Degradation of n-hexadecane and its metabolites by Pseudomonas aeruginosa under microaerobic and anaerobic denitrifying conditions

Abstract

A strategy for sequential hydrocarbon bioremediation is proposed. The initial O(2)-requiring transformation is effected by aerobic resting cells, thus avoiding a high oxygen demand. The oxygenated metabolites can then be degraded even under anaerobic conditions when supplemented with a highly water-soluble alternative electron acceptor, such as nitrate. To develop the new strategy, some phenomena were studied by examining Pseudomonas aeruginosa fermentation. The effects of dissolved oxygen (DO) concentration on n-hexadecane biodegradation were investigated first. Under microaerobic conditions, the denitrification rate decreased as the DO concentration decreased, implying that the O(2)-requiring reactions were rate limiting. The effects of different nitrate and nitrite concentrations were examined next. When cultivated aerobically in tryptic soy broth supplemented with 0 to 0.35 g of NO(2)(-)-N per liter, cells grew in all systems, but the lag phase was longer in the presence of higher nitrite concentrations. However, under anaerobic denitrifying conditions, even 0.1 g of NO(2)(-)-N per liter totally inhibited cell growth. Growth was also inhibited by high nitrate concentrations (>1 g of NO(3)(-)-N per liter). Cells were found to be more sensitive to nitrate or nitrite inhibition under denitrifying conditions than under aerobic conditions. Sequential hexadecane biodegradation by P. aeruginosa was then investigated. The initial fermentation was aerobic for cell growth and hydrocarbon oxidation to oxygenated metabolites, as confirmed by increasing dissolved total organic carbon (TOC) concentrations. The culture was then supplemented with nitrate and purged with nitrogen (N(2)). Nitrate was consumed rapidly initially. The live cell concentration, however, also decreased. The aqueous-phase TOC level decreased by about 40% during the initial active period but remained high after this period. Additional experiments confirmed that only about one-half of the derived TOC was readily consumable under anaerobic denitrifying conditions.

FIG. 1

Increase of the denitrification rate with increasing DO concentration during n-hexadecane metabolism by P. aeruginosa under microaerobic conditions. Symbols: ●, vial 1; ■, vial 2; ▾, vial 3; ◊, vial 4; □, vial 5; ▵, vial 6.

FIG. 2

Proposed dependence of the denitrification rate of P. aeruginosa on DO concentration for different substrates. In addition to the well-known oxygen repression and inhibition, the denitrification rate is also subject to the limitation of electron donation from O₂-requiring hydrocarbon oxidation at very low DO concentrations.

FIG. 3

Effect of nitrite concentration on aerobic growth of P. aeruginosa in TSB. OD₄₆₀, optical density at 460 nm.

FIG. 4

Effects of nitrite and/or nitrate concentrations on growth of P. aeruginosa in a glucose-based medium under anaerobic denitrifying conditions. OD₄₆₀, optical density at 460 nm.

FIG. 5

Results of a typical experiment to examine n-hexadecane metabolism by P. aeruginosa under sequential aerobic and anaerobic denitrifying conditions.

FIG. 6

Cell growth, TOC consumption, and transient nitrite accumulation profiles obtained in experiments performed with the high-TOC medium containing different initial nitrate concentrations. Symbols: ●, 2.7 g of NO₃⁻-N per liter; ▴, 0.8 g of NO₃⁻-N per liter; ■, 0.4 g of NO₃⁻-N per liter.

FIG. 7

Cell growth, TOC consumption, and transient nitrite accumulation profiles obtained in experiments performed with the low-TOC medium containing different initial nitrate concentrations. Symbols: ●, 3.3 g of NO₃⁻-N per liter; ■, 1.3 g of NO₃⁻-N per liter; ▴, 0.7 g of NO₃⁻-N per liter; ◊, 0.35 g of NO₃⁻-N per liter.