made counting impossible were rejected .
The mean total colony forming units per milliliter and the corresponding error (s) were calculated using the following relations :
where Ci = individual colonies, and Vi = volume of original sample.
2.5. Bacterial Quantification in Biofilm
To determine the concentration of bacteria in the biofilm, the pipe was agitated with 14 mL of drinking water in the respective pipes. This small volume was used because the emphasis was on the biofilm and not the bulk water phase. It was assumed that most, if not all, of the bacteria present would be released into this volume of water. For each pipe, diluted solutions were prepared from the 14 mL water. The enumeration procedure for total coliform, E. coli and HPC bacteria were the same as those described for the water phase above.
3. Results and Discussions
3.1. Heterotrophic Plate Count (HPC) Bacteria
The results of the study are presented in Tables 3-6. For the HPC bacteria, the errors of the mean values (s) range from 3.0% - 9.8% (except two means values whose errors are above 20%), which fall within the range indi-
Table 3. Average concentrations of HPC bacteria in response to various acetate concentrations in pipes and bottles at 37˚C for maximum 21 days retention time of water.
Table 4. Average concentrations of total coliform in response to various acetate concentrations in pipes and bottles at 37˚C for maximum 21 days retention time of water.
Table 5. Average concentrations of E. coli in response to various acetate concentrations in pipes and bottles at 37˚C for maximum 21 days retention time of water.
cated by Niemelä . The results obtained show a general increase in bacterial concentration after the introduction of the nutrient. In pipe 1, the bacteria increased 2.5 fold after 24-hour residence time (Table 3).
On reaching its peak after six days (Figure 1(a)), the bacteria had multiplied 33 times compared to the dose initially introduced. There was a gradual decrease in bacterial population afterwards, until it dropped to about 1.2 times the initial concentration. Similarly, concentrations of 10 μg ac-C eq/L (pipe 2) and 25 μg ac-C eq/L (pipe 3) respectively resulted in HPC bacterial growth by 19 and 29 times the initial concentration after 24- hour residence time in the pipes. There was a continuous growth thereafter until maximum concentrations of 6.7 × 106 CFU/mL and 9.6 × 106 CFU/mL were attained (Table 3), respectively representing 1.5 × 103 and 2.2 × 103 times the initial concentration. It is interesting to note that on reaching their respective peaks, bacteria in pipes 2 and 3 with acetate concentrations of 10 μg ac-C eq/L and 25 μg ac-C eq/L respectively, had grown 46 and 65 times more than those in pipe 1 with no nutrients.
The results show that increased nutrient concentration corresponds to higher growth rate and subsequent increase in bacterial levels. In all the cases, the HPC bacteria growth reached its peak on days 5 and 6, by which time all the acetate might have been consumed. The maximum yield (Ymax) obtained in pipes with acetate concentrations of 10 μg ac-C eq/L and 25 μg ac-C eq/L were 6.7 × 1014 CFU/g ac-C and 3.8 × 1014 CFU/g ac-C respectively.
Figure 1. Response of HPC bacteria to different acetate concentrations in (a) pipes and (b) bottles at 37˚C. Pipe 1/Bottle 1 (0 μg ac-C eq/L), Pipe 2/Bottle 2 (10 μg ac-C eq/L) and Pipe 3/Bottle 3 (25 μg ac-C eq/L).
The HPC bacteria in the control bottles responded somewhat the same way as those in the pipes (Figure 1(b)). After 24 hours residence time, the bacteria increased from 4390 CFU/mL to 9.4 × 104 CFU/mL, 8.0 × 105 CFU/mL and 1.0 × 106 CFU/mL for acetate concentrations of 0 μg ac-C eq/L, 10 μg ac-C eq/L and 25 μg ac-C eq/L respectively. The maximum growth of 1.0 × 105 CFU/mL, 6.5 × 106 CFU/mL and 8.4 × 106 CFU/mL respectively was attained on days 5 and 6, representing growth factors of 23, 1.5 × 103 and 1.9 × 103 respectively (Table 3).
