Insights into the mechanisms of desiccation resistance of the Patagonian PAH-degrading strain Sphingobium sp. 22B.

AIM: To analyse the physiological response of Sphingobium sp. 22B to water stress. METHODS AND RESULTS: The strain was grown under excess of carbon source and then subjected to low (60RH) and high (18RH) water stress conditions for 96 h. Quantification of trehalose, glycogen, polyhydroxybutyrate (PHB) and transmission electron microscopy (TEM) was studied. Genes linked with desiccation were searched in Sphingobium sp. 22B and Sphingomonas 'sensu latu' genomes and their transcripts were quantified by real-time PCR. Results showed that, in the absence of water stress, strain 22B accumulated 4·76 ± 1·41% of glycogen, 0·84 ± 1·62% of trehalose and 44·9 ± 6·4% of PHB per cellular dry weight. Glycogen and trehalose were mobilized under water stressed conditions, this mobilization was significantly higher in 60RH in comparison to 18RH. Gene treY was upregulated sixfold in 60RH relative to control condition. TEM and quantification of PHB revealed that PHB was mobilized under 60RH condition accompanied by the downregulation of the phbB gene. TEM images showed an extracellular amorphous matrix in 18RH and 60RH. Major differences were found in the presence of aqpZ and trehalose genes between strain 22B and Sphingomonas genomes. CONCLUSION: Strain 22B showed a carbon conservative metabolism capable of accumulation of three types of endogenous carbon sources. The strain responds to water stress by changing the expression pattern of genes related to desiccation, formation of an extracellular amorphous matrix and mobilization of the carbon sources according to the degree of water stress. Trehalose, glycogen and PHB may have multiple functions in different degrees of desiccation. The robust endowment of molecular responses to desiccation shown in Sphingobium sp. 22B could explain its survival in semi-arid soil. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the physiology implicated in the toleration of the PAH-degrading strain Sphingobium sp 22B to environmental desiccation may improve the bioaugmentation technologies in semi-arid hydrocarbon-contaminated soils.

Insights into the genome and proteome of Sphingomonas paucimobilis strain 20006FA involved in the regulation of polycyclic aromatic hydrocarbon degradation.

In order to study the mechanisms regulating the phenanthrene degradation pathway and the intermediate-metabolite accumulation in strain S. paucimobilis 20006FA, we sequenced the genome and compared the genome-based predictions to experimental proteomic analyses. Physiological studies indicated that the degradation involved the salicylate and protocatechuate pathways, reaching 56.3% after 15 days. Furthermore, the strain degraded other polycyclic aromatic hydrocarbons (PAH) such as anthracene (13.1%), dibenzothiophene (76.3%), and fluoranthene. The intermediate metabolite 1-hydroxy-2-naphthoic acid (HNA) accumulated during phenanthrene catabolism and inhibited both bacterial growth and phenanthrene degradation, but exogenous-HNA addition did not affect further degradation. Genomic analysis predicted 126 putative genes encoding enzymes for all the steps of phenanthrene degradation, which loci could also participate in the metabolism of other PAH. Proteomic analysis identified enzymes involved in 19 of the 23 steps needed for the transformation of phenanthrene to trichloroacetic-acid intermediates that were upregulated in phenanthrene cultures relative to the levels in glucose cultures. Moreover, the protein-induction pattern was temporal, varying between 24 and 96 h during phenanthrene degradation, with most catabolic proteins being overexpressed at 96 h-e. g., the biphenyl dioxygenase and a multispecies (2Fe-2S)-binding protein. These results provided the first clues about regulation of expression of phenanthrene degradative enzymes in strain 20006FA and enabled an elucidation of the metabolic pathway utilized by the bacterium. To our knowledge the present work represents the first investigation of genomic, proteomic, and physiological studies of a PAH-degrading Sphingomonas strain.

Draft Whole-Genome Sequence of Sphingobium sp. 22B, a Polycyclic Aromatic Hydrocarbon-Degrading Bacterium from Semiarid Patagonia, Argentina.

Sphingobium sp. 22B is a polycyclic aromatic hydrocarbon-degrading strain isolated from Patagonia, Argentina, with capabilities to withstand the environmental factors of that semiarid region. The draft genome shows the presence of genes related with responses to carbon starvation and drying environmental conditions.

Application of the knowledge-based approach to strain selection for a bioaugmentation process of phenanthrene- and Cr(VI)-contaminated soil.

