Degradation of Pentachlorophenol by a Flavobacterium Species ...

Vol. 56, No. 2

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1990, p. 541-544

0099-2240/90/020541-04$02.00/0 Copyright C) 1990, American Society for Microbiology

Degradation of Pentachlorophenol by a Flavobacterium Species Grown in Continuous Culture under Various Nutrient Limitationst EDWARD TOPPl* AND RICHARD S. HANSON2 Land Resource Research Centre, Central Experimental Farm, Agriculture Canada, Ottawa, Ontario KJA 0C6,

Canada,'

and Gray Freshwater Biological Institute, University of Minnesota, Navarre, Minnesota 553922 Received 6 September 1989/Accepted 13 November 1989

A Flavobacterium sp. was grown in continuous culture limited for growth with ammonium, phosphate, sulfate, glucose, glucose + pentachlorophenol (PCP) (0.065 h-1), or PCP. Cells were harvested, washed, and suspended to 3 x 107 cells ml-' in shake flasks containing a complete mineral salts medium without added carbon or supplemented with 50 mg of PCP ml-' or 50 mg of PCP ml-' + 100 mg of glucose ml-'. The PCP concentration and the viable cell density were determined periodically. Cells that were grown under phosphate, glucose, or glucose + PCP limitation were more sensitive to PCP and took longer to degrade 50 mg of PCP ml-' than did cells that were grown under ammonium, sulfate, or PCP limitation. Glucose stimulated viability and PCP degradation in all cases except when the cells were grown under carbon limitation with glucose and PCP added together as the carbon source. These results indicate that there is a relationship between nutrient limitation, phenotypic variation, and the sensitivity to and degradation of PCP by this organism.

250; and FeSO4 6H20, 1. The concentrations of the carbon sources provided for carbon-limited growth were as follows (in milligrams per liter): glucose, 500; PCP, 450; or glucose, 500, and PCP, 85, provided together. For nitrogen-limited growth the medium composition was as follows (in milligrams per liter): K2HPO4, 315; KH2PO4, 95; MgSO4 7H20, 100; NH4Cl, 70; FeSO4 6H20, 1; glucose, 5,000. For phosphorus-limited growth the medium composition was as follows (in milligrams per liter): K2HPO4, 17; KH2PO4, 4; MgSO4 7H20, 100; NH4Cl, 250; KCl, 300; FeSO4 6H20, 1; glucose, 5,000. For sulfur-limited growth the medium composition was as follows (in milligrams per liter): K2HPO4, 315; KH2PO4, 95; MgSO4 7H20, 6; MgCl2, 80; NH4Cl, 250; FeSO4 6H20, 1. We routinely checked for contamination by plating samples from the chemostat onto nutrient agar. At least three volume changes were effected following a change in medium before we used the cells for experimentation. Experimental incubations. Cells were harvested by centrifuging (12,000 x g for 12 min) fluid that was taken directly from the chemostat vessel, and the pellet was washed with mineral salts medium (MS-NH4 medium [26]) and suspended in MS-NH4 medium to a cell density of 3 x 107 cells ml-', as estimated by optical density determination (an A600 of 1 corresponded to 5 x 109 CFU ml-1). Portions of 20 ml were then dispensed into 125-ml Erlenmeyer flasks and incubated as described previously (26). Quantitation of PCP and 2,4,6trichlorophenol and viable cell enumeration on nutrient agar were done as described previously (26). All experiments were repeated at least twice.

Pentachlorophenol (PCP) is a biocide that is used primarily for wood preservation (6). It is now a ubiquitous environmental pollutant (19, 22). It is degraded aerobically by a number of bacterial isolates (1, 4, 8, 9, 23, 25, 28), including a Flavobacterium sp. (18, 21). One approach for decontaminating polluted soil or water is by inoculation with pollutantdegrading microorganisms (5, 9, 14, 15). The degradation of PCP in soil or water by inoculated Flavobacterium cells is influenced by a number of factors, including inoculum density, pH, temperature (5, 15), other available carbon sources (26), and other pollutants such as creosote or copperchromate-arsenate (unpublished data). The compositions of bacterial cells vary according to the environmental conditions (for representative reviews, see references 2, 3, 11, and 12). Such phenotypic variation can influence the sensitivity to toxic agents, including the halogenated phenols (10). The degradation of pollutants can be inhibited by their toxicity at high concentrations (13, 27). In this context, we wished to examine whether the sensitivity to and degradation of PCP by a Flavobacterium sp. was influenced by the conditions of growth. We grew the Flavobacterium sp. at a constant growth rate in continuous culture in mineral media limited for carbon (glucose, PCP, or glucose and PCP added together), nitrogen, phosphate, or sulfur. Cells grown under these various conditions were harvested and incubated in batch cultures with all nutrients in excess. Viability and PCP degradation kinetics were then determined. MATERIALS AND METHODS Continuous culture. A Flavobacterium sp. was grown in a chemostat (Bioflo model C30; New Brunswick Scientific Co., Inc., Edison, N.J.) as described previously (26). The dilution rate for growth on PCP as the sole carbon source was 0.023 h-1. For all other growth conditions the dilution rate was 0.065 h-1. For carbon-limited growth, the medium composition was as follows (in milligrams per liter): K2HPO4, 315; KH2PO4, 95; MgSO4. 7H20, 100; NH4Cl, * Corresponding author. t Land Resource Research Centre contribution

