Bacteria isolated from explosive contaminated environments transform pentaerythritol tetranitrate (PETN) under aerobic and anaerobic conditions.

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.

Bioremediation of heavy oily sludge: a microcosms study.

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.

Metagenomic and genomic characterization of heavy metal tolerance and resistance genes in the rhizosphere microbiome of Avicennia germinans in a semi-arid mangrove forest in the tropics.

Mangroves are often exposed to heavy metals that accumulate in the food chain, generate toxicity to mangrove plants and affect microbial diversity. This study determined the abundance of genes associated with resistance and tolerance to heavy metals in the rhizosphere microbiome of Avicennia germinans from a semi-arid mangrove of La Guajira-Colombia by metagenomics and genomics approach. Twenty-eight genes associated with tolerance and 49 genes related to resistance to heavy metals were detected. Genes associated with tolerance and resistance to Cu, especially cusA and copA, were the most abundant. The highest number of genes for tolerance and resistance were for Zn and Co, respectively. The isolate Vibrio fluvialis showed the ability to tolerate Cu, Ni, Zn, and Cd. This work used a complementary approach of metagenomics and genomics to characterize the potential of mangrove microorganisms to tolerate and resist heavy metals and the influence of salinity on their abundance.

Metagenomic Analysis of Biochemical Passive Reactors During Acid Mine Drainage Bioremediation Reveals Key Co-selected Metabolic Functions.

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.

Liver biomarkers response of the neotropical fish Aequidens metae to environmental stressors associated with the oil industry.

The Acacias River in Colombia receives large volumes of industrial effluents mostly derived from the oil industry. To contribute to the study of the possible effects of industrial wastewaters on the aquatic environment and particularly on fish populations, a native neotropical fish, Aequidens metae was used as a sentinel species. Wild specimens of A. metae were caught at three different places of the Acacias River taking as reference the point of discharge of an oil industry effluent; upstream, downstream, and at the vicinity of the discharge pipe. A fourth sampling site was chosen as a reference site away from urban settlements. Samplings were performed twice, during the rainy and dry seasons. After anesthesia animals were weighted and measured, and humanely sacrificed. Livers were extracted, frozen on site and transported to the laboratory. Condition indices were calculated. Total protein content and the detoxification 7-ethoxyresorufin-O-deethylase (EROD) enzyme activity were estimated. Histopathological alterations were also evaluated. Water quality was estimated through the measurement of several variables. Results obtained evidenced that the highest induction in EROD activity and the strongest histological alterations in liver of the monitored fish appeared during the dry seasons at the discharge site and downstream to this point.

Transformation of TNT, 2,4-DNT, and PETN by Raoultella planticola M30b and Rhizobium radiobacter M109 and exploration of the associated enzymes.

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.

Effect of hydraulic retention time on microbial community in biochemical passive reactors during treatment of acid mine drainage.

The effect of hydraulic retention time (HRT) on the microbial community during acid mine drainage (AMD) treatment was investigated. Physicochemical and molecular (illumina and qPCR) analyses were performed on reactive mixtures collected from seven bioreactors in three-operation period (8, 17 and 36weeks). Long HRT (4day) favored the relative abundance of SRB, causing the increase of residual sulfides and short HRT (1day) affected the anaerobic conditions of the bioreactors and favored the presence the acidophilic chemolithotrophic microorganisms. Besides qPCR indicated that genes related to cellulose degradation were present in low copy numbers and were affected by the HRT. Finally, environmental factors (pH, organic source, metal sulfides, and sulfate concentrations) had significant impact on relative abundance of the phylogenetic lineages, rather than the types of lineages present in the reactive mixture. The findings of this study indicate that HRT affects the stability of passive bioreactors and their microbial communities.

Biochemical passive reactors for treatment of acid mine drainage: Effect of hydraulic retention time on changes in efficiency, composition of reactive mixture, and microbial activity.

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.

Persistence of pentolite (PETN and TNT) in soil microcosms and microbial enrichment cultures.

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.

Prevalence of the gene trzN and biogeographic patterns among atrazine-degrading bacteria isolated from 13 Colombian agricultural soils.

The following study evaluated the diversity and biogeography of 83 new atrazine-degrading bacteria and the composition of their atrazine degradation genes. These strains were isolated from 13 agricultural soils and grouped according to rep-PCR genomic fingerprinting into 11 major clusters, which showed biogeographic patterns. Three clusters (54 strains) belonged to the genus Arthrobacter, seven clusters (28 strains) were similar to the genus Nocardioides and only one strain was a gram-negative from the genus Ancylobacter. PCR assays for the detection of the genes atzA, B, C, D, E, F and trzN conducted with each of the 83 strains revealed that 82 strains (all gram positive) possessed trzN, 74 of them possessed the combination of trzN, atzB and atzC, while only the gram-negative strain had atzA. A similar PCR assay for the two analogous genes, atzA and trzN, responsible for the first step of atrazine degradation, was performed with DNA extracted directly from the enrichment cultures and microcosms spiked with atrazine. In these assays, the gene trzN was detected in each culture, while atzA was detected in only six out of 13 soils. These results raise an interesting hypothesis on the evolutionary ecology of the two atrazine chlorohydrolase genes (i.e. atzA and trzN) and about the biogeography of atrazine-degrading bacteria.

Improved purification and PCR amplification of DNA from environmental samples.

