© 2018 The Authors H2Open Journal Vol 1 No 2

160 doi: 10.2166/h2oj.2018.014

Detection of drinking water contamination by an optical real-time

bacteria sensor

B. Højrisa,*, S. N. Kornholtb, S. C. B. Christensenc, H.-J. Albrechtsend and L. S. Olesena

a
GRUNDFOS Holding A/S, Poul Due Jensens Vej 7, DK-8850 Bjerringbro, Denmark

b
Aarhus Vand A/S, Viby J, Denmark

c
HOFOR A/S, København S, Denmark

d
Department of Environmental Engineering, Technical University of Denmark, Kgs. Lyngby, Denmark

*Corresponding author. E-mail: [email protected]

Abstract

In a drinking water distribution system, little is known about the characteristics of a microbiological pollution,
how it enters the system, and how it can be detected. The drinking water industry has relied on various pollution
indicators, through grab sampling and laboratory analyses, revealing results long after the water has been used.
To be able to react more proactively to pollution events, many drinking water distributors supplement grab
sampling with proportional sampling and/or real-time sensors. We have tested the ability of a new bacteria moni-
tor to detect four different pollution events: wastewater intrusion, rainwater runoff, resuspension of drinking
water sediments, and bird droppings entering the distribution system. The monitor response, in terms of bacteria
and abiotic particle concentrations, was compared with traditional laboratory methods. The results illustrate the
benefits of using such real-time bacteria sensors for monitoring the dynamics of drinking water microbiology and
for early warning of potential pollution events.

Key words: bacteria monitor, drinking water, early warning, real-time sensor, pollution detection

INTRODUCTION

Clean healthy drinking water is commonly characterized as water that complies with local regulation.
In most cases, monitoring of water quality is carried out through grab sampling, representing individ-
ual points in time and space, combined with laborious laboratory analyses yielding results within days.
The probability of catching a periodic pollution event is thus very small (Besmer & Hammes 2016)
and the chance of acting on it even smaller.
What utilities lack to be able to react proactively on pollution events is real-time monitoring
methods with a high timely resolution going beyond current legislations. Fast methods measuring,
e.g. conductivity, oxygen level, pH, and turbidity, are already implemented in many systems, and
they are continuously becoming more robust and maintenance free (Raich 2013; Banna et al.
2014). However, they do not measure changes in microbiological load, but merely provide indicators
that may or may not correlate with the number and activity of microorganisms. Methods that target

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY-NC-SA 4.0), which permits
copying, adaptation and redistribution for non-commercial purposes, provided the contribution is distributed under the same licence as the
original, and the original work is properly cited (http://creativecommons.org/licenses/by-nc-sa/4.0/).

Downloaded from https://iwaponline.com/h2open/article-pdf/1/2/160/518746/0010160.pdf

by guest
 H2Open Journal Vol 1 No 2
161 doi: 10.2166/h2oj.2018.014

microorganisms more directly do exist, though they are mostly laboratory-based methods or require
handling of samples prior to analysis (Lopez-Roldan et al. 2013). Some of the most recent methods
that have been tested in the field include flow cytometry (Prest et al. 2013), ATP (Vang et al.
2014), and optical bacteria detection (Højris et al. 2016). Both flow cytometry and ATP monitoring
require sensitive chemicals, create waste, and require regular maintenance. The latter optical technol-
ogy is, on the other hand, chemical free and requires little maintenance. It was recently demonstrated
in laboratory and field tests to be able to monitor the concentration of bacteria and abiotic particles
with a 10-minute resolution (Højris et al. 2016).
We have tested the ability of this optical bacteria monitor to detect different types of potential
drinking water pollutants and compared this ability to that of traditional laboratory-based methods
and turbidity measurements. As model pollutants, we have applied municipal wastewater,
surface water runoff/soil, distribution system sediments, and bird excrement. Unintended cross-
connection between drinking water and wastewater systems (Vestergaard et al. 2007) and bird
excrement from partly open storage facilities, have each been associated with at least one
major pollution event in Denmark, and surface water runoff is known to cause periodic events
of elevated plate counts and possibly presence of E. coli during the shift from dry to wet periods
(Kistemann et al. 2002). Sediments in drinking water distribution systems may be loosened
during pressure shocks and could influence monitoring of both bacteria and abiotic particles
(Christensen 2011).

