FIELD OF INVENTION

The present invention relates to an observed conversion of the herbicide degradation product 2,6-dιchlorobenzamιde (BAM) in samples taken from the environment. The invention provides a 2,6-dιchlorobenzamιde degrading or converting activity, which may form the basis of means and methods of bioremediation of polluting 2,6-dιchlorobenzamιde in the environment. The invention further provides a method for obtaining the 2,6- dichlorobenzamide degrading or converting activity as well as compositions and micro¬ organisms harboring the activity and methods for employing these in bioremediation

BACKGROUND OF THE INVENTION

Dichlobenil (2,6-dιchlorobenzonιtπle) and chlorthiamide (2,6- dichlorobenzenecarbothioamide) are active ingredients of the herbicides Prefix G and Casoron G that have been prohibited in Denmark since 1997; but still used in other countries. In soil dichlobenil is mainly degraded to the persistent metabolite 2,6- dichlorobenzamide (BAM) which has been detected in 19% of the 5.096 investigated ground water supply wells in Denmark; in 7.5% of these wells the concentration exceeds the EU limit of 0.1 μg I \ Denmark has as the only country included measurements of this metabolite in the national ground water monitoring on a large scale. In Germany the concentration of the metabolite exceeds the EU limit in 15% of 359 ground water samples and in Sweden BAM has been detected below the EU limit in 10 out of 24 ground water samples [3,4]. It seems possible that BAM is a problem in ground water of other countries as well.

Degradation of BAM in nature

BAM is accumulated when dichlobenil is degraded by three bacterial cultures tested in other laboratories [5-7]. Recently, we tested eight bacteria whereof seven were able to degrade dichlobenil but again BAM was accumulated and not degraded (Holtze et al. unpublished results). So far BAM has not been degraded or mineralized in pure cultures of bacteria or fungi but indications of BAM mineralization in environmental samples have been observed. E.g. 5.6% of 14C-BAM added to pond water and sediment was mineralized to 14CO2 in 40 days [6]. A recent study indicates a microbial degradation of BAM in sediments where the mineralization was estimated indirectly to be in the order of 5-27% during 436 days [8]. Other investigations of Danish groundwater sediments indicate that BAM is not degraded at all under different redox conditions during an experimental period of one year [9-11].

Toxicity of BAM

Danish authorities have defined BAM as a Class 1 compound, meaning that, in principle, the metabolite must not be present in drinking water since it is believed to constitute a hazard to public health. However, a thorough investigation of the toxicity of BAM has never been conducted and knowledge on its toxicity is sparse. Studies have shown that in fresh water BAM is moderately toxic to daphnia, an algeal species and two piscine species. According to the Danish environmental authorities BAM is classified as moderately toxic to aquatic animals and it is known to cause lowered body weight, increased weight of the liver, changes in blood composition and decreased progeny weight.

In light of the continued use of dichlobenil-containing pesticides in many countries and the increasing concern with regard to the public health issues associated with pesticides and their metabolites, the presence of BAM in the environment is likely to gain more focus in the coming years. In particular the presence of BAM in sources of drinking water appear problematic since clean drinking water is becoming an increasingly scarce resource in most areas of the world.

For other types of pollution, bioremediation, relying on the use of biological organisms in removing hazardous substances from polluted areas has proven very efficient. However, developing means and procedures for reducing the levels of BAM in the environment obviously relies on the identification of microorganisms capable of converting BAM to non- hazardous compounds.

SUMMARY OF THE INVENTION

The primary aspect of the invention provides an enzyme or an activity, such as an enzyme activity, which is present in an environmental sample and/or in an enrichment sample derived from said environmental sample and results in the conversion over a time of 100 days of at least 5% of any 2,6-dichlorobenzamide initially present in or added to said environmental sample or to said enrichment sample. In particular, the enzyme or enzyme activity is capable of converting 2,6-dichlorobenzamide in amounts corresponding to at least 10% of the amount present in said environmental sample over a time period of from 1 to 30 days and with 2,6-dichlorobenzamide being present in concentrations of from 0.001 μg/l to 500 mg/l. Further aspects of the invention provide a bacterial strain expressing an enzyme with an activity according to the invention and a composition comprising an enzyme or enzyme activity according to the invention.

Also, within the area of the invention is a method for evaluating the persistence of 2,6- dichlorobenzamide and/or 2,6-dιchlorobenzonιtrιle present at a given location, said method comprising determining any presence of an enzyme activity, an enzyme or a bacterial strain according to the invention on said location or in a sample derived there from.

The present invention further provides a method of obtaining and/or increasing an enzyme or enzyme activity according to the invention said method comprising the step of obtaining an environmental sample, and one or more steps of: (a) adding to said environmental sample an amount of one or more compounds selected from the group consisting of chlorthiamide, 2,6-dιchlorobenzonιtrιle and 2,6-dιchlorobenzamιde, and/or (b) adding to said environmental sample one or more nitrogen sources other than 2,6-dιchlorobenzamιde and 2,6-dιchlorobenzonιtπle, and/or (c) adding to said environmental sample one or more carbon sources other than 2,6-dιchlorobenzamιde and 2,6-dιchlorobenzonιtπle, and/or (d) isolating one or more microbial species from said environmental sample, and/or (e) cultuπng the microbial species from step (d), and/or (f) isolating one or more molecular species originating from said environmental sample or from microbial species isolated there from.

Another aspect of the invention pertains to the use of a composition or a bacterial strain according to the invention for reducing the amounts of 2,6-dιchlorobenzamιde and/or the amount of 2,6-dιchlorobenzonιtπle at a location or in an environmental sample derived there from.

Finally, the invention relates to a method for reducing the amount of 2,6- dichlorobenzamide and/or the amount of 2,6-dιchlorobenzonιtπle at a given location, said method comprising providing to said location an amount of a composition or a bacterial strain according to the invention

DETAILED DESCRIPTION OF THE INVENTION The present invention related to the surprising observation that 2,6-dichlorobenzamide (BAM) is degraded at a significant rate in environmental samples taken from locations where one or both of the two herbicides, Prefix G and Casoron G, have been used. This is contrary to previous studies showing limited degradation or virtually no degradation at all 5 of 2,6-dichlorobenzamide. All of the previous studies showing that BAM is not degraded have been performed with a 14C-BAM labeled at the carbonyl carbon. In contrast, the present invention is based on experiments using 14C-labelled dichlobenil and BAM, which was labeled more conservatively in the aromatic ring structure. No degradation or conversion of either dichlobenil or BAM was seen in three sandy agricultural soils in our 10 laboratory when using these conservative-labeled compounds (Rasmussen et al. unpublished results). In contrast, when dichlobenil-contaminated soils were tested using this technology a rapid degradation of BAM was observed.

A first main aspect of the present invention pertains to an enzyme or an activity such as an 15 enzyme activity resulting in the degradation or conversion to other molecular species of a significant amount of 2,6-dichlorobenzamide (BAM) over a certain period of time. More specifically, this activity when present in an environmental sample or in an enrichment culture derived from the environmental sample results in the conversion over a time period of a certain fraction of any BAM initially present in or added to the environmental sample 20 or to the enrichment culture. The time period referred to will be determined to a certain extent by the exact conditions under which the degradation or conversion occurs, such as the temperature, pH-value and availability of nutrients in the environment as well as the nature and origin of the environmental sample in general. The period of time may extend over single days, months or years, such as from 1 to 5, 2 to 10, 5 to 25, 10 to 50, 25 to 25 100, 50 to 200, 100 to 400 days, such as 1, 2, 5, 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 175, 180, 190, 200, 250, 300, 350 or 400 days, or such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 months, or such as 1, 2, 3, 4 or 5 years. 30 The term "enrichment culture" refers to a sample or a culture, which is generated and maintained in order to increase the proportion of a desirable constituent. In the present context, the term refers specifically to a culture of micro-organisms which is maintained under conditions which favour those micro-organisms possessing an activity according to 35 the invention.

It appears that the amount of 2,6-dichlorobenzamide which is converted or degraded by the enzyme or enzyme activity according to the invention may correspond to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 54, 56, 58, 60, 65, 70, 75, 80, 85, 90, 95 or 100% of any 2,6-dichlorobenzamide initially present in or added to said environmental sample.

5 In one preferred embodiment of the invention the enzyme or enzyme activity results in the conversion over a time of 100 days of at least 5% of any 2,6-dichlorobenzamide initially present in or added to said environmental sample.

In a more preferred embodiment of the invention the activity results in the conversion of 10 50 to 60% of any 2,6-dichlorobenzamide initially present in or added to said environmental sample over a time of 40 to 45 days.

In a further preferred embodiment of the invention the enzyme or enzyme activity is, when present in an environmental sample or in a sample derived from an environmental 15 sample, capable of converting 2,6-dichlorobenzamide in amounts corresponding to at least 10% of the amount present in said environmental sample, such as at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 54, 56, 58, 60, 65, 70, 75, 80, 85, 90, 95 or 100% over a time period of from 1 to 30 days, such as from 1 to 5, 2 to 10, 5 to 20 25, 10 to 30 or 20 to 30 days and with 2,6-dichlorobenzamide being present in concentrations of from 0.1 ng/l to 500 mg/l such as from 1 ng/l to 50 mg/l, from 0.01 μg/l to 10 mg/l, from 0,lμg/l to 1 mg/l, from below 1 μg/l to 100 μg/l, and from 2 μg/l to 50 μg/i-

25 In a currently most preferred embodiment, the enzyme or enzyme activity is capable of converting 2,6-dichlorobenzamide in amounts corresponding to at least 70% of the amount present in said environmental sample over a time period of 15 days with 2,6- dichlorobenzamide being initially present at a concentration of 50 mg/l.

30 From the above it can be seen that the average amounts of 2,6-dichlorobenzamide which is converted or degraded may correspond to at least 0,001, 0,005, 0,01, 0,05, 0,1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 54, 56, 58, 60, 65, 70, 75, 80, 85, 90, 95 or 100% per day, per month, or per 2, 3, 4, 5, 35 6, 7, 8, 9, 10 or 12 months or per year of any 2,6-dichlorobenzamide initially present in or added to said environmental sample.

In the present context the term "environmental sample" designates any volume and/or amount of constituents of a material obtained initially from an outdoor location comprising, for instance, soil and/or sediment and/or water. In particular, the material may comprise top soil and/or water from an aquifer, in particular a source of drinking water.

The degradation or conversion of BAM may result from from one or more abiotic processes, such as one or more chemical processes as well as from biotic processes or from a combination of any of these types of processes. In particular the conversion or degradation of 2,6-dichlorobenzamide may occur via microbially catalysed hydrolysis to a limited extend in combination with abiotic conversion or degradation.

In the present context the term "biotic processes" refers to processes caused or produced by living beings. Abiotic processes, on the other hand, refers to processes in which a chemical in the environment is altered by non-biological mechanisms (such as by exposure to sunlight).

The term "chemical process" designates any process determined by the atomic and molecular composition and structure of the substances involved.

It is to be understood that the conversion or degradation of BAM according to the invention may occur under anaerobic as well as under aerobic conditions. In a preferred embodiment, the activity occurs under aerobic conditions.

