Biodegradation of the emerging contaminant 3-nitro-1,2,4-triazol-5-one and its product 3-amino-1,2,4-triazol-5-one in perlite/soil columns.

3-Nitro-1,2,4-triazol-5-one (NTO) is an ingredient of new safer-to-handle military insensitive munitions formulations. NTO can be microbially reduced to 3-amino-1,2,4-triazol-5-one (ATO) under anaerobic conditions if an electron donor is available. Conversely, ATO can undergo aerobic biodegradation. Previously, our research group developed an anaerobic enrichment culture that reduces NTO to ATO. A second culture could aerobically mineralize ATO. This study aimed to combine anaerobic/aerobic conditions within a down-flow perlite/soil column for simultaneous NTO reduction and ATO oxidation. Acetate biostimulation was investigated to promote oxygen depletion and create anaerobic micro-niches for NTO reduction, whereas perlite increased soil porosity and oxygen convection, allowing ATO oxidation. Two columns packed with a perlite/soil mixture (70:30, wet wt.%) or 100% perlite were operated aerobically and inoculated with the NTO- and ATO-degrading cultures. Initially, the influent consisted of ∼280 µM ATO, and after 30 days, the feeding was switched to ∼260 µM NTO and ∼250 µM acetate. By progressively increasing acetate from 250 to 4000 µM, the NTO removal gradually improved in both columns. The perlite/soil column reached a 100% NTO removal after 4000 µM acetate was supplemented. Additionally, there was no ATO accumulation, and inorganic nitrogen was produced, indicating ATO mineralization. Although NH4+ was produced following ATO oxidation, most nitrogen was recovered as NO3- likely via nitrification reactions. Microbial community analysis revealed that phylotypes hosted in the enrichment cultures specialized in NTO reduction (e.g., Geobacter) and ATO oxidation (e.g., Hydrogenophaga, Ramlibacter, Terrimonas, and Pseudomonas) were established in the columns. Besides, the predominant genera (Azohydromonas, Zoogloea, and Azospirillum) are linked to nitrogen cycling by performing nitrogen fixation, NO3- reduction, and nitroaromatics degradation. This study applied a bulking agent (perlite) and acetate biostimulation to achieve simultaneous NTO reduction and ATO oxidation in a single column. Such a strategy can assist with real-world applications of NTO and ATO biodegradation mechanisms.

Elucidating the mechanisms associated with the anaerobic biotransformation of the emerging contaminant nitroguanidine.

Nitroguanidine (NQ) is a constituent of gas generators for automobile airbags, smokeless pyrotechnics, insecticides, propellants, and new insensitive munitions formulations applied by the military. During its manufacture and use, NQ can easily spread in soils, ground, and surface waters due to its high aqueous solubility. Very little is known about the microbial biotransformation of NQ. This study aimed to elucidate important mechanisms operating during NQ anaerobic biotransformation. To achieve this goal, we developed an anaerobic enrichment culture able to reduce NQ to nitrosoguanidine (NsoQ), which was further abiotically transformed to cyanamide. Effective electron donors for NQ biotransformation were lactate and, to a lesser extent, pyruvate. The results demonstrate that the enrichment process selected a sulfate-reducing culture that utilized lactate as its electron donor and sulfate as its electron acceptor while competing with NQ as an electron sink. A unique property of the culture was its requirement for exogenous nitrogen (e.g., from yeast extract or NH4Cl) for NQ biotransformation since NQ itself did not serve as a nitrogen source. The main phylogenetic groups associated with the NQ-reducing culture were sulfate-reducing and fermentative bacteria, namely Cupidesulfovibrio oxamicus (63.1% relative abundance), Dendrosporobacter spp. (12.0%), and Raoultibacter massiliens (10.9%). The molecular ecology results corresponded to measurable physiological properties of the most abundant members. The results establish the conditions for NQ anaerobic biotransformation and the microbial community associated with the process, improving our present comprehension of NQ environmental fate and assisting the development of NQ remediation strategies.

The key role of biogenic arsenic sulfides in the removal of soluble arsenic and propagation of arsenic mineralizing communities.

