Summary
A chlorobenzoate-degrading Alcaligenes strain, BR60, was introduced to flowthrough lake microcosms and exposed to 3-chlorobenzoate (3Cba) concentrations from 0 to 25 μM. A DNA probe specific for BR60 chlorobenzoate catabolic genes was used with the most probable number (MPN) technique to enumerate bacteria harboring this genetic information. This MPN-DNA hybridization method combined with [U-14C]3Cba uptake rate measurements allowed the correlation of the size and activity of a specific catabolic population in a natural mixed community for the first time. An experiment involving the release of a streptomycin-resistant strain of BR60 indicated that estimates of bacteria carrying the introduced catabolic genotype often outnumbered plate count estimates of viable BR60 by as much as 3 orders of magnitude, particularly when 3Cba inputs were high. The MPN-DNA hybridization method provided catabolic population estimates highly correlated to 3Cba exposure levels and the [U-14C]3Cba uptake rates in the microcosms. Plate counts of BR60 were poorly correlated with both 3Cba exposure levels and uptake rates. In the absence of chlorobenzoate selection, the catabolic genotype declined to very low levels by the MPN-DNA hybridization technique after 8 weeks in the microcosms.
Methodology
Alcaligenes sp. strain BR60 was isolated from sediment of Bloody Run Creek, a chlorobenzoate- and chlorophenol-contaminated creek draining the Hyde Park industrial landfill site in Niagara Falls, N.Y. Details on the isolation and genetic makeup of this organism have been published (24, 25). BR60 was one of a few species isolated on 3Cba as the sole carbon and energy source that harbored an unstable 85-kilobase-pair plasmid, pBR60, carrying the catabolic genotype. A mobile 14-kilobase-pair region of pBR60 (the dpb region) harbors genes central to chlorobenzoate metabolism.
Queen's University Biological Station is situated on Lake Opinicon, a marble-granite-based lake found on the Frontenac Axis of eastern Ontario. The toxicology facility at this station was used to set up 12 microcosms representative of an epilimnetic sediment-water interface. The microcosms were large aquaria (72 by 40 by 22 cm [height]) containing surficial sediments, complete with small macrophytes, detritus, and snails, taken directly from a shallow bay of the lake. A 5-cm layer of epilimnetic sediment was allowed to settle and was not subsequently disturbed. The 50 liters of overlying water was aerated gently with porous stones. Epilimnetic lake water was continuously pumped from an intake pipe in the middle of an adjacent 2-m-deep bay into an overflowing header tank before delivery to the various microcosms at a controlled dilution rate. 3-Cba sterile aqueous solutions were also delivered at fixed rates to selected tanks. Overflow water from experimental tanks was passed through a carbon filter before it entered a seepage bed. No nutrients were added, and all experiments took place at ambient temperatures.
In July and August of 1987, microcosms were set up and dosed with 3Cba at four different approximate concentrations. Lake water was siphoned from the header tank to the microcosms by using aquarium air tubing constricted with plastic clamps such that the dilution rate approximated 0.05/h. 3Cba was delivered from sterile 4 mM reservoirs using commercially available intravenous apparatus. Four microcosms received 3Cba at 0.04 ml/min, and four received 3Cba at 0.4 ml/min. Two microcosms were dosed by floating gauze bags of powdered 3Cba in the water. Two microcosms received no 3Cba. These treatments exposed microcosms to mean 3Cba concentrations ranging from 0 to 36 μM with a high level of variation due to imprecise water flow rates.
Alcaligenes sp. strain BR60 was introduced to one-half of the tanks (representative of the full range of 3Cba concentrations) on 29 June 1987. Cultures in 100 ml of medium A (23) and 3 mM 3Cba plus approximately 20 g of white, sterilized sand were grown to stationary stage and then used to inoculate the microcosms. All microcosms were monitored for 8 weeks.