Ten millilitres of enrichment culture showing degradation of FE was transferred to 100 mL fresh MSM

containing 50 mg L−1 FE and incubated for 7 days. Four rounds of enrichment were performed and the concentration of FE was raised to 200 mg L−1. The final enrichment culture was serially diluted and spread on MSM plates containing 100 mg L−1 FE and 1.8% agar. After being incubated at 30 °C for 3 days, the colonies surrounded by transparent halos and with different morphologies were selected for analysis of their degradation capabilities. One strain, designated T1, was selected for further investigation. The degradation of FA, CDHB and HPP by the enrichment culture was studied ABT-737 molecular weight in the MSM containing 50 mg L−1 FA, CDHB and HPP as the sole carbon source and Carfilzomib 5% (v:v) of enrichment culture was inoculated. Strain T1 was identified based on 16S rRNA gene sequence analysis and morphological, physiological and biochemical

tests referenced in Bergey’s Manual of Determinative Bacteriology (Holt et al., 1994). Total genomic DNA was prepared from strain T1 by high-salt precipitation (Miller et al., 1988). The universal primers 27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492r (5′-TACCTTGTTACGACTT-3′) were used to amplify 16S rRNA gene. The purified PCR fragments were ligated into the linearised vector pMD19-T (TaKaRa Biotechnology, Dalian, China) and transformed into E. coli DH5α. An automatic sequencer (Applied Biosystems, model No.3730) was used to obtain the 16S rRNA gene sequences using sequencing primers M13-47 (5′-CGCCAGGGTTTTCCCAGTCACGAC-3′) and RV-M (5′-GAGCGGATAACAATTTCACACAGG-3′) (Jia et al., 2006). The National Centre for Biotechnology Information (NCBI) database’s blast program was used to analyse the DNA for similarity to other 16S rRNA gene sequences (Altschul et al., 1990). Alignment of the different 16S rRNA gene sequences from GenBank was performed using clustalx 1.8.3 with default settings. Phylogenesis was analysed using mega version 4.1 software. Distances were calculated using the Kimura two-parameter distance model. Unrooted trees were built using the Neighbour Joining method. Dataset was bootstrapped 1000 times.

Strain T1 was precultured in Luria–Bertani medium (LB, containing tryptone 10.0 g L−1, Phospholipase D1 yeast extract 5.0 g L−1 and NaCl 10.0 g L−1, pH 7.0), harvested by centrifugation at 6000 g for 5 min, washed with sterilised MSM. Then the optical density of cells at 600 nm was adjusted to 1.0 (corresponding to 4.6 × 108 cells mL−1). For all experiments, the cells were inoculated at a 5% (v:v) level into 10 mL MSM (pH 7.0) containing 100 mg L−1 FE before being incubated at 30 °C and 180 rpm in a rotary shaker unless otherwise stated. The stock solution of FE (10 mg mL−1 dissolved by methanol) was added to the flasks (50 mL), and the methanol was allowed to evaporate before addition of MSM media. For controls, media without inoculation was maintained and tested in the same manner as above.