Magdalena Grifoll

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.

Microbial community structure of a heavy fuel oil-degrading marine consortium: linking microbial dynamics with polycyclic aromatic hydrocarbon utilization.

A marine microbial consortium obtained from a beach contaminated by the Prestige oil spill proved highly efficient in removing the different hydrocarbon families present in this heavy fuel oil. Seawater cultures showed a complete removal of all the linear and branched alkanes, an extensive attack on three to five-ring polycyclic aromatic hydrocarbons [PAHs; including anthracene, fluoranthene, pyrene, benzo(a)anthracene, chrysene, and benzo(a)pyrene] (30-100%), and a considerable depletion of their alkyl derivatives. Community dynamics analysis revealed that Alcanivorax species, known alkane degraders, predominated in the initial stages. This was followed by an increase in Alphaproteobacteria (i.e. Maricaulis, Roseovarius), which coincided with the depletion of low molecular PAHs. Finally, these were succeeded by Gammaproteobacteria (mainly Marinobacter and Methylophaga), which were involved in the degradation of the high molecular-weight PAHs. The role of these populations in the removal of the specific components was confirmed by the analysis of subcultures established using the aliphatic or the aromatic fraction of the fuel oil, or single PAHs, as carbon sources. The genus Marinobacter seemed to play a major role in the degradation of a variety of hydrocarbons, as several members of this group were isolated from the different enrichment cultures and grew on plates with hexadecane or single PAHs as sole carbon sources.

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

Selective aerobic degradation of linear alkylbenzenes by pure microbial cultures

Abstract Aerobic degradation in the laboratory of C11–C14 linear alkylbenzenes by pure cultures of bacterial strains ( Pseudomonas sp ) revealed that biodegradation of individual isomers increases when the phenyl group is closer to the end of the alkyl chain. This result contributes to the understanding of the fate of these compounds in the aquatic environment and proves that the similar selective biodegradation observed within the isomeric components in ABS surfactants is not constrained by the presence of the sulphonate group in the molecule as previously suggested.

Bacterial PAH degradation in marine and terrestrial habitats.

Cycling of pollutants is essential to preserve functional marine and terrestrial ecosystems. Progress in optimizing these natural biological processes relies on the identification of the underlying microbial actors and deciphering their interactions at molecular, cellular, community, and ecosystem level. Novel advances on PAH biodegradation are built on a progressive approach that span from pure cultures to environmental communities, illustrating the complex metabolic networks within a single cell, and their further implications in higher complexity systems. Recent analytical chemistry and molecular tools allow a deeper insight into the active microbial processes actually occurring in situ, identifying active functions, metabolic pathways and key players. Understanding these processes will provide new tools to assess biodegradation occurrence and, as a final outcome, predict the success of bioremediation thus reducing its uncertainties, the main drawback of this environmental biotechnology.

Microbial degradation of marine evaporitic crude oils

Abstract The composition of alkylbenzenes and organosulphur compounds in crude oils is strongly modified by biodegradation, even at low levels of transformation where the composition of steranes, hopanes, alkylnaphthalenes, and alkylphenanthrenes has not been altered. These conclusions emerge from the comparison of preserved and biodegraded crude oils of marine evaporitic origin (Amposta and Varadero oils) and controlled experiments of microbial transformation of oils containing high amounts of aryl hydrocarbons and organic sulphur species. The compositions of straight chain compounds, such as n- alkanes , n- alkylbenzenes , 2,5- di -n- alkylthiolanes , 2,6,- di -n- alkylthianes and 2,4-dialkylbenzo-[b]thiophen are more easily modified than those of compounds with isoprenoid side-chains. The experiments also show that benzo[b]thiophenes are markedly more resistent to microbial attack than thiolanes and thianes.

Metabolism of fluoranthene by Mycobacterium sp. strain AP1

The pyrene-degrading Mycobacterium strain AP1 was found to utilize fluoranthene as a sole source of carbon and energy. Identification of metabolites formed from fluoranthene (by growing cells and washed-cell suspensions), the kinetics of metabolite accumulation, and metabolite-feeding studies all indicated that strain AP1 oxidizes fluoranthene using three alternative routes. The first route is initiated by dioxygenation at C-7 and C-8 and, following meta cleavage and pyruvate release, produces a hydroxyacenaphthoic acid that is decarboxylated to acenaphthenone (V). Monooxygenation of this ketone to the quinone and subsequent hydrolysis generates naphthalene-1,8-dicarboxylic acid (IV), which is further degraded via benzene-1,2,3-tricarboxylic acid (III). A second route involves dioxygenation at C-1 and C-2, followed by dehydrogenation and meta cleavage of the resulting diol. A two-carbon fragment excision of the meta cleavage product yields 9-fluorenone-1-carboxylic acid (II), which appears to undergo angular dioxygenation and further degradation to produce benzene-1,2,3-tricarboxylic acid (III), merging this route with the 7,8-dioxygenation route. Decarboxylation of benzene-1,2,3-tricarboxylic acid to phthalate (VIII), as well as further oxidation of the latter, would connect both routes with the central metabolism. The identification of Z-9-carboxymethylenefluorene-1-carboxylic acid (I) suggests a third route for fluoranthene degradation involving dioxygenation at C-2, C-3, and ortho cleavage. There is no evidence of any further degradation of this compound.

