Jordi Sabaté

Bacterial Community Dynamics and Polycyclic Aromatic Hydrocarbon Degradation during Bioremediation of Heavily Creosote-Contaminated Soil

ABSTRACT Bacterial community dynamics and biodegradation processes were examined in a highly creosote-contaminated soil undergoing a range of laboratory-based bioremediation treatments. The dynamics of the eubacterial community, the number of heterotrophs and polycyclic aromatic hydrocarbon (PAH) degraders, and the total petroleum hydrocarbon (TPH) and PAH concentrations were monitored during the bioremediation process. TPH and PAHs were significantly degraded in all treatments (72 to 79% and 83 to 87%, respectively), and the biodegradation values were higher when nutrients were not added, especially for benzo(a)anthracene and chrysene. The moisture content and aeration were determined to be the key factors associated with PAH bioremediation. Neither biosurfactant addition, bioaugmentation, nor ferric octate addition led to differences in PAH or TPH biodegradation compared to biodegradation with nutrient treatment. All treatments resulted in a high first-order degradation rate during the first 45 days, which was markedly reduced after 90 days. A sharp increase in the size of the heterotrophic and PAH-degrading microbial populations was observed, which coincided with the highest rates of TPH and PAH biodegradation. At the end of the incubation period, PAH degraders were more prevalent in samples to which nutrients had not been added. Denaturing gradient gel electrophoresis analysis and principal-component analysis confirmed that there was a remarkable shift in the composition of the bacterial community due to both the biodegradation process and the addition of nutrients. At early stages of biodegradation, the α-Proteobacteria group (genera Sphingomonas and Azospirillum) was the dominant group in all treatments. At later stages, the γ-Proteobacteria group (genus Xanthomonas), the α-Proteobacteria group (genus Sphingomonas), and the Cytophaga-Flexibacter-Bacteroides group (Bacteroidetes) were the dominant groups in the nonnutrient treatment, while the γ-Proteobacteria group (genus Xathomonas), the β-Proteobacteria group (genera Alcaligenes and Achromobacter), and the α-Proteobacteria group (genus Sphingomonas) were the dominant groups in the nutrient treatment. This study shows that specific bacterial phylotypes are associated both with different phases of PAH degradation and with nutrient addition in a preadapted PAH-contaminated soil. Our findings also suggest that there are complex interactions between bacterial species and medium conditions that influence the biodegradation capacity of the microbial communities involved in bioremediation processes.

Identification of a Novel Metabolite in the Degradation of Pyrene by Mycobacterium sp. Strain AP1: Actions of the Isolate on Two- and Three-Ring Polycyclic Aromatic Hydrocarbons

ABSTRACT Mycobacterium sp. strain AP1 grew with pyrene as a sole source of carbon and energy. The identification of metabolites accumulating during growth suggests that this strain initiates its attack on pyrene by either monooxygenation or dioxygenation at its C-4, C-5 positions to give trans- orcis-4,5-dihydroxy-4,5-dihydropyrene, respectively. Dehydrogenation of the latter, ortho cleavage of the resulting diol to form phenanthrene 4,5-dicarboxylic acid, and subsequent decarboxylation to phenanthrene 4-carboxylic acid lead to degradation of the phenanthrene 4-carboxylic acid via phthalate. A novel metabolite identified as 6,6′-dihydroxy-2,2′-biphenyl dicarboxylic acid demonstrates a new branch in the pathway that involves the cleavage of both central rings of pyrene. In addition to pyrene, strain AP1 utilized hexadecane, phenanthrene, and fluoranthene for growth. Pyrene-grown cells oxidized the methylenic groups of fluorene and acenaphthene and catalyzed the dihydroxylation andortho cleavage of one of the rings of naphthalene and phenanthrene to give 2-carboxycinnamic and diphenic acids, respectively. The catabolic versatility of strain AP1 and its use ofortho cleavage mechanisms during the degradation of polycyclic aromatic hydrocarbons (PAHs) give new insight into the role that pyrene-degrading bacterial strains may play in the environmental fate of PAH mixtures.

Enhanced biodegradation of Casablanca crude oil by a microbial consortium in presence of a rhamnolipid produced by Pseudomonas aeruginosa AT10

The biodegradation of oil products in the environment is often limited by their low water solubility and dissolution rate. Rhamnolipids produced by Pseudomonas aeruginosa AT10 were investigated for their potential to enhance bioavailability and hence the biodegradation of crude oil by a microbial consortium in liquid medium. The characterization of the rhamnolipids produced by strain AT10 showed the effectiveness of emulsification of complex mixtures. The addition of rhamnolipids accelerates the biodegradation of total petroleum hydrocarbons from 32% to 61% at 10 days of incubation. Nevertheless, the enhancement of biosurfactant addition was more noticeable in the case of the group of isoprenoids from the aliphatic fraction and the alkylated polycyclic aromatic hydrocarbons (PHAS) from the aromatic fraction. The biodegradation of some targeted isoprenoids increased from 16% to 70% and for some alkylated PAHs from 9% to 44%.

