Metaproteomic and gene expression analysis of interspecies interactions in a PAH-degrading synthetic microbial consortium constructed with the key microbes of a natural consortium.

Polycyclic Aromatic Hydrocarbons (PAHs) impose adverse effects on the environment and human life. The use of synthetic microbial consortia is promising in bioremediation of contaminated sites with these pollutants. However, the design of consortia taking advantage of natural interactions has been poorly explored. In this study, a dual synthetic bacterial consortium (DSC_AB) was constructed with two key members (Sphingobium sp. AM and Burkholderia sp. Bk), of a natural PAH degrading consortium. DSC_AB showed significantly enhanced degradation of PAHs and toxic intermediary metabolites relative to the axenic cultures, indicating the existence of synergistic relationships. Metaproteomic and gene-expression analyses were applied to obtain a view of bacterial performance during phenanthrene removal. Overexpression of the Bk genes, naph, biph, tol and sal and the AM gene, ahdB, in DSC_AB relative to axenic cultures, demonstrated that both strains are actively participating in degradation, which gave evidence of cross-feeding. Several proteins related to stress response were under-expressed in DSC_AB relative to axenic cultures, indicating that the division of labour reduces cellular stress, increasing the efficiency of degradation. This is the one of the first works revealing bacterial relationships during PAH removal in a synthetic consortium applying an omics approach. Our findings could be used to develop criteria for evaluating the potential effectiveness of synthetic bacterial consortia in bioremediation.

Co-treatment of an oily sludge and aged contaminated soil: permanganate oxidation followed by bioremediation.

The bioremediation of an oily sludge (321 ± 30 mg of polycyclic aromatic hydrocarbons/kgDRY SLUDGE and 13420 ± 1300 mg of aliphatic hydrocarbons/kgDRY SLUDGE) by mixture with contaminated soil (23 ± 2 mg of polycyclic aromatic hydrocarbons/kgDRY SOIL and 98 ± 10 mg of aliphatic hydrocarbons/kgDRY SOIL) was studied. Furthermore, the effect of oxidative pretreatments (persulfate and permanganate) on the performance of the global process was examined. The treatments reached contamination levels lower than the original residues, indicating the presence of synergic processes between a highly contaminated sludge and soil with a selected hydrocarbon-degrading community. Pretreatment with permanganate significantly improved biodegradation, possibly due to the increase in bioavailability and biodegradability of petroleum hydrocarbons. Two months of incubation was enough to reach the complete elimination of polycyclic aromatic hydrocarbons and 92% elimination of aliphatic hydrocarbons. Monitoring using five parameters (concentration of total petroleum hydrocarbons, total cultivable heterotrophic bacteria count, lipase and dehydrogenase activities, and polycyclic aromatic hydrocarbon-degrading bacteria count) as an approach for a preliminary scanning of the effectiveness of a treatment is proposed based on principal components analysis.

Strategies for oxidation of PAHs in aged contaminated soil by batch reactors.

Polycyclic aromatic hydrocarbons (PAH) are neutral, nonpolar and hydrophobic molecules that tend to sorb onto soil organic matter. Chemical oxidation is a good choice to avoid the limitations of bioremediation. To evaluate the efficiency of different types of oxidation (permanganate, hydrogen peroxide, and persulfate) and activation (heat, alkaline, and iron), batch reactors were prepared. The soil was contaminated with phenanthrene and pyrene (1200 ± 200 and 2800 ± 100mg per kg of dry soil, respectively) and aged for fifteen months. Treatments were prepared with 10g of contaminated dry soil and 20ml of water and incubated at room temperature for 7 days. Analyses of phenanthrene and pyrene concentrations, soil pH and electric conductivity were performed. Counts of heterotrophic cultivable bacteria on R2A medium and PAH-degraders were carried out after 7 days of treatment. The persulfate treatment at room temperature, without the addition of activators, achieved better results than treatments with the same doses of permanganate or hydrogen peroxide. All the strategies to improve persulfate treatments yielded higher degradation of pyrene than the biological control, as expected from the structural description of this compound by Clar's model. The thermal activation of persulfate (65°C for 6h) led to the degradation of more than 90% of both PAHs after 7 days of treatment.

Assessment of Biological Contribution to Natural Recovery of Anthropized Freshwater Sediments From Argentina: Autochthonous Microbiome Structure and Functional Prediction.