Metabolism of fluoranthene by mycobacterial strains isolated by their ability to grow in fluoranthene or pyrene

Mycobacterium sp. strains CP1, CP2, CFt2 and CFt6 were isolated from creosote-contaminated soil due to their ability to grow in pyrene (CP1 and CP2) or fluoranthene (CFt2 and CFt6). All these strains utilized fluoranthene as a sole source of carbon and energy. Strain CP1 exhibited the best growth, with a cellular assimilation of fluoranthene carbon of approximately 45%. Identification of the metabolites accumulated during growth in fluoranthene, the kinetics of metabolites, and metabolite feeding studies, indicated that all these isolates oxidized fluoranthene by the following two routes: the first involves dioxygenation at C-1 and C-2, meta cleavage, and a 2-carbon fragment excision to produce 9-fluorenone-1-carboxylic acid. An angular dioxygenation of the latter yields cis-1,9a-dihydroxy-1-hydrofluorene-9-one-8-carboxylic acid, which is further degraded via 8-hydroxy-3,4-benzocoumarin-1-carboxylic acid, benzene-1,2,3-tricarboxylic acid, and phthalate; the second route involves dioxygenation at C-2 and C-3 and ortho cleavage to give Z-9-carboxymethylenefluorene-1-carboxylic acid. In addition, the pyrene-degrading strains CP1 and CP2 possess a third route initiated by dioxygenation at positions C-7 and C-8, which—following meta cleavage, an aldolase reaction, and a C1-fragment excision—yields acenaphthenone. Monooxygenation of this ketone to the corresponding quinone, and its subsequent hydrolysis, produces naphthalene-1,8-dicarboxylic acid. The results obtained in this study not only complete and confirm the three fluoranthene degradation routes previously proposed for the pyrene-degrading strain Mycobacterium sp. AP1, but also suggest that such routes represent general microbial processes for environmental fluoranthene removal.

Phenol removal from hypersaline wastewaters in a Membrane Biological Reactor (MBR): Operation and microbiological characterisation

In this study, two Membrane Biological Reactors (MBR) with submerged flat membranes, one at lab-scale conditions and the other at pilot-plant conditions, were operated at environmental temperature to treat an industrial wastewater characterised by low phenol concentrations (8-16 mg L(-1)) and high salinity (∼ 150-160 mS cm(-1)). During the operation of both reactors, the phenol loading rate was progressively increased and less than 1mg phenol L(-1) was detected even at very low HRTs (0.5-0.7 days). Membrane fouling was minimized by the cross flow aeration rate inside the MBRs and by intermittent permeation. Microbial community analysis of both reactors revealed that members of the genera Halomonas and Marinobacter (gammaproteobacteria) were major components. Growth-linked phenol degradation by pure cultures of Marinobacter isolates demonstrated that this bacterium played a major role in the removal of phenol from the bioreactors.

Actions of Mycobacterium sp. Strain AP1 on the Saturated- and Aromatic-Hydrocarbon Fractions of Fuel Oil in a Marine Medium†

ABSTRACT The pyrene-degrading Mycobacterium sp. strain AP1 grew in nutrient-supplemented artificial seawater with a heavy fuel oil as the sole carbon source, causing the complete removal of all linear (C12 to C40) and branched alkanes from the aliphatic fraction, as well as an extensive degradation of the three- and four-ring polycyclic aromatic hydrocarbons (PAHs) phenanthrene (95%), anthracene (80%), fluoranthene (80%), pyrene (75%), and benzo(a)anthracene (30%). Alkylated PAHs, which are more abundant in crude oils than the nonsubstituted compounds, were selectively attacked at extents that varied from more than 90% for dimethylnaphthalenes, methylphenanthrenes, methylfluorenes, and methyldibenzothiophenes to about 30% for monomethylated fluoranthenes/pyrenes and trimethylated phenanthrenes and dibenzothiophenes. Identification of key metabolites indicated the utilization of phenanthrene, pyrene, and fluoranthene by known assimilatory metabolic routes, while other components were cooxidized. Detection of mono- and dimethylated phthalic acids demonstrated ring cleavage and further oxidation of alkyl PAHs. The extensive degradation of the alkanes, the two-, three-, and four-ring PAHs, and their 1-, 2-, and 3-methyl derivatives from a complex mixture of hydrocarbons by Mycobacterium sp. strain AP1 illustrates the great substrate versatility of alkane- and PAH-degrading mycobacteria.