Transnational Journal of Science and Technology
October 2012 edition vol.2, No.9
A STUDY OF SOURCES OF MICROBIAL CONTAMINATION
OF PACKAGED WATER
J.O. Jeje
K.T.Oladepo
Department of Civil Engineering, ObafemiAwolowo University, Ile-ife, Nigeria
Abstract:
Cleanness is the real worth of any water, and anyone who is unappreciative of this quality
incurs the wrath of water. Clean water apart from its physical appearance, is one that is free
from all its acquire impurities necessary for a particular use. Chemically, water is a liquid
substance in which two hydrogen atoms combine with one oxygen atom to produce a
compound of formula H2O, the only form that is absolutely pure, but still may attract
contaminants from its storage container through the process of leaching. This means that it is
only the chemical formula that is absolutely pure and not the liquid water. Such level of
purity may only be relevant as a chemical substance rather than as drinking water or water for
other uses. In other words, the level of water purity depends on its desirability for use.
Notwithstanding, water of satisfactory quality should in addition to its chemical and
microbiological qualities be colourless, odourless, and tasteless.
Keywords: Cleanness, nourishment, microbial contamination, packaged water
Introduction
In nature, water is not pure, but acquires contaminants from its surroundings and
those arising from humans and animals as well as other biological activities. Therefore, the
earth’s impurities from the soil, atmosphere, and the environment are freely present in natural
waters. Macroscopic and microscopic organisms many of which are pathogenic together
with organic and inorganic materials do contaminate bodies of water with amount depending
on its source (Anon, 1982). .
Generally, water is very vital for life and life processes, and its value spans domestic,
agricultural, and industrial uses. Every aspect of human life requires water, with each
application requiring special quality in order to achieve the preferred goal. Water for most
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uses involves its bulk property of nutrition, hydration, cooling, carrier, or cleaning action as
application in domestic, agricultural, industrial, and recreational activities. With such
functions, the quality requirements for certain bulk waters are not very stringent.
Consequently, water from springs streams, lakes, and rivers are very applicable in these broad
areas of use. By far, the greatest use of water is for drinking.
Clean drinkable water that is believed to cleanse, refresh, nourish, heal, and
rejuvenate the body and the inner being is called the living water (Mendie, 2001). It is water
imbued with immense spiritual essence coupled with its physic-chemical and physiological
qualities; which enhances total health and imparts longevity to an individual. This type of
water has strong religious connotation, but its adaptation into everyday use is for the purpose
of designating drinking water having extreme level of purity and providing total nourishment
for the body.
Contaminants of packaged waters span the physical, chemical, and microbiological
impurities and the magnitude of each depends on the level of controls of all factors
influencing their manufacture (Mendie, 2001). The physical and chemical contaminants can
easily be prevented at the pre-production stages, but the microbial contaminants need a
disciplined effort sustained by a high level of hygienic sanitation. Generally, the application
of Good Manufacturing and automated process (GMAP) guidelines will reduce to the barest
minimum the level of defects found in such products. Most impurities in packaging water
originate from the raw water, but may persist in the purified water due to poor or inadequate
purification techniques. Extrinsic contaminants however emanate from the environments in
which the water is produced. The macro-environment consists of the production and filling
atmosphere, operatives, and the processing equipment, while the micro-environment involves
that provided by the primary water container. The following sources require rigid controls to
avert product contamination.
Water being the major component in packaged water can also serve as a very
significant source of contamination. Product contamination may arise directly from the
process water, or indirectly from the operatives, cleaning operations, packaging materials, or
cross-contamination from the wet areas of floors, sinks and drains to the processing
equipment (Baird and Petrie, 1981). . The various methods of purification have their unique
ways of inviting contaminations, but the final product must meet the standard requirements
for its intended use. Sources of microbial contamination of the following PW are discussed in
this paper.
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Distilled water immediately after condensation is sterile, and with proper aseptic
collection and storage precautions will maintain this state. In parenteral industry, distilled
water is often maintained at 800c and circulated around a ring system. This practice maintains
the quality of the water by destroying any vegetative organisms that enter as chance
contaminants thereby preventing the generation of pyrogen. This system though effective in
parenteral industry is far too expensive; however, for large-scale manufacture of other
packaged waters (Lec, 1991).