METHODS

The applied sensor utilizes three-dimensional image analysis of individual suspended particles, includ-
ing bacteria, and reports the identified objects as either bacteria or abiotic particles. It analyses
batches of water samples with a time resolution of 10 minutes. A detailed description of the technol-
ogy has been published elsewhere (Højris et al. 2016).
Laboratory tests of the optical sensor were carried out at Technical University of Denmark, Depart-
ment of Environmental Engineering, Kgs. Lyngby, Denmark (DTU), and Grundfos Holding A/S,
Bjerringbro, Denmark (Grundfos). The GRUNDFOS BACMON bacteria monitor (hereafter named
the sensor) and accompanying exchangeable flow cells were supplied by Grundfos Holding A/S,
Denmark.
At DTU, spiking tests were performed using: (a) raw municipal wastewater, (b) surface water runoff
sampled from a puddle of water following a rain event, and (c) sediments from a drinking water dis-
tribution network sampled from flushing a fire hydrant. At Grundfos, spiking tests were performed
using: (a) raw municipal wastewater sampled at Bjerringbro municipal wastewater treatment plant
after sand trap and coarse filtration, (b) surface water runoff simulated by soil taken from 10 cm
depth, and (c) bird excrement sampled at a pigeon-breeding member of Bjerringbro Poultry Associ-
ation. Solid samples were suspended in filter-sterilized (0.2 micrometre) tap water, allowed to
sediment for 1 hour, and decanted.
The pollutants were added to tap water from the two locations. The two tap water qualities were
analysed using the same methods as the spiked samples to establish baseline average and deviation.
Total Direct Cell Count (TDCC) was performed by DAPI (40 ,6-diamidino-2-phenylindole) staining and
epi-fluorescence microscopy (DTU: Olympus BH-2 microscope (Japan), Grundfos: Zeiss Axio Imager
2 (Germany)). Prior to staining, samples containing aggregates were treated with ultrasound (Branson
Sonifier 250) for 1 minute to split the aggregates in individual particles and bacteria. Heterotrophic
Plate Counts (HPC) were carried out by pour plate in yeast extract agar incubated for either 68 + 4
hours at 22 °C or 44 + 4 hours at 37 °C. ATP measurements were performed on an Advance
Coupe luminometer (Celsis) with Lumin(ATE)/Lumin(EX) reagents (Celsis). Turbidity measurements

Downloaded from https://iwaponline.com/h2open/article-pdf/1/2/160/518746/0010160.pdf

by guest
 H2Open Journal Vol 1 No 2
162 doi: 10.2166/h2oj.2018.014

were carried out on a Turb 430 IR field turbidity meter (Wissenschaftlich Technische Werkstätten –
WTW) (at DTU) or a Turbiquant 3000 IR turbidimeter (HACH) (at Grundfos). Conductivity measure-
ments were carried out on a Cond 3310 SET 1 field equipment (WTW), or on a CDM 83 Conductivity
Meter (Radiometer Copenhagen) (both at DTU).