AIMS: The objective of this study was to apply the knowledge-based approach to the selection of an inoculum to be used in bioaugmentation processes to facilitate phenanthrene degradation in phenanthrene- and Cr(VI)-co-contaminated soils. METHODS AND RESULTS: The bacterial community composition of phenanthrene and phenanthrene- and Cr(VI)-co-contaminated microcosms, determined by denaturing gradient gel electrophoresis analysis, showed that members of the Sphingomonadaceae family were the predominant micro-organisms. However, the Cr(VI) contamination produced a selective change of predominant Sphingomonas species, and in co-contaminated soil microcosms, a population closely related to Sphingomonas paucimobilis was naturally selected. The bioaugmentation process was carried out using the phenanthrene-degrading strain S. paucimobilis 20006FA, isolated and characterized in our laboratory. Although the strain showed a low Cr(VI) resistance (0·250 mmol l⁻¹); in liquid culture, it was capable of reducing chromate and degrading phenanthrene simultaneously. CONCLUSION: The inoculation of this strain managed to moderate the effect of the presence of Cr(VI), increasing the biological activity and phenanthrene degradation rate in co-contaminated microcosm. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we have applied a novel approach to the selection of the adequate inoculum to enhance the phenanthrene degradation in phenanthrene- and Cr(VI)-co-contaminated soils.

[Effect of the inoculant strain Sphingomonas paucimobilis 20006FA on the bacterial composition of a phenanthrene-degrading consortium]. / Efecto de la inoculación de la cepa Sphingomonas paucimobilis 20006FA sobre la composición de un consorcio bacteriano degradador de fenantreno.

The effect of the inoculant strain Sphingomonas paucimobilis 20006FA on the bacterial composition of a phenanthrene-degrading consortium obtained from a pristine soil in sequencing batch cultures was studied. Inoculated (F200+1) and non-inoculated (F200) phenanthrene-degrading consortia, were obtained. Bacterial diversity of consortia was studied at cultivable (phenotype and genotype characterization) and non-cultivable (PCR-DGGE) levels. During the successive cultures, a loss in the phenanthrene-degrading capacity and a decrease in the bacterial diversity were observed in both consortia. Although inoculation did not produce any significant changes in the consortia phenanthrene-degrading capacity (29.9% F200 and 27.6% F200+1), it did produce changes in the bacterial composition, showing a differential structural dynamics in the DGGE profiles of the inoculated consortium. In both consortia, a dominant band placed at the same position as that of the DNA of the inoculant strain in the DGGE gel could be observed. However, isolated cultures from the consortia which had an identical band position to that of S. paucimobilis 20006FA in the PCR-DGGE profile showed low similarity with respect to the inoculant strain (RAPD).

Efecto de la inoculación de la cepa Sphingomonas paucimobilis 20006FA sobre la composición de un consorcio bacteriano degradador de fenantreno / Effect of the inoculant strain Sphingomonas paucimobilis 20006FA on the bacterial composition of a phenanthrene-degrading consortium

Se estudió el efecto de la inoculación con la cepa Sphingomonas paucimobilis 20006FA sobre la composición bacteriana de un consorcio degradador de fenantreno en cultivos discontinuos (batch) con 8 repiques sucesivos. El consorcio original se obtuvo a partir de un suelo prístino. A los fines del estudio, se obtuvieron y mantuvieron dos consorcios: uno inoculado (F200+I) y otro sin inocular (F200). Se estudió la diversidad bacteriana de los consorcios mediante el análisis de microorganismos cultivables (por caracterización fenotípica y genotípica) y totales (por PCR-DGGE). A lo largo de los repiques sucesivos pudo observarse en ambos consorcios una tendencia a la pérdida de la capacidad degradadora de fenantreno, acompañada por una disminución de la diversidad bacteriana. Si bien la inoculación no produjo cambios significativos en la capacidad degradadora de fenantreno de los consorcios (29,9% para F200 y 27,6% para F200+I hacia el tercer repique), sí produjo cambios en la composición bacteriana, ya que los perfiles de DGGE revelaron una dinámica estructural diferente en el consorcio inoculado. En ambos consorcios se pudo observar la presencia de una banda intensa posicionada a la misma altura que el ADN del inóculo en el gel de DGGE; sin embargo, los cultivos aislados de los consorcios que presentaban idéntica posición de banda en el perfil PCR-DGGE que la cepa S. paucimobilis 20006FA mostraron baja similitud con la cepa inoculada mediante la técnica de RAPD.