Monitored natural recovery (MNR) is an in situ technique of conventional remediation for the treatment of contaminated sediments that relies on natural processes to reduce the bioavailability or toxicity of contaminants. Metabarcoding and bioinformatics approaches to infer functional prediction were applied in bottom sediments of a tributary drainage channel of Río de La Plata estuary, in order to assess the biological contribution to MNR. Hydrocarbon concentration in water samples and surface sediments was below the detection limit. Surface sediments were represented with high available phosphorous, alkaline pH, and the bacterial classes Anaerolineae, Planctomycetia, and Deltaproteobacteria. The functional prediction in surface sediments showed an increase of metabolic activity, carbon fixation, methanogenesis, and synergistic relationships between Archaeas, Syntrophobacterales, and Desulfobacterales. The prediction in non-surface sediments suggested the capacity to respond to different kinds of environmental stresses (oxidative, osmotic, heat, acid pH, and heavy metals), predicted mostly in Lactobacillales order, and the capacity of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Actinomyces classes to degrade xenobiotic compounds. Canonical correspondence analysis (CCA) suggests that depth, phosphate content, redox potential, and pH were the variables that structured the bacterial community and not the hydrocarbons. The characterization of sediments by metabarcoding and functional prediction approaches, allowed to assess how the microbial activity would contribute to the recovery of the site.

Design and evaluation of synthetic bacterial consortia for optimized phenanthrene degradation through the integration of genomics and shotgun proteomics.

Two synthetic bacterial consortia (SC) composed of bacterial strains Sphingobium sp. (AM), Klebsiella aerogenes (B), Pseudomonas sp. (Bc-h and T), Burkholderia sp. (Bk) and Inquilinus limosus (Inq) isolated from a natural phenanthrene (PHN)-degrading consortium (CON) were developed and evaluated as an alternative approach to PHN biodegradation in bioremediation processes. A metabolic network showing the potential role of strains was reconstructed by in silico study of the six genomes and classification of dioxygenase enzymes using RHObase and AromaDeg databases. Network analysis suggested that AM and Bk were responsible for PHN initial attack, while Inq, B, T and Bc-h would degrade PHN metabolites. The predicted roles were further confirmed by physiological, RT-qPCR and metaproteomic assays. SC-1 with AM as the sole PHN degrader was the most efficient. The ecological roles inferred in this study can be applied to optimize the design of bacterial consortia and tackle the biodegradation of complex environmental pollutants.

Study of the degradation activity and the strategies to promote the bioavailability of phenanthrene by Sphingomonas paucimobilis strain 20006FA.

The present study describes the phenanthrene-degrading activity of Sphingomonas paucimobilis 20006FA and its ability to promote the bioavailability of phenanthrene. S. paucimobilis 20006FA was isolated from a phenanthrene-contaminated soil microcosm. The strain was able to grow in liquid mineral medium saturated with phenanthrene as the sole carbon source, showing high phenanthrene elimination (52.9% of the supplied phenanthrene within 20 days). The accumulation of 1-hydroxy-2-naphthoic acid and salicylic acid as major phenanthrene metabolites and the capacity of the strain to grow with sodium salicylate as the sole source of carbon and energy indicated that the S. paucimobilis 20006FA possesses a complete phenanthrene degradation pathway. However, under the studied conditions, the strain was able to mineralize only the 10% of the consumed phenanthrene. Investigations on the cell ability to promote bioavailability of phenanthrene showed that the S. paucimobilis strain 20006FA exhibited low cell hydrophobicity (0.13), a pronounced chemotaxis toward phenanthrene, and it was able to reduce the surface tension of mineral liquid medium supplemented with phenanthrene as sole carbon source. Scanning electron micrographs revealed that: (1) in suspension cultures, cells formed flocks and showed small vesicles on the cell surface and (2) cells were also able to adhere to phenanthrene crystals and to produce biofilms. Clearly, the strain seems to exhibit two different mechanisms to enhance phenanthrene bioavailability: biosurfactant production and adhesion to the phenanthrene crystals.

Archaeosomes made of Halorubrum tebenquichense total polar lipids: a new source of adjuvancy.

