Caroline Peres

Biodegradation of nitrobenzene by its simultaneous reduction into aniline and mineralization of the aniline formed

Abstract By mixing through a three-reactor system a nitroreducing consortium and an aniline-degrading Comamonas acidovorans, a mixed population was formed which was able to mineralize the nitroaromatic compound nitrobenzene via aniline, its corresponding aminoaromatic compound. The behavior of the mixed population was characterized in batch culture. In the first step, nitrobenzene was reduced to aniline by the reductive consortium and, in the second, oxidative step, aniline was mineralized via catechol and meta cleavage. Even though these two steps may seem incompatible in terms of required redox conditions, they were made to coexist in a single, simple reactor. However, when aeration was optimum for growth, only 16% of the 0.5 mM nitrobenzene introduced was mineralized. Decreasing the aeration led to an increase in the amount of nitrobenzene reduced and decreased its volatilized fraction. A decrease in aeration did not slow down aniline mineralization, although the latter is catalyzed by dioxygenases. This mixed population is thus able to remediate nitrobenzene and also aniline, which is often found with the former in the environment. Using C. acidovorans, which also degrades methylanilines, or other aminoaromatic-compound-degrading organisms, this strategy should be applicable to mineralizing more complex nitroaromatic compounds, like nitrotoluenes or dinitrotoluenes.

Biodegradation of nitroaromatic pollutants: from pathways to remediation.

Nitroaromatic compounds are important contaminants of the environment, mainly of anthropogenic origin. They are produced as intermediates and products in the industrial manufacturing of dyes, explosives, pesticides, etc. Their toxicity has been extensively demonstrated in a whole range of living organisms, and nitroaromatic contamination dating from World War II is the proof of the recalcitrance of such compounds to microbial recycling. In spite of this, bacteria have evolved diverse pathways that allow them to mineralize specific nitroaromatic compounds. Degradation sequences initiated by an oxidation, an attack by a hydride ion, or a partial reduction have been documented. Some of these reactions have been exploited in bioreactors. Although pathways and enzymes involved are rather well understood, the molecular basis of these pathways is still currently under investigation. However, productive metabolism is an exception. As a rule, most bacteria are only able to reduce the nitro group into an amino function. This reduction is cometabolic: the metabolism of exogenous carbon sources is required to provide reducing equivalents. Composting and processes in bioreactors have exploited the easy reduction of the nitroaromatic compounds. In the case an amino-aromatic compound is produced, it is important to incorporate it in the remediation scheme. Some processes dealing with both nitro- and amino-aromatic compounds have been described, the amino derivative being either mineralized by the same or, more often, another microorganism, or immobilized on soil particles. Depending on the nitroaromatic compound and the environment it is contaminating, a whole range of reactions and reactor studies are now available to help devise a successful remediation strategy.

Mineralization of 14-C-U-ring labeled 4-hydroxylamino-2,6-trinitrotoluene by manganese-dependent peroxidase of the white-rot basidiomycete Phlebia radiata

The in vitro biotransformation of 4-hydroxylamino-2,6-dinitrotoluene (4-OHA-2,6-DNT)—the first identified reduction product of 2,4,6-trinitrotoluene (TNT)—was studied in the presence of a cell-free preparation of manganese-dependent peroxidase (MnP) from the white-rot basidiomycete Phlebia radiata. 4-OHA-2,6-DNT was rapidly oxidized to 4-nitroso-2,6-dinitrotoluene (4-NO-2,6-DNT), part of which reacted with the remaining 4-OHA-2,6-DNT to give 4,4′-azoxy-2,2′,6,6′-tetranitrotoluene. 4-NO-2,6-DNT was also slowly transformed to TNT and 4-amino-2,6-dinitrotoluene (4-A-2,6-DNT). In mineralization tests, 4% of the initial 14C-U-ring labeled 4-OHA-2,6-DNT was recovered as 14CO2. In the presence of up to 10 mM of reduced glutathione (GSH), 4-OHA-2,6-DNT was directly reduced to 4-A-2,6-DNT and the mineralization rate reached 27%. At 25 mM GSH, MnP was inhibited, resulting in an insignificant mineralization rate. The inclusion of GSH in the in vitro system led to a 4-OHA-2,6-DNT deficit in the HPLC mass balances not fully accounted for by the degree of mineralization, but corresponding to unidentified polar compound(s) reflecting up to 65% of the initial substrate. This is the first report of 4-OHA-2,6-DNT mineralization by a fungal MnP and the first clear-cut experimental observation of 4-NO-2,6-DNT, the previously postulated intermediate of microbial TNT metabolism.