According to previous observations conversion or degradation of BAM has only been observed in low or insignificant amounts and at low or insignificant rates. The activity according to the invention however, can be characterised by a number of parameters. In one embodiment of the invention the conversion or degradation of 2,6-dichlorobenzamide thus occurs in an environmental sample and/or in an enrichment culture derived from said environmental sample: (a) after supplementing said environmental sample or said enrichment culture with an amount of a buffered nutrient containing medium which has a pH within the range of from 3 to 9, such as within the range of from 3.5 to 8.5, from 4 to 8, from 4.5 to 7.5, from 5 to 7, from 5.5 to 6.5 and which does not comprise any sources of carbon and nitrogen other than BAM, and/or (b) after addition of 2,6-dichlorobenzamide to said environmental sample or said enrichment culture in concentrations exceeding 0.01 μg/l, such as concentrations exceeding 0.1 μg/l such as concentrations exceeding 1 μg/l, such as concentrations exceeding 0.01 mg/l, such as concentrations exceeding 0.1 mg/ml, such as concentrations exceeding 1 mg/ml, and/or (c) after addition of a carbon source other than 2,6-dichlorobenzamide to said environmental sample or said enrichment culture, wherein the amount of carbon added corresponds to from 0.1 to 1000 times the amount of carbon present in the environmental sample in the form of 2,6-dichlorobenzamide, such as from 0.5 to 950, 1 to 900, 2 to 850, 3 to 800, 4 to 750, 5 to 700, 6 to 650, 7 to 600, 8 to 550, 9 to 500, 10 to 450, 20 to 400, 30 to 40, 50 to 300, 60 to 250, 70 to 200, 80 to 150, 90 to 100, such as 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 99, 100, 102, 105, 107, 110, 112, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 190, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000 times the amount of carbon present in the environmental sample in the form of 2,6-dichlorobenzamide, and/or (d) after addition of a nitrogen source other than 2,6-dichlorobenzamide to said environmental sample or said enrichment culture, wherein the amount of nitrogen added corresponds to from 1 to 1000 times the amount of nitrogen present in the environmental sample in the form of 2,6-dichlorobenzamide, such as from 15 to 950, 20 to 900, 25 to 850, 30 to 800, 35 to 750, 40 to 700, 45 to 650, 50 to 600, 55 to 550, 60 to 500, 65 to 450, 70 to 400, 75 to 350, 80 to 300, 85 to 250, 90 to 200, 92 to 150, 95 to 125, 97 to 112, 99 to 102, such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 99, 100, 102, 105, 107, 110, 112, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 190, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000 times the amount of carbon present in the environmental sample in the form of 2,6-dichlorobenzamide, and/or (e) at a pH that is within the range of from 4 - 7.5, such as within the range of from 4.25 to 7.25, from 4.50 to 7, from 4.75 to 6.5, from 5 to 6.25, from 5.25 to 6, from 5.50 to 5.75, and/or (f) at a temperature that is within the range of from 4 to 4O0C, such as from 7 to 39°C, from 10 to 38°C, from 15 to 37, from 20 to 36, from 25 to 35, from 30 to 34, from 31 to 33°C; and wherein said conversion of 2,6-dichlorobenzamide is inhibited by one or more antibiotics selected form the group comprising penicillin G, ampicillin, streptomycin, tetracyclin and stimulated by one or more antibiotics from the group comprising nystatin, and cycloheximide.

As for the buffered nutrient-containing medium it may be added in amounts such that it accounts for from 1 to 99%, such as from 2 to 98, 5 to 95, 7 to 93, 10 to 90, 15 to 85, 20 to 80, 25 to 75, 30 to 70, 35 to 65, 40 to 60, or 50 to 55% of the environmental sample after addition of the supplements mentioned. When referring to the amount of 2,6-dichlorobenzamide present in the environmental sample or to the carbon or nitrogen present in the environmental sample in the form of 2,6-dichlorobenzamide it is to be understood that reference is made to the total amount of 2,6-dichlorobenzamide present in the sample including the 2,6-dichlorobenzamide initially present in the sample and any 2,6-dichlorobenzamide which has been added to the sample, such as 2,6-dichlorobenzamide added for experimental purposes.

It will be understood that for the activity according to any of the embodiments of the invention a set of conditions can be determined at which the conversion or degradation of 2,6-dichlorobenzamide or the rate at which 2,6-dichlorobenzamide is converted or degraded reaches a maximum. In a presently preferred embodiment of the invention the activity according to the invention is characterised in that the amount of 2,6- dichlorobenzamide converted relative to the amount of 2,6-dichlorobenzamide initially present in an environmental sample supplemented with 5 volumes or less of a Mineral Salt Medium reaches a maximum and/or wherein the conversion of 2,6-dichlorobenzamide occurs at a maximum rate: (a) after addition of a carbon source other than 2,6-dichlorobenzamide to said environmental sample, wherein the amount of carbon added corresponds to 10 times the amount of carbon present in the environmental sample in the form of 2,6-dichlorobenzamide, such as from 1 to 100, from 2 to 90, from 3 to 80, from 4 to 70, from 5 to 60, from 7 to 50, from 7.5 to 40, from 8 to 30, from 8.5 to 20, from 9 to 15, from 9.5 to 12.5 times the amount of carbon present in the environmental sample in the form of 2,6-dichlorobenzamide, and/or (b) after addition of a nitrogen source other than 2,6-dichlorobenzamide to said environmental sample, wherein the amount of nitrogen added corresponds to approximately 100 times the amount of nitrogen present in the environmental sample in the form of 2,6-dichlorobenzamide, such as from 10 to 1000, from 20 to 900, from 30 to 800, from 40 to 700, from 50 to 600, from 70 to 500, from 75 to 400, from 80 to 300, from 85 to 200, from 92 to 150, from 95 to 125 or from 97 to 112 times the amount of carbon present in the environmental sample in the form of 2,6-dichlorobenzamide and/or (e) after addition to said environmental sample of 30 mg/l, such as from 5 to 100 mg/ml, 10 to 70, 15 to 50, 20 to 40, 25 to 35 mg/l of one or more antibiotics selected form the group consisting one or more antibiotics from the group comprising nystatin, and cycloheximide, and/or (f) at a pH which is approximately 6.6 such as from 6 to 9, 6.5 to 8.5, or 7 to 8, and/or (g) at a temperature which is 4°C or more such as 5°C or more, 10°C or more, 15°C or more, 200C ore more, 25°C or more, 300C or more, 320C ore more, 35°C or more, or 37°C or more; and after addition to said environmental sample of more than 10 mg/l 2,6- dichlorobenzamide, such as more than 15 mg/l, more than 20 mg/l, more than 25 mg/l, more than 30 mg/l, more than 35 mg/l, more than 40 mg/l, more than 60 mg/l, more than 90 mg/l, or more than 200 mg/l 2,6-dichlorobenzamide.

As mentioned, the environmental sample may be supplemented with a certain amount of a nutrient containing medium, such as a Mineral Salt Medium. The sample may thus be supplemented with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 volumes or less of a Mineral Salt Medium. In particular, the amounts may correspond to 5 volumes or less.

In an embodiment of the invention the enzyme or enzyme activity according to the invention is characterized in that the amount of 2,6-dichlorobenzamide converted relative to the amount of 2,6-dichlorobenzamide initially present in an environmental sample supplemented with 5 volumes or less of a Mineral Salt Medium reaches a maximum and/or wherein the conversion of 2,6-dichlorobenzamide occurs at a maximum rate: (a) after addition of a carbon source other than 2,6-dichlorobenzamide to said environmental sample, wherein the amount of carbon added corresponds to 10 times the amount of carbon present in the environmental sample in the form of 2,6-dichlorobenzamide, and/or (b) after addition of a nitrogen source other than 2,6-dichlorobenzamide to said environmental sample, wherein the amount of nitrogen added corresponds to approximately 100 times the amount of nitrogen present in the environmental sample in the form of 2,6-dichlorobenzamide, and/or (e) after addition to said environmental sample of 30 mg/l antibiotics selected from the group consisting nystatin and cycloheximide, and/or (f) at a pH which is approximately 6.6, and/or (g) at a temperature which is 100C or more; and after addition to said environmental sample of approximately 50 mg/l 2,6- dichlorobenzamide.

As for the carbon source and/or nitrogen source referred to above it is preferred that they are easily accessible carbon or nitrogen sources and/or that they can be defined as primary carbon sources for the microorganisms present in the environmental sample under any of the conditions defined above. Also it is preferred that the carbon source and/or the nitrogen source is a nutrient source alternative to 2,6-dichlorobenzamide. Examples on such nutrient sources are Sodium succinate and Ammonium chloride. Accordingly, in an even more preferred embodiment of the invention the carbon source is Sodium succinate and/or the nitrogen source is Ammonium chloride.

An additional embodiment of the invention provides an activity present in an enrichment sample as described above, wherein said enrichment sample has a total volume of from 5 to 100 ml, such as 5, 10, 15, 20, 25, 30, 40 or 50 ml and/or comprises a Mineral Salt Medium, and/or contains from 1 to 200 mg/l 2,6-dichlorobenzamide, such as 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175 or 200 mg/l 2,6-dichlorobenzamide and/or comprises from 1.000 to 50.000 dpm 14C 2,6-dichlorobenzamide such as 1.000, 2.000, 5.000, 10.000, 25.000 or 50.000 dpm 14C 2,6-dichlorobenzamide and/or has not been supplemented with any source of nitrogen and/or carbon, and/or comprises from 0.1 to 10 g/l sodium succinate, such as 0.1, 0.5. 1, 3, 5, 7,5 or 10 g/l sodium succinate and/or comprises Mineral Salt Medium having a reduced content of nitrogen. In relation to this embodiment it is further understood that the enrichment cultures are initially established by transferring an aliquot of the environmental sample to fresh medium when the mineralization is at the end of the exponential phase. Also, it is to be understood that the activity according to the invention may be present in enrichment cultures generated by serial transfers of culture aliquots to fresh medium once the activity in the culture has reached the end of the exponential phase. Preferably a series of from 2 to 20 transfers are made, such as from 3 - 15, from 4 to 12, from 5 to 10, or from 7 to 8 transfers.

A further embodiment of the invention pertains to an activity as described above wherein the amount of 2,6-dichlorobenzamide converted relative to the amount of 2,6- dichlorobenzamide initially present in an environmental sample supplemented with 5 volumes of Mineral Salt Medium reaches a maximum when all of the following conditions (a) through (g) are fulfilled:

(a) after addition of a carbon source other than 2,6-dichlorobenzamide to said environmental sample, wherein the amount of carbon added corresponds to 10 times the amount of carbon present in the environmental sample in the form of 2,6-dichlorobenzamide, and (b) after addition of a nitrogen source other than 2,6-dichlorobenzamide to said environmental sample, wherein the amount of nitrogen added corresponds to approximately 100 times the amount of nitrogen present in the environmental sample in the form of 2,6-dichlorobenzamide, and (e) after addition to said environmental sample of 30 mg/l antibiotics selected from the group consisting nystatin, and cycloheximide. (f) at a pH which is approximately 6 or 6.6, and (g) at a temperature which is 100C or more; and after addition to said environmental sample of approximately 10 mg/l 2,6- dichlorobenzamide.

5 It is contemplated that a large part of the conversion or degradation of 2,6- dichlorobenzamide occurs in a process of mineralization. BAM is converted to 2,6- dichlorobenzoic acid, which via mineralization of the side chain yields CO2 and one or more presently unidentified residual products. Technically, the determination of the rates at which 2,6-dichlorobenzamide is converted or degraded and the amounts degraded or 10 converted may be based on the amount of 14CO2 generated from 14C-labelled 2,6- dichlorobenzamide added to the sample.

WO 01/07912 describes a hapten-polymer carrier complex, which is useful for immunoassay purposes and in particular for detection of 2,6-dichlorobenzamide with high 15 specificity and sensitivity. A skilled person will appreciate that the technology provided in WO 01/07912 is useful for monitoring the degradation or conversion of 2,6- dichlorobenzamide according to the invention,

Mostly, conversion or degradation of 2,6-dichlorobenzamide is likely to occur in the 20 uppermost layers of soil and/or sediments. In preferred embodiments therefore, the activity according to the invention is found in layers of soil and/or sediment that are found at a depth below ground level that is within the range of 0 - 2.5 metres. In particular the activity may be found in layers of soil and/or sediment that are within 0 - 5 centimeters, 0 to 10 centimeters, 0 to 15 centimeters, 0 to 20 centimeters, 0 to 25 centimeters, 0 to 30 25 centimeters, 0 to 40 centimeters, 0 to 50 centimeters, 0 to 60 centimeters, 0 to 70 centimeters, 0 to 80 centimeters, or 0 to 90 centimeters.

