Figure 3 shows the time concentration profiles of CL-20 and biomass in growing culture of Phanerochaete chrysosporium for a CL-20 initial concentration of 7 mg/L. Substrate disappearance for other initial CL-20 concentrations (1, 3, 100 mg/L) followed virtually the same pattern as shown in Figure 4. The CL-20 concentration remained stable in non-inoculated controls. In all experiments Cl-20 biodegradation initiated after a lag phase of about 65 hours and was nearly fully transformed in less than 95 hours of incubation irrespectively of initial concentration, However, in microcosms containing 500 mg/L of initial concentration, CL-20 depletion did not exceed 10.4 ± 6.8% at an incubation period of 113 hours.

Dry biomass data presented in Table 1 show that the presence of CL-20, up to levels of 500 mg/L, did not adversely affect the biomass production in microcosms that contained varying initial concentrations of the nitramine. This finding suggests that CL-20 does not inhibit the growth of Phanerochaete chrysosporium within the concentration range studied. Similar findings have been reported in RDX studies where no noticeable differences in the produced mycelial mass for RDX levels up to 250 mg/L [27]. In contrast to CL-20, however, Phanerochaete chrysosporium was not able to biodegrade RDX above its solubility limit (approximately 80 mg/L). Therefore, CL-20 appears to be more amenable to biodegradation by the fungus, compared to RDX.

The logistic model [28], employed successfully in fungal systems [29], was used to fit the experimental data (Figure 3).

In its differential form, the logistic equation is presented as follows: (1) d X d t   =   r . X   ( 1 − X X max )

Equation (1) can be solved to obtain: (2) X   =   X max 1 + ( X max − X o X o )   exp − r twhere r is the maximum specific growth rate in a particular environment, X is the population density, X₀ is the initial population density, X_max is the maximum population density achievable in that environment, and t is time. The substrate disappearance rate is related to biomass growth by: (3) d S d t   =   − k ⋅ S ⋅ X max 1 + ( X max − X o X o )   exp − r twhere S is the substrate concentration.

Integration of the equation (3) yields: (4) S   =   S o [ φ ( exp r ⋅ t − 1 ) + 1 ] − k / rwhere φ   =   X o X max and k   =   V max   X max K m

V_max is the maximum specific reaction rate and K_m is the half-saturation constant.

Elapsed time, t in Equation (4) was adjusted by subtracting the observed lag times, t_L, to account for the period during which no noticeable CL-20 utilization occurs. A regression analysis was used to derive the kinetic parameters in Equations (2) and (4) using CL-20 and biomass concentration data obtained from experiments conducted at an initial energetic concentration of 7 mg/L with 10 g/L glycerol and 0.2 g/L ammonium sulfate with initial and maximum biomass concentrations of 0.00034 g/L and 1.7 g/L, respectively. The values of k and r are obtained as 13.5 h⁻¹ and 0.145 h⁻¹, respectively. Using the obtained parameters, a general model for CL-20 biodegradation could be written as: (5) S   =   S 0 [ 0.00034 X max   ( exp 0.145   t − 1 ) + 1 ] − 93.1 (6) X   =   X max 1 + ( X max − 0.00034 0.00034 )   exp − 0.145       t

Figure 4 shows that the logistic growth kinetic model (Equation 5) fits well the CL-20 biodegradation time concentration profiles over a wide range of initial CL-20 concentrations (1–100 mg/L).

The various carbon to nitrogen (C:N) ratios used in the fungal experiments are shown in Table 2. The experimental results are shown in Figures 5, 6 and 7.

The results presented in Figures 5a,b show that the CL-20 degradation by growing cultures of Phanerochaete chrysosporium does not require carbon starvation. The explosive was almost fully degraded in low C and high C media, however, longer lag phases were observed at higher initial glycerol levels. Biodegradation in low C media did not appear to initiate in response to carbon limitation. At the 0.5 or 1 g/L levels, glycerol was below detectable limits within 60 and 83 hours of incubation, respectively. At the 10 g/L level, 20% of glycerol was consumed, almost linearly, within 134 hours. The corresponding growth curves (Figure 5c) indicate that providing higher amounts of glycerol results in substantially higher fungal biomass growth.