Purification and PCR amplification procedures for DNA extracted from environmental samples (soil, compost, and river sediment) were improved by introducing three modifications: precipitation of DNA with 5% polyethylene glycol 8000 (PEG) and 0.6 M NaCl; filtration with a Sepharose 4B-polyvinylpolypyrrolidone (PVPP) spin column; and addition of skim milk (0.3% w/v) to the PCR reaction solution. Humic substances' concentration after precipitation with 5% PEG was 2.57-, 5.3-, and 78.9-fold lower than precipitation with 7.5% PEG, 10% PEG, and isopropanol, respectively. After PEG precipitation, Sepharose, PVPP and the combined (Sepharose-PVPP) column removed 92.3%, 89.5%, and 98%, respectively, of the remaining humic materials. Each of the above-mentioned modifications improved PCR amplification of the 16S rRNA gene. DNA extracted by the proposed protocol is cleaner than DNA extracted by a commercial kit. Nevertheless, the improvement of DNA purification did not improve the detection limit of atrazine degradation gene atzA.

Treatment of high-strength dairy wastewater in an anaerobic deep reservoir: analysis of the methanogenic fermentation pathway and the rate-limiting step.

The wastewater of the largest dairy factory in Israel (Tnuva, Tel-Yosef), discharging approximately 6000 tons BOD per year, is treated in two serial, deep reservoirs (anaerobic/facultative). In this study, which focused on the anaerobic reservoir, we combined in situ measurements (over 18 months) and supporting lab experiments, in order to evaluate its efficiency and to identify the rate-limiting step of the methanogenic fermentation pathway. The anaerobic reservoir could remove above 75% of the BOD and COD all year round, but this was not enough to prevent malodors during the winter. Acetate and propionate, products of lactose fermentation, were the predominant intermediate metabolites in the reservoir and their concentrations were strongly dependent on the temperature and the organic load. The combined effects of colder winter temperatures and seasonal increase of organic load, resulted in a decreased rate of propionate oxidation and a consequent accumulation of soluble BOD and COD. Laboratory batch experiments, conducted during this season, found propionate oxidation to be the rate-limiting step in the process, characterized by a lag period preceding its degradation.

Reductive dehalogenation of tetrabromobisphenol-A by sediment from a contaminated ephemeral streambed and an enrichment culture.

This study was aimed at improving our understanding of the physiology of the microorganisms that reductively dehalogenate tetrabromobisphenol-A (TBBPA). Activity was followed in contaminated sediments from a polluted streambed as well as from fracture filling material underlying the stream. Reductive dehalogenation was observed in surface sediments but not in fracture filling samples from a depth of 3m. Likewise, anaerobic microbial activity, represented by sulphate reduction, was much higher in the surface sediment. In the culture enriched from the surface sediment, optimal microbial debromination of TBBPA took place at a salinity of 2% and 3% NaCl, temperature of 30 degrees Celsius, and pH of 7-8. Ethanol, pyruvate and the combination of hydrogen with acetate were the most suitable electron donors and carbon sources for this culture. Alternative electron acceptors like Fe(3+), SO(4)(2-), SO(3)(2-), NO(3)(-) and 2,4,6-tribromophenol inhibited TBBPA debromination. The debrominating bacteria were heat sensitive (80 degrees Celsius, for 10min) but were not inhibited by bromoethanesulphonate or molybdate. This study allowed optimisation of our culturing conditions, but was also important for understanding the factors which influence TBBPA debromination in situ.

Enrichment of a microbial culture capable of reductive debromination of the flame retardant tetrabromobisphenol-A, and identification of the intermediate metabolites produced in the process.

Tetrabromobisphenol-A is a reactive flame retardant used in the production of many plastic polymers. In previous research, it was demonstrated that anaerobic microorganisms from contaminated sediment debrominate tetrabromobisphenol-A to bisphenol-A, but an enrichment culture was not established. The current study was carried out to identify the intermediate metabolites in this process and to determine the factors facilitating enrichment of debrominating microorganisms. During the enrichment process in an anaerobic semi-continuous batch reactor, tetrabromobisphenol-A debromination gradually slowed down with concurrent accumulation of three intermediate products. These compounds were tentatively identified using GC-MS as tri-, di-, and mono-brominated bisphenol-A. GC-MS and HPLC analyses showed one dominant metabolite of dibromobisphenol-A, and NMR analysis identified it as 2,2'-dibromobisphenol-A. Addition of sterile sediment (15% wt/wt) to the reactor stimulated debromination of tetrabromobisphenol-A. Furthermore, different solid amendments such as surface soil and pulverized gray chalk from the site subsurface (100 m below ground) were also stimulating agents. We conclude that organic matter is involved in stimulation since the stimulation effect of the sediment, soil and gray chalk was abolished after it was heat-treated to 550 degrees C. Our study suggests that the debrominating culture requires some organic components found in the sediment, soil, and chalk in order to sustain activity and perhaps to survive. The possible mechanisms of stimulation by these solids are discussed.

Metabolism of diphenylurea by a Marinobacter sp. isolated from a contaminated ephemeral stream bed in the Negev Desert.

A moderate halophilic Marinobacter sp. (designated strain DPUZ) able to metabolize 1,3-diphenylurea (DPU) was isolated from a contaminated ephemeral desert stream bed near an industrial complex in the northern part of the Negev Desert (Israel). Metabolism of DPU was accompanied by a transient accumulation of a metabolite identified as aniline using gas chromatography-mass spectrometry, thus indicating a metabolic pathway involving cleavage of the urea bridge between the phenyl structures. Aniline was further degraded without detection of other metabolites suggesting a complete degradation. Strain DPUZ grows at NaCl concentrations between 0.2 and 2.6 M with an optimum at 0.51 M. It grows at a temperature range between 20 and 40 degrees C with an optimum at 35 degrees C. This is the first study on bacterial metabolism of DPU.