background
levels, and 1.5 log removal was observed. Corrosion on the surface of the iron may be a possible
reason for this result.
With the exception of iron, the level of B. globigii detected on the coupon dropped below the
background levels by day 14 or 42. In the day 28 clay sample, no spores were detected on the
sampled coupons. It is difficult to explain why the level of spores dropped below the
background levels. It could be that there was wide variability in the adhesion of spores to
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individual coupon materials due to different amounts of adhered biofilm and organic matter. It is
also possible that downstream water swirling and spiraling patterns in the wastewater flow
caused adhesion variability. Also, the constant fluctuation of secondary effluent composition
may also have contributed to the variability of the test results. The average pH and temperature
in the unfiltered secondary effluent flow are summarized in Table 5. Note that temperature
during the Phase 1 Test 1 period was considerably higher than the other two tests, although it is
unclear if this influenced the experimental results. In summary, although there is variability in
the results, the data suggests that most B. globigii spores injected into the pipes are not persistent
on the coupon material over 14 or 42 days. The only exception is iron, where spores remained
above background levels up to 42 days.
Table 4. Average Bacillus globigii Concentrations Adhered to Infrastructure Materials
Time after B.
globigii
injection
Phase 1; Average/?, globigii Concentration (CFU/in2)
Test 1
Test 2
Brick
Concrete
HDPE
Clay
Iron
PVC
Rubber
0 (pre-injection)
5.2E+02
1.2E+02
2.0E+02
1.3E+02
2.5E+01
2.5E+02
1.3E+02
1 hr
5.8E+02
1.2E+04
8.2E+03
1.9E+04
8.1E+03
1.2E+04
1.2E+04
4 lir
1.2E+02
7.0E+02
1.3E+03
2.0E+03
3.7E+03
2.1E+03
2.0E+03
Day 1
1.4E+02
7.4E+02
2.3E+02
5.8E+02
5.0E+02
3.5E+02
2.0E+03
Day 2
2.8E+01
1.8E+02
3.3E+02
3.4E+02
1.1E+03
6.4E+02
9.8E+02
Day 3
2.8E+01
1.3E+02
1.9E+01
1.7E+02
2.5E+02
2.1E+02
3.6E+02
Day 4
8.7E+01
4.0E+02
3.6E+01
9.8E+02
1.0E+02
7.1E+02
5.9E+02
Day 7
2.5E+02
5.0E+01
1.0E+01
1.6E+02
6.5E+02
2.6E+02
9.4E+01
Day 10
2.8E+01
5.0E+01
1.6E+01
0.0E+00
1.5E+02
1.3E+02
2.4E+01
Day 14
2.5E+02
5.0E+00
5.0E+00
3.5E+01
4.6E+02
1.6E+02
6.5E+01
Day 21



2.5E+01
3.0E+02
6.0E+01
3.5E+01
Day 28



0.0E+00
5.6E+02
3.8E+01
1.6E+02
Day 35



1.5E+01
1.6E+02
3.0E+01
2.8E+01
Day 42



2.5E+00
2.5E+02
2.5E+00
0.0E+00
HDPE, high density polyethylene
Table 5. Water Quality Measured via Online Sensors
Phase and Number
of Tests
Testing Date
Average pH
(pH units)
Average
Temperature
(°C)
Phase 1 Test 1
Jul. 18th -Aug. 01st 2016
6.9
26
Phase 1 Test 2
Oct. 24th-Dec. 01st 2016
7.0
19
Phase 2 Test 1
Apr. 11th 2017
7.2
21
12

