Miljøprojekt, 991 – Bakterievækst og tilsætningsstoffer

Growth of Bacteria and Additives

Summary and conclusions

This report deals with growth of bacteria in drinking well water and whether the growth is due to the chemical products used during the production of the well itself. In this report these chemical products are termed additives, corresponding to the usage in the Danish language of tilsætningsstoffer. Appendix F in the report is a Danish language glossary.

When drinking water wells are established, additives are used for the following purposes:

1. as drilling mud and additives to it
2. lubrication of drilling equipment
3. construction of the well's filter and sealing of the hole
4. disinfection
5. improvement of capacity and regeneration.

The quality of Danish drinking water in general is very good, and problems of bacterial growth in the water are usually not encountered when using groundwater as drinking water. However, some Danish well drilling companies have experienced that the groundwater from newly established wells has a bacterial count that is higher than the waterworks are willing to accept. Without being able to explain these counts, the well drillers attempt to reduce them by various means, such as disinfection with hypochlorite, carrying out "sterile" drilling (avoiding all access of surface soil to the well), or extending the duration of the test pumping to several weeks. The well drillers, the waterworks, and the Danish EPA all want to be able to understand why the bacterial counts rise. Thus, the EPA initiated this project, with the Danish Toxicology Centre as the project leader.

One plausible explanation of the growth of bacteria in the fresh groundwater can be that the bacteria that occur naturally in the groundwater use the drilling additives as a carbon source, and they propagate in the water. This project set out the evaluate whether, in fact, the additives can be carbon sources for groundwater bacteria.

The project consisted of two parts, which were conducted by the Danish Toxicology Centre and by Environment & Resources (E&R DTU) at the Technical University of Denmark. In the first part of the project, a survey was conducted of the additives now in use by Danish well-drilling companies. These data were compared with those presented by the EPA in 1995 (Weber at al. 1995). In the second part of this project, two of the additives were assayed in laboratory experiments for their potential as carbon sources for groundwater bacteria.

In the first part of the project, in January 2004 a questionnaire was submitted to six Danish well-drilling companies as a means of getting an overview of the additives that are in use in Denmark today. Four of the companies responded, and these data are to be found in the report's Tables 1 and 2. The tables present the additives in use, their suppliers, and the rough quantities of each that are used.

Since the EPA survey in 1995, there has not been much change in which additives are used. However, because the 1995 survey only contains a few data on quantities, it is not possible to conclude anything about increased or reduced consumption of the individual additives.

Microbiologically, it is very probable that bacteria will be present in the water from the prolonged test pumping of any new well. Bacteria are natural inhabitants of the groundwater environment, and a few of the additives are suitable as carbon sources for growth of these bacteria.

On the other hand, the large volume of water flowing during the test pumping can defeat bacterial growth. For instance, during the final period of test pumping, the concentration of a given additive in the water would most probably be so low that it would be inaccessible to the metabolic machinery of the bacteria.

In the second part of the project, the project group chose to investigate the additives bentonite (Wyoming bentonite API) and carboxymethyl cellulose (CMC) for their potential as carbon sources for groundwater bacteria. These studies were done at the E&R DTU laboratory. Because bentonite is a product of an igneous rock, its carbon content is extremely low, but it is used in very large quantities. For CMC two qualities were investigated: the drilling technical quality Gabrosa P 300 G and a very pure quality Akucell AF 1985, which is used as a food additive. The technical quality CMC can contain 5-20 % sodium acetate as an unreacted residue from the chemical synthesis. Each additive was tested at a very low concentration and at a 100-times higher concentration. For bentonite, these were 50 and 5000 g /L water, and for both CMC qualities, 10 og 1000 g /L water. The experimental conditions for the investigations were chosen roughly to resemble those in the groundwater environment. All experiments were conducted in triplicate.

For the microbial inoculum that potentially might grow on the additives, two different types of inocula were chosen. One of these consisted of two, well-defined strains of bacteria (Pseudomonas fluorescence P17 and Aquaspirillum sp. NOX) that are known to grow well in the groundwater environment and to grow despite very low concentrations of carbon (e.g. 10 g /L). Use of these two strains allowed extrapolation from the growth results of these experiments to the results of earlier, published experiments, in which the strains had known and very low concentrations of acetate to grow on. This way, results with the drilling additives could be correlated directly with definite concentrations of acetate carbon, or acetate-C equivalents. This method is assumed to assay assimilable organic carbon (AOC), or the total carbon in the water that is accessible to microorganisms. In practice, the growth of the two bacterial strains is assayed on a single agar substrate, on which their colonies have different morphologies. The AOC of each additive at each concentration is then expressed as g acetate-C equivalents/L water.

The other type of inoculum for the growth experiments was a broad and undefined population extracted from filter sand from the municipal water plant in Lyngby, Denmark, which the E&R DTU laboratory has investigated extensively. In these experiment the additives' potential for regrowth was studied. The regrowth potential is an expression of the maximal bacterial growth attainable with the additive as a carbon source in drinking water. The regrowth potential was assayed both as colony-forming units (CFU) on agar plates and as measurement of ATP (adenosine triphosphate).

The results of all experiments showed that neither bentonite nor CMC will affect the concentration of bacteria in the water pumped up from a new well. However, there was an exception in the high concentration (1000 g/L) of the technical quality CMC (Gabrosa P 300 G). Here, an AOC of 15 g acetate-C equivalents/L was measured, which is 3 times above the control samples. The pure quality CMC at the high concentration did not result in bacterial growth. This is presumably due to the absence of acetate in this quality.

In conclusion, it is not probable that the additives currently in use in Denmark for the establishing of drinking wells contribute the bacterial counts seen in water from new wells. The use of large quantities of technical quality CMC might, however, result in microbial growth if the additive contains an appreciable concentration of acetate as an impurity.

Thus, this project is not able to explain the elevated CFU's observed by the well drillers of new drinkingwater wells.