Effect of the Substrate on 4NCB Reduction.
The microbial community plays an important role in the biodegradation of organic xenobiotics in soil . A novel approach was previously established that could effectively enhance the biodegradation rate of nitroaromatic compounds using pure strains [10, 11]; however, complex microbial populations and complex soil compounds exist in the soil. Here, the intent was to evaluate effect on 4NCB degradation using an additional compound in two systems (MSM, soil).
The effect of the additional substrate on the 4NCB removal and the OD values of soil microorganisms were observed in the MSM after 3 days. At a concentration of 100 mg/L 4NCB, minimal indigenous microorganisms existed that could utilize 4NCB as carbon and nitrogen sources to grow, as shown in Fig. 1A (0.160 ± 0.022, the OD600 value for the indigenous microorganisms of the control group), and higher levels of 4NCB would be difficult to remove by indigenous microorganisms (the 4NCB degradation for the control = 16.9%). Thus, an increase in the biomass of the indigenous microorganisms could be a key factor affecting 4NCB biodegradation. Therefore, testing the effect of a normal substrate on the biomass of the indigenous microorganisms to enhance 4NCB removal was required in the next stage of the experiment. First, with additional carbon (sucrose, starch), The biomass of indigenous microorganisms (OD600 values = 0.301 ± 0.083 and 0.456 ± 0.122, respectively) and the degradation of 4NCB residue (19.2% and 17.5%, respectively) indicated minimal increases compared with the data for the control (Fig. 1A). The results indicated that the indigenous microorganisms could not sufficiently utilize the additional carbon to enhance the strain’s growth and therefore did not produce an increase in the 4NCB biodegradation rate. Second, with inorganic nitrogen sources (ammonium nitrate or urea) (OD600 values = 0.274 ± 0.022 and 0.3446 ± 0.0607, respectively), an increase in the 4NCB biodegradation rate was not apparent compared with the control data; however, the 4NCB data (11.7% and 14.0%, respectively) indicated that the biodegradation rates had been slightly suppressed. Finally, the effect of adding inorganic nitrogen sources on the 4NCB biodegradation rates was opposite to that of indigenous strains from soil. Supplementation with organic nitrogen (peptone) markedly improved the OD600 value (1.54), and the 4NCB degradation was 62.9% after 3 days. The results indicated that the substantial enhancement of indigenous microbial growth was induced organically and resulted in increased 4NCB biodegradation.
For all the biostimulated substrates in soil, the highest level of 4NCB degradation (78.3%) was found to be a result of supplementation with peptone. No substantial difference was observed in 4NCB degradation (51.9% and 46.2%, respectively) with a carbon source (sucrose or starch), and the degradation (48.8% and 43.1%, respectively) was limited with an inorganic source (urea or ammonium nitrate). Considering these results, peptone was determined to be the optimal primary substrate for our subsequent experiments.
Effect of the Reductant on 4NCB Reduction.
4NCB is resistant to microbial degradation due to the simultaneous existence of chlorine and nitro groups, which may induce a partial reduction of 4NCB in the initial steps. Thus, a reductant could enhance 4NCB biodegradation by pure strains (11). Therefore, we tested the effect of an additional reductant on the 4NCB removal and the OD values of the soil microorganisms. Figure 1A shows that were no statistically significant differences between the OD600 values for the additional reductant and those for the control group. This finding refutes the assumption that a reductant cannot be utilized as a substrate to support microbial growth. When reductants (D-glucose, mannitol, ascorbic acid (VC), and FeSO4 (Fe2+) were added to the MSM, the concentrations of the 4NCB residue were 75.6 mg/L, 70.9 mg/L, 75.8 mg/L, and 83.7 mg/L, respectively. These results indicated that the additional reductant could catalyze the 4NCB biodegradation rate of soil microorganisms. However, in soil, the additional reductant had a negligible effect on 4NCB reduction due to the complex compounds in the soil (Fig. 1B). The 4NCB biodegradation rate ranged from 47.1–54.3% with reductant compared with 50.4% for the control.
However, the utilization of biostimulation with a substrate or a reductant to enhance 4NCB removal from soil warrants further evaluation.
Synergistic Effect of the Reductant and Peptone on 4NCB Reduction.
With the synergistic effect of the optimal mixture, the 4NCB and nitrobenzene biodegradation rates for target strains from various environments were effectively enhanced . Thus, it was important to evaluate whether the approach could be utilized to effectively enhance 4NCB biodegradation rates using soil microorganisms. When supplementing MSM with peptone and reductants (control, D-glucose, ascorbic acid, mannitol and Fe2+), the 4NCB residue concentrations from an initial concentration of 100 mg /L 4NCB were 83.1 ± 3.9 mg/L, 36.5 ± 1.2 mg/L, 1.1 ± 0.9 mg/L, 11.9 ± 2.5 mg/L and 18.4 ± 3.5 mg/L, respectively, after 3 days. In addition, the OD600 data were 0.160 ± 0.017, 1.29 ± 0.105, 1.404 ± 0.015, 1.48 ± 0.5244 and 1.49 ± 0.102, respectively. When soil was amended with peptone and reductants (control, peptone, peptone + D-glucose, ascorbic acid + peptone, mannitol + peptone and Fe2+ + peptone), the 4NCB residue concentrations from an initial concentration of 100 mg kg− 1 4NCB were 49.6 ± 2.8 mg/kg, 21.7 ± 3.2 mg/kg, 22.5 ± 4.5 mg/kg, 3.3 ± 1.6 mg/kg, 12.7 ± 2.4 mg/kg and 10.2 ± 2.2 mg/kg, respectively. These results show that the optimal mixture (ascorbic acid + peptone) could be effectively utilized to enhance 4NCB removal in soil with a low initial content of 4NCB. The variations of residual 4NCB concentrations in soil were examined when the initial 4NCB concentration was 1 g/kg 4NCB (Fig. 4). After 4–8 days, the level of 4NCB treatment using the control was negligible. The treatment maintained a level of 72.9% (729.2 ± 71.2 mg/kg) of the initial concentration. As verification of the approach, the 4NCB content in soil had decreased from 1 g kg− 1 to 43.8 ± 14.6 mg kg− 1 after 6 days (Fig. 4), and additional 4NCB was completely degraded after 8 days. Thus, the approach may be a potent technique to treat soil contaminated with 4NCB.
To identify the co-metabolic pathway in soil that contributed to effectively utilizing the approach, GC/MS analysis of 1 g/kg 4NCB biodegraded products in soil was performed using the organic extracts of the isolate. The retention time and m/z spectra of the identified co-metabolites are shown in Fig. 5. Two major co-metabolites were found: 4-chloroaniline and 4-chloroformanilide. No previous reports have described the 4-chloroformanilide metabolite.