The effect of nutrient nitrogen concentration on CL-20 disappearance at initial concentrations of glycerol (10 g/L) and CL-20 (7 mg/L) is shown in Figure 6. CL-20 was consumed simultaneously with ammonium sulfate in growing cultures of the fungus in high N medium (1 g/L ammonium sulfate). In low N media (0.1 g/L and 0.2 g/L ammonium sulfate), degradation occurred as soon as the nitrogen was exhausted. In the latter case, secondary metabolism was probably initiated in response to limitation of nitrogen.

Approximately 50% of ammonium sulfate was utilized in cultures that contained 1 g/L of the chemical. It can be concluded that CL-20 biodegradation by Phanerochaete chrysosporium does not necessarily require nitrogen limitation; however the process is delayed as the amount of the nitrogen in the medium is increased. Conversely, as shown in Figure 6, increasing the concentration of ammonium sulfate in the nutrient medium significantly enhanced the growth of the fungi.

While the general observation is that the optimal production of the ligninolytic enzymes requires nutrient limitation, there are many studies reporting biodegradation of pollutants under both limited and rich nitrogen and/or carbon conditions. The results indicate that, the amounts of supplied nutrient nitrogen and carbon are important factors that control the CL-20 biodegradation in growing cultures of Phanerochaete chrysosporium. The absence of nitrogen limitation requirement may be attributed to the possible dependence of CL-20 degradation on the MnP enzyme, rather than LiP; since MnP can be produced in nitrogen sufficient media [30]. This is supported by the findings of Fournier et al. [9], which indicated direct degradation of CL-20 by MnP. The principal role of Mn²⁺ dependent peroxidase is related to oxidation of Mn²⁺ to Mn³⁺, which then binds to an appropriate ligand, which in turn becomes a strong oxidant and diffuses into the substrate structure. Although the precise role and mechanism of MnPs in degradation of secondary metabolites is not clear, high levels of these lignonoytic enzymes usually correspond to the ability of white rot fungi to attack the nitrated energetic molecules. Our results indicate that cells supplemented with high nitrogen attack a CL-20 for more nitrogen, although their normal physiology is to degrade large molecules when they are nitrogen starved. This could be explained by the work of Kapich et al. [31]. They observed very low MnP activity in the absence of peptone whereas the addition of this organic nitrogen source increased considerably the activity of MnP. They reported that high concentration of organic nitrogen (up to 3–4 g/L of peptone) did not repress MnP production in P. chrysosporium but instead stimulated it.

On the other hand, Stahl et al. [27] reported that the amount of RDX degraded by growing Phanerochaete chrysosporium cultures was approximately 10 times higher under nitrogen-limited conditions as compared to nitrogen-sufficient (non-ligninolytic) conditions. They also reported that RDX was directly amenable to degradation by MnP, but not LiP [25].

The effect of the type nitrogen source (organic vs. inorganic) on the CL-20 biotransformation is shown in Figure 7. Ammonium sulfate and yeast extract (1 g/L) were selected to represent the two different nitrogen sources. The yeast extract, having a lower nitrogen content (about 10%) than ammonium sulfate (21%), was supplied at the maximum nitrogen requirement of the fungus for the given conditions, based on the data obtained from experiments with ammonium sulfate. The use of yeast extract resulted in comparable maximum biomass production (measured as 2.14 ± 0.16 g/L pellets dry weight at 134 hours of incubation) but significantly slower disappearance of CL-20 in the growing cultures, in comparison to ammonium sulfate (Figure 7). Both nitrogen sources resulted in comparable initial biotransformation rates, however, after 14 days of incubation, 11.6% of the initial CL-20 (7 mg/L) still remained in microcosms containing yeast extract. The slower biodegradation rates observed when using yeast extract, as the nitrogen source, may be attributed to the possible alteration of the ligninolytic enzyme system of the fungus. Sensitivity of these enzymes to various nitrogenous compounds has been reported in the literature. For example, Vahabzadeh et al. [32] reported that when urea was used as a nitrogen source, decolorizing ability of Phanerochaete chrysosporium was affected significantly and no MnP or LiP activities were detected. Also, non-ligninolytic conditions were observed using malt extract in the nutrient medium [33].