In particular embodiments of the invention the conversion or degradation of 2,6- dichlorobenzamide is observed in environmental samples taken from a depth of 0 - 30, 0 - 30 20, 10 - 20, 10 - 30, 10 - 50 and 30 - 50 cm below ground level. In other particular embodiments of the invention, the most significant conversion or degradation of 2,6- dichlorobenzamide is seen in samples taken from 0 - 30, 70 - 80, 80 - 100, 100 - 120, 120 - 140, 140 - 160, 160 - 180, or 180 - 200 cm below ground level.

35 According to an interesting embodiment of the invention, an activity may be found at relatively deep levels, which is capable of converting 2,6-dichlorobenzamide present at very low concentrations, such as concentrations below 0.1 μg/l, below 0.01 μg/l, below 0.001 μg/l, below 0.1 ng/l or below 0.01 ng/l. As a part of the inventive concept of the present invention it is contemplated that the activity according to the invention has developed during exposure of the environment to either chlorthiamide (2,6-dιchlorobenzenecarbothιoamιde) or dichlobenil (2,6- dichlorobenzonitπle), or both, over a period of time. In particular, it is plausible that the 5 BAM degrading or BAM converting activity is found in soil or sediments at locations such as plant nurseries, plantations, public areas such as parks, gardens or other recreational areas as well as gardens surrounding private homes. Accordingly, an activity of the invention may be found in particular in environmental samples, which are obtained from locations, which have previously been supplied with chlorthiamide and/or dichlobenil in 10 amounts leading to an accumulation of dichlobenil and 2,6-dιchlorobenzamιde, which exceeds a certain level. For dichlobenil this level may be 0.1, 1, 10, 100, 200, 300, 400, 500, 600, 700, 800 or 900 μg/kg or from 1 to 5 mg/kg. For 2,6-dιchlorobenzamιde this level may be 1, 10, or 100 ng/kg or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 300, 400, or 500 μg/kg soil. 15 In preferred embodiments, the invention thus pertains to an enzyme or enzyme activity which is obtainable from a bacterial culture which is isolated by a process comprising obtaining or establishing a bacterial culture from soil collected from one or more locations where a herbicide comprising Chlorthiamide (2,6-dιchlorobenzenecarbothιoamιde) and/or 20 Dichlobenil (2,6-dιchlorobenzonιtrιle) as an active ingredient, have been used.

Typically, residual concentrations of 2,6-dιchlorobenzamιde are lower than 100 μg/kg, such as 10, 20, 30, 40, 50, 60, 70, 80, or 90 μg/kg. On particular locations, however, higher concentrations of 2,6-dιchlorobenzamιde have been found, such as 200, 300, 400, 500, 25 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 1800, 1900, 2000, 3000, 4000 or 5000 μg/kg soil. An enzyme or enzyme activity of the invention may be found in particular in environmental samples, which are obtained from locations, at which 2,6-dιchlorobenzamιde is present or has been present in any of these concentrations.

30 In the most well characterized embodiments of the invention the activity leading to conversion or degradation of BAM is found in an environmental sample, which has been collected at a location selected from the group consisting of: a walking path close to Vestergade and Ellenet, DK-5960 Marstal, Denmark, a courtyard of a plant nursery, Brostykkevej 182-212, DK-2650 Hvidovre, Denmark, a tennis court close to Vestergade 35 and Ellenet, DK-5960 Marstal, Denmark, a trench on Brostykkevej 182-212, DK-2650 Hvidovre, Denmark, a private dump site in Sengeløse on Ingersvej 4, DK-2640 Hedehusene, Denmark, and a court yard on Solhøjvej 44, DK-2640 Hedehusene, Denmark. According to the underlying inventive concept of the invention, however, the activity leading to the conversion or degradation of BAM is primarily found in an environmental sample taken from an environment or a location that has previously been exposed to the herbicides chlorthiamide and dichlobenil. It is thus contemplated that the exact nature of the activity according to the invention may vary with the extent to which these locations or environments have been exposed to the herbicides. In particular, the rates at which BAM is degraded or converted may correlate to the amounts of the pesticides that have been added to the environment. As mentioned, both the uses of chlorthiamide and dichlobenil has been prohibited at the aforementioned locations but the compounds are still being used in many countries world-wide and in particular in the western world. It is therefore to be expected that an activity leading to the conversion or degradation of BAM at rates that are higher than those according to the present invention may be found at locations where the herbicides are still in use.

As mentioned, degradation or conversion of 2,6-dichlorobenzamide, such as the very limited degradation of BAM, which has previously been observed, is believed to occur via conversion of BAM to 2,6-dichlorobenzoic acid. This conversion is most likely catalysed by enzymes belonging to the amidase complex or to the nitrilhydratase/amidase complex or by enzymes belonging to the group of ring cleaving enzymes. Accordingly in a preferred embodiment of the invention, the activity according to the invention leading to the conversion of 2,6-dichlorobenzamide comprises a process catalysed by an enzyme or enzyme activity belonging to the amidase complex or to the nitrilhydratase/amidase complex or from the group comprising ring-cleaving enzymes. The term "amidase complex" is understood to comprise enzymes that that catalyzes the hydrolysis of monocarboxylic amides to free acid plus NH3. The term "nitrilhydratase/amidase complex" refers to nitrile hydratases and amidases, which are hydrolytic enzymes responsible for the sequential metabolism of nitrile compounds. Nitrile hydratases are mononuclear iron or (non-corrinoid) cobalt enzymes that catalyse the hydration of nitriles to their corresponding amides.

The term "ring cleaving enzymes" is understood to comprise enzymes that catalyse the degradation or conversion of aromatic compounds, more specifically the fission of the aromatic ring. The ring cleavage enzymes are classified into two groups based on the site of ring fission. Enzymes catalyzing ring fission between two hydroxyl groups are designated intradiol (ortho-) cleavage enzymes. Enzymes that cleave at a bond proximal to one of the two adjacent hydroxyl groups are designated extradiol (meta-) cleavage enzymes.

Since under most circumstances no or only little conversion of BAM has previously been observed it is believed that the activity according to the invention develops as a result of the adaptation of enzymatic processes to the presence of BAM and its precursors. In an embodiment of the invention the conversion of 2,6-dichlorobenzamide is mediated by a catalytic activity resulting from an altered substrate specificity of a known enzyme belonging to the amidase complex or the nitrilhydratase/amidase complex or from the group comprising ring cleaving enzymes.

In accordance with the above a further characteristic of this embodiment is a conversion of 2,6-dichlorobenzamide, which comprises the formation of 2,6-dichlorobenzoic acid or products resulting from ring cleavage. Such products may be chlorinated cathechols, which again may be rapidly converted or degraded.

In an equally preferred embodiment of the invention, the conversion of 2,6- dichlorobenzamide by the activity of the invention is a process of mineralization. In the present context, the term "mineralization" refers to the conversion of any kind of organically bound element into one or more inorganic forms. The term "organic" refers broadly to substances belonging to the family of compounds characterized by chains or rings of carbon atoms that are linked to atoms of hydrogen and sometimes oxygen, nitrogen, and other elements. The term "inorganic" is used to describe chemical compounds that contain no carbon, excluding the oxides of carbon, carbon disulfide, cyanides, and their associated acids and salts.

As a further characteristic of the enzyme or enzyme activity of the present invention, the conversion of 2,6-dichlorobenzamide leads to the formation, such as the transient formation, of one or more degradation products selected from the group consisting of: DCBA (2,6-dichlorobenzoic acid), O-BAM (ortήo-chlorobenzamide), ortΛo-chlorobenzoic acid, benzamide and benzoic acid. In addition, products such as ortho-chlorobenzonitrile and benzonitrile may be formed under saturated conditions.

According to one aspect of the invention the conversion of 2,6-dichlorobenzamide occurs as part of a metabolic or co-metabolic process within a microbial organism. The term "metabolic process" refers to the physical and chemical processes by which living organized substance is produced and maintained (anabolism), and also the transformation by which energy is made available for the uses of the organism (catabolism). By "co- metabolic process" is meant a process mediated by one or more enzymes in parallel with one or more metabolic processes mediated by the same enzyme or enzymes. The term "microbial organism" refers broadly to a minute living organism, a microphyte or microzoon, including in particular bacteria, algae, protozoa, and fungi. In a particular embodiment of the invention the microbial organism is a bacterial or fungal species.

It may be a specific characteristic of the activity according to the invention that the conversion of 2,6-dichlorobenzamide is growth related in the presence of of 2,6- dichlorobenzamide that exceed a threshold value of 0.001 mg/l to 100 mg/l, such as from 0.05 mg/l to 50 mg/l, 0.01 mg/l to 5 mg/l, 0.05 mg/l to 2.5 mg/l, 0.1 - 1 mg/l, 0.25 mg/l to 0.75 rng/l such as 0,001, 0,01, 0.05, 0.1, 0.25, 0.75, 1, 5, 10, 50 or 100 mg/l, while not being related to growth in the presence of 2,6-dichlorobenzamide at concentrations below said threshold value. According to at present most detailed description of the invention, said threshold value is from 0.1 - 1 mg/l.

Preferably, the said one or more microbial organisms belongs to the genera Aminobacter, Pseudomonas, Rhizobium, Rhodococcus, Sphingomonas and Variovorax.

More specifically, the said one or more microbial organisms are selected from the group consisting of Rhizobium radiobacter, Rhodoccocus erythropolis, Pseudomonas flourescens, Aminobacter aganoensis, Aminobacter niigataensis and Pseudomonas putida.

For many of the embodiments relating to the commercial exploitation of the enzyme, enzyme activity and bacterial species according to the present invention it is preferred that a pure culture of the bacterial species is established. According to a presently most preferred embodiment of the invention, the enzyme or enzyme activity is therefore obtainable from a bacterial pure culture which is isolated by a process comprising obtaining or establishing a bacterial enrichment culture from soil collected from one or more locations where a herbicide comprising Chlorthiamide (2,6- dichlorobenzenecarbothioamide) and/or Dichlobenil (2,6-dichlorobenzonitrile) as an active ingredient, have been used. Considerations regarding the amounts of chlorthiamide and dichlobenil used and the resulting amounts of dichlobenil and 2,6-dichlorobenzamide.

The bacterial pure culture may be obtained from soil collected from a depth below ground level that is within the range of 0 - 2.5 meters, such as at a depth that is within the range of 0 - 5 centimeters, 0 to 10 centimeters, 0 to 15 centimeters, 0 to 20 centimeters, 0 to 25 centimeters, 0 to 30 centimeters, 0 to 40 centimeters, 0 to 50 centimeters, 0 to 60 centimeters, 0 to 70 centimeters, 0 to 80 centimeters, or 0 to 90 centimeters. In particular, the soil may be collected from a depth of 10 - 20, 10 - 30, 10 - 50, 30 - 50, 40 - 60, 50 - 80, 70 - 80, 80 - 100, 100 - 120, 120 - 140, 140 - 160, 160 - 180, 180 - 200, and 200 - 250 cm below ground level. The process by which the enzyme or enzyme activity according to the invention is obtainable, may comprise the steps of a. establishing a bacterial culture from the soil; b. transferring a volume from the bacterial culture to fresh culture medium in order to obtain an enrichment culture; c. transferring a volume from the resulting enrichment culture to an agar plate, and d. screening colonies on the plate for their ability to convert 2,6-dichlorobenzamide.

In order to obtain a pure culture it may be preferred to conduct a number of serial transfers or dilutions of the enrichment culture in fresh culture medium. For each step of dilution a bacterial culture aliquot of is transferred to fresh culture medium and cultured preferably until entering the non-exponential growth phase before an aliquot of the resulting culture is collected for further dilution. Accordingly, the process may further comprise one or more successive steps of transferring a volume from the enrichment culture obtained in step b to fresh culture medium prior to transferring the bacterial culture to agar plates. In a preferred embodiment, 5 to 10 successive steps of transfer/dilution are carried out.

