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Pentaerythritol tetranitrate (PETN) is a nitrate ester explosive that may be persistent with scarce reports on its environmental fate and impacts. Our main objective was to isolate and characterize bacteria that transform PETN under aerobic and anaerobic conditions. Biotransformation of PETN (100 mg L-1) was evaluated using mineral medium with (M + C) and without (M - C) additional carbon sources under aerobic conditions and with additional carbon sources under anaerobic conditions. Here, we report on the isolation of 12 PETN-transforming cultures (4 pure and 8 co-cultures) from environmental samples collected at an explosive manufacturing plant. The highest transformation of PETN was observed for cultures in M + C under aerobic conditions, reaching up to 91% ± 2% in 2 d. Under this condition, PETN biotransformation was observed in conjunction with the release of nitrites and bacterial growth. No substantial transformation of PETN (<45%) was observed during 21 d in M - C under aerobic conditions. Under anaerobic conditions, five cultures could transform PETN (up to 52% ± 13%) as the sole nitrogen source, concurrent with the formation of two unidentified metabolites. PETN-transforming cultures belonged to Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Actinobacteria. In conclusion, we isolated 12 PETN-transforming cultures belonging to diverse taxa, suggesting that PETN transformation is phylogenetically widespread.

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Oily sludge is a residue from the petroleum industry composed of a mixture of sand, water, metals, and high content of hydrocarbons (HCs). The heavy oily sludge used in this study originated from Colombian crude oil with high density and low American Petroleum Institute (API) gravity. The residual waste from heavy oil processing was subject to thermal and centrifugal extraction, resulting in heavy oily sludge with very high density and viscosity. Biodegradation of the total petroleum hydrocarbons (TPH) was tested in microcosms using several bioremediation approaches, including: biostimulation with bulking agents and nutrients, the surfactant Tween 80, and bioaugmentation. Select HC degrading bacteria were isolated based on their ability to grow and produce clear zones on different HCs. Degradation of TPH in the microcosms was monitored gravimetrically and with gas chromatography (GC). The TPH removal in all treatments ranged between 2 and 67%, regardless of the addition of microbial consortiums, amendments, or surfactants within the tested parameters. The results of this study demonstrated that bioremediation of heavy oily sludge presents greater challenges to achieve regulatory requirements. Additional physicochemical treatments analysis to remediate this recalcitrant material may be required to achieve a desirable degradation rate.

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Acid mine drainage (AMD) is the major pollutant generated by the mining industry, and it is characterized by low pH and high concentration of metals and sulfate. The use of biochemical passive reactors (BPRs) is a promising strategy for its bioremediation. To date, there are various studies describing the taxonomical composition of BPR microbial communities, generally consisting of an assemblage of sulfate-reducing organisms inside Deltaproteobacteria, and a diverse set of anaerobic (ligno)cellulolytic bacteria; however, insights about its functional metagenomic content are still scarce. In previous studies, a laboratory-scale AMD bioremediation using biochemical passive reactors was designed and performed, tracking operation parameters, chemical composition, and changes, together with taxonomic composition of the microbiomes harbored in these systems. In order to reveal the main functional content of these communities, we used shotgun metagenomics analyses to explore genes of higher relative frequencies and their inferred functions during the AMD bioremediation from three BPRs representing the main microbiome compositions detected in the system. Remarkably, genes encoding for two-component regulatory systems and ABC transporters related to metal and inorganic ions, cellulose degradation enzymes, dicarboxylic acid production, and sulfite reduction complex were all detected at increased frequency. Our results evidenced that higher taxonomic diversity of the microbiome was arising together with a functional redundancy of the specific metabolic roles, indicating its co-selection and suggesting that its enrichment on BPRs may be implicated in the cumulative efficiency of these systems.

