Biotransformation and Biodegradation of N-Substituted Aromatics in Methanogenic Granular Sludge.

N-substituted aromatic compounds are environmental contaminants associated with the production and use of dyes, explosives, pesticides and pharmaceuticals among others. Nitro- and azo-substituted aromatic compounds with strong electron withdrawing groups are poorly biodegradable in aerobic treatment systems. Therefore anaerobic treatment technologies were considered in this research. The toxicity of these compounds to methanogenic bacteria was studied. Batch toxicity assays indicated that nitroaromatics and azo dyes were highly inhibitory to acetoclastic <strong></strong> methanogenic bacteria, with 50% inhibiting concentrations (50% IC) as low as 14 to 538 μM. However, the corresponding aromatic amines were several orders of magnitude less toxic.The biodegradability of eighteen N-substituted aromatic and six alkylphenol compounds under methanogenic conditions was assessed in batch assays with unadapted and 2-nitrophenol (2NP) adapted granular sludge. Net methane production indicated that all three isomers of aminobenzoate, 2-aminophenol (2AP) and 4-cresol were found to be completely mineralized by the unadapted sludge. All the other compounds tested were not degraded under the experimental conditions employed. The 2NP- adapted granular sludge showed a similar degradation spectrum but also cross acclimatized with other compounds as it was able to also mineralize 4-aminophenol and 5-aminosalicylic acid (5ASA).The facile reduction of the nitro- and azo-electron withdrawing groups was used as a detoxification strategy in continuous laboratory scale (160 mL) upward-flow anaerobic sludge bed reactors (UASB), supplied with either a mixture of volatile fatty acids (VFA) or glucose and selected nitroaromatic compounds. The nitroaromatics tested included: 2NP, 4-nitrophenol, 2,4-dinitrophenol, 2,4-dinitrotoluene, 4-nitrobenzoic acid (4NBc), 5-nitrosalicylic acid (5NSA) and nitrobenzene. All compounds were efficiently reduced to their corresponding aromatic amines and the primary substrate chemical oxygen demand (COD) was efficiently converted to methane even at influent nitroaromatic concentrations exceeding the 50% IC values by up to 30-fold. After long term reactor operation (several months), aromatic amines were no longer observed to accumulate as products of 4NBc, 2NP and 5NSA elimination. The granular sludge sampled from these reactors were able to fully mineralize 4- aminobenzoate, 2AP and 5ASA to methane when these were offered as the sole carbon and energy source in anaerobic biodegradability assays. These results suggest that 4NBc, 2NP and 5NSA were completely biodegraded in the continuous reactors at nitroaromatic loading rates up to 312, 910 and 553 mg/L-d, respectively.Continuous <strong></strong> UASB reactors were also run with the azo dye Mordant Orange 1 [MO1, 5-(4- nitrophenylazo)salicylic acid] with either no primary substrate, glucose or VFA. Except for the first few weeks, no elimination of azo dye was evident in the column receiving no primary substrates. On the other hand, MO I was readily cleaved in the reactors (>99%) receiving glucose and VFA at MO I loading rates up to 295 and 161 mg/L-d, respectively. In these reactors, both 1,4-phenyIenediamine (1,4PDA) and 5ASA were detected as products of MO1 cleavage. After 180 days, 5ASA arising from MO1 cleavage could only be detected at trace concentrations in the glucose fed reactor. The sampled sludge was able to rapidly mineralize 5ASA to methane in the anaerobic biodegradability assay. The results suggest that MO1 was cleaved into 1,4PDA and 5ASA; and that 5ASA was fully degraded by the anaerobic consortia; whereas, 1,4PDA persisted. Azodisalicylate (ADS), a pharmaceutical azo dye constructed from two 5ASA units, .vas completely mineralized in UASB reactors at ADS loading rates up to 225 mg/L-d even in the absence of cosubstrate, indicating that the metabolism of 5ASA could provide the reducing equivalents needed for the azo reduction. Batch experiments confirmed the ADS mineralization.The results of this research demonstrated that anaerobic treatment is a feasible technology for the treatment of highly toxic nitroaromatics and azo dyes. It was also shown that some nitroaromatic compounds and azo dyes can be completely mineralized and serve serve as a carbon, energy and nitrogen source for anaerobic bacteria, in contrast to the common assumption that they arc only biotransformed to mutagenic and carcinogenic aromatic amine