Bottled water microbiology b technical

1. Bottled water microbiology
M. Salgot
University of Barcelona

2. BASIC FACTS
Natural mineral waters (NMW), like all subterranean waters
(groundwater), contain a natural bacterial flora
The presence of the normal flora of mineral water has given rise to a
number of questions and debates about its effect on health
Since it is compulsory not to treat these waters, they contain
microorganisms. NMW cannot be disinfected

3. Groundwater microorganisms use existing organic carbon
(usually scarce) in the aquifers
Usually, the reactions are mainly without oxygen
Other components which can influence the reactions are:
N derivatives (nitrates); manganese (IV), iron (III),
sulphate (SO4
2+), CO2
The accumulation of CO2 leads to effervescent mineral
springs
Usually, in the aquifers, the supply of utilizable carbon and
energy is very small

4. In the groundwater habitat, all life forms larger than
microorganisms are excluded (because of the physical
characteristics of the aquifers: pore diameters)

5. Microbiological analysis of Natural Mineral Waters (NMW)
has always revealed the presence of some bacteria
that are capable of growth and can form colonies on
appropriate culture media (once extracted ad
analyzed)

6. - The colony counts of the water are less than 1-4 CFU/mL
- In the storage tank and immediately after bottling they
are, in average, only slightly higher
- During storage at 20ºC, bacterial populations increase in
numbers, reaching a peak of more than 105 CFU/mL
by the end of one week
- During the next four weeks, the bacterial counts
decrease slowly or remain fairly constant
- At the end of 2 years of storage, colony counts are still
about 103 CFU/mL
CFU: a single bacteria reproduces to form a colony

7. About the bacteria of NMW
- are psychrotropic (can grow at temperatures as low
as 5 ºC)
- their maximum growth temperature is 35 ºC
- do not require growth factors (vitamins, amino
acids…)
- seems to have different growth potential
depending on the bottle material and size
- growth do not depend on the storage temperature
- reduce its size to be more efficient in sequestering
nutrients (eating)
Primarily aerobic, saprophytic, Gram-negative rods

8. Many mineral waters contain 0.1 – 0.7 mg/L of dissolved
organic carbon (half of it is not easily biodegradable:
humic and fulvic acids)
It is then theoretically possible to predict the number of
culturable bacteria in a bottled mineral water
(coincidence with the indicated numbers)
Other limiting factors may exist (e.g. phosphorus)
Usually there are only bacteria, but occasionally fungi can
be found

9. The food chain in aquifers is primarily heterotrophic (“eat”
organic matter), reliant either upon an influx of
dissolved organic carbon along a given hydrological
path (outside origin) or organic compounds of
sedimentary (internal) origin that become degraded

10. Several species
- Pseudomonas
- Burkholderia
- Ralstonia
- Comamonas
- Acidovorax
- Stenotrophomonas
- Brevundimonas
- Sphingomonas
- Acinetobacter
- Cytophaga
- Flavobacterium
- Arthrobacter
- Corynebacterium

11. Carbonated water has lower than usual counts
The metabolism and nutrition of NMW bacteria indicate
that they are not pathogen for human (are not
adapted to living in humans or animals)

12. The bottle habitat / bottling procedure
Compressed air is used in virtually all stages of the water-bottling
process: the microbiological quality of the process air must
be of a very high standard
The complexed organic matter present in low concentration can be
dramatically modified though bottling, under the influence of
increasing temperature and oxygenation
A high increase of cell numbers with the bottling procedure is
described

13. There is a relation with the surface/volume area of the flask: small
containers provide considerably more area. The greater the
surface area in relation to the volume of water, the more
rapidly growth of bacteria takes place
The explanation for this is that nutrients present in low concentration
are adsorbed and concentrated onto the surfaces and, thus,
can be more available for the bacteria
Flask surface adsorption of organic matter is the basis for the
adhesion of bacteria to solid surfaces; because of the
increased concentration, the nutrients are more available

14. Photodegradation of dissolved organic matter is a common
phenomenon
The exposure time of recalcitrant organic substances in water to
daylight, and moreover to sunlight, may again stimulate the
growth of microorganisms since complexed substances
(carbohydrates, fatty acids, and amino acids) may become
bioavailable. Photochemical processes generate low
molecular mass, readily biodegradable molecules
VBNC (Viable but non culturable) dormant cells resuscitate when
bottled