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The nitrated compounds 2,4-dinitrotoluene (2,4-DNT), 2,4,6-trinitrotoluene (TNT), and pentaerythritol tetranitrate (PETN) are toxic xenobiotics widely used in various industries. They often coexist as environmental contaminants. The aims of this study were to evaluate the transformation of 100 mg L-1 of TNT, 2,4-DNT, and PETN by Raoultella planticola M30b and Rhizobium radiobacter M109c and identify enzymes that may participate in the transformation. These strains were selected from 34 TNT transforming bacteria. Cupriavidus metallidurans DNT was used as a reference strain for comparison purposes. Strains DNT, M30b and M109c transformed 2,4-DNT (100%), TNT (100, 94.7 and 63.6%, respectively), and PETN (72.7, 69.3 and 90.7%, respectively). However, the presence of TNT negatively affects 2,4-DNT and PETN transformation (inhibition > 40%) in strains DNT and M109c and fully inhibited (100% inhibition) 2,4-DNT transformation in R. planticola M30b.Genomes of R. planticola M30b and R. radiobacter M109c were sequenced to identify genes related with 2,4-DNT, TNT or PETN transformation. None of the tested strains presented DNT oxygenase, which has been previously reported in the transformation of 2,4-DNT. Thus, unidentified novel enzymes in these strains are involved in 2,4-DNT transformation. Genes encoding enzymes homologous to the previously reported TNT and PETN-transforming enzymes were identified in both genomes. R. planticola M30b have homologous genes of PETN reductase and xenobiotic reductase B, while R. radiobacter M109c have homologous genes to GTN reductase and PnrA nitroreductase. The ability of these strains to transform explosive mixtures has a potentially biotechnological application in the bioremediation of contaminated environments.

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ResumenLa explotación del petróleo y el uso de sus derivados han contribuido para el desarrollo tecnológico a nivel mundial. Esta actividad, no obstante, genera muchos subproductos los cuales atentan contra la salud del ambiente. Las aguas de producción, por ejemplo, pueden contener trazas de metales pesados e hidrocarburos, razón por la cual deben ser reincorporadas de forma adecuada al ambiente. En este trabajo, se aislaron, caracterizaron e identificaron molecularmente ocho bacterias en base a su capacidad para degradar fenol. Primero, se evaluó el crecimiento y la degradación de fenol bajo diferentes concentraciones (500, 800 y 1 200 mg / L), y posteriormente se emplearon diseños estadísticos para la selección de condiciones óptimas de degradación. Los resultados mostraron que las ocho cepas evaluadas fueron capaces de usar el fenol como fuente única de carbono; no obstante, las cinéticas de degradación y la máxima tolerancia de las cepas al fenol variaron ampliamente. Debido a su tolerancia y capacidad para metabolizar fenol, la cepa Pseudomonas sp. Sps1 fue seleccionada para posteriores estudios. Mediante el uso de un diseño de Plackett Burman y un diseño factorial fraccionado en el cual diferentes fuentes de carbono, nitrógeno, y fósforo fueron evaluadas y degradación de fenol fue usado como variable de respuesta, fue posible seleccionar las condiciones óptimas para la degradación de fenol por Sps1. Finalmente, se emplearon estas condiciones para evaluar la degradación de fenol en aguas de re-uso provenientes directamente de la industria petrolera. Los resultados, mostraron que la bioestimulación favoreció el proceso de degradación del fenol comparado con el control. En conclusión, en este estudio se demostró que la bioaumentación con bacterias nativas acompañada de bioestimulación generan una tecnología económica y ambientalmente amigable para la remoción de contaminantes en aguas residuales tratadas de la industria petrolera.

AbstractOil exploitation and the usage of its derivatives have undeniably contributed to the technological advance worldwide. This industrial activity, however, generates several by-products that can threaten environmental sustainability. Seawage, for example, can contain organic pollutants and heavy metals; therefore, its deposition must be preceded by adequate water treatment. In this study, we isolated, characterized, and molecularly identified eight bacteria on the basis of their capability to degrade phenol. First, we determined the rates of bacterial growth and phenol degradation using different concentrations of the aromatic hydrocarbon (500, 800 and 1 200 mg / L), and then used sequential statistical designs to select optimal conditions for its degradation. Results showed that all isolated strains were capable of degrading phenol as the sole carbon source; the degradation kinetics and phenol tolerance, however, widely varied among strains. We chose the strain Pseudomonas sp. Sps1 for further studies due to its remarkable tolerance and capability to degrade phenol. By using a Plackett Burman design, followed by a fractionated factorial design in which several carbon, nitrogen, and phosphorus sources were evaluated, and phenol degradation was used as the response variable, we found optimal conditions for phenol degradation by Sps1. Finally, we compared phenol degradation in seawage by the use or not of the optimal conditions established by the statistical methods. Results showed that biostimulation dramatically increased phenol degradation compared with the control. In conclusion, we found that bioaugmentation with the native bacteria Sps1 and a statistically-based biostimulation approach provided an economically and environmentally friendly alternative for the removal of pollutants from oil industry sewage.