According to a preferred embodiment of the invention, it is contemplated that the enzyme or enzyme activity according to the invention is obtainable by culturing a bacterial strain of the α-proteobacter class and further that the enzyme or enzyme activity is obtainable by culturing a bacterial strain of the Aminobacter genus or, alternatively phrased, that the enzyme or enzyme activity is present in bacteria from said genus and/or class.

More specifically, it is contemplated that enzyme or enzyme activity is obtainable by culturing a bacterial strain of the Aminobacter aganoensis and/or the Aminobacter niigataensiε species or that it is present in said strains.

According to a preferred embodiment of the present invention the enzyme or enzyme activity further has the ability to degrade 2,6-dichlorobenzonitrile, when present in an environmental sample. The enzyme or enzyme activity may lead to degradation of 2,6- dichlorobenzonitrile in amounts corresponding to at least 10% of the amount present in said environmental sample, such as at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 54, 56, 58, 60, 65, 70, 75, 80, 85, 90, 95 or 100% over a time period of from 1 to 30 days, such as from 1 to 5, 2 to 10, 5 to 25, 10 to 30 or 20 to 30 days and with 2,6-dichlorobenzonitrile being present in concentrations of from 0.1 ng/l to 500 mg/l such as from 1 ng/l to 50 mg/l, from 0.01 μg/l to 10 mg/l, from 0,lμg/l to 1 mg/l, from below 1 μg/l to 100 μg/l, and from 2 μg/l to 50 μg/l. Finally, the enzyme or enzyme activity may be obtainable by culturing a bacterial strain or a bacterial species, Aminobacter aganoensis/Aminobacter niigataensis ASI-I, which is deposited according to the Budapest Treaty on 7 July 2005 under the accession number DSM 17439 with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg Ib, 38124 Braunschweig, Germany

Another main aspect of the invention is a microbial species, which is capable of exerting a 2,6-dichlorobenzamide degrading or converting activity as described above. Also in preferred embodiments of this aspect of the invention the microbial species is a bacterial species or a fungal species.

In further specific embodiments the microbial species of the invention belong to any of the genera Pseudomonas, Rhizobium, Rhodococcus, Sphingomonas, Aminobacter and Variovorax. In still more specific embodiments of the invention the microbial species are selected from the group consisting of Aminobacter aganoensis, Aminobacter niigataensis, Rhizobium radiobacter, Rhodoccocus erythropolis, Pseudomonas flourescens and Pseudomonas putida.

A preferred embodiment of this aspect of the invention pertains to a bacterial strain or a microbial species as described above, which also has the ability to degrade one or more compounds selected from the group consisting of: DCBA (2,6-dichlorobenzoic acid), ortho- chlorobenzonitrile, O-BAM (Ortfto-chlorobenzamide), ortho-chlorobenzoic acid, benzonitrile, benzamide and benzoic acid.

The currently most preferred embodiment provides a bacterial strain or a bacterial species, Aminobacter aganoensis/Aminobacter niigataensis ASI-I, which is deposited according to the Budapest Treaty on 7 July 2005 under the accession number DSM 17439 with DSMZ- Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg Ib, 38124 Braunschweig, Germany

A further preferred embodiment of the invention provides a bacterial strain as described above, which when present in an environmental sample, also has the ability to degrade 2,6-dichlorobenzonitrile in amounts corresponding to at least 10% of the amount present in said environmental sample over a time period of from 1 to 30 days and with 2,6- dichlorobenzonitrile being present in concentrations of from 0.001 μg/l to 500 mg/l.

Another prominent aspect of the invention provides a composition comprising an activity according to the invention. The composition may further comprise agents or substances that have a positive effect on the conversion or degradation of 2,6-dichlorobenzamide to its desired breakdown products in the environment being in contact with the composition. Non-limiting examples of such agents or substances are agents capable of stabilising the pH of the environment, such as buffering agents, acid agents or alkaline agents. In addition relevant substances may be antibiotics such as nystatin and cycloheximide. Substances may be compositions of nutrients such as complete or incomplete nutrient rich media such as the Mineral Salt Medium used in the present invention.

The composition may comprise one ore more added sources of nutrients such as easily accessible nutrients. In particular the composition may comprise alternative primary nutrient sources to 2,6-dichlorobenzamide, such as alternative carbon and/or nitrogen sources. In a particular embodiment the composition thus further comprise one or more nitrogen sources other than 2,6-dichlorobenzamide, and/or one or more carbon sources other than 2,6-dichlorobenzamide.

The composition may comprise the activity in isolated or partly isolated form, such as in the form of a culture of a microbial organism and in particular in the form of a pure and/or homogenous culture of a microbial organism. Alternatively, the composition may comprise the activity in the form of one or more isolated and purified or partly isolated and purified molecular species, such as one or more enzymes. However, in preferred embodiments, said composition further comprises one or more microbial species as defined above.

Potentially, a composition as described in the preceding paragraphs can be used in bioremediation, such as bioremediation of contaminated soil, sediment or aquifer such as a source of ground water.

The term "Bioremediation" refers to the use of biological organisms such as plants or microbes to aid in removing hazardous substances from an area. In particular, it refers to

the treatment of pollutants or waste as for instance in an chemical spill, contaminated soil, sediment or aquifer, or industrial process by the use of micro-organisms such as bacteria that are capable of breaking down the undesirable substances. Bioremediation is the environmental cleanup by biological agents comprising the use of biological means to restore or clean up contaminated land or water, for example, by adding bacteria and/or other organisms that consume or neutralize contaminants in the soil, sediment or water.

Bioremediation has previously proved to be a very useful technique in the handling of for instance oil spills, and it is contemplated that bioremediation of the accumulation of 2,6- dichlorobenzamide may be a technically and economically superior alternative to for instance cleaning of drinking water by the use of active carbon filters. Bioremediation using the composition of the present invention may be performed at the very locations that are polluted with the precursors of 2,6-dichlorobenzamide before the pollution spreads to for instance sources of drinking water. Therefore, using a composition of the present invention in the correction of accumulated 2,6-dichlorobenzamide may also be preferred for political reasons. In particular this may apply if it has been decided that drinking water may not be purified to achieve the desired quality.

In accordance with the above-described utility of the composition according to the invention, another aspect of the invention pertains to the use of said composition according for reducing the amounts of 2,6-dichlorobenzamide and/or the amounts of 2,6- dichlorobenzonitrile at a location or in an environmental sample derived therefrom. It will appear that bioremediation of 2,6-dichlorobenzamide or 2,6-dichlorobenzonitrile pollution using the composition of the invention may be performed alone or in combination with other means for reducing the levels of 2,6-dichlorobenzamide and/or the levels of 2,6- dichlorobenzonitrile.

Monitoring the conversion or degradation of 2,6-dichlorobenzamide and/or the degradatioin of 2,6-dichlorobenzonitrile as provided for above may be incorporated as a step in the bioremediation process. Accordingly, one embodiment of the invention provides a method for reducing the amount of 2,6-dichlorobenzamide and/or the amounts of 2,6- dichlorobenzonitrile at a given location, said method comprising providing to said location an amount of a composition or a bacterial strain as described above.

In a preferred embodiment the method further comprises a step of determining the amounts of 2,6-dichlorobenzamide and/or the amounts of 2,6-dichlorobenzonitrile converted or the rate at which 2,6-dichlorobenzamide is converted.

Techniques for measuring the amounts of 2,6-dichlorobenzamide and 2,6- dichlorobenzonitrile may as described above rely on the use of non-radioactive and radioactive labelling techniques and in particular on the use of 14C-labelled 2,6- dichlorobenzamide and measurements of the amounts Of CO2 produced.

Again, it may be preferred that the composition or the bacterial strain is applied to filtering equipment, including the equipment which is already in place at waterworks or water processing facilities. Accordingly, the composition or the bacterial strain may be applied to conventional sandfilters already used by many waterworks or, alternatively, biofilters may be established on the basis of the composition or the bacterial strain. An important aspect of the invention pertains to the use of an enzyme activity, an enzyme and/or an enzyme complex derived from an environmental sample, which has been exposed to a herbicide and/or a pesticide and/or products from the degradation or conversion of a herbicide and/or a pesticide, in environmental remediation. It is implicated that any or all of the above-described characteristics may apply to the enzyme activity according to this aspect of the invention. In particular embodiments of this aspect of the invention, the enzyme activity, an enzyme and/or an enzyme complex is produced recombinantly using standard techniques known in the art. In a preferred embodiment of this aspect of the invention the enzyme activity, an enzyme and/or an enzyme complex is capable of degrading and/or converting 2,6-dichlorobenzamide and/or 2,6- dichlorobenzonitrile.

The invention further pertains to the use of a composition or a bacterial strain as described above for reducing the amounts of 2,6-dichlorobenzamide and/or the amount of 2,6- dichlorobenzonitrile at a location or in an environmental sample derived there from. It may be preferred for certain applications to rely on a bacterial strain or a composition, which has the ability to convert 2,6-dichlorobenzonitrile in addition to the ability to convert 2,6- dichlorobenzamide. This is the case, for instance, when aiming at reducing the levels of 2,6-dichlorobenzamide in soil exposed to Casoron G or Prefix G, since the soil will be expected to also contain considerable amounts of 2,6-dichlorobenzonitrile.

When using the term "location" in the context of the present invention, it is to be understood that this term may refer to any environment which is or has been exposed to 2,6 - Dichlorobenzenecarbothioamide and/or 2,6-dichlorobenzonitrile and any environment in which 2,6-dichlorobenzamide may be present in undesirable amounts. Thus, a location may for example be a landfill, a plant nursery, a railway bed, an agricultural field, a private garden and a public park as well as soil taken from such locations. A location may also be an aquifer, such as a lake or a fresh water reservoir or water taken from such sources as well as the term will cover a well and well water, including well water or surface water intended for drinking.

The modes by which the composition and the bacterial strain according to the present invention may be applied are numerous. When aiming at reducing the levels of 2,6- dichlorobenzamide and 2,6-dichlorobenzonitrile in soil or sediment at a given location the composition may be applied directly to the soil or sediment. When used in the treatment of water such as well water or surface water from a reservoir the composition or said bacterial strain is applied to filtering equipment at a waterworks or a water processing facility. The term "environmental remediation" refers to removing hazardous substances from an area. In particular, it refers to the treatment of pollutants or waste as for instance in an chemical spill, contaminated soil, sediment or aquifer, or industrial process by the use of micro-organisms such as bacteria that are capable of breaking down the undesirable substances.

Another main aspect of the invention pertains to a method for evaluating the persistence of 2,6-dichlorobenzamide and/or 2,6-dichlorobenzonitrile present at a given location. The method comprises determining any presence of an activity or a bacterial strain or species as specified above according to any of on said location or in a sample derived therefrom. Any of the above-mentioned specifications may apply to the sample.

In particular, the method may involve the determination of the presence of said activity via an approach, which comprises the steps of (a) determining the rate at which 2,6-dichlorobenzamide and/or 2,6- dichlorobenzonitrile is converted, and/or (b) determining the amounts of 2,6-dichlorobenzamide and/or 2,6- dichlorobenzonitrile which are converted, and/or (b) determining a change in the number of micro-organisms present in response to addition of 2,6-dichlorobenzamide and/or addition of 2,6-dichlorobenzonitrile, and/or (c) determining a change in the diversity of micro-organisms present in response to addition of 2,6-dichlorobenzamide and/or 2,6-dichlorobenzonitrile.