Water produced by reverse osmosis can be sterile and pyrogen-free as it is forced by
osmotic pressure through a semi-permeable membrane, which allows only substances of
molecular weight less than 250 daltons to diffuse through. Post ro contamination can occur
because of ingress of microorganisms downstream of the membrane into the storage vessel of
distribution system.
Purified water prepared by deionization is the most common form of process water
for non-sterile pharmaceutical, food, and cosmetic products. Deionization, method has
perhaps the highest potential for contamination.
The source of water for the production of
DI water normally municipal water of potable quality, bore hole or well. DI water systems
may involve some combinations of the following units, all of which can harbor
microorganisms. (a) Carbon filters (b) Water softeners (c) Cation and anion exchangers as
either twin or mixed beds and (d) A storage and distribution system (Heiering, 1970); (Hunter
and Burge, 1993); (Jackman, 1980).
Carbon filters are effective in removing chlorine, oxygen, and lower molecular weight
hydrocarbons; but are less effective in removing high molecular weight organic materials
such as humic and fulvic acids which are common to surface water supplies. Carbons filters
are usually included to minimized irreversible fouling of the deonizing resins; and organic
molecules adsorbed onto, and retained within the activated carbon particles will support
microbial growth. In most instances, the highest numbers of contaminating organism occurs
towards the bottom of the bed because residual chlorine is removed in the top portion.
Organisms normally recorvered from carbon filters include: Coliforms, arthrobacter,
alcaligenes, micrococcus, corynebacter, and pseudomonaspecies (Favero, Carson, Bond,
andPerterson, 1971). .
Water softener are required when the raw water has a high calcium and magnesium
contents. They are generally more prone to microbial contamination than cation or anion
exchange resins since the latter are regenerated with strong acid and alkali, which have strong
bactericidal effects. Sodium chloride regeneration solutions for the softeners do not provide
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the necessary periodic bactericidal effects. Infact, the brine make-up tanks are usually
contaminated with halophiles and other salt-tolerant organisms unless adequate precautions
are in place (Baird and Shooter, 1978).
Ion-Exchange Resins,when properly maintained do not present much problem of
microbial contamination. However, after a prolonged period of inactivity or the presence of
certain type of organic material in the input water may introduce the problem of microbial
contamination. Organism s most associated with deionizers areAcinetobacter, Alcaligenes,
and Pseudomonas species (Baird and Shooter, 1978). Incorporation of UV lamps operating
at 254nm may used to control the microbial load provided they are correctly sized to cope
with the flow rate, optical clarity, and the expected bioburden of the water. In addition, the
filtration of the deionized water through 0.45 or 0.20 microns membrane filters s widely used
to reduce microbial burden. Other problems of ion-resins are the shedding of particles or
resin fines into the process water clogging of Millipore membrane filters.
Water purification systems must have the capacity to supply the volume needed at the
rate required, including peak exigencies. The greater the volume of water required per unit
time, the larger and more expensive generally, the water purification equipment which
permits storage of purified water purification equipment. The use of storage tanks
compliments the installation of smaller processing equipment which permits storage of
purified water at period of low demands for availability during intervals of high usage. Water
storage tanks are mainly stainless steel. Polythene storage tanks which is fashionable in the
third world countries has not been well studied particularly for the release of extractives into
the purified water and should be used with care (Craun and McCbe,1993). .
Generally, water storage tanks have been known to contribute to the problem of
organisms’ profileration particularly when they are not completely full as their walls are
susceptible to such growths. The inclusions of UV light into the ceiling of tanks have been
shown to produce low organisms counts. Also, to minimize microbial proliferation, he
contents of water storage tanks should be circulated at a rate of about two turnovers per hour.
Particulate matter generation during storage of water arising from improper pretreatments or
post ozone treatment can be controlled by adequate filtration.