RESULTS AND DISCUSSION

Spiking tap water with three potential pollution sources, surface water runoff, domestic wastewater
and pigeon faeces, showed at which concentrations the pollution could be detected (Figure 1). For
each series of measurements, including sensor responses, turbidity, and total direct cell counts
(DAPI), the average and standard deviations of spiked samples were compared to the average and
standard deviation of the tap water itself.
Applying the student T-test (α ¼ 0.015) to the spikes and background measurements, gave the levels
at which the added pollution source could be significantly detected. The results of the T-tests are
shown in Figure 2. For surface water runoff, the abiotic particle variable detected the pollution at
2 mg/L dry soil, total cell counts at 6.7 mg/L and turbidity and the bacteria variable both at 20 mg/L.
For wastewater, the abiotic particle variable detected the pollution at 0.1 mg/L dry soil, total cell
counts and the bacteria variable both at 0.3 mg/L and turbidity at 3 mg/L. For pigeon faeces, all
except turbidity detected the pollution at 0.5 mg/L dry faeces, whereas turbidity only detected it at
5 mg/L.
Spiking tests performed at DTU (see Figure 3) all significantly differed from the background (T-test,
α ¼ 0.05). Background levels in the un-spiked tap water were: Bacteria (sensor) 6,440 + 1,330 mL–1,
Abiotic particles 22,000 + 2,060, HPC 37 °C 9.9 + 1.7 CFU/mL, HPC 22 °C 9.9 + 1.7 CFU/mL, Tur-
bidity 0.2654 + 0.04 NTU, ATP 1.81 + 0.24 pg ATP/mL (average + one standard deviation). In all
spiking tests, a good linearity (R2 . 0.975) was observed for all parameters measured.
To address the accuracy of single measurements, a series of tests was made at DTU in which the
water sample was kept stagnant within the flow cell during several measurements (data not shown).
The lower values measured for bacteria in tap water samples expressed a higher deviation (15–30%)
compared to bacteria in spiked samples (9–14%) and abiotic particles in all samples (4–13%).
The total number of particles detected by the sensor (the sum of bacteria and abiotic particles) is
expected to corelate with the turbidity of the water for non-coloured samples. The lower threshold
of the sensor in terms of particle size does, however, leave out particles smaller than approximately
500 nm, which to some extent limits this correlation (Højris et al. 2016). Further, as the turbidity con-
tribution is different for different particle sizes and diffractive indexes (Melik & Folger 1983; Cheng
et al. 2010), the correlation may be further compromised.
Comparing the sum of bacteria and abiotic particles measured by the sensor to turbidity for each
individual pollution simulation (see Figure 4) shows good correlation between the total concentration
of particles (bacteria þ abiotic particles) and turbidity. However, the slope differs significantly
between the different pollution sources. The two tests simulating pollution from surface water
runoff, carried out at two different locations and analysed at two different laboratories, have similar
slopes, whereas the two tests using municipal wastewater show different slopes. This observation is in
good agreement with previous studies (McCoy & Olson 1986).
The number of bacteria measured by the sensor is also expected to corelate with ATP, given the
cells involved have similar amounts of ATP. Comparing bacteria measured by the sensor and ATP,
for the two tests where ATP was measured (see Figure 5), shows a good correlation, which in the
two cases is independent on the type of pollution. However, it may be expected that bacterial cells
from other more nutrient rich pollution sources, like faeces, have a higher ATP content and that
the slope in such cases are different.

Downloaded from https://iwaponline.com/h2open/article-pdf/1/2/160/518746/0010160.pdf

by guest
 H2Open Journal Vol 1 No 2
163 doi: 10.2166/h2oj.2018.014

Figure 1 | Results from spiking drinking water (at Grundfos) with three potential pollution sources: domestic wastewater (top/
right), surface water runoff simulated by soil suspension (middle), and pigeon faeces (bottom/left). Error bars indicate + one
standard deviation. Horizontal bands represent background levels spanned by averages + one standard deviation.

Downloaded from https://iwaponline.com/h2open/article-pdf/1/2/160/518746/0010160.pdf

by guest
 H2Open Journal Vol 1 No 2
164 doi: 10.2166/h2oj.2018.014

Figure 2 | Significant deviation from background levels based on students T-test (α ¼ 0.015). ‘ þ ’ indicates when the responses
of the different methods are significantly higher than the background. ‘(þ)’ indicates responses significantly lower than the
background. ‘na’ indicates that analysis was not possible. The bacteria and abiotic particles are measured by the sensor and it is
seen that it reacts 10–100 times before the turbidity meter and at the same contamination levels as total direct cell counts
(TDCC).

Assuming HPC reveal relevant fractions of the total populations, and that these fractions are com-
parable, some correlation could be expected between the bacteria measured by the sensor and plate
counts. Comparing the results from the two tests where both parameters were measured (see Figure 6)
does show a relatively good correlation. The fraction of the total cell number as measured by the
sensor that can be identified with HPC is, however, different depending on the pollution level. As
the measured plate counts spans four decades, the total number of bacteria as measured by the
sensor only spans two. Clearly, there are different fractions of bacteria that are not detected by the
plate count method, which is also the result of several previous studies (Byrd et al. 1991; Allen
et al. 2004; Chowdhury 2012).
As published earlier (Højris et al. 2016), there is a relatively good correlation between the number
of bacteria detected by the sensor and the TDCC performed manually on a microscope. The results of
the presented tests support this as there is close to a 1:1 relationship between the two measures (see
Figure 7). In the test simulating surface water runoff performed at Grundfos, the total direct cell
counts were, however, higher than the numbers reported by the sensor, still with a good linear corre-
lation. A plausible explanation could be that the cells in the suspended soil used for the test were
clustered and that the clusters were broken up prior to analysing the TDCC analyses.
Using plate counts as a tool to identify pollution of drinking water has been practiced for many
years, regardless the increasing acceptance that the method only reveals an unknown fraction of
the entire population (Byrd et al. 1991; Allen et al. 2004; Chowdhury 2012). In the two tests

