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IOA Conference and Exhibition Valencia, Spain - October 29 – 31, 2007
6.4 - 1
Use of ozone for Legionella reduction in water systems
B. Ruiz
1
, J. Bauzá
1
, J. Benito
2
and A. Pascual
1
1
Quality, Food Safety and Environment Department, Research Association on Food Industry (ainia
Technological Centre). Parque Tecnológico de Valencia, C. Benjamin Franklin, 5-11. E-46980
Paterna (Valencia). Spain. (E-mails: bruiz@ainia.es; jbauza@ainia.es; apascual@ainia.es)
2
Implantación de Tratamientos y Diseños Industriales, S.L. (ITDI). Puebla de Valverde, 5 bajo.
46014 Valencia. Spain. (E-mail: jbenito@grupoitdi.com)
Abstract
Ozone is reported to be effective against Legionella in water systems such cooling towers and tap
water pipelines. Until now, most of the studies performed to asses the efficiency of disinfectants
against Legionella were made in vitro or in existing water systems. In vitro studies do not take into
account the higher resistance of this bacterium to biocides due to the protection offered by biofilms.
On the other hand, studies in existing water systems do not offer repeatability of the results. In
order to evaluate the efficiency of ozone to eliminate Legionella strains in water pipelines, a pilot
plant has been designed aiming at simulating the microbiological growth of Legionella erythra and
the disinfection capacity of ozone under these conditions. The plant, consisting of 9 independent
water pipelines, has been used to compare treatment efficiency under equivalent conditions of
system design, materials (i.e. iron, PVC and polypropylene) and effect of biofilms. Performance
evaluation of ozone is based on the ability to reduce not only Legionella, but also biofilms, which
contribute to the establishment and dissemination of these bacteria in water systems, and their
resistance to treatments.
Technical issues about the application of ozone for Legionella decontamination in water systems
are discussed in this paper, including injection methods, automation system, liquid and gas phase
on-line measurements and interaction of ozone with the structure of the facility. Safety
considerations and the environmental impact of the ozonitation are also been considered.
Key-words: ozone, Legionella sp., disinfection
Legionella
Legionella is a Gram-negative bacterium, including species that cause legionellosis or
Legionnaires disease, most notably L. pneumophilia. Legionella are common in many
environments, with at least 50 species and 70 serogroups identified.
Legionella species are the causative agent of the human Legionnaires disease and the lesser
form, Pontiac fever. Legionella transmission is via aerosols by the inhalation of mist droplets
containing the bacteria. Common sources include cooling towers, domestic hot-water systems and
fountains. Natural sources of Legionella include freshwater ponds and creeks. Outbreaks of
legionnaires disease have occurred in or near large building complexes such as hotels, hospitals,
offices and factories.
Initial symptoms are flu-like, including fever, chills, and dry cough. Advanced stages of the disease
cause problems with the gastrointestinal tract and the nervous system and lead to diarrhoea and
nausea. Other advanced symptoms of pneumonia may also present. Legionnaires disease can be
very serious and can cause death in up to 5% to 30% of cases.
Application of ozone as biocide to control Legionella
Over the course of the past decade, the use of ozone as the sole method of treating recirculating
cooling tower water has gained much attention. Currently there are several hundred of such systems
operating in the United States, and their success and failures have widely been published (11).
The interest in ozone as an alternative to chlorine and other chemical disinfectants is based on its
high biocidal efficacy, wide antimicrobial spectrum, absence of by-products that are detrimental to
health and the ability to generate it on demand, ‘in situ’, without needing to store it for later use.
6.4 - 2
Mechanism of ozone as biocide
Ozone is a powerful broad-spectrum antimicrobial agent that is active against bacteria, fungi,
viruses, protozoa, and bacterial and fungal spores (13). When molecular ozone dissolves in water,
the molecule can remain as O
3
or decompose by a variety of mechanisms, ultimately producing the
hydroxyl free radical (HO*) even stronger oxidizing agent than ozone (19).
Those agents oxidise all the molecules capable to be oxidized around them starting from the easiest.
Biological cells are not an exception and the components of their membranes (proteins, lipids and
polysaccharides) become also oxidized and structurally modified giving cell lysis as result.
Inactivation by ozone is a complex process that attacks various cell membrane and wall
constituents (e.g. unsaturated fats) and cell content constituents (e.g. enzymes and nucleic acids).