Results and Discussion
A typical atmospheric air sample (Table 1) shows that particles ≤1µm constitute about
99% of the total particulate load, but only 3% by weight that is significant in air filtration
system. With an atmospheric particulate concentration in the range 10 8-1013m-3, and a viable66
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October 2012 edition vol.2, No.9
nonviable percentage of between 0.0005-0.02%, which may be higher in tropical conditions,
this level of contamination, is totally unacceptable in packaged water production. It therefore
demands the use of a clean room for production.
The viable particulate matters include bacteria, moulds, and yeasts particularly those
that can tolerate desiccation and drought. Even though the air is not their natural
environment. As it contains no nutrient and moisture in a form that can be utilized for growth
and reproduction,
Table 1: Size distribution of a typical atmospheric dust sample
Particle size
Proportionate
Percent particle
Range (µm)
particle count
Count
Percent by weight
by (%w/w)
number(%)
10-30
10000
0.005
28
5.0-10
35000
0.175
52
3.0-5
500000
0.250
11
1.0-3
214000
1.070
6
0.5-1
1352000
6.780
2
0-0.5
18280000
91.720
1
microorganisms commonly isolated from the air are the spore formers e.gbacillus sppand
Clostridium
spp;
the
non-sporing
bacteria
Staph.spp,
Streptococcus
spp,
and
Corynebacterspp; Moulds e.g. Penicilliumspp, Clasdoporiumspp, Aspergillusspp, Mucorspp
as well as yeasts.
The type and number of microorganism found in the atmosphere depends on the
activity in the environment; and the amount of dust generated, such as during filling and
sealing of water containers. A water production factory that involves many operatives in
manual operations such as filling and sealing will surely have a higher aerial microbial count
than a semi or fully automatic plant with fewer personnel. In fact, a dirty and untidy room
will also exhibit higher count compared to a clean room. Factor such as temperature and
humanity that impinges on the comfort of the operatives will also affect level of
microorganisms in the production environment positively. A controlled damp environment
usually contains fewer organisms than a dry one, hence, the need for adequate airconditioning rather than fans in water factories.
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Microorganisms are carried into the atmosphere suspended on particles of dust, skin
scales, clothing or droplets of moisture, following talking, sneezing or coughing. The size of
the particles to which the organisms are attached in addition to humidity of the air determines
the rate at which they will settle out. Unattached microbes will settle down gradually, the rate
being dependent upon air currents caused by ventilation, air extraction, convection currents,
and the activity in the rooms. Packaging materials e.g LDPE, bottles, etc generate a lot of
dust bearing microorganisms, which consequently contaminate the final products. Most
particles in an uncontrolled environment range in size from 0.1-1µm and are small enough to
be retained almost indefinitely. Larger particles of about 3µm however interfere with
gravitational fall (Table 2). However, as draughts or movements of occupants disturb the air,
sedimentation is reduced, fresh dust become air-borne and level of contamination of product
increases.
Table 2: Sedimentation rates of individual spherical particles of unit density in air at
200C.
Diameter (µm)
Rate of fall(mm/s)
0.2
0.00225
1
0.035
10
3.0
100
250.0
1000
3850.0
i.e. a 1µm particle takes about an hour to
fall 125 mm
Microbiologically, the quality of the manufacturing air can be determined quickly using Agar
settling method. Here, petridish containing nutrient agar are exposed to the atmosphere for a
given period of time. This method relies upon microorganism or dust particles bearing them
to settle on the surface of nutrient agar. Air sampling methods e.g using slit, centrifugal or
membrane filter sampler are more accurate and reliable. Each method depends on a measured
volume of sucked air from the atmosphere that impinges on a nutrient agar; or drawn through
membrane filter which is then in areas of low microbial contamination, particularly if
samples are taken close to the working areas.
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For sterile products such as Water for infections, which are not terminally sterilized by
autoclaving, the microbial contents of manufacturing air should be very low about < 10
cfu/ml organisms per 1000 liters of air, i.e Class A (M1.5-2.0) clean room of preservatives,
they can be manufactured in Class100 (M3.5) clean rooms i.e 100 particles per 3.5 liters of
air. Packaged drinking water production that requires no preservative, are not terminally
sterilized, will therefore be prepared in either Class 10 or 100 clean rooms to prevent aerial
contamination depending on the type.
