Secondary succession of bacterial communities and co-occurrence of phylotypes in oil-polluted Populus rhizosphere Shinjini Mukherjee a , Mirja Heinonen a , Magali Dequvire a , Timo Sipilä a , Pertti Pulkkinen b , Kim Yrjälä a, * a Molecular Environmental Microbiology-group, Department of Biosciences, University of Helsinki, Helsinki, Finland b Finnish Forest Research Institute (Metla), Haapastensyrjä Research Unit, Haapastensyrjäntie 34, FI-12600, Läyliäinen, Finland article info Article history: Received 30 August 2012 Received in revised form 22 November 2012 Accepted 22 November 2012 Available online 10 December 2012 Keywords: Bacterial community structure Secondary succession Functional diversity Most-probable number (MPN) Terminal restriction fragment length polymorphism (T-RFLP) abstract Oil pollution can be considered as a driver for bacterial secondary succession. A greenhouse study was conducted using forest mineral soil contaminated by heavy fuel oil and planted with hybrid aspen. The aim was to compare the bacterial succession in oil- and plant-amended ecosystems. Samples were taken weekly from bulk and rhizosphere soil. The oil degrading bacteria were enumerated by most-probable number assay and the bacterial community structures were determined by terminal restriction fragment length polymorphism (T-RFLP) of the 16S rRNA and extradiol dioxygenase genes. Hierarchical clustering of phylotypes coupled with heatmap analysis of T-RFLP fingerprints revealed patterns of co-occurrence of bacterial groups. Co-occurring OTUs present only in the oil treatments were observed from both 16S rRNA and extradiol dioxygenase gene heatmaps, which represent the bacteria adapted to oil pollution. A rhizosphere effect was detected in oil-contaminated soil with elevated bacterial 16S rRNA gene diversity and MPN counts. The forest soil was dominated by two phyla, Acidobacteria and Proteobacteria, while Betaproteobacteria were clearly activated by addition of oil. Directional succession of bacterial communities was unique to oil treatments, shown by the 16S rRNA gene community structure. These results advance our understanding of secondary succession of bacterial communities in bio- and rhizoremediation. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Anthropogenic activities such as oil extraction, refinement and transportation have resulted in surface and near-subsurface soil contamination with petroleum hydrocarbons, including crude (or synthetic crude) oil, gasoline, diesel and creosote (Robertson et al., 2007). Oil pollution contributes to environmental stress, which disturbs the ecological balance by causing damage to sensitive organisms. On the community level, relatively vulnerable species may be reduced in abundance or eliminated, leading to weakened ecosystem functioning (Sousa, 1984). The microbial dynamics in the pollution process can be thought of as secondary succession of bacteria in a disturbed ecosystem where secondary succession is defined as a pattern of change in the community composition after a radical disturbance in the physical environment (Connell and Slatyer, 1977; Cook et al., 2005; Horn, 1974). Little experimental evidence exists to indicate that predictable patterns in microbial community structure or composition occur during secondary succession or ecosystem restoration (Banning et al., 2011). Since bacteria have the functional potential to bioremediate a polluted habitat (Thompson et al., 2005), understanding the dynamics of bacterial communities in pollution is an important aspect of ecological restoration of polluted sites (Ager et al., 2010). Rhizoremediation is one of the most effective means to reme- diate organic contaminants (Dietz and Schnoor, 2001; Gerhardt et al., 2009; Germida et al., 2002; Kuiper et al., 2004; Mackova et al., 2006; Susarla et al., 2002). The interest in the use of woody plants for rhizoremediation gained momentum with the demand of biomass production for energy (Golan-Goldhirsh et al., 2004). Plant species and soil type have a substantial influence on the structure and function of rhizosphere-associated microbial populations (Badri et al., 2009; Berg and Smalla, 2009; Cebron et al., 2009; el Zahar Haichar et al., 2008). Microbial community structure in the rhizosphere has been studied using both culture dependent (Yousaf et al., 2010) and culture independent (DNA based fingerprinting) methods (Nocker et al., 2007; Nunes da Rocha et al., 2009; Singh et al., 2004). Terminal restriction fragment length polymorphism (T-RFLP) allows reproducible analysis of a large number of samples, also those containing high diversity, and the subsequent in silico * Corresponding author. Tel.: þ358 (0) 9 19159220; fax: þ358 (0) 9 19159262. E-mail address: kim.yrjala@helsinki.fi (K. Yrjälä). Contents lists available at SciVerse ScienceDirect Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio 0038-0717/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.soilbio.2012.11.018 Soil Biology & Biochemistry 58 (2013) 188e197