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Biochemical passive treatment represents a promising option for the remediation of acid mine drainage. This study determined the effect of three hydraulic retention times (1, 2, and 4 days) on changes in system efficiency, reactive mixture, and microbial activity in bioreactors under upward flow conditions. Bioreactors were sacrificed in the weeks 8, 17 and 36, and the reactive mixture was sampled at the bottom, middle, and top layers. Physicochemical analyses were performed on reactive mixture post-treatment and correlated with sulfate-reducing bacteria and cellulolytic and dehydrogenase activity. All hydraulic retention times were efficient at increasing pH and alkalinity and removing sulfate (>60%) and metals (85-99% for Fe(2+) and 70-100% for Zn(2+)), except for Mn(2+). The longest hydraulic retention time (4 days) increased residual sulfides, deteriorated the quality of treated effluent and negatively impacted sulfate-reducing bacteria. Shortest hydraulic retention time (1 day) washed out biomass and increased input of dissolved oxygen in the reactors, leading to higher redox potential and decreasing metal removal efficiency. Concentrations of iron, zinc and metal sulfides were high in the bottom layer, especially with 2 day of hydraulic retention time. Sulfate-reducing bacteria, cellulolytic and dehydrogenase activity were higher in the middle layer at 4 days of hydraulic retention time. Hydraulic retention time had a strong influence on overall performance of passive reactors.

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Pentolite is a mixture (1:1) of 2,4,6-trinitrotoluene (TNT) and pentaerythritol tetranitrate (PETN), and little is known about its fate in the environment. This study was aimed to determine the dissipation of pentolite in soils under laboratory conditions. Microcosm experiments conducted with two soils demonstrated that dissipation rate of PETN was significantly slower than that of TNT. Interestingly, the dissipation of PETN was enhanced by the presence of TNT, while PETN did not enhanced the dissipation of TNT. Pentolite dissipation rate was significantly faster under biostimulation treatment (addition of carbon source) in soil from the artificial wetland, while no such stimulation was observed in soil from detonation field. In addition, the dissipation rate of TNT and PETN in soil from artificial wetland under biostimulation was significantly faster than the equivalent abiotic control, although it seems that non-biological processes might also be important for the dissipation of TNT and PETN. Transformation of PETN was also slower during establishment of enrichment culture using pentolite as the sole nitrogen source. In addition, transformation of these explosives was gradually reduced and practically stopped after the forth cultures transfer (80 days). DGGE analysis of bacterial communities from these cultures indicates that all consortia were dominated by bacteria from the order Burkholderiales and Rhodanobacter. In conclusion, our results suggest that PETN might be more persistent than TNT.

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Se evaluó el proceso de bioestimulación por nutrientes utilizando fertilizantes inorgánicos compuestos (FIC) N:P:K 28:12:7 y sales inorgánicas simples (SIS) NH4NO3 y K2HPO4 en suelos contaminados con hidrocarburos utilizando respirometría. El suelo fue contaminado con lodos aceitosos a una concentración 40.000 mgTPH/kgps. Para cuantificar el consumo de oxígeno se utilizaron dos respirómetros de medición manométrica HACH® 2173b y OXITOP® PF600 durante ensayos de 13 días (n=3). Se evaluaron dos tratamientos (FIC y SIS) y tres controles (abiótico, sustrato de referencia y sin nutrientes). Se analizaron parámetros físico-químicos (pH, nutrientes y TPH) y microbiológicos (heterótrofos y degradadores) al inicio y al final de cada ensayo. SIS y el control sin nutrientes presentaron las mayores tasas de respiración, en el equipo HACH se obtuvieron valores de 802,28 y 850,72 mgO2kgps-1d-1 respectivamente, y en OXITOP fueron de 936,65 y 502,05 mgO2kgps-1d-1, respectivamente, indicando que los nutrientes de SIS estimularon el metabolismo microbiano. Por otro lado, FIC presentó los recuentos y tasas de respiración más bajas (188,18 y 139,87 mgO2kgps-1d-1 en HACH y OXITOP, respectivamente), esto pudo estar relacionado a un efecto inhibitorio generado por la acumulación de amoniaco, limitando el crecimiento de la población degradadora.