Continuous degradation of mixtures of 4-nitrobenzoate and 4-aminobenzoate by immobilized cells of Burkholderia cepacia strain PB4

Abstract Although isolated on 4-aminobenzoate, Burkholderia cepacia strain PB4 is also able to grow on 4-nitrobenzoate. Degradation of an equimolar mixture of the nitroaromatic compound 4-nitrobenzoate and its corresponding aminoaromatic derivative 4-aminobenzoate by this strain was investigated. Batch experiments showed that, irrespective of preculturing conditions, both compounds were degraded simultaneously. The mixture-degrading ability of B. cepacia strain PB4 was subsequently tested in continuous packed bed reactors (PBR) with the strain immobilized on Celite grade R-633 or R-635. Higher degradation rates were achieved with the larger particles of Celite R-635. Maximum simultaneous degradation rates per liter of packed bed of 0.925 mmol l−1 h−1 4-nitrobenzoate and 4-aminobenzoate were obtained for an applied loading rate of the same value (0.925 mmol l−1 h−1 of each compound). Even when the applied load was not removed in its entirety, neither of the two compounds was degraded preferentially but a percentage of both of them was mineralized. The present study shows the possibility for a pure strain to biodegrade not only a nitroaromatic compound (4-nitrobenzoate) but also its corresponding amino derivative (4-aminobenzoate) continuously and simultaneously.

Cross Induction of 4-Nitrobenzoate and 4-Aminobenzoate Degradation by Burkholderia Cepacia Strain PB4

A variety of industrial activities (production of explosives, pesticides, polymers, etc.) have introduced into the environment several nitroaromatic compounds (NACs) which are toxic and recalcitrant to biodegradation by the microorganisms commonly found in nature. The most common reaction undergone by these compounds is the reduction of the nitro group, the aminoaromatic compound (AAC) then produced generally not being further degraded. Due to this ubiquitous nitroreductase activity, AACs are often found together with NACs at contaminated sites or effluents. An efficient bioremediation process should combine treatment of both classes of compounds. Burkholderia cepacia, isolated on 4-aminobenzoate as sole source of C, N and energy, was found to be also able to grow on 4-nitrobenzoate. This pure strain has thus the property to mineralize both a NAC and the corresponding AAC. Metabolic pathway studies with B. cepacia have shown that it mineralizes 4-nitrobenzoate and 4aminobenzoate through independent but converging pathways. Curiously, 4-nitrobenzoate degradation was induced by 4-aminobenzoate and vice-versa. The degradation of an equimolar mixture of these two compounds (1 mM each) by washed cells of B. cepacia grown on 4nitrobenzoate, 4-aminobenzoate or glucose was studied in batch cultures. When induced on either 4-nitrobenzoate or 4-aminobenzoate, B. cepacia metabolized the inducer faster than the second compound. When grown on glucose, the microorganism mineralized both compounds simultaneously after a short lag. No diauxic growth was observed but rather a normal growth curve. The two pathways of degradation were found to be working simultaneously and such an organism might be appropriate for the bioremediation of a NAC, in this case 4-nitrobenzoate, together with its corresponding AAC, 4-aminobenzoate.