Carl E. Cerniglia

Biodegradation of polycyclic aromatic hydrocarbons

The intent of this review is to provide an outline of the microbial degradation of polycyclic aromatic hydrocarbons. A catabolically diverse microbial community, consisting of bacteria, fungi and algae, metabolizes aromatic compounds. Molecular oxygen is essential for the initial hydroxylation of polycyclic aromatic hydrocarbons by microorganisms. In contrast to bacteria, filamentous fungi use hydroxylation as a prelude to detoxification rather than to catabolism and assimilation. The biochemical principles underlying the degradation of polycyclic aromatic hydrocarbons are examined in some detail. The pathways of polycyclic aromatic hydrocarbon catabolism are discussed. Studies are presented on the relationship between the chemical structure of the polycyclic aromatic hydrocarbon and the rate of polycyclic aromatic hydrocarbon biodegradation in aquatic and terrestrial ecosystems.

Microbial Metabolism of Polycyclic Aromatic Hydrocarbons

Publisher Summary This chapter deals with the microbial transformation of polycyclic aromatic hydrocarbons (PAHs). The similarities and differences between the microbial and mammalian metabolism are described. Bacteria, filamentous fungi, yeasts, cyanobacteria, diatoms, and other eukaryotic algae have the enzymatic capacity to oxidize PAHs that range in size from naphthalene to benzo[ a ]pyrene. The hydroxylation of PAHs always involves the incorporation of molecular oxygen; however, there are differences in the mechanism of hydroxylation of PAHs by prokaryotic and eukaryotic microorganisms. Bacteria oxygenate PAHs to form a dihydrodiol with a cis configuration. The genes for the initial oxidation of PAHs are localized on plasmids. In contrast to bacteria, fungi oxidize PAHs via a cytochrome P-450 monooxygenase to form arene oxide, which can isomerize to phenols or undergo enzymatic hydration to yield trans-dihydrodiols. Multiple oxidative pathways may be involved in the cyanobacterial metabolism of PAHs. Studies on PAH metabolism are entering a new era; biochemical genetic techniques such as gene cloning and transposon mutagenesis will provide new insight into the biochemistry and regulation of PAH degradative pathways.

The Reduction of Azo Dyes by the Intestinal Microflora

Azo dyes are widely used in the textile, printing, paper manufacturing, pharmaceutical, and food industries and also in research laboratories. When these compounds either inadvertently or by design enter the body through ingestion, they are metabolized to aromatic amines by intestinal microorganisms. Reductive enzymes in the liver can also catalyze the reductive cleavage of the azo linkage to produce aromatic amines. However, evidence indicates that the intestinal microbial azoreductase may be more important than the liver enzymes in azo reduction. In this article, we examine the significance of the capacity of intestinal bacteria to reduce azo dyes and the conditions of azo reduction. Many azo dyes, such as Acid Yellow, Amaranth, Azodisalicylate, Chicago Sky Blue, Congo Red, Direct Black 38, Direct Blue 6, Direct Blue 15, Direct Brown 95, Fast Yellow, Lithol Red, Methyl Orange, Methyl Red, Methyl Yellow, Naphthalene Fast Orange 2G, Neoprontosil, New Coccine, Orange II, Phenylazo-2-naphthol, Ponceau 3R, Ponceau SX, Red 2G, Red 10B, Salicylazosulphapyridine, Sunset Yellow, Tartrazine, and Trypan Blue, are included in this article. A wide variety of anaerobic bacteria isolated from caecal or fecal contents from experimental animals and humans have the ability to cleave the azo linkage(s) to produce aromatic amines. Azoreductase(s) catalyze these reactions and have been found to be oxygen sensitive and to require flavins for optimal activity. The azoreductase activity in a variety of intestinal preparations was affected by various dietary factors such as cellulose, proteins, fibers, antibiotics, or supplementation with live cultures of lactobacilli.

Mutagenicity of azo dyes: Structure-activity relationships

Azo dyes are extensively used in textile, printing, leather, paper making, drug and food industries. Following oral exposure, azo dyes are metabolized to aromatic amines by intestinal microflora or liver azoreductases. Aromatic amines are further metabolized to genotoxic compounds by mammalian microsomal enzymes. Many of these aromatic amines are mutagenic in the Ames Salmonella/microsomal assay system. The chemical structure of many mutagenic azo dyes was reviewed, and we found that the biologically active dyes are mainly limited to those compounds containing p-phenylenediamine and benzidine moieties. It was found that for the phenylenediamine moiety, methylation or substitution of a nitro group for an amino group does not decrease mutagenicity. However, sulfonation, carboxylation, deamination, or substitution of an ethyl alcohol or an acetyl group for the hydrogen in the amino groups leads to a decrease in the mutagenic activity. For the benzidine moiety, methylation, methoxylation, halogenation or substitution of an acetyl group for hydrogen in the amino group does not affect mutagenicity, but complexation with copper ions diminishes mutagenicity. The mutagenicity of benzidine or its derivatives is also decreased when in the form of a hydrochloride salt with only one exception. Mutagenicity of azo dyes can, therefore, be predicted by these structure-activity relationships.

Fungal metabolism of polycyclic aromatic hydrocarbons: past, present and future applications in bioremediation

This article examines the importance of non-ligninolytic and ligninolytic fungi in the bioremediation of polycyclic aromatic hydrocarbon contaminated wastes. The research from the initial studies in Dave Gibson’s laboratory to the present are discussed.