Various approaches for determining the amounts of 2,6-dichlorobenzamide, which are converted or degraded, may be contemplated. Such methods may rely on means for detection, which involve radioactive and/or nonradioactive techniques. The methods may further involve measuring the amounts of 2,6-dichlorobenzamide and/or any of the molecular species resulting from the conversion or degradation of 2,6-dichlorobenzamide. In a preferred embodiment of the invention the determination of the presence of the activity according to the invention comprises a determination of the amounts of CO2 produced by the conversion of 2,6-dichlorobenzamide and/or the rate at which said CO2 is produced.

Another important aspect of the invention pertains to a method of obtaining and/or increasing an activity according to the invention, said method comprising the step of obtaining an environmental sample as described above, and one or more steps of: (a) adding to said environmental sample an amount of one or more compounds selected from the group consisting of chlorthiamide, dichlobeinil and 2,6- dichlorobenzamide, and/or (b) adding to said environmental sample one or more nitrogen sources other than 2,6-dichlorobenzamide, and/or 2,6-dichlorobenzonitrile, (c) adding to said environmental sample one or more carbon sources other than 2,6-dichlorobenzamide, and/or 2,6-dichlorobenzonitrile, (d) isolating one or more microbial species from said environmental sample, and/or (e) culturing the microbial species from step (d), and/or (f) isolating one or more molecular species originating from said environmental sample or from microbial species isolated there from.

Broadly speaking, the method comprises one or more steps of generating a selection pressure by repeated treatment of an environmental sample with a given amount or concentration of 2,6-dichlorobenzamide and/or 2,6-dichlorobenzonitrile.

It is contemplated that, when seeking to obtain or increase an activity according to the invention, one may aim at providing condition which are believed to favor the conversion or degradation of 2,6-dichlorobenzamide and/or 2,6-dichlorobenzonitrile. Therefore it is to be understood that in a preferred embodiment the method of obtaining and/or increasing an activity according to the invention may involve subjecting the environmental sample to any or all of the conditions described above.

A related aspect of the invention pertains to a product, which is obtained by the above described method of obtaining and/or increasing an activity according to the invention, or a product, which is obtainable by the described method. Other related aspects of the invention pertain to products in general, which are obtained or obtainable by the method of obtaining and/or increasing an activity according to the invention, such as compositions comprising the activity according to the invention. In particular, a product, which is obtained or obtainable by the described method, may comprise a pure and/or homogenous culture of a microbial organism in particular a bacteria, which is capable of degrading, converting and/or mineralizing 2,6-dichlorobenzamide and/or 2,6-dichlorobenzonitrile.

For all aspects of the invention and in particular for the aspects of the invention pertaining to a method for evaluating the persistence of 2,6-dichlorobenzamide and/or 2,6- dichlorobenzonitrile present at a given location and to a method of obtaining and/or increasing an activity it is further understood that in terms of size and/or composition the sample may have any of the above mentioned characteristics. In particular, a certain amount of soil or sediment may be used ranging for instance from 0.1 gram to 100 grams, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 grams. The sample may be supplemented with any given amount of nutrient containing medium, ranging for instance from 0.5 to 500 ml, such as 5 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 and 500 ml. The set-up or system may rely on the use of any open or closed or sealed type of container or bottle having a volume ranging from for instance 2 to 2000 ml, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 10 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 ml. In a preferred embodiment the activity according to the invention is obtained and/or increased in an environmental sample which consists of 1 g soil, is supplemented with 5 ml Mineral Salt Medium and is contained within a sealed container having a volume of 20 ml.

15 A presently preferred embodiment is a set-up as illustrated in figure 2 comprising a sealed glass bottle in which a glass vial containing an alkaline solution has been inserted together with the sample.

Approaches for determining the presence of an activity according to the invention and for 20 characterizing said activity logically involve the use of an experimental set-up or system of specific characteristics. A particular amount of soil or sediment may be used, ranging for instance from 0.1 gram to 100 grams, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 grams. Again, it is to be understood that when the activity is present in the form of culture of micro-organisms such 25 as a homogenous culture of micro-organisms or in the form of one or more isolated and/or purified or partly purified enzymes the set-up or system this setup or system may not contain any soil or sediment or it may not contain any substantial amounts of soil or sediment. The sample may be supplemented with any given amount of nutrient containing medium, ranging for instance from 0.5 to 500 ml, such as 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 30 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 and 500 ml. The set-up or system may rely on the use of any open or closed type of container or bottle having a volume ranging from for instance 2 to 2000 ml, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 35 900, 1000, 1500, or 2000 ml. In a preferred embodiment of the invention the activity according to the invention and the conversion of 2,6-dichlorobenzamide occurs in an environmental sample which consists of 1 g soil, is supplemented with 5 ml Mineral Salt Medium and is contained within a sealed container having a volume of 20 ml. As the sample size is not an absolutely critical factor influencing the activity according to the invention the sample may also comprise 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 Mineral Salt Medium or more.

With respect to the above description of the various aspects of the present invention and of the specific embodiments of these aspects it should be understood that any feature and characteristic described or mentioned above in connection with one aspect and/or one embodiment of an aspect of the invention also apply by analogy to any or all other aspects and/or embodiments of the invention described.

When, in the present application, an object according to the present invention or one of its features or characteristics is referred to in singular this also refers to the object or its features or characteristics in plural. As an example, when referring to "a microbial species" it is to be understood as referring to one or more microbial species.

Throughout the present specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The following examples are included to demonstrate particular embodiments of the inven¬ tion. However, those of skill in the art should, in view of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. The following examples are offered by way of illustration and are not intended to limit the invention in any way. The invention will now be described in further details in the following non-limiting examples and figures 1 to

FIGURE LEGENDS

Figure 1: Proposed degradation scheme for dichlobenil.

Figure 2: Outline of experimental set-up for determining the conversion or degradation of of 2,6-dichlorobenzamide.

Figure 3: A) Mineralization of 2,6-dichlorobenzamide in a sample taken from a former plant nursery. B) Mineralization of 2,6-dichlorobenzamide in a sample taken from a former plant nursery after re-addition of 2,6-dichlorobenzamide. Figure 4: Degradation of BAM (A) and accumulation of degradation products (B) in soil from Hvidovre. BAM (D); BAM in sterile soil (■); O-BAM (O); DCBA ( - ).

Figure 5: A) Graphical representation of the mineralization of 2,6-dichlorobenzamide in samples that were tested positive in the initial screening of soil samples. B and C) triple determinations of mineralization of 2,6-dichlorobenzamide in samples from two of the locations tasting positive in the experiment depicted in panel A).

Figure 6: A) Relative amounts of 2,6-dichlorobenzamide mineralized over time in samples taken from specified depths at specified locations. B) Triplicate determinations of the relative amounts of 2,6-dichlorobenzamide mineralized over time in samples taken from a depth of 10 - 30 cm at a specified location.

Figure 7: Depth profile of the mineralization of 2,6-dichlorobenzamide at a single location.

Figure 8: A and B) Heterogeneity study showing variations in the mineralization of 2,6- dichlorobenzamide in multiple samples taken from the same locations.

Figure 9: Relative amounts of BAM mineralised over time at different concentrations of added BAM. Numbers in the figure legends refer to the concentration of unlabelled BAM present in the slurries and numbers in brackets refer to the experiment number. To all slurries 0.008 mg/L 14C-labelled BAM has been added. Each point in the graph represents the average of three single determinations and deviations from the mean are calculated as standard error.

Figure 10. The graph shows the relative amounts of BAM being mineralised over time after addition of various amounts of carbon. The values of the legend refer to the fold increase in the carbon levels as compared to the amounts of carbon added in the form of BAM. Numbers in brackets refer to the experiment number. Each data point represents the mean of three single determinations from which the deviation is determined as standard error.

Figure 11. The graph indicates the relative amounts of BAM being mineralised over time after addition of various amounts of nitrogen. The values of the legend refer to the fold increase in the nitrogen levels as compared to the amounts of nitrogen added in the form of BAM. Numbers in brackets refer to the experiment number. Each data point represents the mean of three single determinations from which the deviation is determined as standard error. Figure 12. The graph shows the relative amounts of BAM being mineralised over time at different temperatures. Each data point represents the mean of three single determinations from which the deviation is determined as standard error. •

Figure 13. The figure illustrates the relative amount of BAM being mineralised at different pH over time. Values of the legends refer to the pH of the medium immediately prior to addition of the soil. Each data point represents the mean of triplicate determinations and the deviation from the mean is calculated as standard error.

Figure 14. The graph illustrates the mineralization of 2,6-dichlorobenzamide in the presence of antibiotics. Each data point represents the mean of triplicate determinations and the deviation from the mean is calculated as standard error.

Figures 15 to 19. The graphs all illustrate the mineralization of 2,6-dichlorobenzamide in enrichment cultures.

Figure 20. Mineralisation of BAM by Aminobacter strain ASI-I in MS medium. Each curve show mineralisation of BAM following transfer of the culture to a fresh BAM-containing medium.

Figure 21. Growth of Aminobacter strain ASI-I in a complex medium (LB). From the curve a doubling time of 11 h was calculated.

Figure 22. Mineralisation of high BAM concentrations by Aminobacter strain ASI-I. BAM was added the MS medium in concentrations of 1000 mg/l (■ ) 500 mg/l ( -*■ ) 100 mg/l (D) and 50 mg/l (Δ).

Figure 23. Mineralisation of low BAM concentrations by Aminobacter strain ASI-I. BAM was added the MS medium in concentrations of 5 mg/l (■ ) 2.5 mg/l ( ^ ) I mg/l (D), 0.1 mg/l (Δ) and 0.01 mg/l (•).

Figure 24. Degradation of dichlobenil (DCB) and BAM by Aminobacter sp. ASIl and a mixed enrichment culture, culture 2, from Hvidovre soil. The open symbols indicate DCB (0), BAM (D) and 2,6-dichlorobenzoic acid (Δ) whereas the corresponding closed symbols are sterile controls. Data points represent the mean of triplicate measurements and error bars indicate standard error. Figure 25. Mineralisation of 50 mg I"1 dichlobenil (A) and BAM (B) by MSHl obtained from the mixed enrichment culture, culture 2, by plating on R2A agar plates. The curves represent duplicate flasks.

5 EXAMPLES

Example 1: Mineralization of dichlobenil and BAM in soil

Chemicals

Analytical-grade 2,6-dichlorobenzonitrile (Dichlobenil, 99.5% purity, 18.0 mg I"1 solubility 10 at 200C) and 2,6-dichlorobenzamide (BAM, 99.5% purity, 2.7 g I"1 solubility at 23°C) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Dichlobenil and BAM were added to the microbiological experiments from stock solutions of 5 g I"1 dissolved in dimethylsulfoxide (DMSO; C2H6OS, Merck, Darmstadt, Germany). 14C-ring-labelled dichlobenil (162.5 mCi g"1, >95% chemical and radiochemical purity) and BAM (25.2 mCi 15 mmol"1, >95% chemical and radiochemical purity) was purchased from Izotop, Institute of Isotopes, Budapest, Hungary. 14C-labelled NaHCO3" was purchased from Amersham Pharmacia Biotech UK Limited, Buckinghamshire, UK (628 μCi mg"1).

MS medium

20 A mineral salt medium (MS) used for soil slurries was modified from the earlier described HCMM2 medium by excluding KNO3 and (NH4)SO4 and adding 1.0 ml of filter-sterilized FeCI3 • 6H2O solution (5.14 mg I"1) after the medium was autoclaved [12-14].

MS medium without Nitrogen: 25 Buffer solution 68.0 g KH2PO4 1.36 g/l 89.0 g Na2HPO4.2H2O 1.78 g/l 500 ml MiIIiQ water 30 Nutrient solution

2.5 g MgSO4.7H2O 0.05 g/l 0.66 g CaCI2.2H2O 0.0132 g/l 35 500 ml MiIIiQ water Trace element solution 2.5 g H3BO3 2.86 mg/l 0.77 g MnSO4-H2O 1.54 mg/l 0.0195 g CuSO4.5H2O 0.039 mg/l 0.0105 g ZnCI2 0.021 mg/l 0.0205 g CoCI2.6H2O 0.041 mg/l 0.0125 g Na2MoO4.2H2O 0.025 mg/l 500 ml MiIhQ water

Iron solution 0.514 g FeCI3.6H2O 5.14 mg/l 100 ml MiIhQ water

Preparation of MS medium

10 ml buffer solution 10 ml nutrient solution 1 ml trace element solution MIIIIQ water ad 1000 ml pH adjusted to 7.5 (1.0 NaOH) Solution is autoclaved at 121°C, 20 mm.