The acetate concentrations in bottles 2 and 3 produced maximum yields (Ymax) of 6.5 × 1014 CFU/g ac-C and 3.4 × 1014 CFU/g ac-C respectively. According to Boe-Hansen , the yield of indigenous bacteria is within the range of 4.1 × 1012 CFU/g ac-C and 1.32 × 1013 CFU/g ac-C. Compared to what is reported in literature , our values are slightly higher by a factor of about 102. However, bacteria growth in the pipes was comparatively higher than the bottled samples by an average factor of 1.2. The observed difference in growth might have been caused by the biofilm in the pipes. The growth in pipe 1 could be attributed to the use of existing substrate in the pipe by bacteria as source of energy, whereas the observed growth in pipes 2 and 3 could be ascribed to the bacteria’s ability to use the available nutrients to build more cells and as source of energy for growth . The biofilm may provide additional organic and inorganic compounds for the bacteria to grow and later be released to the water phase. The growth in bottle 1 could be attributed to two factors, namely; 1) higher water temperature [17,18] and 2) release of dead cells into the water that were hydrolyzed and used by other bacteria for growth. In all the scenarios described above, the decline in the curves shows a decrease in the growth rate of the bacteria after a certain time. The conditions in the bottles were such that, there were no biofilms to accommodate the bacteria and that the decline could be caused by the possible death of those bacteria that could not survive. In the case of the pipes, the depletion of the nutrient in the water phase might have caused the bacteria to migrate to the biofilm.
3.2. Total Coliforms
The initial coliform concentration in each pipe or bottle was 7.2 × 103 CFU/100 mL. From 0 to 24 hours, the coliform bacteria in the pipes increased to 1.3 × 105 CFU/100 mL, 2.2 × 105 CFU/100 mL and 3.7 × 105 CFU/100 mL respectively for acetate concentrations of 0 μg ac-C eq/L, 10 μg ac-C eq/L and 25 μg ac-C eq/L, and then increased at different rates until attaining maximum growths of 1.9 × 105 CFU/100 mL, 3.6 × 105 CFU/100 mL and 4.4 × 105 CFU/100 mL respectively on day 3 (Table 4, Figure 2(a)). At their peak growths, the coliform bacteria had grown by factors of 26, 50 and 61 respectively, compared to the initial concentration. Maximum coliform yields were respectively 3.6 × 1011 CFU/g ac-C and 1.8 × 1011 CFU/g ac-C for 10 μg ac-C eq/L and 25 μg ac-C eq/L. This was followed by a decline in coliform levels.
The bottles also showed similar trends like the pipes, with a steady increase from an initial coliform concentration of 7.2 × 103 CFU/100 mL to peak levels of 1.6 × 105 CFU/100 mL, 3.5 × 105 CFU/100 mL, and 4.4 × 105 CFU/100 mL respectively for 0 mg ac-C eq/L, 10 μg ac-C eq/L and 25 μg ac-C eq/L (Table 4 and Figure 2(b)). These growth patterns resulted in maximum yields of 3.5 × 1011 CFU/g ac-C and 1.8 × 1011 CFU/g ac-C in bottles 2 and 3 respectively. After attaining the maximum growth on day 3, there was a continuous decline in the growth.
In both pipes and bottles, the decline in coliform concentration continued until no coliform was detected in
the water phase from day 10 for pipe 1/bottle 1, and day 14 for pipe 2/bottle 2 and pipe 3/bottle 3 (Table 4 and Figure 2). However, in situations where the bacteria concentration fell below the detection limit of 3.3 CFU/100 mL, the latter was used in the graphical representation.
A comparison of the pipes and bottles revealed a slightly higher growth in the pipes than in the bottles by an average factor of 1.2. Similarly, the decrease in coliform levels was more pronounced in the bottles than the pipes. These observed trends were probably due to the presence of biofilm which initially enhanced the growth and subsequently sustained it in the pipes. The observed bacterial growth in pipe 1, which did not contain any acetate, could be explained by the higher water temperature [11,17,18], until the bacteria could no longer withstand this effect and then began to die, resulting in the declining pattern of the growth. The growth patterns of total coliform in pipes 2 and 3 were most likely influenced by the combined effects of temperature and nutrient. The higher acetate concentration probably facilitated the growth of coliform bacteria in both the pipes and the bottles. The results support the findings by LeChevallier  and LeChevallier et al.  who reported that acetate is utilized by coliform bacteria to produce new cellular material and as energy source for survival and growth. However, the bacteria are able to grow in the biofilm even if nutrient concentration is low .
