Abstract
Colony hybridizations with a gene probe for enumeration of 2,4-dichlorophenoxy-acetic acid (2,4-D)-degrading bacteria were compared with classical enrichment and radiolabel most-probable-number (MPN) assay methods. Two natural water samples (rivers) and raw sewage were tested by each method. UV scans of enrichment cultures revealed 2,4-D degradation with raw sewage occurred in 4–11 days, 4–>22 days with Mary's River water, and 5–>22 days with Willamette River water. [14C]-2,4-D MPN analysis, measuring release of14CO2, yielded estimates of bacteria per milliliter able to degrade 2,4-D. Raw sewage estimates were 1.4 × 105 2,4-D degraders/ml, Mary's River >1.6 × 105/ml, and Willamette River water 1.6 × 104/ml. Activities noted by UV scan enrichment data supported these results.
Autoradiograms of colony blots were also used to estimate numbers of 2,4-D-degrading bacteria. These estimates were also supported by the UV scan data from enrichment cultures. Raw sewage gave counts between 5 × 104 and 2.9 × 105 2,4-D-degrading bacteria/ml, which correlates well with the estimates obtained by14C-MPN analyses. River waters, both much lower in total bacterial counts and organic carbon than raw sewage, yielded fewer 2,4-D-degrading bacteria than estimated by14C-MPN. Media composition and cometabolism may account for discrepancies in estimates for 2,4-D-degrading bacteria observed when colony blot and14C-MPN analyses were compared.
Replica plating made it possible to test for 2,4-D biodegradation from colonies reactive in autoradiograms. Five of 12 (42%) colonies reacting in the colony hybridization exhibited biodegradation activities. Nonreactive colonies failed to degrade 2,4-D.
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Literature Cited
Aaronson S (1970) Experimental microbial ecology. New York: Academic Press, pp 91–92
Alexander M (1969) Microbial degradation and biological effects of pesticides in soil. Soil Biol Rev Res Nat Resour Res (UNESCO) 9:209–240
Amy PS, Schulke JW, Frazier LM, Seidler RJ (1985) Characterization of aquatic bacteria and cloning of genes specifying partial degradation of 2,4-dichlorophenoxyacetic acid. Appl Environ Microbiol 49:1237–1245
Bell GR (1957) Some morphological and biochemical characteristics of a soil bacterium which decomposes 2,4-dichlorophenoxyacetic acid. Can J Microbiol 3:821–840
Don RH, Pemberton JM (1981) Properties of six pesticide degradation plasmids isolated fromAlcaligenes paradoxus andAlcaligenes eutrophus. J Bacteriol 145:681–686
Evans WC, Smith BWS, Fernley HN, Davies JI (1971) Bacterial metabolism of 2,4-dichlorophenoxyacetic acid. Biochem J 122:543–551
Fisher PR, Appleton J, Pemberton JM (1978) Isolation and characterization of the pesticide-degrading plasmid pJP1 fromAlcaligens aradoxus. J Bacteriol 135:798–804
Ghosal D, You IS, Chatterjee DK, Chakrabarty AM (1985) Microbial degradation of halogenated compounds. Science 228:135–142
Karns JS, Dilbane JJ, Chatterjee DK, Chakrabarty (1984) Microbial biodegradation of 2,4,5-trichlorophenoxyacetic acid and chlorophenols. In: Omenn GS, Hollaender A (eds), Genetic control of environmental pollutants. New York: Plenum Press, pp 3–21
Kaufman DD, Kearney PC (1976) Microbial transformations in the soil. In: Audus LJ (ed), Herbicides, physiology, biochemistry, and ecology. London: Academic Press Inc., pp 29–64
Lehmicke LG, Williams RT, Crawford RL (1979)14CO2-most-probable-number method for enumeration of active heterotrophic microorganisms in natural water. Appl Environ Microbiol 38:644–649
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory
Pemberton JM (1981) Genetic engineering and biological detoxification of environmental pollutants. Residue Rev 78:1–11
Reasoner DJ, Geldreich EE (1985) A new medium for the enumeration and subculture of bacteria from potable water. Appl Environ Microbiol 49:1–7
Rodina AG (1972) Methods in aquatic microbiology. Baltimore: University Park Press, p 179
Standard methods for the examination of water and wastewater, water, 16th edition. (1985) Washington, DC: American Public Health Assoc
Steenson TI, Walker N (1957) The pathway of breakdown of 2,4-dichloro-and 4-chloro-2 methylphenoxyacetic acid by bacteria. J Gen Microbiol 16:146
Tiedje JM, Alexander M (1969) Enzymatic cleavage of the ether bond of 2,4-dichlorophenoxyacetate. J Agric Food Chem 17:1080–1084
Tiedje JM, Duxburry MM, Alexander M, Dawson JE (1969) 2,4-D metabolism: pathway of degradation of chlorocatechols byArthrobacter sp. J Agric Food Chem 17:1021–1026
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Amy, P.S., Staudaher, M.V. & Seidler, R.J. Comparison of a gene probe with classical methods for detecting 2,4-dichlorophenoxyacetic acid (2,4-D)-biodegrading bacteria in natural waters. Current Microbiology 21, 95–101 (1990). https://doi.org/10.1007/BF02091826
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DOI: https://doi.org/10.1007/BF02091826