ENRICHMENT SYSTEMS

Several methods of enrichment were used to isolate a microorganism or a group of microorganisms that were capable of growing on 3,6-dich- loropicolinate (36DCPA) or other carbon sources.

2.4.1 Direct Enrichment

For direct enrichment 5g of a peaty loam soil that had been sprayed once with 36DCPA at field application rates (1.2kg ha- 1 , supplied by The Dow Chemical Co., Kings Lynn, Norfolk, UK) was placed in a sterile flask containing defined medium with 3,6- dichloropicolinate at a concentration of 0.5g carbon l-^ as a sole source of carbon and energy. The flask was shaken at 30°C and sampled regularly for organisms which could grow on solid medium containing 36DCPA at 0.1, 0.25 and 0.5g carbon l-^.

2.4.2 Soil Columns

An adaptation of the method’ used by Macura A MAlek (1958) was developed to continually percolate soil with 36DCPA defined medium.

2.4.2.1 Preparation of Soil Particles

Soils were air dried for two days at room temperature and particulate matter betweem 3mm and 5mm diameter was picked out to fill the columns.

2.4.2.2 Continuous-Flow Enrichment Apparatus

Figure 2.2 shows a schematic representation of the apparatus. The influent reservoir was a 101 bottle containing defined medium supplemented with organic carbon sources. Medium was pumped onto the column by a Watson Marlow flow inducer (MHRE 2) (Falmouth, Cornwall, U.K.) at a constant rate of 8ml h“ ^. Humidified carbon dioxide-free air was fed to the column at 50ml min- 1 . The system was maintained at room temperature.

Figure 2.2 Schematic representation of the soil enrichment columns.

A, Air in flow; B, Manometer; C, Humidifier; D, Nylon Gauze,; E, Column packed with soil particles 3-5mm diameter; F, Watson Marlow flow inducer; G, Medium reservoir; H, Effluent reservoir; J, Waste; K, Air pressure equalisation filter.

2.4.2.3 Sampling Procedure

Samples for microbial population analysis were taken from the effluent culture or directly from the soil column. 0.1ml of culture was plated directly onto agar plates containing defined medium with a variety of carbon sources. In addition 1ml samples were inoculated into shake flasks containing defined media with a variety of carbon sources, incubated for 1 week at 30°C and 0.1ml of this culture was plated out.

Soil was sampled from the column by removing soil aggregates with a sterile inoculating loop. The aggregates were shaken in • sterile 0.2M phosphate buffer (section 2.2.1) and the suspension

was treated in the same way as the column effluent.

2.4.3 Analogue Enrichment. (Focht & Alexander, 1970)

Samples of soil from the soil column (section 2.4.2) and samples of untreated soil were used as an inoculum into a 11 fermenter (LHE series 500 system) containing defined medium with unsubstituted picolinate as the sole source of carbon at 0.5g carbon l“1. The samples were grown as a closed culture for several days before initiating a flow of fresh medium. To expose organisms capable of growing on picolinate to 36DCPA a method was devised having two media reservoirs connected by a Y piece. This enabled the medium flowing into the culture vessel to be changed from picolinate alone to a mixture of picolinate plus 36DCPA by loosening and clamping off the respective tubing.

2.5 MINERALISATION OF [1l*C]-3,6-DICHL0R0PIC0LINATE

Mineralisation of [1**C]-3,6-dichloropicolinate was measured using a method similar to Goswami & Koch (1976). The [ 1**C] 36DCPA (with aspecific activity, 10.6 pCi pmol“ 1) was labelled in the carbon 2 and 6 positions and was kindly supplied by The Dow Chemical Co., Kings Lynn, Norfolk, U.K.

2.5.1 Degradation Apparatus

Figure 2.3 shows a schematic representation of the apparatus used to measure the degradation of [ ^C]-36DCPA. Air was sparged through water to humidify it and through potassium hydroxide to remove any carbon dioxide present. The air was sterilised by passing through a sterile glass fibre filter. [^C]-carbon dioxide formed by degradation of the herbicide was forced out of the reaction vessel, through a drying bottle and into 8ml of a carbon dioxide trapping solution (section 2.5.3) by the incoming air. The apparatus was maintained at 30°C. The reaction vessel was not continually flushed with air because this caused an unacceptably high amount of absorption reagent into the vapour state, and hence was lost. The vessel was flushed for at least 24h with air before a sample was to be measured for 1l*C02 ,

2.5.2 Preparation of Soil

Soils were collected, sealed in polythene bags to maintain the collection moisture content and stored in cardboard fibre boxes at 4°C. 100g of soil was weighed into a 250ml conical flask and a field application of 2,6-[ 1*iC]-36DCPA, namely 25 Ug, that is, 0.25 ug g-1 (0.25 p.p.m.) was added. An identical flask was established with soil that had been heat sterilised at 160°C for I6h, and reconstituted to the correct moisture content by the addition of sterile distilled water. Table 2.1 shows the relevant data on soils used as supplied by the Dow Chemical Co. Ltd.

2.5.3 Absorption Reagent amd Scintillant

The carbon dioxide absorption reagent consisted of 30} (v/v) ethanolamine in 2-methoxyethanol. 8ml of the reagent was added to a scintillation vial and was used to trap the carbon dioxide released.

The scintillant used contained 0.02} (w/v) P0P0P (1,*ldi—2—(5 phenyloxazolyl)-benzene), 0.4} (w/v) PP0 (2,5 diphenyl-oxazole), 3 volumes of sulphur-free toluene and 2 volumes of ethoxyethanol. 12ml of the reagent was added to each scintillation vial.

Table 2.1

Data on soil supplied by Dow Chemical Co. for degradation studies.

Soil Type Location ! pH

M 46 Peaty Loam Mississippi I 6.2

M 47 Sandy California ! 6.5

M 51 Peaty Illinois ! 5.8

M 53 Clay Montana i 7.7

Figure 2.3 Schematic representation of the apparatus used to measure the breakdown of [ ^ C ] - 3,6-dichloropicolinate.

A, Air inflow; B, Humidifier; C, KOH solution; D, Vessel containing soil; E, Concentrated HgSOij for drying gas; F, Carbon dioxide absorbing reagent; G, Sterile air filter.