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*+ -








O Brick







~ Clay





X

X Concrete





~
3 -
~ HDPE



~



0 Iron

n


&
V

* PVC

i—i

X



A Rubber


~
X
A
2 -

v
x
0
~
~
~

f
V
~

~
0


s
A

X



1 -
x a
w
o
$
0
o
0

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procedures used by wastewater utilities.
With respect to chlorine disinfection of adhered B. globigii, brick and clay were the only
materials that had more B. globigii removal at the 50 mg/L chlorine level compared to the
removal observed after 4 hours in secondary effluent flow during Phase 1. After 4 hours of flow
in Phase 1, spore removals of 0.7 and 1.0 log were observed for brick and clay, respectively
(Figure 4). After chlorination at the 10 mg/L, then 25 mg/L, and finally 50 mg/L level, spore
removals were 1.5 log for both materials (Figure 5). However, it is unknown if the spores
removed during chlorination were the same fraction of spores removed due to shear forces from
the flow.
For all other materials tested, more spore removal was observed at 4 hours with secondary
effluent flow only, compared to the 50 mg/L level of chlorination (Table 4 and Table 6). Even
though direct comparison of data from the current secondary effluent study to past drinking
water studies was difficult, previous research has reported that biofilm adhered organisms had
significantly increased resistance to chlorine disinfection (Miller et al. 2015). It is possible that
this resistance carries over to wastewater environments.
It is difficult to explain the reason for the lower log inactivation of B. globigii in PVC at 50 mg/L
chlorine than 25 mg/L chlorine. This could again be a result of lower adhesion of B. globigii to
some coupons due to the water flow variations at the time of B. globigii injection, or the
composition of the secondary effluent. Additionally, variation in the accumulated organic
matter on the coupons surfaces may result in variable chlorine efficacy.
Table 6. Average Bacillus globigii Concentrations Before and After Chlorination
Coupon Collection
Time /
Calculated Chlorine
Concentration /
Measured Chlorine
Concentration
Phase 2; Average/?, globigii Concentration (CFU/in2)
Brick
Concrete
HDPE
Clay
Iron
PVC
Rubber
0 hr/NA/ND
0.0E+00
2.5E+01
5.3E+02
1.3E+02
9.5E+02
5.0E+00
1.8E+02
1 hr/NA/ND
3.2E+04
2.1E+03
1.9E+03
2.8E+04
2.6E+03
8.6E+02
3.9E+03
1.5 hr/10 mg/L/ND
2.3E+04
8.4E+03
5.0E+03
4.1E+03
8.0E+03
3.1E+03
2.8E+03
2.0 hr/25 mg/L/ND
2.3E+03
2.9E+03
1.2E+03
1.8E+03
1.4E+03
6.3E+02
2.2E+03
2.5 hr/50 mg/L/27 mg/L
1.0E+03
1.6E+03
5.5E+02
9.0E+02
1.4E+03
1.4E+03
1.3E+03
*CFU, colony forming units; HDPE, high-density polyethylene; NA, not applicable; ND, non detect
14
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c
o
CC
2.0
1.5 -
1.0 -
^ 0.5 -
C3
O
hJ
W
0
o
Brick
w
Clay
X
Concrete
~
HDPE
0
Iron
~
PVC
A
Rubber
o.o -
-0.5 -
-1.0
£
o
X
X
10
20
30
40
50
60
Chlorine Concentration (mg L"1)
Figure 5. Impact of chlorine on Bacillus globigii adhered to infrastructure materials.
4.0 Conclusions
The following conclusions can be drawn from the data collected during Phase 1 of the
experimentation, which observed spore persistence in the presence of continuous secondary
effluent flow:
•	The data suggest that shear force from water flow (45 gpm in a 6-inch diameter open
channel) is capable of 2 to 4 log removal of spores on all materials tested except iron.
Log removals of 3.2 and 3.4 via water flow were observed at 14 days for HDPE and
concrete. Flow removed 1.9, 2.3 and 2.7 log of the adhered spores from PVC, rubber and
clay, respectively, at 14 days of exposure.
•	Few spores adhere to brick above the background levels, and adhered spore levels
dropped below the background concentration by 4 hours after spore injection
•	At 42 days of exposure to secondary effluent flow, log removals of 3.7, 3.8 and >4.0
were observed for PVC, clay and rubber.
•	For PVC, rubber, clay, HDPE and concrete, the number of spores detected at the end of
the experiment were below the background levels. This may be due to variations in spore
adherence to coupons due to different levels of biofilm and organic matter accumulation
on the coupons, variations in the secondary effluent concentration, or variations in flow
15
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patterns in the test bed channels.
•	Spores were observed to remain adhered to iron above background levels for at least 42
days after injection. Log removal due to shear forces from the flow was 1.2 at day 14 and
1.5 at day 42. Persistence on iron might be due to corrosion products on the surface of
the iron.
The following conclusions can be drawn from the data collected during Phase 2 of the