BACKGROUND: Archaeosomes (ARC), vesicles prepared from total polar lipids (TPL) extracted from selected genera and species from the Archaea domain, elicit both antibody and cell-mediated immunity to the entrapped antigen, as well as efficient cross priming of exogenous antigens, evoking a profound memory response. Screening for unexplored Archaea genus as new sources of adjuvancy, here we report the presence of two new Halorubrum tebenquichense strains isolated from grey crystals (GC) and black mood (BM) strata from a littoral Argentinean Patagonia salt flat. Cytotoxicity, intracellular transit and immune response induced by two subcutaneous (sc) administrations (days 0 and 21) with BSA entrapped in ARC made of TPL either form BM (ARC-BM) and from GC (ARC-GC) at 2% w/w (BSA/lipids), to C3H/HeN mice (25 microg BSA, 1.3 mg of archaeal lipids per mouse) and boosted on day 180 with 25 microg of bare BSA, were determined. RESULTS: DNA G+C content (59.5 and 61.7% mol BM and GC, respectively), 16S rDNA sequentiation, DNA-DNA hybridization, arbitrarily primed fingerprint assay and biochemical data confirmed that BM and GC isolates were two non-previously described strains of H. tebenquichense. Both multilamellar ARC mean size were 564 +/- 22 nm, with -50 mV zeta-potential, and were not cytotoxic on Vero cells up to 1 mg/ml and up to 0.1 mg/ml of lipids on J-774 macrophages (XTT method). ARC inner aqueous content remained inside the phago-lysosomal system of J-774 cells beyond the first incubation hour at 37 degrees C, as revealed by pyranine loaded in ARC. Upon subcutaneous immunization of C3H/HeN mice, BSA entrapped in ARC-BM or ARC-GC elicited a strong and sustained primary antibody response, as well as improved specific humoral immunity after boosting with the bare antigen. Both IgG1 and IgG2a enhanced antibody titers could be demonstrated in long-term (200 days) recall suggesting induction of a mixed Th1/Th2 response. CONCLUSION: We herein report the finding of new H. tebenquichense non alkaliphilic strains in Argentinean Patagonia together with the adjuvant properties of ARC after sc administration in mice. Our results indicate that archaeosomes prepared with TPL from these two strains could be successfully used as vaccine delivery vehicles.

Dynamics of microbial community during bioremediation of phenanthrene and chromium(VI)-contaminated soil microcosms.

The combined effect of phenanthrene and Cr(VI) on soil microbial activity, community composition and on the efficiency of bioremediation processes has been studied. Biometer flask systems and soil microcosm systems contaminated with 2,000 mg of phenanthrene per kg of dry soil and different Cr(VI) concentrations were investigated. Temperature, soil moisture and oxygen availability were controlled to support bioremediation. Cr(VI) inhibited the phenanthrene mineralization (CO(2) production) and cultivable PAH degrading bacteria at levels of 500-2,600 mg kg(-1). In the bioremediation experiments in soil microcosms the degradation of phenanthrene, the dehydrogenase activity and the increase in PAH degrading bacteria counts were retarded by the presence of Cr(VI) at all studied concentrations (25, 50 and 100 mg kg(-1)). These negative effects did not show a correlation with Cr(VI) concentration. Whereas the presence of Cr(VI) had a negative effect on the phenanthrene elimination rate, co-contamination with phenanthrene reduced the residual Cr(VI) concentration in the water exchangeable Cr(VI) fraction (WEF) in comparison with the soil microcosm contaminated only with Cr(VI). Clear differences were found between the denaturing gradient gel electrophoresis (DGGE) patterns of each soil microcosm, showing that the presence of different Cr(VI) concentrations did modulate the community response to phenanthrene and caused perdurable changes in the structure of the microbial soil community.

Assessing interactions, predicting function, and increasing degradation potential of a PAH-degrading bacterial consortium by effect of an inoculant strain.

A natural phenanthrene-degrading consortium CON was inoculated with an exogenous strain Sphingobium sp. (ex Sp. paucimobilis) 20006FA yielding the consortium called I-CON, in order to study ecological interactions into the bacterial community. DGGE and proteomic profiles and analyses by HTS (High-Throughput Sequencing) technologies demonstrated inoculant establishment and changes on CON composition. Inoculation increased degradation efficiency in I-CON and prevented intermediate HNA accumulation. This could be explained not only by the inoculation, but also by enrichment in Achromobacter genus at expense of a decrease in Klebsiella genus. After inoculation, cooperation between Sphingobium and Achromobacter genera were improved, thereby, some competition could have been generated, and as a consequence, species in minor proportion (cheaters), as Inquilinus sp. and Luteibacter sp., were not detected. Sequences of Sphingobium (corresponding to the inoculated strain) did not vary. PICRUSt predicted a network with bacterial phylotypes connected with enzymes, showing functional redundancy in the phenanthrene pathway, with exception of the first enzymes biphenyl-2,3-diol 1,2-dioxygenase and protocatechuate 4,5-dioxygenase that were only encoded in Sphingobium sp. This is the first report where a natural consortium that has been characterized by HTS technologies is inoculated with an exogenous strain in order to study competitiveness and interactions.