Downloaded from https://iwaponline.com/h2open/article-pdf/1/2/160/518746/0010160.pdf

by guest
 H2Open Journal Vol 1 No 2
165 doi: 10.2166/h2oj.2018.014

Figure 3 | Results from spiking drinking water (at DTU) with three potential pollution sources: domestic wastewater (top),
surface water runoff simulated by puddle water (middle), and sediment from a drinking water distribution system (bottom).
Error bars indicate + one standard deviation. Background levels have been subtracted and combined standard deviations
calculated.

Downloaded from https://iwaponline.com/h2open/article-pdf/1/2/160/518746/0010160.pdf

by guest
 H2Open Journal Vol 1 No 2
166 doi: 10.2166/h2oj.2018.014

Figure 4 | Total particle concentration as the sum of bacteria and abiotic particle (both measured by the sensor) plotted against
turbidity for each spiking test. The total particle concentration correlates well with turbidity within each test, but the coefficient
differs among the different pollution sources. The two surface water run-off simulations express the same coefficient, whereas
the two wastewater simulations are different.

Figure 5 | Bacteria concentration (measured by the sensor) plotted against ATP for wastewater and surface water run-off (both
performed at DTU). In both tests a good correlation was found between the bacteria and ATP concentrations. Note that the
regression lines of the two different pollution sources coincide.

Figure 6 | Bacteria concentration (measured by the sensor) plotted against heterotrophic plate counts (HPC) at 22 and 37 °C for
wastewater and surface water run-off (both performed at DTU).

shown in Figure 6, one colony on a plate may thus represent a number of bacteria between four and
650. Similar observations may be made regarding the use of turbidity as an approximate or indirect
measure of bacterial pollution. Though a good correlation has been shown for each pollution

Downloaded from https://iwaponline.com/h2open/article-pdf/1/2/160/518746/0010160.pdf

by guest
 H2Open Journal Vol 1 No 2
167 doi: 10.2166/h2oj.2018.014

Figure 7 | Bacteria concentration (measured by the sensor) plotted against total direct cell counts (DAPI) for wastewater,
surface water run-off, and pigeon faeces (all performed at Grundfos).

source, it is not possible to estimate the number of bacteria and abiotic particles based on a turbidity
measurement, unless the type of pollution and the corresponding correlation is known beforehand.
TDCC or number of bacteria as measured by the sensor seems to be a more precise measure of bac-
terial pollution of drinking water. With the several days’ turnover for plate counts or the laborious
manual microscopy of the total direct count, the sensor’s time resolution of 10 minutes approaches
a real-time measure. Though the sensor does not count the individual cells if these are clustered, it
gives a better approximation of the total cell number than both plate counts and turbidity.

CONCLUSIONS

The results show a good correlation between the sensor’s bacteria signal and total direct cell counts
performed in the laboratory. The sensor’s ability to detect different simulated pollution events was
shown to be comparable to laboratory-based total direct cell counts and the concentrations at
which the pollution was detected was one half to two decades lower than the detection limit for tur-
bidity. A good correlation was seen against ATP, but the correlation with turbidity (against the sum of
bacteria and abiotic particles from the sensor) depended upon the pollution source. The applied
sensor and plate counts showed similar responses, but the ratio between the two ranged from 4:1
in the most polluted samples to 650:1 in the diluted ones. These results demonstrate the benefit of
the sensor over turbidity sensors in monitoring bacteria in drinking water, detecting potential pol-
lution events, and illustrate the applicability of the bacteria sensor as early warning of
microbiological pollution of drinking water.