There is no definitive method of class determination or how to establish the air
cleanliness level. However, the European standard contains in addition to the particles counts
per unit volume, the corresponding limit for viable microorganisms in cfu/m 3 (Table 3). The
filling and sealing of packaged drinking water should be performed in a grade B environment
with grade C background, whilst the handling and filling of small and large volume
parenterals should be done in grade A environment with grade B background.
Equipment used in packaged water production should not pose a threat of particulate
contamination. In addition, adequate facilities should be put in place to create particulate-free
controlled environment. This is the main objective of clean room provision.
Table 3: Air classification for manufacture of packaged water based on European
community GMP guide
Grade
Max. permitted number of particle per Max. permitted number of viable
m3 ≥ shown below
microorganisms (cfu/m3)
0.5µm
5µm
A
3500
None
<
B
3500
None
5
C
350000
2000
100
D
3500000
20000
500
The operation of clean rooms depends on adequate air filtration, and particles of 0.5
microns or larger are removed using High Efficiency Particulate Air (HEPA) filters, Aseptic
facility for packaged water production requires air having very low concentration of 0.5um
particles, and the absence of 5up particles. Practically, air of this quality will be free from
viable contaminants, since microorganisms are almost consistently linked with larger,
inanimate particles that protect them from dehydration in the atmosphere. High-level particle
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extraction is achieved through coarse filtration stage or electrostatic field method followed by
HEPA filters.Filtered air may be used to purge the whole room or it may be confined to a
specific area as in laminar flow hood where operations can be carried out in a gentle current
of air.
In order to enhance efficiency, all filters must be kept dry, since moisture can aid
movement of microorganisms through damp filters; and the integrity of air filtration panels
must be checked regularly.
Air fumigation is occasionally applied to keep microbial level
low. Agents such as propylene glycol at 0.05mg/1 and quaternary ammonium compounds at
0.075% have produced good results. Ultraviolet irradiation at 240-280nm wavelength is used
to reduce bacterial contamination of air, but within a short distance, though certain spores are
known to be resistant to such treatment.
Most often, production and filling equipment is purchased with little consideration for
easy cleaning and hygienic considerations are frequently neglected in most locally fabricated
equipment. Equipment should be made of materials capable of withstanding conventional
cleaning methods such as disinfectant treatment and stream.
Water filling and sealing
equipment should be made up of stainless steel instead of iron, which will impart rust
particles into the process water. Hoses, that are old and rotten, and equipment with crevices
are very difficult to clean and sanitize properly. Water pumps that are old or rusted constitute
a major source of contamination. The rails, gaskets and other fittings should be regularly
cleaned and sanitized. Unsuitable and inefficient equipment provides reservoir for microbial
contamination because of faulty design, and products made with them readily acquire these
contaminants.
The presence of personnel and their movements or activities correlates air-borne
microbial contamination. Fewer operatives should be located within production floor, or
filling cubides should be created for individual operators. Manually produced drinking water
creates more incentives for microbial contamination compared to automatic or semiautomatic filling production lines. Improper cleaning methods for plastic bottles or bags
may introduce contaminants. A high microbial quality for water rinses for containers is
desirable and should contain <10cfu/ml to eliminate product contamination may be
encountered include: poor supervision, poor hygiene design of equipment and layout, rapid
staff turnover, changes in cleaning procedure introduced to reduce cost.
Primary packaging material has a dual role in containing the material and in
preventing contamination with microorganism and ingress of volatile gases that may result in
spoilage. The packaging can also act as a source of microbial contamination if not properly
sanitized. In practice, when used for non-sterile products, of preserved liquid, the packaging
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often contributes significantly to the total bio-burden of the product. The microflora of
packaging material is dependent upon both its composition and storage conditions.
