Biodegradation of the high explosive hexanitrohexaazaiso-wurtzitane (CL-20)

Abstract

The aerobic biodegradability of the high explosive CL-20 by activated sludge and the white rot fungus Phanerochaete chrysosporium has been investigated. Although activated sludge is not effective in degrading CL-20 directly, it can mineralize the alkaline hydrolysis products. Phanerochaete chrysosporium degrades CL-20 in the presence of supplementary carbon and nitrogen sources. Biodegradation studies were conducted using various nutrient media under diverse conditions. Variables included the CL-20 concentration; levels of carbon (as glycerol) and ammonium sulfate and yeast extract as sources of nitrogen. Cultures that received CL-20 at the time of inoculation transformed CL-20 completely under all nutrient conditions studied. When CL-20 was added to pre-grown cultures, degradation was limited. The extent of mineralization was monitored by the (14)CO(2) time evolution; up to 51% mineralization was achieved when the fungus was incubated with [(14)C]-CL-20. The kinetics of CL-20 biodegradation by Phanerochaete chrysosporium follows the logistic kinetic growth model.

Keywords: CL-20; Hexanitrohexaazaisowurtzitane; Phanerochaete chrysosporium; activated sludge; biodegradation; logistic growth model; mineralization.

Figure 1.

Molecular structures of RDX, HMX and CL-20.

Figure 2.

Mineralization of the hydrolyzed [¹⁴C]-CL-20 by activated sludge.

Figure 3.

Time course profile for the degradation of CL-20 and biomass build-up in growing cultures of Phanerochaete chrysosporium with 10 g/L glycerol and 0.2 g/L ammonium sulfate at an initial CL-20 concentration of 7 mg/L; (•) pellets dry weight, (∇) CL-20 concentration, (—) logistic model (Equations 5 and 6).

Figure 4.

Time course profile for the degradation of CL-20 in growing cultures of Phanerochaete chrysosporium with 10 g/L glycerol and 0.2 g/L ammonium sulfate at initial CL-20 concentrations of 1, 3, 7 and 100 mg/L; data were fitted to the logistic model (Equation 5).

Figure 5.

Time course profile for: (a) carbon source (glycerol) consumption, (b) removal of CL-20, and (c) biomass build-up in growing cultures of Phanerochaete chrysosporium incubated at low C-low N: (•) 0.5 g/L or (○) 1 g/L glycerol and 0.2 g/L of ammonium sulfate; and high C-low N: (▾) 10 g/L glycerol and 0.2 g/L of ammonium sulfate (—) logistic model (Equations 5 and 6).

Figure 6.

Time course profile for the biomass build-up, nitrogen source (ammonium sulfate) consumption and removal of CL-20, in growing cultures of Phanerochaete chrysosporium incubated at high C-low N: 10 g/L glycerol and (•) 0.1 g/L or (○) 0.2 g/L of ammonium sulfate; and high C-high N: (▾) 10 g/L glycerol and 1 g/L of ammonium sulfate (—) logistic model (Equations 5 and 6).

Figure 7.

Time course profile for the degradation of CL-20 in growing cultures of Phanerochaete chrysosporium incubated in high C-high N media (10 g/L glycerol and 1 g/L ammonium sulfate or yeast extract).

Figure 8.

Mineralization of CL-20 by growing cultures of Phanerochaete chrysosporium incubated at various nutrient conditions: (▪) high C-high N: 10 g/L glycerol and 1 g/L yeast extract, (∇) high C-high N: 10 g/L glycerol and 1 g/L ammonium sulfate, (▾) high C-low N: 10 g/L glycerol and 0.2 g/L ammonium sulfate, (○) high C-low N: 10 g/L glycerol and 0.1 g/L ammonium sulfate, (□) low C-low N: 1 g/L glycerol and 0.2 g/L ammonium sulfate, (•) non-inoculated control.

Figure 9.

CL-20 disappearance profile in nitrogen-limited grown (100-hours old) cultures of Phanerochaete chrysosporium, using liquid fraction and pellets only, or both.

Figure 10.

CL-20 disappearance profile in 6 days-old Phanerochaete chrysosporium cultures with different nutrient C:N ratios (•) high C-low N: 10 g/L glycerol and 0.2 g/L ammonium sulfate, (○) low C-low N: 1 g/L glycerol and 0.2 g/L ammonium sulfate, (▾) low C-low N: 1 g/L glycerol and 0.2 g/L ammonium sulfate (re-supplemented with 10 g/L glycerol during CL-20 addition), (∇) high C-high N: 10 g/L glycerol and 1 g/L ammonium sulfate.

Figure 11.

CL-20 degradation profile in nitrogen-limited pre-grown Phanerochaete chrysosporium cultures of various ages (a) 5-days (b) 6-days (c) 10-days (d) 18-days (Solid lines represent the non-linear regression curve fits, assuming pseudo-first order kinetics).