Evaluation of toxic effects of aeration and trichloroethylene oxidation on methanotrophic bacteria grown with different nitrogen sources

Abstract

In this study we evaluated specific and nonspecific toxic effects of aeration and trichloroethylene (TCE) oxidation on methanotrophic bacteria grown with different nitrogen sources (nitrate, ammonia, and molecular nitrogen). The specific toxic effects, exerted directly on soluble methane monooxygenase (sMMO), were evaluated by comparing changes in methane uptake rates and naphthalene oxidation rates following aeration and/or TCE oxidation. Nonspecific toxic effects, defined as general cellular damage, were examined by using a combination of epifluorescent cellular stains to measure viable cell numbers based on respiratory activity and measuring formate oxidation activities following aeration and TCE transformation. Our results suggest that aeration damages predominantly sMMO rather than other general cellular components, whereas TCE oxidation exerts a broad range of toxic effects that damage both specific and nonspecific cellular functions. TCE oxidation caused sMMO-catalyzed activity and respiratory activity to decrease linearly with the amount of substrate degraded. Severe TCE oxidation toxicity resulted in total cessation of the methane, naphthalene, and formate oxidation activities and a 95% decrease in the respiratory activity of methanotrophs. The failure of cells to recover even after 7 days of incubation with methane suggests that cellular recovery following severe TCE product toxicity is not always possible. Our evidence suggests that generation of greater amounts of sMMO per cell due to nitrogen fixation may be responsible for enhanced TCE oxidation activities of nitrogen-fixing methanotrophs rather than enzymatic protection mechanisms associated with the nitrogenase enzymes.

FIG. 1

Comparison of methane uptake rates (a), CTC/DTAF and naphthalene oxidation rates (b), and formate oxidation rates (c) in nitrate-supplied M. trichosporium OB3b fresh cells, acetylene-exposed cells, air-exposed cells, and TCE-exposed cells. The error bars show the range of values obtained with duplicate vials in panels b and c. (a and c) , fresh cells; , acetylene-exposed cells; , air-exposed cells; ∗, TCE-exposed cells. (b) , CTC/DTAF ratios; , naphthalene oxidation rates. VSS, volatile suspended solids; d, day.

FIG. 2

Methane uptake rates and naphthalene oxidation rates of nitrogen-fixing M. trichosporium OB3b cells. Fresh cells were compared with air-exposed and N₂-exposed cells after 4 h of incubation. The error bars show the ranges of values obtained with duplicate vials. ░⃞, CH₄ uptake rates; , naphthalene oxidation rates. d, day.

FIG. 3

Decreases in methane uptake and naphthalene oxidation activities following 2 h of aeration for M. trichosporium OB3b cells (a) and CAC-2 cells (b). The error bars show the ranges of values obtained with duplicate vials. , CH₄ uptake rates; , naphthalene oxidation rates.

FIG. 4

Methane uptake rates and naphthalene oxidation rates of M. trichosporium OB3b (a) and CAC-2 (b) TCE-free cells and TCE-exposed cells following 90 min of incubation. The error bars show the ranges of values obtained with duplicate vials. , CH₄ uptake rates of TCE-free cells; , CH₄ uptake rates of TCE-exposed cells; , naphthalene oxidation rates of TCE-free cells; , naphthalene oxidation rates of TCE-exposed cells. d, day.

FIG. 5

Decreases in methane uptake and naphthalene oxidation activities due to TCE product toxic effects on M. trichosporium OB3b cells (a) and CAC-2 cells (b) following 90 min of TCE oxidation. The error bars show the ranges of values obtained with duplicate vials. , CH₄ uptake rates; , naphthalene oxidation rates; ▩, CTC/DTAF ratios.

FIG. 6

Decreases in TCE and naphthalene oxidation activities of nitrogen-fixing OB3b cells due to oxidation of different amounts of TCE. The mass of TCE that corresponded to the measured T_c of the cells is indicated. The error bars show the ranges of values obtained with duplicate vials. □, TCE oxidation rates, ∗, naphthalene oxidation rates. The lines are the linear regression lines for the data.

FIG. 7

Decreases in respiratory activity as measured by CTC/DTAF ratios of nitrogen-fixing OB3b cells following oxidation of different amounts of TCE during a 4-h incubation. The mass of TCE that corresponded to the measured T_c of the cells is indicated. The error bars show the ranges of values obtained with duplicate vials. The data points are CTC/DTAF ratios, and the line is the linear regression line for the data.