Efficiency of surfactant-enhanced bioremediation of aged polycyclic aromatic hydrocarbon-contaminated soil: Link with bioavailability and the dynamics of the bacterial community.

Shifts in the bacterial-community dynamics, bioavailability, and biodegradation of polycyclic aromatic hydrocarbons (PAHs) of chronically contaminated soil were analyzed in Triton X-100-treated microcosms at the critical micelle concentration (T-CMC) and at two sub-CMC doses. Only the sub-CMC-dose microcosms reached sorbed-PAH concentrations significantly lower than the control: 166±32 and 135±4mgkg-1 dry soil versus 266±51mgkg-1; consequently an increase in high- and low-molecular-weight PAHs biodegradation was observed. After 63days of incubation pyrosequencing data evidenced differences in diversity and composition between the surfactant-modified microcosms and the control, with those with sub-CMC doses containing a predominance of the orders Sphingomonadales, Acidobacteriales, and Gemmatimonadales (groups of known PAHs-degrading capability). The T-CMC microcosm exhibited a lower richness and diversity index with a marked predominance of the order Xanthomonadales, mainly represented by the Stenotrophomonas genus, a PAHs- and Triton X-100-degrading bacterium. In the T-CMC microcosm, whereas the initial surface tension was 35mNm-1, after 63days of incubation an increase up to 40mNm-1 was registered. The previous observation and the gas-chromatography data indicated that the surfactant may have been degraded at the CMC by a highly selective bacterial community with a consequent negative impact on PAHs biodegradation. This work obtained strong evidence for the involvement of physicochemical and biologic influences determining the different behaviors of the studied microcosms. The results reported here contribute significantly to an optimization of, surfactant-enhanced bioremediation strategies for chronically contaminated soil since the application of doses below the CMC would reduce the overall costs.

Assigning ecological roles to the populations belonging to a phenanthrene-degrading bacterial consortium using omic approaches.

The present study describes the behavior of a natural phenanthrene-degrading consortium (CON), a synthetic consortium (constructed with isolated strains from CON) and an isolated strain form CON (Sphingobium sp. AM) in phenanthrene cultures to understand the interactions among the microorganisms present in the natural consortium during phenanthrene degradation as a sole carbon and energy source in liquid cultures. In the contaminant degradation assay, the defined consortium not only achieved a major phenanthrene degradation percentage (> 95%) but also showed a more efficient elimination of the intermediate metabolite. The opposite behavior occurred in the CON culture where the lowest phenanthrene degradation and the highest HNA accumulation were observed, which suggests the presence of positive and also negative interaction in CON. To consider the uncultured bacteria present in CON, a metagenomic library was constructed with total CON DNA. One of the resulting scaffolds (S1P3) was affiliated with the Betaproteobacteria class and resulted in a significant similarity with a genome fragment from Burkholderia sp. HB1 chromosome 1. A complete gene cluster, which is related to one of the lower pathways (meta-cleavage of catechol) involved in PAH degradation (ORF 31-43), mobile genetic elements and associated proteins, was found. These results suggest the presence of at least one other microorganism in CON besides Sphingobium sp. AM, which is capable of degrading PAH through the meta-cleavage pathway. Burkholderiales order was further found, along with Sphingomonadales order, by a metaproteomic approach, which indicated that both orders were metabolically active in CON. Our results show the presence of negative interactions between bacterial populations found in a natural consortium selected by enrichment techniques; moreover, the synthetic syntrophic processing chain with only one microorganism with the capability of degrading phenanthrene was more efficient in contaminant and intermediate metabolite degradation than a generalist strain (Sphingobium sp. AM).

Monitoring the impact of bioaugmentation with a PAH-degrading strain on different soil microbiomes using pyrosequencing.