experimentation, which observed spore persistence in the presence of chlorine:
•	Secondary effluent may represent a dilute raw wastewater, but it still exerted a large free
chlorine demand. When enough free chlorine was added to achieve 10 and 25 mg/L in
the secondary effluent, no chlorine was detected in the flow. When enough chlorine was
added to achieve 50 mg/L in the secondary effluent, 27 mg/L was detected. This demand
is due to the organic and inorganic compounds in the secondary effluent, and will likely
be more pronounced in raw wastewater.
•	Adding chlorine to disinfect B. globigii spores adhered to wastewater infrastructure
coupons was ineffective. Log removals of 1.5 were observed on both brick and clay after
the 50 mg/L chlorine injection compared to 0.7 and 1.0 log removal for brick and clay,
respectively, after 4 hours in secondary flow only (in Phase 1). All other materials had
more spore removal after 4 hours in secondary effluent flow only compared to
chlorination. However, it is unknown if the spores removed during chlorination were the
same fraction of spores removed due to shear forces from the flow.
Should Bacillus spore contamination flow into a sewer during a real contamination event, the
data show that most of the spores will flow with the water. Spores that do adhere to
infrastructure do so in a largely transient manner, and most are washed off of the infrastructure
material in the days after the contamination event. There could be spores adhered to the
infrastructure for at least 42 days on clay, PVC, rubber and especially on iron, where the spores
were most persistent. It is possible that spores might persist longer, but times frames beyond 42
days were not addressed in this project. Adding chlorine to a wastewater system to
decontaminate spores is largely ineffective. Future work should examine the efficacy of other
disinfectants such as chloramines or peracetic acid, which may not degrade as quickly in
wastewater as free chlorine. Alternatively, levels of free chlorine above 50 mg/L could also be
tested, or the amount of contact time at 50 mg/L could be increased. However, users of this data
must also consider that adding chlorine to wastewater in a sewer may have unintended
consequences, such as formation of disinfection by products through reaction with organic
matter, or trapped chlorine vapors in the collection system.
16
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5.0 References
Coroller L., Leguerinel I. and Mafart P. 2001. Effect of water activities of the heating and the
recovery media on the apparent heat resistance of Bacillus cereus spores. Applied and
Environmental Microbiology, 67(1), 317-322.
Chu C., Lu C., Lee C.M. and Tasi C. 2003. Effects of chlorine level on growth of biofilm in
drinking water pipes. Water Science and Technology, Water Supply, 3(1-2): 171—177.
De Beer D., Srinivasan R. and Stewart P.S. 1994. Direct measurement of chlorine penetration
into biofilms during disinfection. Applied and Environmental Microbiology, 60(12),
4339-4344.
Emtiazi F., Schwartz T., Marten S.M., Krolla-Sidenstein P. and Obst U. 2004. Investigation of
natural biofilms formed during the production of drinking water from surface water
embankment filtration. Water Research, 38(5), 1197-1206.
Greyline Instruments. 2017. "Users Guide: Installation and Operation Instructions [for the] Area-
Velocity Flow Meter Model AVFM 5.0."
http://www.grevline.com/images/products/pdf/AVFM-Userguide.pdf accessed
05/26/2017.
Hach. 2005. "Digital D3400 sc conductivity/resistivity sensor data sheet."
https://www.hach.com/contacting-conductivitv-sensor-for-low-conductivitv-k-0-Q5-with-
l-2-kvnar-compression-fitting/product-downloads?id=7640077974. accessed 05/26/2017.
Hach. 2014. "Free chlorine method 8021." https://www.hach.com/asset-get.download-
en.jsa?code=55577, accessed 05/26/2017.
LeChevallier M.W., Cawthon C.D. and Lee R.G. 1988. Factors promoting the survival of
bacteria in chlorinated water supplies. Applied and Environmental Microbiology, 54(3),
649-654.
Miller H.C., Wylie J., Dejean G., Kaksonen A.H., Sutton D., Braun K. and Puzon G.J. 2015.
Reduced efficiency of chlorine disinfection of Naegleria fowleri in a drinking water
distribution biofilm. Environmental Science and Technology, 49(18), 11125-11131.
Szabo J., Rice, E.W. and Bishop, P.L. 2007. Persistence and decontamination of Bacillus
atrophaeus subsp. globigii spores on corroded iron in a model drinking water system.
Applied and Environmental Microbiology, 73(8), 2451-2457.
17
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United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
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Washington, DC 20460
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