The prestige oil spill. I. Biodegradation of a heavy fuel oil under simulated conditions

In vitro biodegradation of the Prestige heavy fuel oil has been carried out using two microbial consortia obtained by enrichment in different substrates to simulate its environmental fate and potential utility for bioremediation. Different conditions, such as incubation time (i.e., 20 or 40 d), oil weathering, and addition of an oleophilic fertilizer (S200), were evaluated. Weathering slowed down the degradation of the fuel oil, probably because of the loss of lower and more labile components, but the addition of S200 enhanced significantly the extension of the biodegradation. n-Alkanes, alkylcyclohexanes, alkylbenzenes, and the two- to three-ring polycyclic aromatic hydrocarbons (PAHs) were degraded in 20 or 40 d of incubation of the original oil, whereas the biodegradation efficiency decreased for higher PAHs and with the increase of alkylation. Molecular markers were degraded according to the following sequence: Acyclic isoprenoids > diasteranes > C27-steranes > betabeta-steranes > homohopanes > monoaromatic steranes > triaromatic steranes. Isomeric selectivity was observed within the C1- and C2-phenanthrenes, dibenzothiophenes, pyrenes, and chrysenes, providing source and weathering indices for the characterization of the heavy oil spill. Acyclic isoprenoids, C27-steranes, C1- and C2-naphthalenes, phenanthrenes, and dibenzothiophenes were degraded completely when S200 was used. The ratios of the C2- and C3-alkyl homologues of fluoranthene/pyrene and chrysene/benzo[a]anthracene are proposed as source ratios in moderately degraded oils. The 4-methylpyrene and 3-methylchrysene were refractory enough to serve as conserved internal markers in assessing the degradation of the aromatic fraction in a manner similar to that of hopane, as used for the aliphatic fraction.

Biodegradation of a crude oil by three microbial consortia of different origins and metabolic capabilities

Microbial consortia were obtained three by sequential enrichment using different oil products. Consortium F1AA was obtained on a heavily saturated fraction of a degraded crude oil; consortium TD, by enrichment on diesel and consortium AM, on a mixture of five polycyclic aromatic hydrocarbons [PAHs]. The three consortia were incubated with a crude oil in order to elucidate their metabolic capabilities and to investigate possible differences in the biodegradation of these complex hydrocarbon mixtures in relation to their origin. The efficiency of the three consortia in removing the saturated fraction was 60% (F1AA), 48% (TD) and 34% (AM), depending on the carbon sources used in the enrichment procedures. Consortia F1AA and TD removed 100% of n-alkanes and branched alkanes, whereas with consortium AM, 91% of branched alkanes remained. Efficiency on the polyaromatic fraction was 19% (AM), 11% (TD) and 7% (F1AA). The increase in aromaticity of the polyaromatic fraction during degradation of the crude oil by consortium F1AA suggested that this consortium metabolized the aromatic compounds primarily by oxidation of the alkylic chains. The 500-fold amplification of the inocula from the consortia by subculturing in rich media, necessary for use of the consortia in bioremediation experiments, showed no significant decrease in their degradation capability. Journal of Industrial Microbiology & Biotechnology (2002) 28, 252–260 DOI: 10.1038/sj/jim/7000236

Photolysis of PAHs in aqueous phase by UV irradiation

The photooxidation of polycyclic aromatic hydrocarbons (PAHs) was investigated in an aqueous ethanolic solution irradiated with a medium-pressure mercury lamp in laboratory photoreactors equipped with a quartz immersion well. Degradation photolysis of fluorene was more efficient than sensitized photolytic oxidation in the presence of TiO2 suspensions. Photolysis kinetics was dependent on molecular weight and the presence and type of substituents. During the photolytic degradation of fluorene and its derivatives, 9-fluorenone and its corresponding derivatives, which were more resistant to photolysis, were formed.

Isolation and characterization of a 2-methylphenanthrene utilizing bacterium: identification of ring cleavage metabolites

Abstract A bacterial strain capable of utilizing 2-methylphenanthrene (2-MP) as its sole source of carbon and energy for growth was isolated from creosote contaminated soil. The isolate was identified as a strain of Sphingomonas sp. and was designated strain JS5. Utilization of 2-MP by strain JS5 was demonstrated by an increase in bacterial biomass concomitant with a decrease of 2-MP in liquid mineral medium with this compound as sole source of carbon and energy. Growth yield indicated a 23% assimilation of 2-MP carbon. Washed-cell suspensions of strain JS5 incubated with 2-MP accumulated a major metabolite identified as 1-hydroxy-6-methyl-2-naphtoic acid, according to its UV, mass and NMR spectra, and a minor compound with HPLC Rt and UV spectrum indistinguishable from 5-methylsalicylate. The identification of those metabolites, and the demonstration of 2,3-catechol dioxygenase activity in 2-MP induced cells show that the biodegradation of 2-MP by strain JS5 is initiated via dioxygenation and meta-cleavage of the non-methylated aromatic ring, and then proceeds by reactions similar to those reported for phenanthrene. Incubation of the strain with a MP-containing mixture from a pyrolytic fuel oil demonstrates that strain JS5 also acts on other methylated phenanthrenes.