Tapan K. Dutta

Biodegradation of n-Alkylcycloalkanes and n-Alkylbenzenes via New Pathways in Alcanivorax sp. Strain MBIC 4326

ABSTRACT The degradation of long-chain n-alkylbenzenes andn-alkylcyclohexanes by Alcanivorax sp. strain MBIC 4326 was investigated. The alkyl side chain of these compounds was mainly processed by β-oxidation. In the degradation ofn-alkylcyclohexanes, cyclohexanecarboxylic acid was formed as an intermediate. This compound was further transformed to benzoic acid via 1-cyclohexene-1-carboxylic acid.

Current State of Knowledge in Microbial Degradation of Polycyclic Aromatic Hydrocarbons (PAHs): A Review

Polycyclic aromatic hydrocarbons (PAHs) include a group of organic priority pollutants of critical environmental and public health concern due to their toxic, genotoxic, mutagenic and/or carcinogenic properties and their ubiquitous occurrence as well as recalcitrance. The increased awareness of their various adverse effects on ecosystem and human health has led to a dramatic increase in research aimed toward removing PAHs from the environment. PAHs may undergo adsorption, volatilization, photolysis, and chemical oxidation, although transformation by microorganisms is the major neutralization process of PAH-contaminated sites in an ecologically accepted manner. Microbial degradation of PAHs depends on various environmental conditions, such as nutrients, number and kind of the microorganisms, nature as well as chemical property of the PAH being degraded. A wide variety of bacterial, fungal and algal species have the potential to degrade/transform PAHs, among which bacteria and fungi mediated degradation has been studied most extensively. In last few decades microbial community analysis, biochemical pathway for PAHs degradation, gene organization, enzyme system, genetic regulation for PAH degradation have been explored in great detail. Although, xenobiotic-degrading microorganisms have incredible potential to restore contaminated environments inexpensively yet effectively, but new advancements are required to make such microbes effective and more powerful in removing those compounds, which were once thought to be recalcitrant. Recent analytical chemistry and genetic engineering tools might help to improve the efficiency of degradation of PAHs by microorganisms, and minimize uncertainties of successful bioremediation. However, appropriate implementation of the potential of naturally occurring microorganisms for field bioremediation could be considerably enhanced by optimizing certain factors such as bioavailability, adsorption and mass transfer of PAHs. The main purpose of this review is to provide an overview of current knowledge of bacteria, halophilic archaea, fungi and algae mediated degradation/transformation of PAHs. In addition, factors affecting PAHs degradation in the environment, recent advancement in genetic, genomic, proteomic and metabolomic techniques are also highlighted with an aim to facilitate the development of a new insight into the bioremediation of PAH in the environment.

Metabolism of butyl benzyl phthalate by Gordonia sp. strain MTCC 4818

The microbial degradative characteristics of butyl benzyl phthalate (BBP) were investigated by the Gordonia sp. strain MTCC 4818 isolated from creosote-contaminated soil. The test organism can utilize a number of phthalate esters as sole sources of carbon and energy, where BBP was totally degraded within 4 days under shake culture conditions. High performance liquid chromatography profile of the metabolites isolated from spent culture indicated the accumulation of two major products apart from phthalic acid (PA), which were characterized by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy as mono-n-butyl phthalate (MBuP) and monobenzyl phthalate (MBzP). Neither of the metabolites, MBuP, MBzP or PA, supported growth of the test organism, while in resting cell transformation, the monoesters were hydrolyzed to PA to a very minor extent, which was found to be a dead-end product in the degradation process. On the other hand, the test organism grew well on benzyl alcohol and butanol, the hydrolyzed products of BBP. The esterase(s) was found to be inducible in nature and can hydrolyze in vitro the seven different phthalate diesters tested to their corresponding monoesters irrespective of their support to the growth of the test organism.