Both molecular ozone and the free radicals produced by its breakdown play a part in this
inactivation mechanism but there is no consensus on which of them is more decisive. The micro-
organism is killed by cell envelope disruption or disintegration leading to leakage of the cell
contents. Disruption or lysis is a faster inactivation mechanism than other disinfectants which
require the disinfectant agent to permeate through the cell membrane in order to be effective.
As regards the spectrum of action, each micro-organism has an inherent sensitivity to ozone.
Bacteria are more sensitive than yeasts and fungi. Gram-positive bacteria are more sensitive to
ozone than Gram-negative organisms and spores are more resistant than vegetative cells.
Due to the mechanism of the ozone action, which destroys the micro-organism through cell lysis, it
cannot lead to micro-organism resistance.
Legionella colonization and biofilm
Legionella species are aquatic bacteria widespread in nature which have been found in water over
a wide temperature range but growing the best between 30°C and 40°C (24, 1, 17). Their tolerance
to relatively high temperatures, probably help them to colonise some artificial water systems that
are often above ambient temperatures. Legionella are prevalent in artificial water systems, and
then is transmitted via aerosols and occasionally by direct aspiration.
Several strains of Legionella have been known for some time to live within amoebae in the natural
environment (11, 20). In addition, Legionella pneumophila developed into amoebas are more
resistant to chemical and other biocides than in-vitro Legionella (2, 3, 4).
The biofilm formed by Legionella and other microorganims is capable to fix on the surface of the
water systems. This phenomenon has been observed in PVC, stainless steel, rubber, wood and
softly in copper (20). The biofilm formation can especially be found on the surface of water pipes,
tower basins and heat exchangers.
Background of water contamination
As the cooling effect works by evaporation of circulation water itself, an increase of total dissolved
solids (TDS) is registered on water samples; reaching levels of 1500mg/L of TDS after 3 cycles of
circulation (11) due to the concentration of the dust of the circuits in the water. The gas washing
effect leads also to a permanent admixing of all kinds of biological germs from the operation air into
the water, so that a constant recontamination of the water can not be avoided during the operation
of the systems. The growth of biomass, especially bacteria, in the cooling system is supported by
the increase of TDS, due to the increase of available nutrients. In addition, temperatures around 30
– 36°C, especially in Mediterranean countries, offers ideal growing conditions for micro-organisms
living in water suspensions or biofilm.
Cooling towers, which are wet or evaporative condensers that form part of an air conditioning
system, can present a particular hazard because they readily generate fine water droplets and
there is an air current to transport them. As they are usually located on rooftops there is a potential
for infecting large numbers of people. The bacteria may also colonise hot and cold water systems.
For instance, showers and spa baths have been associated with infection.
In summary cooling towers and evaporative condensers have been implicated as the most
common source of Legionella spp. and linked with most of the outbreaks of legionellosis (7).
Hence, the HSE (Health and Safety Executive) recommends, at least quarterly, sampling of cooling
towers and routine sampling of hot and cold water systems if the recommended temperature
regime is not followed or has failed or if the local risk assessment suggests it is needed.
6.4 - 3
Description of real systems for Legionella inactivation based in ozone treatment
Ozone is one of the most powerful oxidizing agents available for treatment on industrial wastewater
and legionary disease prevention. The main problem of the using ozone, is the instability of the
compound (must be produced in situ) and the difficulty to solubilise it in water (19) in acceptable
concentrations enough to kill bacteria.
Ozone has been applied on several water systems in industries and hospitals however the
scientific conclusions regarding the effectiveness of those treatments are still not clear.
Cooling towers
Cooling towers are industrial devices for lowering the temperature of water by evaporative cooling
in which atmospheric air is in contact with falling water, thereby exchanging heat. The term also
includes those devices which incorporate a water-refrigerant or water-water heat exchanger.
The lack of maintenance, the accumulation of COD (Chemical Oxigen Demand), dust and micro-
organisms create ideal conditions for the development of biological aerobic growth (including
Legionella) in industrial cooling towers.
Besides several biocides as chlorine, electro-chlorination, chlorine dioxide, monochloramine,
copper/silver copper and chlorine; ozone has been found as a suitable biocide to reduce the
numbers of presumptive Legionella pneumophila in test cooling towers, offering several
environmental benefits.
There are numerous developed systems using ozone, applied to keep cooling towers free of dust
and microbiological growth. For example a system called Coolzon which applies ozone in a
separate treatment loop. It consists in a filtration step followed by ozonation of the cleaned water.
The first step aims to reduce the dust and COD content giving a reduction of available nutrients for
biological growth. The second one oxidizes the developed biomass and the rest of dust attached to
the circuit walls (12).