Degradation of Phenanthrene and Anthracene by Cell Suspensions of Mycobacterium sp. Strain PYR-1

ABSTRACT Cultures of Mycobacterium sp. strain PYR-1 were dosed with anthracene or phenanthrene and after 14 days of incubation had degraded 92 and 90% of the added anthracene and phenanthrene, respectively. The metabolites were extracted and identified by UV-visible light absorption, high-pressure liquid chromatography retention times, mass spectrometry, 1H and 13C nuclear magnetic resonance spectrometry, and comparison to authentic compounds and literature data. Neutral-pH ethyl acetate extracts from anthracene-incubated cells showed four metabolites, identified ascis-1,2-dihydroxy-1,2-dihydroanthracene, 6,7-benzocoumarin, 1-methoxy-2-hydroxyanthracene, and 9,10-anthraquinone. A novel anthracene ring fission product was isolated from acidified culture media and was identified as 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid. 6,7-Benzocoumarin was also found in that extract. When Mycobacterium sp. strain PYR-1 was grown in the presence of phenanthrene, three neutral metabolites were identified as cis- andtrans-9,10-dihydroxy-9,10-dihydrophenanthrene andcis-3,4-dihydroxy-3,4-dihydrophenanthrene. Phenanthrene ring fission products, isolated from acid extracts, were identified as 2,2′-diphenic acid, 1-hydroxynaphthoic acid, and phthalic acid. The data point to the existence, next to already known routes for both gram-negative and gram-positive bacteria, of alternative pathways that might be due to the presence of different dioxygenases or to a relaxed specificity of the same dioxygenase for initial attack on polycyclic aromatic hydrocarbons.

Environmental aspects of PAH biodegradation.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants, some of which are on the US Environmental Protection Agency priority pollutant list. Consequently, timely clean-up of contaminated sites is important. The lower-mol-wt PAHs are amenable to bioremediation; however, higher-mol-wt PAHs seem to be recalcitrant to microbial degradation. The rates of biodegradation of PAHs are highly variable and are dependent not only on PAH structure, but also on the physicochemical parameters of the site as well as the number and types of microorganisms present. PAHs sorb to organic matter in soils and sediments, and the rate of their desorption strongly influences the rate at which microorganisms can degrade the pollutants. Much of the current PAH research focuses on techniques to enhance the bioavailability and, therefore, the degradation rates of PAHs at polluted sites. Degradation products of PAHs are, however, not necessarily less toxic than the parent compounds. Therefore, toxicity assays need to be incorporated into the procedures used to monitor the effectiveness of PAH bioremediation. In addition, this article highlights areas of PAH research that require further investigation.

Biotransformation of Malachite Green by the Fungus Cunninghamella elegans

ABSTRACT The filamentous fungus Cunninghamella elegans ATCC 36112 metabolized the triphenylmethane dye malachite green with a first-order rate constant of 0.029 μmol h−1 (mg of cells)−1. Malachite green was enzymatically reduced to leucomalachite green and also converted to N-demethylated and N-oxidized metabolites, including primary and secondary arylamines. Inhibition studies suggested that the cytochrome P450 system mediated both the reduction and the N-demethylation reactions.

A universal protocol for PCR detection of 13 species of foodborne pathogens in foods

A universal protocol for PCR detection of 13 species of foodborne pathogens in foods wasdeveloped. The protocol used a universal culture medium and the same PCR conditions with 13sets of specific primers. The 13 species of foodborne pathogens examined were Escherichiacoli, E. coli‐ETEC, E. coli‐O157:H7, Shigella spp., Salmonella spp., Yersinia enterocolitica, Y. pseudotuberculosis, Vibrio cholerae, V.parahaemolyticus, V. vulnificus, Listeria monocytogenes, Staphylococcus aureusand Bacillus cereus. No interference was observed using the PCR assay when foodsample was artificially inoculated with each individual bacterial species. Twelve different seafoodsamples and two soft cheese samples without artificial inoculation were examined by thisprotocol. Vibriovulnificus, Salmonella spp.,E. coli,Listeria monocytogenes and Bacillus cereus were detected in some foods.Internal probe hybridization and nested PCR procedures were used to confirm the above findings.

Complete and integrated pyrene degradation pathway in Mycobacterium vanbaalenii PYR-1 based on systems biology

Mycobacterium vanbaalenii PYR-1 was the first bacterium isolated by virtue of its ability to metabolize the high-molecular-weight polycyclic aromatic hydrocarbon (PAH) pyrene. We used metabolic, genomic, and proteomic approaches in this investigation to construct a complete and integrated pyrene degradation pathway for M. vanbaalenii PYR-1. Genome sequence analyses identified genes involved in the pyrene degradation pathway that we have proposed for this bacterium. To identify proteins involved in the degradation, we conducted a proteome analysis of cells exposed to pyrene using one-dimensional gel electrophoresis in combination with liquid chromatography-tandem mass spectrometry. Database searching performed with the M. vanbaalenii PYR-1 genome resulted in identification of 1,028 proteins with a protein false discovery rate of <1%. Based on both genomic and proteomic data, we identified 27 enzymes necessary for constructing a complete pathway for pyrene degradation. Our analyses indicate that this bacterium degrades pyrene to central intermediates through o-phthalate and the beta-ketoadipate pathway. Proteomic analysis also revealed that 18 enzymes in the pathway were upregulated more than twofold, as indicated by peptide counting when the organism was grown with pyrene; three copies of the terminal subunits of ring-hydroxylating oxygenase (NidAB2, MvanDraft_0817/0818, and PhtAaAb), dihydrodiol dehydrogenase (MvanDraft_0815), and ring cleavage dioxygenase (MvanDraft_3242) were detected only in pyrene-grown cells. The results presented here provide a comprehensive picture of pyrene metabolism in M. vanbaalenii PYR-1 and a useful framework for understanding cellular processes involved in PAH degradation.