Plastic materials such as polythene, polypropylene, and polyvinyl chloride plastics
have smooth impression surfaces, and carry low surface microbial counts. The process of
creating water bags may introduce a lot of incidental organisms, which may be resistant to
sanitization.Improper storage, packing and transportation in any unhygienic packaging
material or conveying vehicle may introduce mould spores. Generally, plastic bottles are
more prone to contamination with sporing bacilli and moulds. Moist heat or chemicals should
be sued to sterilized or sanitize the primary packaging containers approximately before use.
By far the greatest source of transfer of microorganisms to water products is through the
operatives. Particularly in manual factories employing large number of water bag makers and
filters, the preponderance to contamination cannot be quantified. Apart from the sheer level
of activity on the production and filling floors, which generates aerial contaminants, the
operatives are directly associated with transfer of contaminants to products. Resident skin
flora including Staphylococusspp, Diphtheroids, Mimaspp, and Akaligenesspp are common
contaminants of packaged drinking water. In fatty and waxy skin lipophilic yeasts, mainly
Pityrosporumovale from the scalp and P. orbicular from the glabrous skin. Dermatophytes
such as Epidermophyton spp.Microsporonspp, and Tricbopbytonspp, as well as saprophytes
from ear secretions are possible contaminants, including the tribe Enterobacteriaceae,
Clostridium spp Bacillus spp, enterococci, micrococci and streptococci.
Large number of droplets expelled from the respiratory tract by coughing and sneezing may
contain organisms from the nose, mouth, throat, and lungs.
Healthy carriers often spread
Staph. aureus, Strep. Pyrogenes, Strep. vividians, and even Mycobacterium tuberculosis
through this route. Furthermore, contaminants from nasal passages and ear secretions e.g.
Staph.aureus, Strep.salivarus, Haemopbilus influenza, and K. pneumonia continually pose
veritable reservoir of infections that constitute potential culprits for product contamination.
The nose fingering, ear or scalp scratching habits, deteriorations in personal hygiene, and
inadequate conveniences in the factory are inimical to production of wholesome product. A
comprehensive training programme in personal hygiene in addition to regular medical checkups including food handlers tests certification and provision of hygienic industrial
conveniences for operatives are fundamental inclusions of GMAP in water industry.
Because of the proliferation of PDW products, serious concern has been expressed on
their quality and safety. Most particularly, are the qualities of the satcheted-packaged water
in which a lot of indignation have been expressed as to their unwholesomeness. Indeed, the
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literature is replete with reports on disease outbreaks associated with packaged drinking
water6. Many of these reports implicate gastroenteritis including diarrhea, typhoid fever,
dysentery, and cholera. Cholera outbreak in Portugal traceable to the consumption of bottled
mineral water in which 48 persons died and 2467 bacteriological confirmed hospitalized
cases11.
Also of concerned are the potentials for contamination by pathogens such as Giandia,
Cryptosporidium, and enteric viruses that cannot be easily isolated from water, even when
such water has been suspected. Even when analytical facilities are available, large volumes
of water are required thus making the routine sampling of bottled water from retail outlets
impracticable.
Viruses such as hepatitis A and poliovirus have been shown to survive for
longer than 120 days in bottled mineral water (Jurank, Taylor, and Feachem, (1998). There
have also been outbreaks of giardiasis due to gross water contamination (Craun and McCbe
1993). Autochthonous water flora such as Pseudomonas spp can also cause disease in
humans. Ps.cepaciais increasingly identified as a cause of serious chest infections in children
with cystic fibrosis. Straints of Pseudomonas spp isolated from bottled water in Boston,
U.S.A. were shown to be resistant to several antimicrobial agents. A pigmented water
pathogen, Flabobacteriummeningoseticum has been associated with severe generalized sepsis
in infants (Parker, 1972). .
Any microorganism can contaminate and survive in packaged water. However, those
commonly isolated are resident within the production environment. Coliform organisms are
seldom found in good quality packaged water, all the same, regular testing should be carried
out to detect their survival being a primary indicator organism for low quality water products.
