Introduction In Denmark, herbicides like the phenoxy acids, phenylureas and especially the degradation product of dichlobenil; 2,6-dichloro-benzamide (BAM), have closed down numerous groundwater wells due to their presence above the 0.1 µg/L threshold limit. In recent years a number of microorganisms capable of degrading these classes of pesticides at relevant low concentrations have been isolated and described. We are currently investigating if these microorganisms can be used to purify groundwater either at remediation wells or during simple biological water treatment at waterworks.

Conclusions:

Bacteria degrading various pesticides have been isolated and

bio-filters may be used to purify polluted groundwater

The MSH1 strain can degrade BAM to below threshold value in

laboratory setups

Problems may exist regarding fast degradation at low conc.

and competition with indigenous microorganisms

A currently running field experiment will clarify the potential of

this technology further

Rapid degradation of pesticides at low concentrations - the possibility of using biodegradation to purify polluted groundwater during sand filtration

C.N. Albers*, S.R. Sørensen, O.S. Jacobsen, J. Aamand Geological Survey of Denmark and Greenland, Øster Voldgade 10, DK-1350 Copenhagen, Denmark

* E-mail: cal@geus.dk

Results – lab experiments Field experiment – prel. results

Methods

Batch experiments showed that Aminobacter sp. MSH1 can degrade and mineralize BAM in concentrations from less than 1 µg/L to 50 mg/L at temperatures from 10-25 °C. E.g. the incubation of a moderate number of cells (6*107 cells/mL) resulted in the complete degradation of 1 µg/L BAM in less than 0.5 hours at 10 °C (Fig. 1).

Perspectives of the technology include simple water treatment at waterworks and pump&treat remediation plants treating contaminated water before recharging into the aquifer.

Column experiments show that the MSH1 strain adheres well to common filter materials like sand, chalk and expanded clay and that the strain degrades BAM well in the first days after inoculation.

Future experiments will focus on: - The tendency that degradation of BAM per cell at very low

concentrations in sand filters is relatively slow, (mass transfer limitations?) which may make it difficult to reach the 0.1 µg/L threshold limit under field conditions.

- The fact that degradation capacity is lost relatively fast including the interaction and competition with other microorganisms including bacteria and protozoa living in the biofilters, that is:

- Can MSH1 compete with indigenous bacteria in this oligotrophic environment and form a stable population?

- Will the added MSH1 be quickly grazed by protozoa?

- Can other filter materials be used to better protect the bacteria?

The strain has been tested in a number of small- and large-scale laboratory filters. At 20°C and a residence time of 1 hour, 2.7 µg/L BAM was initially degraded to below the detection limit but during 25 days most of the degrading activity was lost (Fig. 3a), while at 10°C and shorter residence time, which are more realistic waterworks conditions, only 65% of 2 µg/L BAM was degraded (Fig. 3b). This degradation was however only slightly decreased over several weeks despite backwashing of the filters.

Fig. 1 Pure culture batch experiment with 1 µg/L BAM and 6*107 MSH1-cells / mL.

Pesticide in groundwater

Traditional simple water treatment Remediation plants

Wells

Aeration

Sand filters

Clean water

To consumer

In batch experiments with BAM-degrading Aminobacter sp. MSH1 the degradation of BAM, mineralization to CO2 and formation of degradation products was followed using 14C-BAM.

Glass columns were used to asses adherence of cells and subsequent degradation in a number of filter materials. Distribution of cells in the filters and losses through the outlets were assessed using qPCR and Aminobacter specific primers.

Larger filters were used to treat BAM-contaminated water at close to realistic conditions (2 µg/L BAM, 10°C, 20 min. residence time, regular backwashing)

The glass column setup

Fig. 3 Laboratory sand filter experiments with a) 20°C and 60 minutes residence time and b) 10°C and 25 minutes residence time. The EU threshold limit for pesticides (0.1 µg/L) is shown in the dotted line.

Fig. 2 A) Degradation of 2.7 µg/L BAM at 1 hour residence time 1 and 4 days after filter inoculation with MSH1. B) Distribution of MSH1 in filters after 4 days of operation. Initial inoculation was on average 2*108 cells/g.

The ambiguous lab-scale column experiments made a field experiment attractive to avoid laboratory biases. This is currently running. Initial results show good degradation in the days after inoculation, but also that this degradation is lost within 1 month. Various analyses are currently performed to explain the loss of degradation.

The portable test filters used in the field experiment are build into a container (sketch) and may be operated like actual waterworks filters. Degradation of BAM + a number of parameters are studied including losses of bacteria by flow and grazing, number of cells in filter sand etc.

This study was financially supported by the BIOTREAT

and the Miresowa projects

Discussions and future experiments

Over the years, bacteria have been isolated, which degrade low concentrations of a number of pesticides, including: - Phenoxy acids (e.g. mecoprop, dichlorprop, MCPA, 2,4-D) - Phenylurea herbicides (e.g. diuron, linuron, isoproturon) - Dichlobenil and its degradation product BAM

Fig. 4. Preliminary results from pilot waterworks of a) concentration in inlet and outlets and b) converted to % removal in each outlet. Filter 1 and 2 contain active (used in another filter) and fresh quartz sand respectively. Groundwater temperature is 10°C and the flow was set to a residence time of 20 minutes.