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Undersøgelse af bakterieantal og eftervækstpotentiale i vandværksvand
Danish drinking water is mainly produced from groundwater of high quality with a very
low occurrence of pathogenic micro-organisms. Consequently, disinfection of the drinking
water is not necessary and therefore usually omitted to avoid the production of
disinfection by products. This in turn increases the risk of regrowth of bacteria in the
distribution network.
Regrowth is here defined as an increase in the number of culturable bacteria in the
drinking water on its way from the waterworks to the consumer.
The purpose of this investigation has been to examine the regrowth potential in Danish
drinking water, and to evaluate the methods for determination of the regrowth. In
addition, it has been the purpose to characterise the drinking water by several
microbiological methods and to determine the occurrence of Legionella in drinking
water.
Regrowth potential depends on the method used and is here defined as the increase in
the number of culturable bacteria in a water sample incubated at 15 °C, or as the content
of assimilable organic carbon (AOC).
The investigation has been conducted as a comparative study where the potential for
regrowth in ten types of drinking water has been examined by determination of AOC and by
microbial growth assays. Samples from 7 waterworks were analysed for Legionella. In
addition, the water was characterised by analysis of non-volatile organic carbon (NVOC),
and water quality data were collected from the analyses carried out as routine on the
waterworks.
Water from ten waterworks has been examined. The waterworks extract ground water from
either chalk or from gravel. Both very large and small waterworks and public as well as
private waterworks are represented in the study. The water treatment is traditional,
consisting of aeration and filtration.
On each of the waterworks, three samples were taken. Each sample was divided into three
sub-samples. One to be used for the determination of AOC, one for microbial growth assay
and one for determination of the concentration of NVOC. For two waterworks, where the
regrowth potential was found to be high, the sampling and analyses were repeated, and
included sampling of the raw water as well as the treated water.
In the growth assay, the samples were incubated at 15°C for 14 days. During this
period, the growth of culturable bacteria was determined on different media incubated at
21°C (PCA, Yeast extract agar, Kings Agar B and R2A) and 37°C (PCA, Yeast extract agar,
and R2A), and the total number of bacteria were detected by microscopy (AODC). In addition
the concentration of ATP was determined.
The quality of the water examined in this study was in general good. It can be
characterised as hard water with a high content of CODpermanganate and total
residue, while there was a large variation in the content of iron, nitrate and sulphate.
The analyses showed that the water complies with the Danish drinking water standards with
a few exceptions (iron, turbidity, viable counts at 37°C).
Of the viable counts incubated at 21°C the highest number was obtained on R2A and the
lowest on Kings Agar B. The total number of bacteria, as determined by microscopic
examination, was between 6.5 ·104 per ml and 4.4 · 105 per ml, and
the content of ATP was between 0.4 ng/l and 7.0 ng/l. These results are in compliance with
the literature.
The content of NVOC was between 1.1 mg/l and 5.2 mg/l. This is within the normal range
of NVOC in Danish drinking water, but there is an overrepresentation of samples with a
concentration of NVOC higher than 3 mg/l. The results of the NVOC analyses do not indicate
problems with regrowth.
With regard to assimilable organic carbon, AOC, the water from six of the examined
waterworks had a concentration below 10 µg/l. 10 µg/l is the limit below which the water
is normally considered to be biologically stable. In the water from three waterworks the
AOC concentration was between 20 µg/l and 40 µg/l, indicating a potential for regrowth
in the distribution system. In the water from one of the waterworks, the AOC content was
determined on three different days with results varying between 8,4 µg/l and 660 µg/l.
The results are in accordance with similar results from the Netherlands.
Comparing the results of the AOC determinations and the determinations of NVOC it is
concluded that these two parameters are correlated to some extent, as it was seen that the
AOC concentration was always low when the NVOC concentration was below 2.5 mg/l. On the
other hand, a high concentration of NVOC did not necessarily result in a high AOC
concentration. In average the AOC was 0.39% of the NVOC.
When comparing the results of the AOC determinations and the bacterial counts a weak
correlation was observed. At very low AOC-concentration AOC was correlated to the number
of bacteria (viable counts at 21°C), but at AOC-concentrations above 5 µg/l – 10
µg/l there was no correlation.
The number of viable counts increased in all the growth assays during the 2 weeks of
incubation. In 7 of the waterworks the viable counts on R2A agar at 21°C increased to
between 5.4 · 103 counts/ml and 76 · 103, and the remaining were
above 105 counts/ml. The highest was 6.8 · 105 counts/ml. The
growth assays indicate, as do the AOC determinations that the bacterial growth is limited
when the NVOC is below 2.5 mg/l.
Based on the growth assays it can be concluded that there is a potential for regrowth
in the Danish drinking water. The indigenous bacteria in the water are able to utilise a
part of the carbon that occurs in the water.
There is a fair correlation between the regrowth potentials determined in growth assays
with the different microbial methods. Viable counts at 21°C on the different agar types
are the most sensitive and correlate well. Viable counts at 37°C are in general not
applicable for determination of the regrowth potential, but the growth assays showed that
in some cases there is a regrowth potential for viable counts at 37°C, large enough to
lead to viable counts higher than the maximum allowed concentrations in Danish drinking
water. The determination of ATP and total numbers of bacteria determined by microscopy was
not sensitive enough to be used for determination of the regrowth potential.
For the waterworks with less than 10 µg/l AOC a weak positive correlation with the
regrowth potential determined by the growth assays could be observed. At higher
AOC-concentrations, the yield in the growth assays did not give comparably high results.
The differences between the results from the two methods can probably be explained by the
differences in the biology of the two systems. First, the bacteria in the two systems
probably differ in their substrate selection; secondly, it is likely that only a fraction
of the bacteria, growing in the growth assays, are culturable; third, non pathogenic
protozoans may act as predators on the bacteria in the growth assays and limit the number
bacteria; and fourth, the risk of evolution of a biofilm (and thereby consumption of AOC)
in the growth assays is higher than in the AOC determinations.
The investigation has shown that there is a potential for regrowth in the Danish
drinking water. In the water from three or four out of the ten examined waterworks the
AOC-concentration indicated a risk of regrowth in the distribution system. The conclusions
of the AOC-measurements were supported by the results from the growth assays.
The determination of regrowth potential, as carried out in this investigation, provide
valuable information about the water quality that can not be obtained by traditional
analyses. On the other hand, the investigation does not give actual information of the
relation between the regrowth potential measured in the samples from the waterworks and
the actual regrowth in the distribution systems.
Legionella was not detected in the water from seven of the waterworks.
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