Biogeochemical processes govern the transport and availability of arsenic in sediments. However, little is known about the transition from indigenous communities to cultivable consortia when exposed to high arsenic concentrations. Such cultivable communities could be exploited for arsenic bioremediation of waste streams and polluted sites. Thus, it is crucial to understand the dynamics and selective pressures that shape the communities during the development of customized bacterial consortia. First, from the arsenic partitioning of two sediments with high arsenic concentrations, we found that up to 55% of arsenic was bioavailable because it was associated with the soluble, carbonate, and ionically exchangeable fractions. Next, we prepared sediment enrichment cultures under arsenate- and sulfate-reducing conditions to precipitate arsenic sulfide biominerals and analyze the communities. The produced biominerals were used as the inoculum to develop bacterial consortia via successive transfers. Tracking of the 16S rRNA gene in the fresh sediments, sediment enrichments, biogenic minerals, and bacterial consortia revealed differences in the bacterial communities. Removing the sediment caused a substantial decrease in diversity and shifts toward the dominance of the Firmicutes phylum to the detriment of Proteobacteria. In agreement with the 16S rRNA gene results, the sequencing of the arrA gene confirmed the presence of phylotypes closely related to Desulfosporosinus sp. Y5 (100% similarity), highlighting the pivotal role of this genus in the removal of soluble arsenic. Here, we demonstrated for the first time that besides being important as arsenic sinks, the biogenic arsenic sulfide minerals are reservoirs of arsenic resistant/respiring bacteria and can be used to culture them.

Reductive transformation of the insensitive munitions compound nitroguanidine by different iron-based reactive minerals.

Nitroguanidine (NQ) is an emerging contaminant being used by the military as a constituent of new insensitive munitions. NQ is also used in rocket propellants, smokeless pyrotechnics, and vehicle restraint systems. Its uncontrolled transformation in the environment can generate toxic and potentially mutagenic products, posing hazards that need to be remediated. NQ transformation has only been investigated to a limited extent. Thus, it is crucial to expand the narrow spectrum of NQ remediation strategies and understand its transformation pathways and end products. Iron-based reactive minerals should be investigated for NQ treatment because they are successfully used in existing technologies, such as permeable reactive barriers, for treating a wide range of organic pollutants. This study tested the ability of micron-sized zero-valent iron (m-ZVI), mackinawite, and commercial FeS, to transform NQ under anoxic conditions. NQ transformation followed pseudo-first-order kinetics. The reaction rate constants decreased as follows: commercial FeS > mackinawite > m-ZVI. For the assessed minerals, the NQ transformation started with the reduction of the nitro group forming nitrosoguanidine (NsoQ). Then, aminoguanidine (AQ) was accumulated during the reaction of NQ with m-ZVI, accounting for 86% of the nitrogen mass recovery. When NQ was reacted with commercial FeS, 45% and 20% of nitrogen were recovered as AQ and guanidine, respectively, after 24 h. Nonetheless, NsoQ persisted, contributing to the N-balance. When mackinawite was present, NsoQ disappeared, but AQ was not detected, and guanidine accounted for 11% of the nitrogen recovery. AQ was ultimately transformed into cyanamide, whose dimerization triggered the formation of cyanoguanidine. Alternatively, NsoQ was transformed into guanidine, which reacted with cyanamide to form biguanide. This is the first report systematically investigating the NQ transformation by different iron-based reactive minerals. The evidence indicates that these minerals are attractive alternatives for developing NQ remediation strategies.

Designing bacterial consortia for the complete biodegradation of insensitive munitions compounds in waste streams.

Insensitive munitions compounds (IMCs), such as 2,4-dinitroanisole (DNAN) and 3-nitro-1,2,4-triazol-5-one (NTO), are replacing conventional explosives in munitions formulations. Manufacture and use of IMCs generate waste streams in manufacturing plants and load/assemble/pack facilities. There is a lack of practical experience in executing biodegradation strategies to treat IMCs waste streams. This study establishes a proof-of-concept that bacterial consortia can be designed to mineralize IMCs and co-occurring nitroaromatics in waste streams. First, DNAN, 4-nitroanisole (4-NA), and 4-chloronitrobenzene (4-CNB) in a synthetic DNAN-manufacturing waste stream were biodegraded using an aerobic fluidized-bed reactor (FBR) inoculated with Nocardioides sp. JS 1661 (DNAN degrader), Rhodococcus sp. JS 3073 (4-NA degrader), and Comamonadaceae sp. LW1 (4-CNB degrader). No biodegradation was detected when the FBR was operated under anoxic conditions. Second, DNAN and NTO were biodegraded in a synthetic load/assemble/pack waste stream during a sequential treatment comprising: (i) aerobic DNAN biodegradation in the FBR; (ii) anaerobic NTO biotransformation to 3-amino-1,2,4-triazol-5-one (ATO) by an NTO-respiring enrichment; and (iii) aerobic ATO mineralization by an ATO-oxidizing enrichment. Complete biodegradation relied on switching redox conditions. The results provide the basis for designing consortia to treat mixtures of IMCs and related waste products by incorporating microbes with the required catabolic capabilities.