3.3. E. coli
Prior to the analysis, the initial E. coli concentration in each pipe and bottle was 156 CFU/100 mL. In situations where enumeration did not yield any positive E. coli, a detection limit of 3.3 CFU/100 mL was used to plot the curves. After 2 - 3 days residence time, the results showed an increase in E. coli concentration in the pipes from 156 CFU/100 mL to maximum concentrations of 311 CFU/100 mL, 384 CFU/100 mL and 401 CFU/100 mL respectively for acetate concentrations of 0 μg ac-C eq/L, 10 μg ac-C eq/L and 25 μg ac-C eq/L (Table 5 and Figure 3(a)). The corresponding maximum yields of 3.1 × 108 CFU/g ac-C and 1.3 × 108 CFU/g ac-C for pipes 2 and 3, respectively were obtained. In all the scenarios, the bacteria showed an average increment of 2 times the initial number before reaching their respective peaks. Just like the other figures already discussed, maximum growth was followed by a decline in growth at different rates until no E. coli was detected in the water phase on day 7 and beyond except for pipes 2 and 3, which recorded coliform concentrations on day 7.
The growths of E. coli in the bottles were comparable to those in the pipes. Bottles with concentrations of 10 μg ac-C eq/L and 25 μg ac-C eq/L recorded an average peak growth of 249 CFU/100 mL on day 3, representing maximum yields of 2.5 × 108 CFU/g ac-C and 9.9 × 107 CFU/ g ac-C respectively. In pipe 1/bottle 1 and pipe 3/bottle 3, the peak growths in the bottles were 17 CFU/100 mL and 5 CFU/100 mL respectively more than those in the pipes (Table 5). This could be explained by the fact that the HPC bacteria present in the pipes were probably competing with the E. coli for the available substrate. This is because the concentrations of HPC bacteria were higher than that of E. coli from days 1 - 3 (Figures 3(a), (b)), indicating that the conditions favored the former than the latter in the pipes whilst the reverse occurred in the bottles.
Once the bacteria had attained their respective maximum growths and the nutrient was most likely totally consumed, there was a decline in the concentrations of the bacteria, probably due to starvation which subsequently led to the death of bacteria that could not survive the stress. However, the decline in bacteria concentration was
more pronounced in the pipes/bottles with acetate concentration of 0 μg ac-C eq/L, and to a lesser extent 10 μg ac-C eq/L than in 25 μg ac-C eq/L (Figures 3(a), (b)). This indicates that the latter scenario had considerable amount of acetate present for the bacteria to thrive on and thus prolonged their survival in the pipe. Comparatively, the growth rates in the pipes were higher than those in the control bottles, except for the peak values for acetate concentrations of 0 μg ac-C eq/L and 10 μg ac-C eq/L (Table 5) where the reverse was observed.
3.4. Bacteria Survival in Biofilm Compared to the Water Phase
In drinking-water distribution system, bacteria are known to move to the biofilms [21,22], which are dominated by microbial cells and their excretions , and therefore appear to be more nutritive environments for such bacteria. In this study, samples taken and analyzed on the last day of the experiment revealed a significantly higher bacterial count in the biofilm as compared to the bulk phase. For acetate concentrations of 0 g ac-C eq/L, 10 g ac-C eq/L and 25 g ac-C eq/L, there were respectively 10, 7 and 12 times more HPC bacteria in the biofilm than in the water phase. Thus, after 21 days, there was an average of 10 times more HPC bacteria in the biofilm compared to the water phase. Similarly, coliform bacteria were higher in the biofilm than the water phase by factors of 172, 192 and 231 respectively. In contrast, E. coli bacteria were not detected in both the biofilm and the water phase (Table 6). According to Rompre et al.  and Reynolds , high HPC bacteria growth is likely to interfere in the analysis of E. coli, resulting in false low numbers, and this could explain why E. coli was not detected in both the water phase and the biofilm since the HPC might have overshadowed the presence of the E. coli.
Total HPC bacterial count in the biofilm ranged between 1.2 × 104 CFU/cm2 and 2.7 × 105 CFU/cm2. The lower end of the range is consistent with that of LeChevallier  who showed that HPC densities associated with a variety of pipe surfaces range between
Table 6. Nutrient effect on the survival of bacteria in the water phase and biofilm in pipes at 37˚C on day 21.
1.0 × 104 bacteria/cm2 and 4.7 × 104 bacteria/cm2. Coliform bacterial count ranged from 1.3 × 10–1 bacteria/cm2 to 1.7 × 10–1 bacteria/cm2 (Table 6).