The biostimulation process was evaluated in a hydrocarbon contaminated soil by respirometry after amendment with inorganic compound fertilizer (ICF) (N:P:K 28:12:7) and simple inorganic salts (SIS) (NH4NO3 and K2HPO4). The soil was contaminated with oily sludge (40,000 mgTPH/kgdw). The oxygen uptake was measured using two respirometers (HACH® 2173b and OXITOP® PF600) during thirteen days (n=3). Two treatments (ICF and SIS) and three controls (abiotic, reference substance and without nutrients) were evaluated during the study. Physicochemical (pH, nutrients, and TPH) and microbiological analysis (heterotrophic and hydrocarbon-utilizing microorganisms) were obtained at the beginning and at the end of each assay. Higher respiration rates were recorded in SIS and without nutrient control. Results were 802.28 and 850.72-1d-1,mgO2kgps-1d-1 in HACH, while in OXITOP were 936.65 and 502.05 mgO2kgps respectively. These data indicate that amendment of nutrients stimulated microbial metabolism. ICF had lower respiration rates (188.18 and 139.87 mgO2kgps-1d-1 in HACH and OXITOP, respectively) as well as counts, this could be attributed to ammonia toxicity.

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El presente estudio evaluó el desempeño de dos sales de tetrazolio, una tradicional: INT y una de nueva generación: XTT, para estimar la densidad de microorganismos degradadores de hidrocarburos (HCs) en suelos empleando la técnica del Número Más Probable (NMP). Se analizaron 96 muestras de suelo provenientes de la Ecorregión Cafetera de Colombia. Los microorganismos fueron recuperados en agar mínimo de sales en atmósfera saturada de HCs y la capacidad degradadora fue confirmada por repiques sucesivos utilizando diesel como fuente de carbono. No se observaron diferencias significativas en los recuentos de microorganismos degradadores obtenidos con las dos sales (t de Student, p < 0,05), pero el XTT permitió mejor visualización de los pozos positivos dada la solubilidad del producto reducido, mientras que el INT produjo precipitación, debido al formazán insoluble generado, dificultando su lectura. Se obtuvo un mayor porcentaje de aislamientos empleando XTT (67%), lo cual podría indicar que el tipo de sal es determinante en la viabilidad de estas bacterias. Adicionalmente, se evaluó el límite de detección celular, las condiciones óptimas de concentración de XTT y el tiempo de incubación necesario para la detección de actividad degradadora utilizando la cepa Acinetobacter sp. El aumento en la concentración de XTT de 0,5 mM a 2 mM y el tiempo de incubación tuvieron un efecto inhibitorio y favorable respectivamente, en la recuperación de células viables, adicionalmente, límite de detección de la técnica fue de 10² UFC/ml.

The objective of this study was to evaluate the performance of two tetrazolium indicators: a traditional one: INT and a new generation one: XTT, for the estimation of hydrocarbon (HC) degrading microorganism s density using the Most Probable Number Technique (MPN). Ninety six composite soil samples were taken and analyzed from Ecorregión Cafetera Colombiana. Degrading microorganisms were recovered in minimum salt medium with saturated HC atmosphere. Degrading HC capacity of the microorganisms was confirmed by successive subcultures in the same medium using diesel as only carbon source. Counts obtained with the two salts were not significantly different (Student t test, p < 0,05) but XTT allowed an easier visualization of positive wells due to product solubility of the reduce product. A greater percentage of isolates was obtained using XTT (67%), which suggests that salt type is relevant for recovering of these microorganisms. Additionally, cell detection limit, optimal conditions of XTT concentration and incubation times for detection of activity were evaluated. This evaluation was performed by means of microplate format for hydrocarbon degrading microorganisms using Acinetobacter sp. An inhibitory effect was observed in the recovering of cultivable cells when XTT concentrations increased from 0,5 mM to 2 mM. Incubation time favored this recovering. Detection limit of this technique was established at 10² UFC/ml. Production of the XTT-formazan was positively related with initial cell concentration and negatively with incubation time.