REFERENCES

Allen, M. J., Edberg, S. C. & Reasoner, D. J. 2004 Heterotrophic plate count bacteria – what is their significance in drinking
water? Int. J. Food Microbiol. 92, 265–275.
Banna, M. H., Imran, S., Francisque, A., Najjaran, H., Sadiq, R., Rodriguez, M. & Hoorfar, M. 2014 Online drinking water
quality monitoring: review on available and emerging technologies. Crit. Rev. Environ. Sci. Technol. 44, 1370–1421.
Besmer, M. D. & Hammes, F. 2016 Short-term microbial dynamics in a drinking water plant treating groundwater with
occasional high microbial loads. Water Res. 107, 11–18.
Byrd, J. J., Xu, H.-S. & Colwell, R. R. 1991 Viable but nonculturable bacteria in drinking water. Appl. Environ. Microbiol. 57 (3),
875–878.
Cheng, W. P., Chen, W. Y., Yu, R. F. & Hsein, Y. J. 2010 The relationship between particle size and turbidity fluctuations in
coagulation process. J. Residuals Sci. Technol. 7 (2), 87–94.

Downloaded from https://iwaponline.com/h2open/article-pdf/1/2/160/518746/0010160.pdf

by guest
 H2Open Journal Vol 1 No 2
168 doi: 10.2166/h2oj.2018.014

Chowdhury, S. 2012 Heterotrophic bacteria in drinking water distribution system: a review. Environ. Monit. Assess. 184 (10),
6087–6137.
Christensen, S. C. B. 2011 Asellus Aquaticus and Other Invertebrates in Drinking Water Distribution Systems – Occurrence and
Influence on Microbial Water Quality. PhD Thesis, Technical University of Denmark, Department of Environmental
Engineering.
Højris, B., Christensen, S. C. B., Albrechtsen, H.-J., Smith, C. & Dahlqvist, M. 2016 A novel, optical, on-line bacteria sensor for
monitoring drinking water quality. Sci. Rep. 6, 23935.
Kistemann, T., Claßen, T., Koch, C., Dangendorf, F., Fischeder, R., Gebel, J., Vacata, V. & Exner, M. 2002 Microbial load of
drinking water reservoir tributaries during extreme rainfall and runoff. Appl. Environ. Microbiol. 68 (5), 2188–2197.
Lopez-Roldan, R., Tusell, P., Courtois, S. & Cortina, J. L. 2013 On-line bacteriological detection in water. Trends Anal. Chem.
44, 46–57.
McCoy, W. F. & Olson, B. H. 1986 Relationship among turbidity, particle counts and bacterial quality within water distribution
lines. Water Res. 20 (8), 1023–1029.
Melik, D. H. & Folger, H. S. 1983 Turbidimetric determination of particle size distributions of colloidal systems. J. Colloid
Interface Sci. 92 (1), 161–180.
Prest, E. I., Hammes, F., Kötzsch, S., van Loosdrecht, M. C. M. & Vrouwenvelder, J. S. 2013 Monitoring microbiological
changes in drinking water systems using a fast and reproducible flow cytometric method. Water Res. 47 (19), 7131–7142.
Raich, J. 2013 Review of sensors to monitor water quality, ERNCIP thematic area, Chemical & Biological Risks in the Water
Sector, Deliverable D1 – Task 1, Report EUR 26325 EN. Available from: https://erncip-project.jrc.ec.europa.eu/
documents/review-sensors-monitor-water-quality (accessed April 23, 2018).
Vang, Ó. V., Corfitzen, C. B., Smith, C. & Albrechtsen, H.-J. 2014 Evaluation of ATP measurements to detect microbial ingress
by wastewater and surface water in drinking water. Water Res. 64 (1), 309–320.
Vestergaard, L. S., Olsen, K. E., Stensvold, C. R., Böttiger, B. E., Adelhardt, M., Lisby, M., Mørk, L. & Mølbak, K. 2007 Outbreak
of severe gastroenteritis with multiple aetiologies caused by contaminated drinking water in Denmark. Euro Surveill.
12 (13), 3164–3167.

Downloaded from https://iwaponline.com/h2open/article-pdf/1/2/160/518746/0010160.pdf

by guest