The potential ability of Phanerochaete chrysosporium to obtain its nutrient nitrogen from CL-20 was also investigated. When exposed to CL-20 (500 mg/L) as the sole nitrogen source in the presence of external carbon (glycerol), the fungus was able to grow, but at extremely slow rates with little pellet growth, compared to cultures containing supplemental nitrogen. While no CL-20 depletion or pellet formation was detected up to 22 days, at 35 days 30 ±3% of the nitramine was consumed by the microorganism. This indicates that Phanerochaete chrysosporium may utilize CL-20 as the sole nitrogen source in nitrogen-deficient environments. However, providing an external nitrogen source greatly enhances the rates of fungal growth and CL-20 disappearance.

The experimental results of CL-20 mineralization ability of Phanerochaete chrysosporium using uniformly labeled [¹⁴C]-CL-20 are presented in Figure 8. CL-20 mineralization began after 2 days of incubation in all vials. The explosive was depleted in less than 100 hours after inoculation in high C-low N medium, however, the radioactivity count ratio of the liberated ¹⁴CO₂ to the total initial [¹⁴C]-CL-20 did not exceed 8.5% within this period. When the cultures were incubated for 46 days, considerable mineralization of CL-20, accounting for 47.1% of the initial [¹⁴C]-CL-20 (7 mg/L) was observed. In high C-low N medium, the extent of mineralization observed was 42.9%. When the ammonium sulfate concentration increased to 1 g/L (high C-high N), mineralization was slightly higher (51.2%), within the same incubation period. In general, the cumulative amount of liberated ¹⁴CO₂ was similar at all nitrogen levels and did not significantly change after 28 days of incubation (Figure 8).

Incubation at high N–high C conditions using 1 g/L yeast extract and 10 g/L glycerol resulted in a mineralization level of 46.4%. Despite the differences in transformation rates, comparable mineralization was observed using yeast extract and ammonium sulfate. This suggests that, different enzymatic systems might play role in biotransformation and subsequent mineralization of the metabolites.

On the other hand, in low C-low N medium (1 g/L glycerol and 0.2 g/L ammonium sulfate), only 26.4% mineralization was detected within 46 days (Figure 8). Poor mineralization at low C conditions cannot be attributed to the comparatively little pellet growth (Figure 5), since high C-low N medium (10 g/L glycerol and 0.1 g/L ammonium sulfate) yielded similar biomass levels (Figure 6) but considerably higher ¹⁴CO₂ production. Moreover, mineralization of CL-20 may not be dependent on the lignin degrading enzymes (i.e., MnP), in view of the results obtained by Fournier et al. [9] indicating that the main CL-20 biodegradation product glyoxal is not amenable to mineralization in purified enzyme cultures. Therefore, it may be concluded that presence of excess carbon is necessary to effectively mineralize the CL-20 metabolites by the growing fungus; although transformation of the nitramine appears to be more rapid at low carbon conditions (Figure 5).

During the incubation period (46 days), about 10% mineralization was detected in sterile non-inoculated controls, possibly due to abiotic transformations. The residual [¹⁴C] in the microcosms was determined by transferring the contents of the microcosms into a scintillation vial and analyzing by liquid scintillation counting. When combined with the [¹⁴C] recovered as ¹⁴CO₂, total ¹⁴C recoveries in the range of 90.5 – 102.1% were obtained.

Using growing cultures of Phanerochaete chrysosporium, Fournier et al. [9] reported a considerably higher (80%) maximum mineralization of CL-20, in a nitrogen-limited growth medium containing 1.2 mM ammonium tartrate. Besides the nitrogen source, different incubation conditions, use of glycerol instead of glucose (10 g/L) as a carbon source and/or higher initial CL-20 in our medium (10-fold) may be responsible for the higher mineralization reported by Fournier et al. [9].