Soils

Different dichlobenil-contaminated soils from different parts of Denmark were tested. As an initial screening 77 soil samples (in triplicates) taken at 38 locations from 21 different geographical areas were tested. On 22 locations samples of topsoil (0-30 cm) were taken only whereas samples of soil and sediment from deeper layers (30-400 cm) were taken on the other 16 locations. The soils were not homogenized before the initial screening of all soil samples. In contrast, soils with an initial mineralization potential were homogenized using a 2 mm mesh-size sieve and the potential was investigated further.

To study the heterogeneity of the mineralization potential 10 soil samples were taken in a 2 m wide grid at two locations, Hvidovre courtyard and the Stadion path in Marstal, respectively. These soil samples were homogenized using a 4 mm mesh-size sieve and the samples were shaken for two hours in sterilized glass jars. Mineralization experiments

Mineralization of dichlobenil and BAM was tested adding approximately 10,000 - 20,000 disintegrations per minute (dpm) of 14C-ring-labelled dichlobenil or BAM in addition to 50 mg kg"1 or mg I"1 of unlabelled compound. Both samples of 10 g soil without any MS medium and soil slurries where 25 ml of the MS medium was added to 5 g of soil were tested. Triplicate 100 ml or 20 ml sterilized flasks with airtight lids were incubated at 200C in the dark together with sterile controls added 500 μl 0.5% azide (NaN3). The evolved 14CO2 from dichlobenil or BAM in 100 ml flasks was trapped in a 5 ml test tube holding 2.2 and 2.0 ml of 0.5 M NaOH, respectively, mounted in the flasks. When experiments were run with 20 ml flasks the 14CO2 from BAM was trapped in 2 ml test tubes holding 1 ml of 0.5 M NaOH. Upon sampling, the alkaline solution was replaced with fresh solution at different time intervals. For BAM the entire used solution was mixed with 10 ml of either Ultima Gold™ XR scintillation fluid (Packard Bioscience, Groningen, the Netherlands) or OptiPhase 'HiSafe' 3 (Perkin Elmer Life Sciences, Wallac, Turku, Finland) and counted on a Wallac 1409 liquid scintillation counter. For dichlobenil only 1.0 ml was transferred directly to 10 ml of the scintillation fluid. In order to correct for the evaporation of dichlobenil, we added 1.5 ml of 1.0 M HCI to other 1.0 ml of the alkaline solution resulting in an evaporation of any CO2 present. The solutions were mixed and left for 1 hour before adding scintillation liquid and subsequent counting. As a positive control of this method we included samples with 14C-labelled HCO3". The results were all corrected for quenching and background radioactivity.

High Pressure Liquid chromatography (HPLC) Quantification of dichlobenil, BAM, DCBA, ortho-chlorobenzonitrile, O-BAM, ortho- chlorobenzoic acid, benzonitrile, benzamide and benzoic acid was done using a Hewlett- Packard Series 1050 HPLC System equipped with a UV detector (Phenomenex, Cheshire, UK). Analytes were separated on a gradient using a Waters Xterra® RP18 column (2.1 x 100 mm, 3.5 μm particle size, Milford, MA, USA). The mobile phase was acetonitrile + phosphate buffer (0.7898 g/L Na2HPO4 adjusted to pH 2.38 with HCI). The gradient was initiated with 12% acetonitrile (0-6.5 min) and risen to 65% (6.5-9 min) where after this level was kept till the end of the run (9-14.5 min) followed by a post time of 10 min. The flow rate was 0.300 ml min"1, the injected volume was 10 μl and the temperature of the column was 400C. Seven of the compounds were quantified at 203 nm whereas benzamide and benzonitrile were quantified at 223 nm Results: Mineralization of dichlobenil and BAM in soil

Mineralization of BAM and dichlobenil in a soil from Hvidovre

In soil from a Court Yard of a former plant nursery in Hvidovre (Copenhagen, Denmark) where pesticides containing dichlobenil has been used extensively 60% of the added 14C- BAM was mineralized after 30 days in both soil and soil slurries (Fig. 3A). BAM was used for growth since the mineralization occurred even more rapid and with no lag-phase when BAM was re-added to the slurries with soil from Hvidovre resulting in that 60% of the added BAM was mineralized in only 14 days (Fig. 3B). Furthermore, no mineralization was seen in the sterile controls with sodium azide indicating that the mineralization was a microbiologically mediated process (Fig. 3B). The fact that a lag phase is absent after re- addition of BAM indicates that the number of microorganisms has increased and that the mineralisation of BAM occurs as a part of the metabolic or co-metabolic processes in the microorganisms

HPLC-analysis showed that BAM was removed completely from the soil (Fig. 4A). During the degradation a temporary accumulation of 2,6 dichlorobenzoic acid (DCBA) and ortho- chlorobenzamide (O-BAM) was seen (Fig. 4B).

It has also been shown that dichlobenil is mineralized in the soil from Hvidovre as well (45% mineralization in 70 days, data not shown). During dichlobenil degradation temporary accumulation of both BAM and O-BAM was seen. Based on these results we propose that dichlobenil is degraded to BAM which is either hydrolyzed further to DCBA or dechloπnated to O-BAM. O-BAM may constitute a hazard to public health as it is potentially carcinogenic (Guoguang et al., 2001 [I]).

The initial screening of 77 soil samples

A rapid screening (without homogenizing the soil) of 77 different soil samples from 38 different locations situated in 21 areas showed that BAM was mineralized in six soil samples, namely: Stadion Path Marstal (walking path close to Vestergade and Ellenet, DK- 5960 Marstal, Denmark) 0-30 cm, Tennis Court Marstal (tennis court close to Vestergade and Ellenet, DK-5960 Marstal, Denmark) 10-30 cm, Tennis Court Marstal 30-50 cm, Trench Hvidovre (Brostykkevej 182-212, DK-2650 Hvidovre, Denmark) 10-20 cm, Private Dump Site Sengeløse (Ingersvej 4, DK-2640 Hedehusene, Denmark) 0-20 cm and Court Yard Hedehusene (Solhøjvej 44, DK-2640 Hedehusene, Denmark) 0-20 cm (Fig. 5). Hence, in total a potential for BAM mineralization was seen in six soil samples representing five locations and four areas, All though a rapid mineralization, as observed in the soil sample Court yard Hvidovre mentioned above, was seen in the soil sample Stadium Path Marstal 0-30 cm only.

The four soils with the highest mineralization potential found during the initial screening experiment were further homogenized by sieving the soil samples through a 2 mm mesh- size sieve and a new mineralization experiment adding BAM was set up. The soil sample "Court Yard Hvidovre" was included. This experiment showed that the mineralization potential was very similar when soil from the stadium path (Marstal) and the court yard (Hvidovre) was used (Fig. 6A); 50-60% of the added BAM was mineralized to CO2 in 45-55 days. However, no accumulation of either O-BAM or DCBA was seen in the soil from Marstal as it was seen for the Hvidovre soil (Fig 4). The kinetics of the mineralization in both the soil from Hvidovre and Marstal followed a sigmoid curve (S-shape) indicating that the microbial degradation of BAM was growth-related. In contrast, no mineralization was seen in the other four soils included (Fig. 6A). This could be due to a larger heterogeneity of the mineralization potential in these two soils which is indicated by a low degree of reproducibility (large deviations) as shown in Fig. 5. The large deviations in Fig. 6B for the soils Trench Hvidovre and Private Dump Site Sengeløse are due to only one of three replicates being able to mineralize BAM. Hence, by chance it should be possible to obtain three replicates without a mineralization potential.

Furthermore, soil samples from deeper layers from the Stadium path (Marstal) were screened for their mineralization potential. As seen on Fig. 7 mineralization occurred in soil samples from the 0-30 and 70-200 cm depths. No mineralization was seen in soil samples from 30-70 cm and 200-280 cm depths.

Heterogeneity study of soil samples from Hvidovre and Marstal The on-location spatial heterogeneity of the mineralization potential was tested with soil samples from the two locations with the growth-related mineralization, namely: Stadium Path Marstal and Court Yard Hvidovre. This showed that the potential was heterogeneous since the mineralization in the 10 soil samples from each of the two locations followed different mineralization patterns (Fig. 8): The soil samples will be characterized using the following parameters: pH, conductivity, content of N, C, S and P, and concentration of BAM. Correlation will be made with these parameters and the mineralization potential.

Example 2: Characterization of BAM mineralizing populations and parameters influencing mineralization

Materials and methods Reference is made to the materials and method section of example 1 and furthermore the following materials and methods were used.

Parameters influencing the mineralization of BAM

The BAM mineralization was characterized in triplicate under 25 different sets of conditions. A number o f distinct parameters apply to each set of conditions as presented in table 1. For each triplicate determination of the BAM activity only one parameter is changed relative to standard c onditions. Since results from the experiments in example 1 showed that abiotic mineralization of BAM only contributes to a minor extent to the overall mineralization of BAM the following experiments do not include sterile controls.

Table 1: Variation of parameters in experiments for the characterization of the BAM mineralization.

The mineralization experiments were conducted in autoclaved 20 ml glass vials with 1.0 g soil added to each vial together with 5 ml of the mineral salt (MS) medium. Under standard conditions 50 mg I"1 BAM is added to the MS medium. In four of the experiments other amounts of BAM were added as indicated in table 1.

In experiments 9-11 and 12-14 different amounts of carbon (sodium succinate C4H4O4Na2-GH2O, Sigma-Aldrich ST Louis, MO, USA) and nitrogen (ammonium chloride, NH4CI, Merck, Darmstadt, Germany) was added. In experiment 15-18 the pH of the medium was adjusted to the specified pH using 1.0 mM HCI and 0.5 mM NaOH, respectively. The pH values are determined in the suspensions 24 hours after addition of soil to the medium. In experiments 19-21 and 22-25 antibiotics were added in amounts of 30 mg I"1 when the mineralization reached the exponential phase. The antibiotics used were penicillin (stock solution of 10 mg/l), ampicillin (stock solution of 10 mg/l), streptomycin (stock solution of 10 mg/l), tetracycline (stock solution of 5 mg/l), nystatin (stock solution of 50 mg/l), and cycloheximide (stock solution of 10 mg/l).

DNA extractions, PCR and DGGE

Extraction of DNA from soil.

The following is based on the manufacturers instructions for using the FastDNA Spin Kit for Soil (BiolOl, Vista, California, USA) but minor modifications have been made.

• An aliquot of 978 μl Sodium phosphate buffer is added to 0.50 grams of soil in a MULTIMEX 2 tissue vial and 122 μl MT buffer is added. • The sample is mixed by manual shaking and subsequently by shaking 4 times 30 seconds in a FastPrep machine at lowest speed. • The sample is centrifuged at 13.000 rpm for 1 min. • 700 μl supernatant is transferred to Eppendorf tubes and left at -8O0C for at least 1 hour and subsequently thawed at 300C for 30 minutes. • An aliquot of 200 μl PPS is added and the sample content is mixed by inversion of the sample • The sample is centrifuged 13,000 rpm for 5 mm. • 850 μl is transferred to a new Eppendorf tube and combined with 850 μl binding matrix. • Sample is mixed by inversion for 2 mm. • A 600 μl aliquot of the binding matrix/sample is transferred to a spin filter with Catch tube and centrifuged at 13,000 rpm for 1 mm. • The catch tube is emptied and centrifugation is repeated until all binding matrix/sample has been processed. • An aliquot of 500 μl is added and the sample is centrifuged at 13,000 rpm for 1 mm. • The catch tube is emptied and the centrifugation is repeated to dry the matrix. • The spin filter is further dried at room temperature. • An aliquot of 100 μl RNase/DNase free water is added for elution of the DNA. • The filter is centrifuged at 13,000 rpm for 1 mm. • The matrix is resuspended in the eluate and centrifugation is repeated.