Quinone Moieties Link the Microbial Respiration of Natural Organic Matter to the Chemical Reduction of Diverse Nitroaromatic Compounds.

Insensitive munitions compounds (IMCs) are emerging nitroaromatic contaminants developed by the military as safer-to-handle alternatives to conventional explosives. Biotransformation of nitroaromatics via microbial respiration has only been reported for a limited number of substrates. Important soil microorganisms can respire natural organic matter (NOM) by reducing its quinone moieties to hydroquinones. Thus, we investigated the NOM respiration combined with the abiotic reduction of nitroaromatics by the hydroquinones formed. First, we established nitroaromatic concentration ranges that were nontoxic to the quinone respiration. Then, an enrichment culture dominated by Geobacter anodireducens could indirectly reduce a broad array of nitroaromatics by first respiring NOM components or the NOM surrogate anthraquinone-2,6-disulfonate (AQDS). Without quinones, no nitroaromatic tested was reduced except for the IMC 3-nitro-1,2,4-triazol-5-one (NTO). Thus, the quinone respiration expanded the spectrum of nitroaromatics susceptible to transformation. The system functioned with very low quinone concentrations because NOM was recycled by the nitroaromatic reduction. A metatranscriptomic analysis demonstrated that the microorganisms obtained energy from quinone or NTO reduction since respiratory genes were upregulated when AQDS or NTO was the electron acceptor. The results indicated microbial NOM respiration sustained by the nitroaromatic-dependent cycling of quinones. This process can be applied as a nitroaromatic remediation strategy, provided that a quinone pool is available for microorganisms.

Dissolution and final fate of arsenic associated with gypsum, calcite, and ferrihydrite: Influence of microbial reduction of As(V), sulfate, and Fe(III).

Several studies have demonstrated that gypsum (CaSO4·2H2O) and calcite (CaCO3) can be important hosts of arsenic in contaminated hydrogeological systems. However, the extent to which microbial reducing processes contribute to the dissolution and transformation of carbonate and sulfate minerals and, thereby, to arsenic mobilization is poorly understood. These processes are likely to have a strong impact on arsenic mobility in iron-poor environments and in reducing aquifers where iron oxyhydroxides become unstable. Anoxic batch bioassays with arsenate (As(V)) coprecipitated with calcite, gypsum, or ferrihydrite (Fe(OH)3) were conducted in the presence of sulfate or molybdate to examine the impact of bioprocesses (i.e. As(V), sulfate, and Fe(III)-reduction) on arsenic dissolution, speciation, and eventual remineralization. Microbial reduction of As(V)-bearing calcite caused an important dissolution of arsenite, As(III), which remained in solution up to the end of the experiment (30 days). The reduction of As(V) from gypsum-As(V) also led to the release of As(III), which was subsequently remineralized, possibly as arsenic sulfides. The presence of sulfate triggered arsenic dissolution in the bioassays with ferrihydrite-As(V). This study showed that although gypsum and calcite have a lower capacity to bind arsenic, compared to iron oxides, they can play a critical role in the biogeochemical cycle of arsenic in natural calcareous and gypsiferous systems depleted of iron since they can be a source of electron acceptors for reducing bioprocesses.

Role of indigenous microbiota from heavily contaminated sediments in the bioprecipitation of arsenic.

High arsenic concentrations have been detected in alluvial aquifers of arid and semi-arid zones in Mexico. This work describes the potential of microbial arsenate reduction of the indigenous community present in sediments from an arsenic contaminated aquifer. Microcosms assays were conducted to evaluate arsenate and sulfate-reducing activities of the native microbiota. Two different sediments were used as inoculum in the assays amended with lactate (10mM) as electron donor and with sulfate and arsenate (10mM each) as electron acceptors. Sediments were distinguished by their concentration of total arsenic 238.3±4.1mg/kg or 2263.1±167.7mg/kg, which may be considered as highly contaminated sediments with arsenic. Microbial communities present in both sediments were able to carry out arsenate reduction, accomplished within 4days, with the corresponding formation of arsenite; sulfate reduction took place as well. Both reducing activities occurred without previous acclimation period or enrichment, even at potential inhibitory concentrations of arsenate as high as 750mg/L (10mM). The formation of a yellowish colloidal precipitate was evident when both reducing processes occurred in the microcosm, which contributed to remove between 52 and 90.9% of As(III) from the liquid phase by bioprecipitation of arsenic as arsenic sulfide.