Degradation of phenanthrene via meta-cleavage of 2-hydroxy-1-naphthoic acid by Ochrobactrum sp. strain PWTJD

The present study describes the assimilation of phenanthrene by an aerobic bacterium, Ochrobactrum sp. strain PWTJD, isolated from municipal waste-contaminated soil sample utilizing phenanthrene as a sole source of carbon and energy. The isolate was identified as Ochrobactrum sp. based on the morphological, nutritional and biochemical characteristics as well as 16S rRNA gene sequence analysis. A combination of chromatographic analyses, oxygen uptake assay and enzymatic studies confirmed the degradation of phenanthrene by the strain PWTJD via 2-hydroxy-1-naphthoic acid, salicylic acid and catechol. The strain PWTJD could also utilize 2-hydroxy-1-naphthoic acid and salicylic acid, while the former was metabolized by a ferric-dependent meta-cleavage dioxygenase. In the lower pathway, salicylic acid was metabolized to catechol and was further degraded by catechol 2,3-dioxygenase to 2-hydroxymuconoaldehyde acid, ultimately leading to tricarboxylic acid cycle intermediates. This is the first report of the complete degradation of a polycyclic aromatic hydrocarbon molecule by Gram-negative Ochrobactrum sp. describing the involvement of the meta-cleavage pathway of 2-hydroxy-1-naphthoic acid in phenanthrene assimilation.

Complete degradation of di-n-octyl phthalate by Gordonia sp. strain Dop5.

The present study describes the assimilation of di-n-octyl phthalate by an aerobic bacterium, isolated from municipal waste-contaminated soil sample utilizing di-n-octyl phthalate as the sole source of carbon and energy. The isolate was identified as Gordonia sp. based on the morphological, nutritional and biochemical characteristics as well as 16S rRNA gene sequence analysis. A combination of chromatographic and spectrometric analyses revealed a complete di-n-octyl assimilation pathway. In the degradation process, mono-n-octyl phthalate, phthalic acid, protocatechuic acid and 1-octanol were identified as the degradation products of di-n-octyl phthalate. Furthermore, phthalic acid was metabolized via protocatechuic acid involving protocatechuate 3,4-dioxygenase while 1-octanol was metabolized by NAD(+)-dependent dehydrogenases to 1-octanoic acid, which was subsequently degraded via β-oxidation, ultimately, leading to tricarboxylic acid cycle intermediates. Apart from phthalic acid and 1-octanol metabolizing pathway enzymes, two esterases, di-n-octyl phthalate hydrolase and mono-n-octyl phthalate hydrolase involved in di-n-octyl phthalate degradation were found to be inducible in nature. This is the first report on the metabolic pathway involved in the complete degradation of di-n-octyl phthalate by a single bacterial isolate, which is also capable of efficiently degrading other phthalate esters of environmental concern having either shorter or longer alkyl chains.

Role of oxygenases in guiding diverse metabolic pathways in the bacterial degradation of low-molecular-weight polycyclic aromatic hydrocarbons: a review.

Widespread environmental pollution by polycyclic aromatic hydrocarbons (PAHs) poses an immense risk to the environment. Bacteria-mediated attenuation has a great potential for the restoration of PAH-contaminated environment in an ecologically accepted manner. Bacterial degradation of PAHs has been extensively studied and mining of biodiversity is ever expanding the biodegradative potentials with intelligent manipulation of catabolic genes and adaptive evolution to generate multiple catabolic pathways. The present review of bacterial degradation of low-molecular-weight (LMW) PAHs describes the current knowledge about the diverse metabolic pathways depicting novel metabolites, enzyme-substrate/metabolite relationships, the role of oxygenases and their distribution in phylogenetically diverse bacterial species.

meta-Cleavage of hydroxynaphthoic acids in the degradation of phenanthrene by Sphingobium sp. strain PNB

Polycyclic aromatic hydrocarbons (PAHs) comprise a group of priority organic pollutants that are toxic and/or carcinogenic. Phenanthrene, the simplest PAH among recognized priority pollutants, is commonly used as a model compound for the study of PAH biodegradation. Sphingobium sp. strain PNB, capable of degrading phenanthrene as a sole carbon and energy source, was isolated from a municipal waste-contaminated soil sample. A combination of chromatographic and spectrometric analyses, together with oxygen uptake and enzyme activity studies, suggested the presence of phenanthrene degradation pathways in this strain. Identification of metabolites suggested that initial dioxygenation of phenanthrene took place at both 3,4- and 1,2-carbon positions; meta-cleavage of resultant diols led to the formation of 1-hydroxy-2-naphthoic acid and 2-hydroxy-1-naphthoic acid, respectively. The hydroxynaphthoic acids, in turn, were metabolized by a meta-cleavage pathway(s), leading to the formation of 2,2-dicarboxychromene and 2-hydroxychromene-2-glyoxylic acid, respectively. These metabolites were subsequently transformed to catechol via salicylic acid, which further proceeds towards the tricarboxylic acid cycle leading to complete mineralization of the compound phenanthrene. The present study establishes the metabolism of hydroxynaphthoic acids by a meta-cleavage pathway in the degradation of phenanthrene, expanding our current understanding of microbial degradation of PAHs.