Genetic diversity was determined after extraction of DNA and amplification of the bacterial 16S rDNA by PCR followed by analysis by Denaturing Gradient Gel Electrophoresis (DGGE).

DNA was extracted from different soil samples using FastDNA Spin Kit for Soil (BiolOl, Vista, CA, USA). The experimental procedure deviates from that described above when DNA is extracted from slurries as 1 ml slurry is used instead of the recommended 0.50 grams of soil combined with 987μl Na-phosphate buffer (steps 1 and 2). Furthermore the beat conditions in step 6 are in all instances changed to four cycles of 30 seconds at a speed of 4.0 in a Fastprep apparatus (PF120-Bιol01, Savant Instruments, NY, USA). Finally, after step 8 a step of freezing and thawing is added, the Eppendorf tubes being placed at -800C for 60 minutes and subsequently thawed for 30 minutes.

From the extracted DNA fragments of 16S rDNA were amplified using PCR techniques as follows. The amplification is performed in a volume of 25 μl containing 1 μl template, combined with autoclaved water and 1 μl each of the forward and reverse primers. The forward primer used contains a GC-πch sequence at the 5 'end preventing the complete melting of the PCR product when analyzed on the DGGE gel (Muyzer et al. 1993). Conditions for the amplification were: 94°C for 3 minutes followed by 35 cycles of 94°C for 30 seconds, 5°C for 30 seconds and 72°C for 1 minute. Subsequently 4 μl of the sample was loaded on an agarose gel using standard conditions (Ellmgsøe and Johnsen 2002). Using the DGGE approach RNA fragments of equal length but having different nucleic acid sequences can be separated. The separation is based on the lowering of the electrophoretic mobility of a partially melted RNA in a polyacrylamide gel as compared to a complete double helix. Minor differences in the 16S rDNA nucleic acid sequences affect the melting temperature leading to the separation of the DNAs as distinct bands on the gel (Muyzer et al. 1993). DGGE was performed using equipment from D-CODE™ System (BioRad). 15 μl of the PCR product was combined with 5 μl loading buffer and loaded on a 7.5% polyacrylamide gel with a denaturing gradient of 30 to 70%. Electrophoresis was performed at 600C and 70 V for 17 hours. Subsequently the gel was stained using SYBR Gold for one hour.

Results and discussion of characterizing parameters influencing the mineralizing potential

BAM concentration

From Fig. 9 it is evident that a mineralization has occurred over course of the experiment. It should be noted that rates of mineralization (the slopes of the curves) for the three lowest concentrations of added BAM (0, 0.01 and 0.1 mg/L) decreased at an early point during the experiment and the mineralization was very low. A significant mineralization was only seen when the three highest concentrations of BAM were added (0,1, 1 and 10 mg/l). The mineralization curves using these three concentrations showed a sigmoidal pattern.

A number of facts are of relevance considering that BAM acts as a nutrient for microbial organisms. When the concentration of a nutrient is high, the diffusion of the nutrient to the cell surface is also high. The high concentration results in plentiful supply of nutrient for maintaining basal metabolism as well as for processes leading to growth and cell division. Conversely, at lower nutrient concentrations there is a relatively larger proportion of nutrient available to the cell is used in the basal metabolism and a smaller proportion is used for growth. At still lower nutrient concentrations, no nutrients remain available for growth processes. For that reason, the latter nutrient concentrations can be seen as a threshold value. Above the threshold value the mineralization is growth related (sigmoid curve), while this is not the case below the threshold value where the time course of the mineralization will be represented by a linear curve (Alexander 1994).

From Fig. 9 it can be seen that the rate at which BAM is converted to CO2 is accelerating at the concentrations 0.1, 1 and 10 mg/L. The curve representing conversion at the 1 mg/L concentration, however, is linear all though the mineralization rate is very slow. Accordingly, the threshold value between growth related and non-growth related BAM mineralization must be assumed to lie in between 0.1 and 1 mg/L BAM. Previously, the threshold value between growth related and non-growth related conversion of other organic compounds has been found to be approximately 0.2 μg/L (Torang et al. 2003). Torang et al. also mentions that the change in kinetics for the biodegradation has only been investigated for a limited number of compounds for instance in ground water, sludge and water from lakes and rivers. In soil thresholds of 0.010-0.1 mg kg"1 have been found (Alexander 1994) meaning that the threshold assumed from the present study is realistic. Residual concentrations of BAM are typically below 0.1 mg/kg soil on locations where dichlobenil was previously used. On particular locations, however, residual concentrations of up to 1320 μg/kg soil have been found. In the soil layer extending to a depth of 1 meter the average concentration of dichlobenil is approximately 55 μg/kg soil. It is to be assumed that, over time, all the dichlobenil will be converted to BAM such that BAM is released in the top soil in rather small amounts. In the present experiments using crude soil slurries only minimal mineralization of BAM was determined at concentrations below 1 mg/L. It is therefore relevant to isolate the BAM converting activity and establish its capacity to mineralize low concentrations of BAM when present in the form of a developed product, such as a pure or stable microbial co-culture, consisting of more than one microbial species.

It should be noted, however, that even a non-growth related conversion of BAM is likely to have a highly significant effect on the release of BAM into, for instance, ground water, provided that BAM is present in the soil over a period long enough to allow its conversion. As for the soil from the specified location in Hvidovre the concentration of BAM equals 14 μg/kg soil. Considering the above-established kinetics of the conversion little of this BAM is likely to be converted. However, a significant conversion of BAM is likely to have taken place during the period when dichlobenil as a herbicide was still used at the location.

From investigating the raw data it can be concluded that the large standard errors can be ascribed to differences between the individual replicates. Hence, the experiment is performed in homogenized soil samples using 5 g soil to 25 ml MS medium in order to test whether the trends shown in this preliminary experiment are consistent and statistical significant. The same is valid for the following experiments where other parameters have been changed.

Addition of carbon- and nitrogen sources other than BAM

From Fig. 10 it appears that the most prominent and fastest BAM mineralization takes place after addition of an amount of a carbon source corresponding to 10 times the amount of carbon added in the form of BAM. The addition of 1OxC even results in a faster mineralization than seen in the control. When carbon is added in larger amounts (lOOxC and lOOOxC) no mineralization of BAM was seen.

From Fig. 11 it is evident that mineralization of BAM occurs at approximately the same rate in the control and with addition of an amount of nitrogen corresponding to 10 and 100 times the amount added in the form of BAM. When more nitrogen was added (100OxN) no mineralization was seen.

By adding a carbon or nitrogen source other than BAM the availability of nutrients is increased for microorganisms in the environment possibly leading to increased growth of microorganisms in general. When exposing microorganisms to very large concentrations of a normally favorable nutrient it often appears to have a toxic effect. One can thus argue that the BAM converting microorganisms have a carbon and nitrogen optimum at which the conversion of BAM occurs at the highest rate or that stimulation of the soil microorganisms in general leads to a faster mineralisation. The present analysis indicates that this optimum is approached by addition of an amount of carbon exceeding the amounts added in the form of BAM by 10 times. No such relationship could be found for nitrogen.

An important perspective emerging from these findings relates to the possibility to optimize the conditions when using the BAM converting activity in bioremediation. In related studies it has been shown that it is possible to lower the threshold value between growth related and non-growth related conversion of a substance by a marine bacteria leading to conversion of the substance at much lower concentrations (Alexander 1994).

pH

The pH value of the MS-medium was adjusted to values of 2, 4 or 6 using either hydrochloric acid (HCI) or sodium hydroxide (NaOH) before addition to the soil. In consideration of the buffer capacity of the soil the pH in the suspensions is measured 24 hours after addition of the soil to the MS-medium, giving an indication of the actual pH- value, whereto the microorganisms are exposed. From table 2 it is evident that the buffer capacity of the soil is capable of increasing the pH-values of the suspensions considerably. Conversely, the buffer capacity of the soil also decreases the pH-values when the medium is basic.

Fig. 13 shows that the greatest mineralization occurred when the pH of the medium was adjusted to 6, the second-best when adjustment to pH 4 was done whereas the third-best mineralization was seen in the control (pH 7) and pH 8 which were not significantly different from each other. At pH 2 no mineralisation was seen.

Table 2. The table shows the results of the pH measurements in experiments 15 to 18 and 22.

For any microorganism growth is possible within a given pH range, a well- defined optimum often being present within the range. The range typically spans 2 to 3 pH units. Most natural environments have a pH between 5 and 9, and organisms with a pH optimum within this range are the predominant. Only few organisms are able to grow at a pH lower than 2 or above 10 (Begon et al. 1996)

The results of the present experiment indicate that the pH optimum of the microorganisms in respect of the mineralization of BAM is around 6, indicating that a pH, which is well above or below this value, will inhibit the mineralization.

Antibiotics

In the present characterization of the BAM mineralizing activity experiments are included to determine whether the predominant mineralization of BAM can be ascribed to the activities of bacteria or fungi. As described in the materials and methods section, the antibiotics were added in amounts of 30 mg/L in experiments 19-21 and 23-25 (see table 1) when the mineralization was in the exponential phase. The following antibiotics were used:

Penicillin - predominantly inhibits Gram-positive bacteria Ampicillin - predominantly inhibits Gram-negative bacteria Streptomycin - predominantly inhibits Gram-negative bacteria and many mycobacteria Tetracydin - inhibits most Gram-positive and Gram-negative bacteria Nystatin - inhibits most fungi, except Oomycetes Cycloheximide - inhibits most fungi

Of these antibiotics Nystatin and Tetracydin led to a faster mineralization than in the control, whereas the remaining antibiotics led to a decreased mineralization (Fig. 14). Since these two antibiotics are known to mainly target fungi and not bacteria the results indicates that the mineralization of BAM is a bacterial mediated process. This is further indicated by the inhibition seen when the bactericides are added.

DGGE Preliminary results using DGGE showed that one specific band was enhanced in BAM mineralizing microcosms with the soil sample Stadion Path Marstal; no such band was seen with the soil sample Court Yard Hvidovre. Further experiments with DNA from more enriched microcosms are needed to confirm that this band represent the BAM-mineralizing bacteria (results not shown).

Example 3

Methods for Enrichment cultures using BAM and final isolation of bacterial pure cultures

References is made to the materials and method section of example 1 and furthermore the following materials and methods were used.

To obtain a pure bacterial culture able to mineralize BAM enrichment cultures using BAM were performed. The idea was that the only species showing significantly growth in the microcosms would be the BAM-mineralizing bacteria since BAM was the only source of C, N or both. By transferring a small volume of the microcosm to a large volume of fresh medium containing BAM all other species would finally, in theory, be diluted from the microcosm and a pure culture would be obtained. The transfers to new medium were done at the end of the exponential phase of the mineralization. 1 or 2.5 ml was transferred from soil slurries with BAM to fresh MS medium containing 50 mg I'1 BAM and approximately 10,000 dpm 14C BAM in a total volume of 25 ml. Different enrichment strategies were used, namely: MS medium containing no C or N, MS medium containing lg/l sodium succinate or MS containing 10% IM relative to the original recipe of the MS medium. Incubations were done at 2O0C. The mineralization was measured as in the soil experiments presented in example 1. When the mineralization again reached the end of the exponential phase another transfer was made. After 5-10 transfers in this manner the transfer was made as a dilution series from lO^-lO"6 of the original sample. The highest dilution that showed a mineralization potential was plated on different agar media and bacteria with different morphology were streaked on separate agar plates, grown in liquid cultures and tested for BAM mineralization as above. If the liquid culture was still able to mineralize BAM the culture was tested for purity. Finally, a pure bacterial culture able to mineralize BAM was obtained.

