Biodegradation of nitroglycerin as a growth substrate: a basis for natural attenuation and bioremediation
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Nitroglycerin (NG) is a toxic explosive commonly found in soil and contaminated groundwater at old manufacturing plants and military ranges. When NG enters an aquifer, it behaves as a dense non-aqueous phase liquid (DNAPL). Nitroglycerin is an impact sensitive explosive and therefore excavating the area to remove or treat the contaminant can be dangerous. In situ bioremediation and natural attenuation of NG have been proposed as remediation alternatives and it is therefore necessary to understand the degradation mechanisms of NG in contaminated soil and groundwater and investigate the potential for using bioremediation at contaminated sites. Many bacteria have been isolated for the ability to transform NG as a source of nitrogen, but no isolates have used NG as a sole source of carbon, nitrogen, and energy. We isolated Arthrobacter JBH1 from NG contaminated soil by selective enrichment with NG as the sole growth substrate. The degradation pathway involves a sequential denitration to 1,2-dinitroglycerin (DNG) and 1-mononitroglycerin (MNG) with simultaneous release of nitrite. Flavoproteins of the Old Yellow Enzyme (OYE) family capable of removing the first and second nitro groups from NG have been studied in the past and we identified an OYE homolog in JBH1 capable of selectively producing the 1 MNG intermediate. To our knowledge, there is no previous report on enzymes capable transforming MNG. Here we show evidence that a glycerol kinase homolog in JBH1 is capable of transforming 1 MNG into 1-nitro-3-phosphoglycerol, which could be later introduced into a widespread pathway, where the last nitro group is removed. Overall, NG is converted to CO2 and biomass and some of the nitrite released during denitration is incorporated into biomass as well. As a result, NG can be now considered a growth substrate, which changes the potential to bioremediate NG contaminated sites. The magnitude of the effect of biodegradation processes in the fate of NG in porous systems was unknown, and we have been able to quantify these effects, determine degradation rates, and have evidence that bioaugmentation with Arthrobacter sp. strain JBH1 could result in complete mineralization in contaminated soil and sediments contaminated with NG, without the addition of other carbon sources. Site specific conditions have the potential to affect NG degradation rates in situ. Experiments were conducted to investigate NG degradation at various pH values and NG concentrations, and the effects of common co-contaminants on NG degradation rates. Arthrobacter JBH1 was capable of growing on NG at pH values as low as 5.1 and NG concentrations as high as 1.2 mM. The presence of explosive co-contaminants at the site such as trinitrotoluene and 2,4-dinitrotoluene lowered NG degradation rates, and could potentially result in NG recalcitrance. Collectively, these results provide the basis for NG bioremediation and natural attenuation at sites contaminated with NG without the addition of other sources of carbon. Nonetheless, careful attention should be paid to site-specific conditions that can affect degradation rates.