Metabolic cooperation of Gordonia sp. strain MTCC 4818 and Arthrobacter sp. strain WY in the utilization of butyl benzyl phthalate: effect of a novel co-culture in the degradation of a mixture of phthalates

Degradation of butyl benzyl phthalate (BBP) by a co-culture of Gordonia sp. strain MTCC 4818 and Arthrobacter sp. strain WY was investigated. In the degradation of BBP by the co-culture, the limitations of the individual species in metabolizing BBP were overcome, leading to the development of a consortium capable of complete utilization of this ester. In the degradation of BBP by the co-culture, the presence of multiple esterases was demonstrated in both species by activity staining of non-denaturing gels, indicating their roles in the degradation process. The esterases were found to be inducible, with unique or broad substrate specificities towards BBP and its monoesters. Moreover, a number of catabolic enzymes other than esterases identified in the metabolism of BBP-degraded intermediates facilitated the co-culture-mediated degradation process. The versatility of the co-culture was further established by the rapid and complete degradation of a mixture of phthalate esters of environmental concern.

An insight into the origin and functional evolution of bacterial aromatic ring-hydroxylating oxygenases

Bacterial aromatic ring-hydroxylating oxygenases (RHOs) are multicomponent enzyme systems which have potential utility in bioremediation of aromatic compounds in the environment. To cope with the enormous diversity of aromatic compounds in the environment, this enzyme family has evolved remarkably exhibiting broad substrate specificity. RHOs are multicomponent enzymes comprising of a homo- or hetero-multimeric terminal oxygenase and one or more electron transport (ET) protein(s). The present study attempts in depicting the evolutionary scenarios that might have occurred during the evolution of RHOs, by analyzing a set of available sequences including those obtained from complete genomes. A modified classification scheme identifying four new RHO types has been suggested on the basis of their evolutionary and functional behaviours, in relation to structural configuration of substrates and preferred oxygenation site(s). The present scheme emphasizes on the fact that the phylogenetic affiliation of RHOs is distributed among four distinct ‘Similarity classes’, independent of the constituent ET components. Similar combination of RHO components that was previously considered to be equivalent and classified together [Kweon et al., BMC Biochemistry 9, 11 (2008)] were found here in distinct similarity classes indicating the role of substrate-binding terminal oxygenase in guiding the evolution of RHOs irrespective of the nature of constituent ET components. Finally, a model for evolution of the multicomponent RHO enzyme system has been proposed, beginning from genesis of the terminal oxygenase components followed by recruitment of constituent ET components, finally evolving into various ‘extant’ RHO types.

Functional characterization of diverse ring-hydroxylating oxygenases and induction of complex aromatic catabolic gene clusters in Sphingobium sp. PNB

Sphingobium sp. PNB, like other sphingomonads, has multiple ring‐hydroxylating oxygenase (RHO) genes. Three different fosmid clones have been sequenced to identify the putative genes responsible for the degradation of various aromatics in this bacterial strain. Comparison of the map of the catabolic genes with that of different sphingomonads revealed a similar arrangement of gene clusters that harbors seven sets of RHO terminal components and a sole set of electron transport (ET) proteins. The presence of distinctly conserved amino acid residues in ferredoxin and in silico molecular docking analyses of ferredoxin with the well characterized terminal oxygenase components indicated the structural uniqueness of the ET component in sphingomonads. The predicted substrate specificities, derived from the phylogenetic relationship of each of the RHOs, were examined based on transformation of putative substrates and their structural homologs by the recombinant strains expressing each of the oxygenases and the sole set of available ET proteins. The RHO AhdA1bA2b was functionally characterized for the first time and was found to be capable of transforming ethylbenzene, propylbenzene, cumene, p‐cymene and biphenyl, in addition to a number of polycyclic aromatic hydrocarbons. Overexpression of aromatic catabolic genes in strain PNB, revealed by real‐time PCR analyses, is a way forward to understand the complex regulation of degradative genes in sphingomonads.