The bacterial pure culture was isolated from an enrichment culture established using top soil (0-10 cm) sampled at a walking path close to Vestergade and Ellenet, DK-5960 Marstal, Denmark. The enrichment culture was established using the MS medium containing no C or N. Following four transfers and serial dilutions as described above the culture was streaked on R2A agar plates. Colonies were removed from the plates and screened for their ability to mineralize BAM in pure culture. The BAM-mineralizing pure culture obtained was designated strain ASI-I. A similar procedure, but using topsoil from a courtyard of a plant nursery, Brostykkevej 182-212, DK-2650 Hvidovre, Denmark, led to the isolation of another strain able to mineralize both BAM and dichlobenil. This strain was designated strain MSH-I.

The purity of ASI-I was confirmed by analysis on a DGGE gel on which only a single band was found. Growth of ASI-I was tested in a complex medium (LB Broth, Miller, Difco) containing per liter: 10 g Bacto Tryptone, 5 g Bacto Yeast Extract and 10 g Sodium Chloride.

Results and discussion

Enrichment cultures The mineralization potential was still present after 5-7 successive transfers when transfers were made to MS medium containing C and for some instances when transfer was made to MS medium containing N or neither C nor N ((Fig. 15 - Fig. 19). Characterisation of ASI 1 ASI 1 was further characterised by Deutsche Sammlung Von Mikroorganismen und Zellkulturen, Braunschweig, Germany and shown to be a gram-negative non-spore- formmg rod with a width of 0.6 to 0.7 μm and a length of 1.5 to 3.0 μm. It is oxidase, catalase, aminopeptidase and urease positive It does not hydrolyse either gelatine or esculine. It is negative in test for denitπfication. It was able to grow on R2A, NA and LA agar and after 6-7 days on R2A at 200C it forms white colonies with a reddish colour at the centre. A whole-cell fatty acid profile revealed that the dominant fatty acids were 67.82 % 18: lw7c, 10.63 % 11 methyl 18: 1 w7c, 7.22 % 16.0 and 3.28 % 17:0 ISO. Based on the profile of the cellular fatty acids the strain was shown to be typical for the α- Proteobacteπa, to which the genus Aminobacter belongs. The partial sequencing of the 16SrDNA of the strain shows a similarity of 99.8 to the type strain of Aminobacter aganoensis and also to the typestrain of Aminobacter niigataensis. Further identification of the strain to one of those type strains was not possible.

Growth of Aminobacter strain ASI-I

The observation that Aminobacter strain ASI-I mineralises BAM added the medium as the sole source of carbon and energy shows that the bacterium use BAM for growth. Serial transfers of ASI-I to the same medium without losing the mineralising activity (figure 20) confirmed this hypothesis.

HPLC analysis showed no accumulation of any of the tested degradation products (DCBA, ortho-chlorobenzonitrile, O-BAM, ortho-chlorobenzoic acid, benzonitπle, benzamide and benzoic acid) during the mineralisation of BAM. Transient accumulation of Ortho- chlorobenzamide, and dichlorobenzoic acid has been observed in soils added dichlobenil, but not in the soil from which strain ASI-I was isolated (Holtze et al., 2005 [2]). Therefore it is expected that ASI-I also have the ability to degrade those compounds.

Growth of Aminobacter strain ASI-I was also seen in a complex medium (LB) where a doubling time of 11 h was observed (figure 20). Following growth in this medium ASI-I still had the ability to mineralise BAM. Growth in complex media is desirable when a large biomass is needed e.g. for clean up of BAM contaminated soil or drinking water.

Mineralisation of BAM by Aminobacter strain ASI-I Aminobacter strain ASI 1 mineralised BAM rapidly and used the compound as a sole source of carbon and energy. In cultures provided 50 mg/l BAM about 75% of the added BAM was mineralised to CO2 within 15 days (figure 21). The mineralisation was slower at higher concentrations, but even at a concentration of 500 mg/l a significant mineralisation was seen (figure 22).

Aminobacter strain ASI-I was also tested for its ability to mineralise BAM at low concentrations and even at the lowest concentration tested (0.01 mg/l) a rapid mineralisation of BAM was seen (figure 23). BAM is often found in low concentrations in drinking water. Therefore, degradation at low concentrations is necessary to clean up BAM contaminated drinking water. Probably Aminobacter strain ASI-I is able to remove BAM from drinking water even at concentrations below the threshold of 0.1 μg I"1 set by EU.

By a similar approach, a bacterial pure culture capable of mineralising both BAM and dichlobenil was obtained. Results from experiments on the ability of this pure culture (Culture 2) to mineralize BAM and dichlobenil are shown in figures 24 and 25. REFERENCES

[1] Guoguang,L, Qιngxιang,Z., and Wenymg,L. (2001) The formation of 2- chlorobenzamide upon hydrolysis of the benzoylphenylurea insecticide l-(2- chlorobenzoyl)-3-(4-chlorophenyl) urea in different water systems. J. Agric. Food Chem. 49: 1304-1308.

[2] Holtze, M.S., Hansen, H.C.B., Juhler R. K., , Sørensen J., Aamand J. (2005) Degradation pathways for the herbicide dichlobenil and its potential metabolites 2,6-dichlorobenzamide (BAM) and 2,6-dichlorobenzoic acid in soil (in prep.)

[4] Wolter, R., Rosenbaum, S. and Hannappel, S. (2001) The German groundwater monitoring network. Proc. MTM III, 277-282.

[5] Meth-Cohn, O. and Wang, M. -X. (1997) An in-depth study of the biotransformation of nitπles into amides and/or acids using Rhodococcus rhodochrous AJ270. J. Chem. Soc. ,Perkιn Trans. 1 8, 1099-1104.

[6] Miyazaki, S., Sikka, H. C. and Lynch, R. S. (1975) Metabolism of dichlobenil by microorganisms in the aquatic environment. J. Agπc. Food Chem. 23, 365-368.

[7] Vosahlova, J., Pavlu, L., Vosahlo, J. and Brenner, V. (1997) Degradation of bromoxynil, loxynil, dichlobenil and their mixtures by Agrobacterium radiobacter 8/4. Pestic. So. 49, 303-306.

[8] Clausen L, Aπldskov NP, Larsen F (2002) Nedbrydning og sorption af dichlobenil og BAM - Litteraturopsamling samt laboratoπeforsøg. Delrapport 3 Miljøstyrelsen. Miljøministeπet, Copenhagen [9] Albrechtsen, H. -J., Mills, M.S., Aamand, J. and Bjerg, P. L. (2001) Degradation of herbicides in shallow Danish aquifers: an integrated laboratory and field study. Pest Manag. Sa. 57, 341-350.

[10] Broholm, M. M., Rugge, K., Tuxen, N., Højberg, A. L., Mosbaek, H. and Bjerg, P. L. (2001) Fate of herbicides in a shallow aerobic aquifer: a continuous field injection experiment (Vejen, Denmark). Water Resour. Res. 37, 3163-3176.

[11] Tuxen, N., Tuchsen, P. L., Rugge, K., Albrechtsen, H. -J. and Bjerg, P. L. (2000) Fate of seven pesticides in an aerobic aquifer studied in column experiments. Chemosphere 41, 1485-1494.

[12] Sørensen, S. R. and Aamand, J. (2003) Rapid mineralisation of the herbicide isoproturon in soil from a previously treated Danish agricultural field. Pest Manag. Sa. 59, 1118-1124.

[ 13] Ridgway, H. F., Safaπk, 3., Phipps, D., Carl, P. and Clark, D. (1990) Identification and catabolic activity of well-derived gasoline-degrading bacteria from a contaminated aquifer. Appl. Environ. Microbiol. 56, 3565-3575.

[14] Sørensen, S. R., Ronen, Z. and Aamand, J. (2001) Isolation from agricultural soil and characterization of a Sphingomonas sp. able to mineralize the phenylurea herbicide isoproturon. Appl. Environ. Microbiol. 67, 5403-5409.

[15] Muyzer, G., de Waal, E. C. and Uitterlmden, A. G. (1993) Profiling af Complex Microbial Populations by Denaturing Gradient Gel Electrophoresis Analysis of Polyperase Chain Reaction-amplified Genes Encoding fro 16S rDNA. Applied and Environmental microbiology 59, 695-700. [16] Olsen, R. A., Bakken, L. R. (1987) Viability of Soil Bacteria: Optimization of Plate- Counting Technique and Comparison Between Total Counts and Plate Counts Within Different Size Groups. Microbial Ecology 13 (1), 59-74.

[17] Powlson, D. S., Hirsch, P. R., Brookes, P. C. (2001) The Role of Soil Microorganisms in Soil Organic Matter Conservation in the tropics. Nutrient Cycling in Agroecosystems, 61, 41-51.

[18] Rasmussen, L. D., Sørensen, S. J. (2001) Effects of mercury contamination on the culturable heterothrophic functional and genetic diversity of the bactreπal community in soil. FEMS Microbiology Ecology 36, 1-9.

[19] Shannon, C E. and Weaver, W. (1949) The matematical Theory of Communication. University of Illinois Press, Urbana, IL.

[20] Torang, L., Nyholm, N. Albrectsen, H. -J. (2003) Shift in Biodegradatioin Kinetics of the Herbicides MCPP and 2,4-D at Low Concentrations in Aerobic Aquifer Materials. Environmental Science & Technology.

[21] Zak, J. C, Willing, M. R., Moorhead, D. L., and Wildman, H. G. (1994) Functional diversity of microbial communities: A quantitative approach. Soil Biol. Biochem. 26, 1101- 1108.

[21] Alexander, M. ( 1994) Threshold, Chapt. 7, plO2-113. In Biodegradation and bioremediation, 1st ed. Alexander M. (ed). Academic Press Inc. London, England.

[22] Bakken, L. R ( 1997) Culturable and non-culturable Bacteria in Soil, p 47-61. In Modern Soil Microbiology. Van Elsas, J. D. and Wellington, M. H. (ed). Blackwell Sciences Ltd., Oxford England. [23] Begon, M., Harper, J. L., and Townsend, C. R. (1996) Ecology - Individuals, populatioins and communities, 3rd ed. Begon, M., Harper, J. L., and Townsend, C. R. (eds.) Blackwell Science Ltd., Oxford England.

[24] Ellmgsøe, P., Johnsen, K. (2002) Influence of soil sample size on the assessment of bacterial community structure. Soil Biology & Biochemistry 34. 17-1707.

[25] Madigan, M. T., Maπnko, J. M. and Parker, J. (2003) Brock Biology of Microorganisms. Truehart, C. (ed.) Person Education, Inc. New Jersey, USA.

LNDICATIONS RELATDfG TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

(PCT Rule 13WJ)

A. The iadic ations made below relate to the deposited mierooigsEJsin or othei biological materia! referred to in the description on page ^Z » line 5*28 . B. IDENTIFICATION OF DEPOSIT Further deposits are identified oα an additional sheet [^] Name of depositary in-nitution DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH

Address of depositary institution (including postal code and country) Mascheroder VVeg 1B D - 38124 Braunschweig Germany

Date of deposit AccessionNumber

C. ADDITIONAL EVDIC ATIOXS (leave blank if not applicable) This formation is continued on aα additional sheet Q

As regards the respective Patent Offices of the respective designated states, the applicants request that a sample of the deposited microorganisms only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn.

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)

E. SEPARATE FUR-VISHING OF IMHCATIONS (leave blank if not applicable) The iadic-tioas listed below will be submitted to the International Bureau later (spec ifr thegenei-al nature of the indications e.g., "Accession Number of Deposit")

Form PCmo/13 fl4 (MJyI 9M98: reføprint January 2004)