There are also in the market other, brands which provide compact equipment for controlling
legionnaires disease in cooling towers and evaporating condensers using ozone. Those systems
are prepared to operate in continue ozone dosage but the efficiency of occasional shock
treatments is not reported.
Evaporative condensers
This kind of systems has shown high risk of Legionella infections especially during maintenance
operations. Inadequacies exist in current biocidal treatment practices for cooling system waters.
Several organic biocides in use today have been shown to be ineffective in controlling Legionella
densities (9, 10), but periodic discharges of cooling system water into local rivers or streams have
begun to restrict the amount of chlorine residual (one of the most used biocides) present in such
discharges (23). Thereby ozone treatments appear as very promising as environmental friendly
alternative.
Several brands (14) already sale customized evaporative condensers coupled with ozone systems
designed to control microbiology and corrosion in the condenser water; however not scientific
results are reported on the efficacy.
Hospital plumbing water (water pipelines)
In vitro studies developed on a 38 L plumbing system using ozone, among other biocides was
evaluated by Maruca et coauthors (15). Those studies showed effectively control of L pneumophila
by a residual concentration of 1 to 2 mg/liter of ozone and the data suggested that ozone could
remove L. pneumophila in a large water distribution system. However, because of the rapid
decomposition of the ozone residual in water, its main utility may be limited as a supplemental
disinfectant (15).
Lack of knowledge
The successful applications of ozone in water systems have been characterized by low corrosion
rates of the tubing materials, very low biological counts (Legionella and other mesophilic micro-
organisms), and scale free achievement of relatively high dissolved ozone concentration (11). The
efficiency of ozone has also been proved in water pipelines and in-vitro models (14).
6.4 - 4
Nevertheless, several problems have been reported in real installations. Numerous operational
shortcomings have been found as contributing factors. Those factors include failures to achieve the
desired residual ozone (due to insufficient system sizing), inadequate or unmaintained equipment,
chemical contamination of the system, high interaction of ozone with the materials or poor mixing
of ozone into the recirculation water. As a result excessive biological growth and/or corrosion,
depending on the particular application and water conditions have been reported.
As summery, no reliable data are available regarding the efficiency of ozone to kill Legionella in
different water in vitro systems (8) as well as at real scale (5). The limitations of this technology
and the key factors responsible in the decay of efficiency observed in some experiences described
on the literature are also under discussion.
Therefore, more information on the subject is needed and the construction of a plant able to
simulate real scale water facilities for Legionella inactivation by ozone treatment has been
considered a significant challenge to overcome.
Pilot plant for Legionella controlling in water pipelines using ozone (OZOLEG project)
Concept
In order to evaluate the efficiency of ozone to eliminate Legionella strains in water pipelines, a pilot
plant has been designed aiming at simulating the microbiological growth of Legionella in biofilms
and the disinfection capacity of ozone under these conditions.
Three materials have been used for the construction of the plant in order to evaluate the behaviour
of biofilms and Legionella for each material under different possible treatments with ozone.
General description of the pilot plant
The plant is composed by 9 independent circuits of water pipelines fixed on vertical panels and
constructed in iron, PVC and polypropylene. Each material is used in 3 of the circuits. All circuits
have identical components and measurement equipments, changing only the material used.
The dimensions of each circuit pipeline are: 14,6 m of length and 2,5 cm of internal diameter
(excepting a 59 cm portion with internal diameter of 5 cm). Each circuit has 10 removable portions
or coupons (8 of 2,5 cm internal diameter and 2 of 5 cm internal diameter) designed to evaluate the
adherence capacity of Legionella in biofilms. Five of those coupons have artificially been modified
making them rough in order compare the capacity of adherence of biofilms to irregular surfaces.
The circuits have been filled with tap water re-circulated by independent pumps which take the
liquid from a 200 L deposit made of polypropylene. The pumps have been fixed on the top of each
deposit where suck the liquid from a vertical pipe with a one-way valve at the bottom, avoiding the
discharge of the pumps when stopped. Five valves are distributed on the circuits in order to take
liquid samples from different points.
The distribution of the components of the circuits is shown in Fig. 1 and the description of them in
Table 1.
The whole pilot plant is controlled by an automaton system and the undertaken on-line
measurements recorded by it.
Ozone generation
Each circuit hold an independent ozone generator able to produce 2 g O
3
/h by a corona electrical
discharge applied on ambient air. The generated ozone is injected into the liquid phase of the
circuits by a PVC venturi. The driving force for the ozone injection is done by the liquid in motion
passing through the venturi thereby ozone injection is only possible to be done when pumps are on
but not always.