Drinking water is not expected to be sterile; water products devoid of microbial contaminants
should be the desired choice. Most water samples studied contain contaminants, ranging
from Staph aureus, Micrococcus species, Bacillus subtilis, and Sal. Typhi. Others include
V.cholera, B. cereus, B. licbeniformis, Escberichia coil, Pseudomonas aerugiosa,
Klebsiellaaerogenes, Streptococcus species, and Chromobacter species, 20% for E. coli and
Klebsiellaaerogenes respectively, and 7% Pseudomonas species. Staphylococci were present
in 3% of the total samples. No salmonella species was isolated. Other workers have isolated
pathogenic fungi, animal parasites and viruses . Instances of typhoid infection traceable to
the consumption of these water-based products have equally been reported (Mendie, 2002);
(Millership andChattopadhyay, 1980); (Scott and Bloom, 1990). Most isolates frequently
found in packaged drinking waters occur in very small number of 5-200 cfu/ml, an indication
of possible extrinsic contamination, and due to growth in poorly nutritive medium. Fewer
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cases have been known to produce heavy growths of the contaminants particularly with Ps.
Aeruginosa and Bacillus subtilisisolates. Generally, it has been found that water stored at
8oC produced significantly (P<0.05) lower counts of the contaminants compared to the ones
stored at 370C or 250C (Fig.1-5), a similar trend which was also observed with pH changes at
these temperatures.
This is in conformity with established fact that storage of products at lower
temperature less than 100C hinders growths and proliferation of microorganisms but enhances
the stability of any product not sensitive to cold.
One peculiar observation of growths of contamination in PDW samples is the peak
and through contours manifested by bacterial contaminants.
including
Bacillus
subtilis,
Escberichia
coli,
Microbial contaminants
Klebsiellaaerogenes,
andPseudomonasaeruginosa, which could have been introduced extrinsically all showed
similar pattern of growth profiles (Figs 1-5). For these organisms, growth progressed from
few colonies, rose to a peak, and then declined to zero. With he exception of Staphylococcus
aureus (Fig 4) which may have been introduced intrinsically due to poor water treatment and
purification techniques, their initial counts increased significantly (P<0.05) to about cfu/ml,
and then decreasing finally towards zero.
Fig.1: : variations in growth of Bacillus
subtilis in pacckaged water stored in
polythene bags
Fig. 2: : variations in growth of Escherichia
coli in pacckaged water stored in polythene
bags
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Fig. 3: Fig. 4: variations in growth of
Klebsiella aerogenes in pacckaged water
stored in polythene bags
October 2012 edition vol.2, No.9
Fig. 4: variations in growth of staph. Aureus
in pacckaged water stored in polythene bags
Fig. 5: variations
in growth of pseudomonas
aeruginosa in pacckaged water stored in polythene bags
For each microbial contaminant studied at a particular storage temperature, it was
generally observed that the highest number of survivors were recorded on the 10 th – 12th day
of sampling, then decreased progressively towards zero, Thereafter, rising again towards the
4th week (Figs 1-d,5). Since drinking water contains no preservatives and has poor nutritive
quality, it is only non-exacting organisms that can survive in such products if poorly
prepared.
The cyclical pattern of growth seen here may be due to marginal survival on the
debris of dead organisms hitherto present in the packaged water.
This is a characteristic feature of post kinetic phase of decline, the so-called phase of
survival. It is important to note that potable water should be produced with the quality target
of containing nil microorganisms, since it has now been established that they can actually
grow over stipulated limit when stored at ambient temperatures. In this regard therefore, all
standards that are geared towards sustaining and enhancing good manufacturing practice
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should be vigorously pursued.
October 2012 edition vol.2, No.9
Ps. Aeruginosa in particular poses a serious challenge to the
quality of packaged waters, as it has been found to proliferate even in distilled water.
Acknowledgement
Authors hereby gratefully acknowledge the assistance of the management and staff of
the Central Science Laboratory, ObafemiAwolowo University, Ile-Ife. They made
painstaking effort to prepare samples from the various bottled and sachet water-types used to
determine various parameters.
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Parker, M.T. (1972).The clinical significance of the presence of microorganisms in
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