In an attempt to determine the impacts of extracellular enzymes, CL-20 was separately exposed to the liquid supernatant and the pellet fractions of Phanerochaete chrysosporium. Figure 9 presents the CL-20 degradation profile in cultures containing liquid fraction or pellets only, or both.

The biodegradation of CL-20 started without any lag phase, in all cases. Microcosms containing both pellets and liquid fraction rapidly transformed the CL-20 within 36 hours. Also, nearly complete degradation of CL-20 was observed when using only cell mass (without extracellular enzymes), within 48 hours. However, the initial rate of CL-20 disappearance was considerably less than the one observed using both extracellular enzymes and biomass, indicating extracellular enzymatic mediated reaction. Transformation rate increased after 24 hours, which indicates that extracellular enzymes were restored in the system. In cultures containing only the liquid fraction, degradation occurred, but slowed remarkably after the first 24 hours and reached a plateau, after an 85% CL-20 depletion. This may suggest a loss of enzymatic activity over time.

CL-20 transforming ability of pre-grown cultures of Phanerochaete chrysosporium, at various nutrient conditions was investigated. Figure 10 shows the CL-20 disappearance profile in 6 days-old cultures, grown 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), and high C-high N (10 g/L glycerol and 1 g/L ammonium sulfate). During CL-20 addition, glycerol levels were replenished in several microcosms which contained low C-low N growth medium, in order to bring the concentration of the substrate to excess levels.

Within 24 hours, CL-20 (7 mg/L) was completely transformed in cultures with high C- low N, however; only 13% of it was degraded at low C-low N conditions, i.e. when glycerol was initially supplied at 1 g/L. In the latter case, the degradation took place mostly up to 8 hours following CL-20 addition, and the remaining CL-20 was more or less the same level at 8 and 24 hours. Replenishing the glycerol level resulted in only little improvement in Phanerochaete chrysosporium degradation ability, achieving 30% biodegradation, within the same period. An excess of ammonium sulfate (high N-high C) suppressed degradation significantly and 56% of the initially added CL-20 remained in the reaction medium 24 hours after the addition of the nitramine. Assuming that MnP is the major degrading enzyme for CL-20, this result is not surprising according to several studies that report maximum MnP activities under nitrogen-limited conditions, in Phanerochaete chrysosporium cultures [34,35].

In conclusion, 6-days old grown cultures of Phanerochaete chrysosporium were effective in biotransformation of CL-20 in growth media containing excess carbon, with depleted nitrogen. This indicates that the continuous activity of the CL-20 transforming mechanism is best maintained at the aforementioned conditions.

Operation of CL-20 remediation systems with Phanerochaete chrysosporium, such as biofilters, rely on the biodegradation ability of the fungal pellets for extended periods, depending on maintenance of high levels of enzymatic activity. Pelleted growth form of the white rot fungi offers the possibility of biomass reuse and thereby continuous operation of such processes. In order to evaluate the potential of aged Phanerochaete chrysosporium pellets to transform CL-20, the nitramine was added into cultures at various ages and its disappearance was monitored. Cultures were initially grown under high C-low N (10 g/L glycerol and 0.2 g/L ammonium sulfate) conditions.

Aged cultures, up to 18 days, were able to fully transform the newly added CL-20 (Figure 11), following pseudo-first order kinetics. Such kinetics for pre-grown cultures has previously been suggested to apply to white rot fungi co-metabolism [18]. First order degradation rate constants computed for 5, 6, 10 and 18-days aged cultures were 0.269 (0.023), 0.200 (0.006), 0.126 (0.038), and 0.112 (0.027) hour⁻¹, respectively, with corresponding correlation coefficients of 0.9967, 0.9995, 0.9470 and 0.9628. The rate constants were determined using non-linear regression analysis. The values in parentheses are standard errors. A drop in biodegradation rate and deviation from pseudo-first order kinetics behavior are observed as the culture age increases from 6 to 10 days.