The effect of bioaugmentation with Sphingobium sp. AM strain on different soils microbiomes, pristine soil (PS), chronically contaminated soil (IPK) and recently contaminated soil (Phe) and their implications in bioremediation efficiency was studied by focusing on the ecology that drives bacterial communities in response to inoculation. AM strain draft genome codifies genes for metabolism of aromatic and aliphatic hydrocarbons. In Phe, the inoculation improved the elimination of phenanthrene during the whole treatment, whereas in IPK no improvement of degradation of any PAH was observed. Through the pyrosequencing analysis, we observed that inoculation managed to increase the richness and diversity in both contaminated microbiomes, therefore, independently of PAH degradation improvement, we observed clues of inoculant establishment, suggesting it may use other resources to survive. On the other hand, the inoculation did not influence the bacterial community of PS. On both contaminated microbiomes, incubation conditions produced a sharp increase on Actinomycetales and Sphingomonadales orders, while inoculation caused a relative decline of Actinomycetales. Inoculation of most diverse microbiomes, PS and Phe, produced a coupled increase of Sphingomonadales, Burkholderiales and Rhizobiales orders, although it may exist a synergy between those genera; our results suggest that this would not be directly related to PAH degradation.

Effect of petrochemical sludge concentrations on microbial communities during soil bioremediation.

Qualitative and quantitative changes of microbial communities in soil microcosms during bioremediation were determined throughout one year. The soil was contaminated with 0%, 2.5%, 5%, 10% (wt/wt) of petrochemical sludge containing polynuclear aromatic hydrocarbons. We analyzed the hydrocarbon concentration in the microcosms, the number of cultivable bacteria using CFU and most probable number assays, the community structure using denaturing gradient gel electrophoresis, and the metabolic activity of soil using dehydrogenase activity and substrate-induced respiration assays. After one year of treatment, the chemical analysis suggested that the hydrocarbon elimination process was over. The biological analysis, however, showed that the contaminated microcosms suffered under long-term disturbance. The number of heterotrophic bacteria that increased after sludge addition (up to 10(8)-10(9) cells ml(-1)) has not returned to the level of the control soil (2-6 x 10(7) cells ml(-1)). The community structure in the contaminated soils differed considerably from that in the control. The substrate-induced respiration of the contaminated soils was significantly lower (approximately 10-fold) and the dehydrogenase activity was significantly higher (20-40-fold) compared to the control. Changes in the community structure of soils depended on the amount of added sludge. The species, which were predominant in the sludge community, could not be detected in the contaminated soils.

Remediation of phenanthrene-contaminated soil by simultaneous persulfate chemical oxidation and biodegradation processes.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous compounds with carcinogenic and/or mutagenic potential. To address the limitations of individual remediation techniques and to achieve better PAH removal efficiencies, the combination of chemical and biological treatments can be used. The degradation of phenanthrene (chosen as a model of PAH) by persulfate in freshly contaminated soil microcosms was studied to assess its impact on the biodegradation process and on soil properties. Soil microcosms contaminated with 140 mg/kgDRY SOIL of phenanthrene were treated with different persulfate (PS) concentrations 0.86-41.7 g/kgDRY SOIL and incubated for 28 days. Analyses of phenanthrene and persulfate concentrations and soil pH were performed. Cultivable heterotrophic bacterial count was carried out after 28 days of treatment. Genetic diversity analysis of the soil microcosm bacterial community was performed by PCR amplification of bacterial 16S rDNA fragments followed by denaturing gradient gel electrophoresis (DGGE). The addition of PS in low concentrations could be an interesting biostimulatory strategy that managed to shorten the lag phase of the phenanthrene biological elimination, without negative effects on the physicochemical and biological soil properties, improving the remediation treatment.

Bio-remediación como alternativa a la incineración de residuos con hidrocarburos aromáticos polinucleares

Presenta la aplicación de un esquema de ensayo de tratabilidad en tres niveles para evaluar y diseñar una técnica de bio-remediación para el tratamiento de barros de fondo de separadores API con un alto contenido de hidrocarburos aromáticos polinucleares generados en el polo petroquímico Ensenada. El esquema de trabajo permite definir los requerimientos de adecuación de sitio, el plan de operaciones, el plan de monitoreo y establecer los tiempos de tratamiento. Esto resulta fundamental para poder definir la estructura de costos que sera la que en definitiva permita comparar la bio-remediación con la incineración

Técnicas de bio-remediación: ensayos de tratabilidad: nivel 1

Cuando se pretende aplicar una técnica de bio-remediación en suelo, los ensayos de Nivel 1 comprenden la caracterización del residuo y del sitio disponible para el tratamiento. Estos ensayos permitirán decidir la potencialidad de aplicación de la técnica y los parámetros a ser controlados en etapas posteriores. Se desarrollaron, implementaron y evaluaron ensayos microbiológicos, analíticos y toxicológicos que permitieron abordar estudios de tratabilidad de Nivel 1 sobre 9 residuos con hidrocarburos