BIOREMEDIATION OF RDX AND HMX CONTAMINATED SOIL AND SEDIMENTS USING JANIBACTER CREMEUS IMMOBILIZED IN CALCITE AND EGG SHELL BASED BIOFORMULATIONS

Abstract

Explosives are toxic compounds predominantly found in the military areas. High explosives, Hexahydro-1,3,5- trinitro-1,3,5-triazine (RDX) and Octahydro-1,3,5,7- tetranitro-1,3,5,7-tatrazocine (HMX) are the most commonly used secondary explosives. They are heterocyclic nitramine compounds which can contaminate both soil and water. They are classified as a potential human carcinogen by United States Environment Protection Agency (USEPA) based on animal studies. Because of their detrimental effects on human and environmental health, the treatment of RDX and HMX contaminated soils is of utmost importance. Many physical and chemical methods have been developed for the treatment of contaminated soil. But, these approaches are expensive, non-specific and can lead to secondary pollution. Hence, a need for eco-friendly approach to mitigate the toxic effects of RDX and HMX has been paved leading to a shift to bioremediation as a possible treatment technology. This study focuses on the use of indigenous bacterial isolates (obtained from explosive contaminated sites) immobilized to develop two novel bioformulations, for achieving the remediation goals. Three isolates, namely, Janibacter cremeus, Pseudomonas mosselii and Pseudomonas entomophila from explosive contaminated sites were subjected to remediation of RDX/ HMX in aqueous phase to confirm their efficacy of degradation. All three isolates were evaluated for their degradation efficiency of 60 mg/L RDX and 6 mg/L HMX in minimal salt medium. Bacterial growth, nitrite released and residual explosive concentrations were monitored throughout the study of 30 days. Also, the first order degradation kinetics were studied for the three isolates and the respective half-lives of both RDX and HMX were calculated. The three isolates exhibited positive growth in presence of RDX/ HMX. Degradation of RDX and HMX by J. cremeus and P. entomophila were accompanied by substantial release of nitrite, whereas, P. mosselii exhibited negligible release of nitrite. RDX degradation was observed to be maximum for J. cremeus (88 %) followed by P. entomophila (83 %) and P. mosselii (80 %). HMX degradation was also observed to be highest for J. cremeus (92 %) followed by P. vi entomophila (89 %) and P. mosselii (76 %). Based on the results obtained, J. cremeus was selected for evaluation of RDX/ HMX degradation in soil and sediments. J. cremeus was immobilized to prepare two novel bioformulations for delivery of microbe to soil/ sediment. Also, the carriers in the bioformulations played a major role in assisting the remediation process. Bioformulation 1 (BF1) was prepared using calcite and cocopeat as carriers. Bioformulation 2 (BF2) was prepared using egg shell powder, cocopeat, tween and sodium bi carbonate as carriers. Both the bioformulations were observed to be viable for six months under storage at 4 C. The bioformulations were tested for its remediation potential in soils contaminated with 65 mg/Kg RDX / 3000 mg/Kg of HMX. The remediation experiments were conducted under saturated as well as unsaturated moisture conditions at 35 C for 35 days. RDX was observed to be degraded by 75 and 60 % under saturated and unsaturated conditions respectively. The saturated treatment sets exhibited better microbial growth during the study in terms of live cell count and total enzyme activity. The bacteria, J. cremeus was observed to exhibit significant release of nitrite under both unsaturated as well as saturated conditions. Mass spectrometric studies showed that, both the conditions lead to the formation of nitroso-derivatives of RDX. But under saturated condition, an intermediate, 5-hydroxy-4-nitro-2,4-diazapentanal was observed which is a precursor to 4-nitro-2,4-diazabuatnal ultimately leading to mineralization to formaldehyde, carbon di oxide and other simpler compounds. HMX on the other hand was observed to be degraded only under saturated conditions by 40 %, The unsaturated conditions exhibited negligible reduction in HMX concentration. Moreover, the microbial activity in the unsaturated treatment sets was observed to decrease continuously. Mass spectrometric (MS) analysis was performed to identify the intermediates formed during HMX degradation. Nitroso derivatives of HMX were observed during the anoxic degradation of HMX. Also, observed was the presence of 5-hydroxy-4-nitro-2,4-diazapentanal, a precursor of 4- nitro-2,4- diazabutanal, which eventually could get mineralized to formaldehyde and other simpler compounds.Sediments from explosive manufacturing facility were remediated using the developed BF 1 and 2. Sediment A and B were acidic and highly contaminated with nitramine explosives. Sediment A was characterized by the presence of RDX. The RDX was observed to be degraded by 87-88 % in 150 days in sediment A. Sediment B was co-contaminated with RDX and HMX. RDX was degraded by 53-55 %, whereas, HMX was degraded by 47-49 % in 90 days. The degradation was observed to be accompanied by release of nitrite. Also, applied bioformulations (BF 1 and 2) lead to an increase in the pH of both the sediments, thereby enhancing the microbial activity. The study demonstrates successful development and application of eco-friendly, economical and highly efficient bioformulations that can play a crucial role in remediation of hazardous nitramine explosive compounds present in soil/ sediments.