The presence of organic material and algae in the biofilm probably enhanced the growth of these bacteria , indicating that there was preferential bacteria growth in the biofilm . The high numbers of background heterotrophic bacteria were probably responsible for the decreasing coliform recovery by membrane filter [3,9,25]. These authors indicated that high numbers of HPC bacteria might also interact in the analysis of coliform bacteria and could possibly be responsible for the non-detection of E. coli in both the water phase and the biofilm.
The results of this experimental study corroborate that of LeChevallier  who discovered significant bacterial growth in distribution systems occurring at assimilable organic carbon (AOC) levels between 10 μg ac-C eq/L and 50 μg ac-C eq/L. Boe-Hansen  also reported considerable microbial growth observed at AOC concentrations less than 10 μg ac-C eq/L. According to Szewzyk et al. , a potential source of degradable organic carbon in drinking-water systems is the use of inappropriate materials such as galvanized pipes, tubes and fittings and the release of organic or inorganic compounds that support the growth of heterotrophic bacteria or fungi. The growth could also be due to the possibility of the biofilms releasing indicator organisms and heterotrophic bacteria into the water phase in the pipes . As was discovered in the present study, Szewzyk et al.  reported that the depletion of the nutrient in the water phase in pipes causes bacteria to migrate to the biofilm, which is dominated by organic and inorganic compounds, making it ideal habitats for the bacteria to survive and even grow. However, the death of bacteria in the pipes could also be ascribed to environmental stress such as starvation which eventually caused the bacteria concentration to decline .
Generally, for all the bacteria under investigation, significant growth was recorded in pipe 1 and bottle 1 even though no nutrient was introduced to them. This observation can be attributed to the temperature  and the presence of substrate in the pipes. Thus, the biofilm provides additional nutrients and favorable conditions for the bacteria, thereby enhancing the growth rate in the pipe compared to the bottle, which has no biofilm. In both cases, bacteria that were not able to survive the temperature and starvation perhaps died and their cells were probably used as nutrients for surviving bacteria to live on. Furthermore, the transfer of cells from the water phase to the inner surface of the pipe causes equilibrium to be reached between the amount of bacteria in the water phase and on the surfaces. The end result is a detachment process influenced by biological factors such as cell motility within the biofilm, synthesis and release of extracellular polymeric substances (EPS) degrading enzymes, cell growth rate, grazing activity and cell death/ lysis . The detached bacteria from the surface are detected in the water phase as suspended bacteria, thereby increasing the bacterial population. This observation is buttressed by the findings of Camper et al. .
Unlike the conditions under which this experiment was conducted, if the sources and mechanisms that introduce these bacteria into drinking water pipes in real time situations are not contained, the bacteria will continue to thrive in the system, even in the water phase. As a result, there may not be a decline in bacterial population with time, contrary to what was observed in this study. It is therefore strongly recommended that drinking water providers put the necessary measures in place to ensure that cross connections are continually checked to prevent contamination from surrounding soil and water bodies. Furthermore, the mechanisms that facilitate the entry of disease-causing viruses, bacteria and protozoa into distribution systems, such as poor maintenance practices, long retention times, and the presence of nutrients should be strictly monitored and regularly checked to avert any contamination and regrowth of coliform and HPC bacteria in the systems.
The main objective of this study was to investigate the impact of nutrient on coliform and HPC bacteria in drinking water pipes. The study has demonstrated the ability of nutrients, in the form of sodium acetate to sustain bacterial growth in drinking water pipes. Our results show that nutrients were used up by bacteria for cell building and growth. This led to an increase in bacterial population that could be detrimental to the quality of the drinking water. Once the nutrient was totally consumed and became depleted in the bulk phase, the bacterial population reached a near stationary level and subsequently declined. The results further demonstrate the migration of bacteria from the water phase to the biofilm since the latter provided a more suitable environment and safe haven for the bacteria to thrive on, thus promoting their growth and prolonging their survival in the system. Consequently, degradation of water quality is likely to last for a much longer time than anticipated. It can also be concluded from this study that the absence of E. coli and coliform bacteria in water does not preclude the existence of these bacteria in drinking water distribution networks.
The authors are thankful to Hans-Jørgen Albrechtsen for his constructive criticism and thought-provoking suggestions which helped improve the contents of this paper.