Biodegradation of variable-chain-length n-alkanes in Rhodococcus opacus R7 and the involvement of an alkane hydroxylase system in the metabolism.

Jessica Zampolli,Elena Collina,Patrizia Di Gennaro,Marina Lasagni

doi:10.1186/s13568-014-0073-4

Abstract

Rhodococcus opacus R7 is a Gram-positive bacterium isolated from a polycyclic aromatic hydrocarbon contaminated soil for its versatile metabolism; indeed the strain is able to grow on naphthalene, o-xylene, and several long- and medium-chain n-alkanes. In this work we determined the degradation of n-alkanes in Rhodococcus opacus R7 in presence of n-dodecane (C12), n-hexadecane (C16), n-eicosane (C20), n-tetracosane (C24) and the metabolic pathway in presence of C12. The consumption rate of C12 was 88%, of C16 was 69%, of C20 was 51% and of C24 it was 78%. The decrement of the degradation rate seems to be correlated to the length of the aliphatic chain of these hydrocarbons. On the basis of the metabolic intermediates determined by the R7 growth on C12, our data indicated that R. opacus R7 metabolizes medium-chain n-alkanes by the primary alcohol formation. This represents a difference in comparison with other Rhodococcus strains, in which a mixture of the two alcohols was observed. By GC-MSD analysis we also identified the monocarboxylic acid, confirming the terminal oxidation.Moreover, the alkB gene cluster from R. opacus R7 was isolated and its involvement in the n-alkane degradation system was investigated by the cloning of this genomic region into a shuttle-vector E. coli-Rhodococcus to evaluate the alkane hydroxylase activity. Our results showed an increased biodegradation of C12 in the recombinant strain R. erythropolis AP (pTipQT1-alkR7) in comparison with the wild type strain R. erythropolis AP. These data supported the involvement of the alkB gene cluster in the n-alkane degradation in the R7 strain.

Highlights

The problems associated with contaminated sites are assuming rising prominence in many countries (Vidali, 2001), due to the increase of anthropogenic pollution in the environment
Rhodococcus opacus R7 is a Gram-positive bacterium isolated from a polycyclic aromatic hydrocarbon contaminated soil for its ability to grow on naphthalene. It is characterized by a versatile metabolism, the strain is able to grow on naphthalene, o-xylene, and several long- and medium-chain n-alkanes
Rhodococcus opacus R7 (CIP 107348), isolated for its ability to grow on naphtalene and o-xlyene (Di Gennaro et al, 2001), was grown on M9 mineral medium (Maniatis et al, 1982), supplemented with naphthalene or o-xylene or different n-alkanes in an atmosphere saturated with these compounds, as the only carbon and energy source

Summary

Introduction

The problems associated with contaminated sites are assuming rising prominence in many countries (Vidali, 2001), due to the increase of anthropogenic pollution in the environment. There are a lot of studies on the metabolism of these compounds in Gramnegative bacteria while Gram-positive bacteria have not Alkane degraders are bacteria that have a very versatile metabolism, so that they can use as carbon source many other compounds in addition to alkanes (Smits et al, 2002; Margesin et al, 2003; Harayama et al, 2004). Linear molecules whose chain length can vary from 1 (in methane) to more than 50 carbon atoms They are the major components of petroleum fuels which can be commonly found in contaminated environments (So and Young, 1999), they constitute about 20-50% of crude oil, depending on the source of the oil. It is a three-component alkane hydroxylase complex consisting of a particulate nonheme integral membrane alkane monooxygenase (AlkB) and two soluble proteins, rubredoxin (AlkG) and rubredoxin reductase (AlkT) (Alonso and Roujeinikova, 2012)

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