The effect of the inoculant strain Sphingomonas paucimobilis 20006FA on the bacterial composition of a phenanthrene-degrading consortium obtained from a pristine soil in sequencing batch cultures was studied. Inoculated (F200+I) and non-inoculated (F200) phenanthrene-degrading consortia, were obtained. Bacterial diversity of consortia was studied at cultivable (phenotype and genotype characterization) and non-cultivable (PCR-DGGE) levels. During the successive cultures, a loss in the phenanthrene-degrading capacity and a decrease in the bacterial diversity were observed in both consortia. Although inoculation did not produce any significant changes in the consortia phenanthrene-degrading capacity (29.9% F200 and 27.6% F200+I), it did produce changes in the bacterial composition, showing a differential structural dynamics in the DGGE profiles of the inoculated consortium. In both consortia, a dominant band placed at the same position as that of the DNA of the inoculant strain in the DGGE gel could be observed. However, isolated cultures from the consortia which had an identical band position to that of S. paucimobilis 20006FA in the PCR-DGGE profile showed low similarity with respect to the inoculant strain (RAPD).

Effects of the inoculant strain Sphingomonas paucimobilis 20006FA on soil bacterial community and biodegradation in phenanthrene-contaminated soil.

The effects of the inoculant strain Sphingomonas paucimobilis 20006FA (isolated from a phenanthrene-contaminated soil) on the dynamics and structure of microbial communities and phenanthrene elimination rate were studied in soil microcosms artificially contaminated with phenanthrene. The inoculant managed to be established from the first inoculation as it was evidenced by denaturing gradient gel electrophoresis analysis, increasing the number of cultivable heterotrophic and PAH-degrading cells and enhancing phenanthrene degradation. These effects were observed only during the inoculation period. Nevertheless, the soil biological activity (dehydrogenase activity and CO(2) production) showed a late increase. Whereas gradual and successive changes in bacterial community structures were caused by phenanthrene contamination, the inoculation provoked immediate, significant, and stable changes on soil bacterial community. In spite of the long-term establishment of the inoculated strain, at the end of the experiment, the bioaugmentation did not produce significant changes in the residual soil phenanthrene concentration and did not improve the residual effects on the microbial soil community.

Effect of petrochemical sludge concentrations of changes in mutagenic activity during soil bioremediation process.

The present study was performed to assess the effect of the petrochemical sludge application rate on the mutagenic activity (Ames test) of soil and the persistence of mutagenic activity during laboratory soil bioremediation process. Sludge-soil systems were prepared at four different sludge application rates (1.25, 2.5, 5, and 10% w/w). Unamended soil was used as a control. Immediately following sludge application, in the absence or presence of S9, a linear correlation between sludge application rates and mutagenicity was found but differed significantly (p < 0.05) from the control system only at higher application rates (5 and 10% w/w). The direct mutagenicity of all systems decreases during the bioremediation process, and after a year of treatment only the 10% system induced a mutagenic response that was significantly different from the control system. On the other hand, an initial increase of the indirect mutagenicity was observed at all application rates. The time required for observing this increase was inversely proportional to the initial sludge concentration. After a year of treatment, the indirect mutagenicity of all sludge-amended soils was not significantly different but was significantly different from the unamended soils. The persistence of the direct mutagenic activity of the sludge-amended soils was related to the sludge concentration, whereas the indirect mutagenic persistence was related to the relationship between easily degradable hydrocarbons and polynuclear aromatic hydrocarbons concentration and independent from the initial application rate.

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

Se 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

Assessment of the toxic potential of hydrocarbon containing sludges.

A short-time period microbial toxicity test-battery was used for the investigation of acute toxicity and genotoxicity of five hydrocarbon containing sludges. Four sludges were obtained from a petrochemical industry and the fifth from a petroleum refinery. Some of the sludges had been stored for long periods. Bioremediation potential assays for soils polluted with each of the sludges were also considered. The sludges did not show acute toxicity in any of the microbial tests performed. However, when the diethylether soluble fractions of these sludges were analyzed some of them showed acute toxicity, for which the clearest results were obtained with the resazurin reduction method. The greatest toxicity detected with the Resazurin based method was found in the diethylether extracts of the freshly collected (not stored) sludges. On the other hand, the diethylether soluble fraction of those sludges that had been stored showed genotoxicity when analyzed with the Salmonella/microsome assay. After the incorporation of the sludges into the soil, increased bacterial counts were noted and substantial hydrocarbon elimination was achieved in 30 days, showing that bioremediation may be a possible technology for cleaning soils polluted with these sludges.