RESULTS AND DISCUSSION The viability of cells that were suspended in batch culture was influenced by the substrate limitation in continuous culture (Fig. 1 to 3). In the absence of added carbon, there was a decline in the populations of suspended cells that were grown in the presence of PCP with or without glucose or under phosphate limitation (Fig. 2 and 3A). Sulfate-limited cells were followed for only 45 h (Fig. 3B). In the case of cells that were grown with PCP as the limiting nutrient, there was a transient loss of viability that was recovered within 50

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FIG. 1. Viability of the Flavobacterium sp. suspended in MS-

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NH4' medium in shake flasks following growth in continuous culture under glucose (A) or ammonium (B) limitation. Cells were incubated without added carbon (0), supplemented with 50 mg of PCP liter-' (Li), or supplemented with 50 mg of PCP liter-' + 100 mg of glucose liter-' (E). Standard deviations of greater than 10% of the mean are indicated.

h. This transient effect was probably due to a change in culturability because of cellular damage rather than cell death. The decline in the viable population in the absence of added carbon was most rapid with cells that were grown under glucose + PCP limitation, in which case the rate of loss was 3.2 x 105 CFU ml-' h-1. 109

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FIG. 2. Viability of the Flavobacterium sp. suspended in MSNH4' medium in shake flasks following growth in continuous

culture under glucose + PCP (A) or PCP (B) limitation. Symbols are the same as those described in the legend to Fig. 1. Standard deviations of greater than 10% of the mean are indicated.

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FIG. 3. Viability of the Flavobacterium sp. suspended in MS-

NH4' medium in shake flasks following growth in continuous culture under phosphate (A) or sulfate (B) limitation. Symbols are the same as those described in the legend to Fig. 1. Standard deviations of greater than 10% of the mean are indicated.

In all cases, provision of 50 mg of PCP liter-' as the sole carbon source effected a transient but significant reduction in the viable cell population. With carbon- or phosphate-limited cells, the population was reduced by approximately 100-fold (Fig. 1A, 2, and 3A). The decline in the viable cell population was much less for cells that were grown under ammonium or sulfate limitation (Fig. 1B and 3B). Cells that were grown under ammonium or sulfate limitation yielded a more flocculant pellet when they were centrifuged, suggesting the production of an extracellular polysaccharide. This material might facilitate PCP degradation by providing a second carbon source in a manner analogous to that of exogenous supplementary carbon (26; this study). In all cases recovery of viable populations was complete within about 2 days. Glucose stimulated the viability of cells that were grown under glucose, ammonium, or sulfate limitation (Fig. 1 and 3B). Glucose was much less stimulatory to cells that were grown under PCP limitation, glucose + PCP limitation, or phosphate limitation (Fig. 2 and 3A). We do not know why growth in continuous culture in the presence of PCP yielded cells that were not stimulated by glucose in the batch incubation. We have previously shown (26) that PCP metabolism by glutamate-grown Flavobacterium cells is stimulated by glucose. The lag preceding the degradation of PCP was variable, depending on the growth conditions (Fig. 4). In the absence of added glucose, cells grown under nitrogen, sulfate, or PCP limitation degraded 50 mg of PCP liter-' in about half the time of that of cells that were grown under the other substrate limitations. Glucose rapidly stimulated PCP degradation by sulfate-, nitrogen-, or PCP-limited cells. PCPlimited cells responded less vigorously to glucose supplementation, and cells grown under glucose + PCP or phosphate limitation were not stimulated during the course

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Kinetics of PCP degradation by the Flavobacterium sp.

suspended in MS-NH4+ medium in shake flasks following growth in continuous culture under the indicated substrate limitation (A to F). Cells were supplemented with 50 mg PCP liter-' (O) or with 50 mg of PCP liter-' + 100 mg of glucose liter-' (H).

of the incubation. The length of the apparent lag corresponded to the time required to get the viable populations in the range of 107 CFU ml-'. The responses of nitrogen- and glucose-limited cells to 50 mg of 2,4,6-trichlorophenol ml-' were similar to those of PCP-limited cells, indicating that the observed phenomena may be general for the polychlorinated phenols metabolized by a Flavobacterium sp. (data not

shown) (24). There is clearly

a relationship between phenotypic variation in the Flavobacterium sp. and its biodegradative efficacy, apparently mediated through differences in the sensitivity to PCP. Cells grown under nitrogen or sulfate limitation degraded PCP the most rapidly. Nutrient limitation may be a parameter which should be optimized in the growth of inoculants destined for use in the decontamination of heavily polluted materials. Our results suggest that the nature of the nutrient limitation in an environment might influence the sensitivity of the microflora to toxic concentrations of an organic pollutant and therefore might influence the acclimation time preceding biodegradation of the pollutant. Substrate limitation can influence the expression of membrane proteins (20), phospholipids (16, 17), and polyglucans (7). An explanation of our observations awaits compositional analysis of cells grown under various conditions.

ACKNOWLEDGMENTS E.T. was the recipient of a postgraduate scholarship from the Natural Sciences and Engineering Research Council of Canada. This work was partially funded by Biotrol Inc. (Chaska, Minn.). We thank Linda Guay and Linda Howe for typing the manuscript.

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