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 newlineexplosives, Hexahydro-1,3,5- trinitro-1,3,5-triazine (RDX) and Octahydro-1,3,5,7- newlinetetranitro-1,3,5,7-tatrazocine (HMX) are the most commonly used secondary newlineexplosives. They are heterocyclic nitramine compounds which can contaminate both newlinesoil and water. They are classified as a potential human carcinogen by United States newlineEnvironment Protection Agency (USEPA) based on animal studies. Because of their newlinedetrimental effects on human and environmental health, the treatment of RDX and newlineHMX contaminated soils is of utmost importance. Many physical and chemical newlinemethods have been developed for the treatment of contaminated soil. But, these newlineapproaches are expensive, non-specific and can lead to secondary pollution. Hence, a newlineneed for eco-friendly approach to mitigate the toxic effects of RDX and HMX has newlinebeen paved leading to a shift to bioremediation as a possible treatment technology. newlineThis study focuses on the use of indigenous bacterial isolates (obtained from newlineexplosive contaminated sites) immobilized to develop two novel bioformulations, for newlineachieving the remediation goals. Three isolates, namely, Janibacter cremeus, newlinePseudomonas mosselii and Pseudomonas entomophila from explosive contaminated newlinesites were subjected to remediation of RDX/ HMX in aqueous phase to confirm their newlineefficacy of degradation. All three isolates were evaluated for their degradation newlineefficiency of 60 mg/L RDX and 6 mg/L HMX in minimal salt medium. Bacterial newlinegrowth, nitrite released and residual explosive concentrations were monitored newlinethroughout the study of 30 days. Also, the first order degradation kinetics were newlinestudied for the three isolates and the respective half-lives of both RDX and HMX newlinewere calculated. The three isolates exhibited positive growth in presence of RDX/ newlineHMX. Degradation of RDX and HMX by J. cremeus and P. entomophila were newlineaccompanied by substantial release of nitrite, whereas, P. mosselii exhibited newlinenegligible release of nitrite. RDX degradation was observed to be maximum for J. newlinecremeus (88 %) followed by P. entomophila (83 %) and P. mosselii (80 %). HMX newlinedegradation was also observed to be highest for J. cremeus (92 %) followed by P. vi newlineentomophila (89 %) and P. mosselii (76 %). Based on the results obtained, J. cremeus newlinewas selected for evaluation of RDX/ HMX degradation in soil and sediments. newlineJ. cremeus was immobilized to prepare two novel bioformulations for delivery of newlinemicrobe to soil/ sediment. Also, the carriers in the bioformulations played a major newlinerole in assisting the remediation process. Bioformulation 1 (BF1) was prepared using newlinecalcite and cocopeat as carriers. Bioformulation 2 (BF2) was prepared using egg shell newlinepowder, cocopeat, tween and sodium bi carbonate as carriers. Both the newlinebioformulations were observed to be viable for six months under storage at 4 and#61616;C. The newlinebioformulations were tested for its remediation potential in soils contaminated with 65 newlinemg/Kg RDX / 3000 mg/Kg of HMX. The remediation experiments were conducted newlineunder saturated as well as unsaturated moisture conditions at 35 and#61616;C for 35 days. RDX newlinewas observed to be degraded by 75 and 60 % under saturated and unsaturated newlineconditions respectively. The saturated treatment sets exhibited better microbial growth newlineduring the study in terms of live cell count and total enzyme activity. The bacteria, J. newlinecremeus was observed to exhibit significant release of nitrite under both unsaturated newlineas well as saturated conditions. Mass spectrometric studies showed that, both the newlineconditions lead to the formation of nitroso-derivatives of RDX. But under saturated newlinecondition, an intermediate, 5-hydroxy-4-nitro-2,4-diazapentanal was observed which newlineis a precursor to 4-nitro-2,4-diazabuatnal ultimately leading to mineralization to newlineformaldehyde, carbon di oxide and other simpler compounds. newlineHMX on the other hand was observed to be degraded only under saturated conditions newlineby 40 %, The unsaturated conditions exhibited negligible reduction in HMX newlineconcentration. Moreover, the microbial activity in the unsaturated treatment sets was newlineobserved to decrease continuously. Mass spectrometric (MS) analysis was performed newlineto identify the intermediates formed during HMX degradation. Nitroso derivatives of newlineHMX were observed during the anoxic degradation of HMX. Also, observed was the newlinepresence of 5-hydroxy-4-nitro-2,4-diazapentanal, a precursor of 4- nitro-2,4- newlinediazabutanal, which eventually could get mineralized to formaldehyde and other newlinesimpler compounds.