The level of dissolved ozone in each circuit is automatically controlled by the automaton system
depending of the correspondent measurement on the liquid phase and the desired level to be
achieved.
Equipment for measurement and automation system
Each circuit hold probes for on-line measurements of pH, conductivity temperature and redox
potential automatically recorded each 15 minutes by the automation system.
6.4 - 5
Four electro-valves collect 2 samples of gas and 2 of liquid from each circuit every 45 minutes for
the corresponding ozone measurements. All the measurements of ozone are undertaken by
ANSEROS equipment; gas phase (Ozomat GM-6000-pro) and liquid phase (Ozomat MP coupled
with ozone disrober Ozomat WP). The control of electro-valves, equipment and recording of data
are automatically controlled by the automaton system.
Microbiological assays
From the pilot plant 2 type of samples, liquid samples and coupons, are investigated in different
points of the applied treatments. In both samples Legionella erythra and total aerobic mesophilic
bacteria have been determined based on plates counting. The method ISO 11731 has been used
for Legionella detection in water samples using BYCE as culture media whereas total aerobic
mesophilic bacteria have been determined at 30 ºC using PCA as culture media.
Counts from coupons require gently ultrasonic extraction (without cell destruction) prior to apply the
method described above for bacteria counting.
Fig. 1. Scheme of the plant for studies of Legionella growth and disinfection using ozone
Safety
In order to reduce the risk of contamination in the pilot plant, a non-pathogenic strain of Legionella
(L. erythra), with a similar behaviour than L. pneumophila regarding inclusion in biofilms, has been
chosen for the inoculation of the circuits.
The whole pilot plant is physically isolated into a restricted area where the air is filtered by an
absolute filter HEPA (mod. PE11ST00) coupled with an air extractor (THLZ-200, 1 CV) giving a
negative pressure in the area. The pilot plant area is also equipped with three UV lamps (36W)
aimed to inactivate any possible generated aerosol of Legionella.
Water samples are automatically collected for ozone detection, the measurements are recorded by
the automaton system and the samples disinfected by chlorination prior to be eliminated. The
chlorination is controlled by an independent system for detection of volume and dosage, calibrated
to reach 50 mg/L of free chlorine on the water samples.
The pilot plant area is equipped with an ambient ozone detector (ANSEROS, Sen 6060-S) coupled
with a remote monitor.
M1
M3
M2
M4
M5
P
O1
O2
C
R
G
B
I
V2
V1
D
T4
T3
T2
T1
T6
T5
H2
H1
T7
T8
L
A1
A2
6.4 - 6
The operators authorised to access in the restricted area, are equipped with integral clothes
(chemical and microbiological protection), goggles, mask filter for microbiological protection, rubber
boots, gloves and a portable ozone detector (ANSEROS, Ozomat SEN-P).
Table 1. Circuit components and the corresponding short form
ref. code
details
D 200 L deposit
B pump
I venturi
O 1 sampling point ozone gas phase 1
G ozone generator
A 1 sampling point ozone liquid phase 1
P pH probe
C conductivity probe
M 1 water sampling point 1
T 1 coupon 1
T 2 coupon 2
L transparent pipeline
M 2 water sampling point 2
T 3 coupon 3
T 4 coupon 4
H 1 coupon (big) 1
H 2 coupon (big) 2
M 3 water sampling point 3
T 5 coupon 5
T 6 coupon 6
M 4 water sampling point 4
R redox probe
T 7 coupon 7
T 8 coupon 8
M 5 water sampling point 5
A 2 sampling point ozone liquid phase 2
O 2 sampling point ozone gas phase 2
Operational conditions during the pilot plant starting up
Each circuit has been filled with at least 58 L of tap water which has been re-recirculated for about
2 weeks without any external inoculation or ozone application in order to stimulate a natural
installation of aerobic biofilms.
After this period, a suspension of L. erythra has been inoculated in each circuit, reaching a
concentration of ca. 1x10
5
cfu/mL in the water of the circuits.
Conclusions
An automated pilot plant has been developed at ainia to operate under safe microbiological
conditions. The plant is automatically recording on-line ozone measurements (liquid and gas), pH,
conductivity, temperature and redox potential without the intervention of the operators.
This plant will allow the investigation of Legionella survival after applying different ozone
treatments. Effect of biofilm formation will also be studied.
6.4 - 7
References
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