Computational protocol: Positive Effects of Bacterial Diversity on Ecosystem Functioning Driven by Complementarity Effects in a Bioremediation Context

Similar protocols

Protocol publication

[…] Bacteria used in this study were isolated from two different aquifers contaminated with crude oil in eastern Colombia: one located near the Cravo Sur petroleum exploitation site (hereafter Cravo Sur) and other located near the Gloria Norte exploitation site (hereafter Gloria Norte), both on the region of Casanare. Sampling was carried out by Biointech (Colombia), a private company with permission from the land owners to collect samples. This study did not involve endangered or protected species. Prior to bacteria isolation approximately one liter of a mixture of crude oil and water was sampled from each site and kept in the dark at room temperature (∼20°C) until strain isolation. Bacterial communities from each site were plated in LB agar plates (10 g of tryptone, 5 g of yeast extract and 10 g of NaCl in one liter of distilled water) and after four days of incubation at 30°C, six bacterial colonies from each site were isolated based on clearly distinct morphology. Identification of the different isolates based on morphologically distinctive characters was required to perform the current study (see below). While probably not representative of the actual composition of petroleum degrading bacteria originally present in the sampled aquifer, together, the isolated strains represented over 70% of the cultured bacterial diversity. Given the full-factorial experimental design (see below), twelve strains was the maximum number of strains we could logistically handle. All twelve isolated strains showed positive growth using crude oil as the only carbon source and were preserved in −80°C in a 50% glycerol solution. The isolates (hereafter genotypes) were identified by means of 16S r-RNA sequencing (). For this, single-colony 16S r-RNA amplification was performed with a PCR System Icycler® BioRad thermocycler using primers specific for bacteria, 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′), yielding a fragment of about 1465 bp. The PCR reactions contained 1.5 μl of cell lysate, 2.5 μl of buffer, 1.25 μl of MgCl2, 2.5 μl of dNTP's, 0.5 μl of each primer and 0.5 μl of Taq DNA polymerase each; the final volume (25 μl) was achieved by topping up with distilled – deionized water. The PCR thermal cycling scheme consisted of 120 s at 95°C, 30 cycles of: 30 s at 95°C, 30 s at 50°C, 45 s at 72°C, and a final extension of 7 min at 72°C. Purification, concentration and sequencing of PCR products were performed by Macrogen Inc. (Korea). We then compared the obtained sequences to the BLAST database and retained for each isolated strain the closest known relative taxa with the highest maximal identity percentage (i.e., reference taxa). The reference taxa of the six genotypes from Cravo Sur were: Burkholderia thailandensis, Stenotrophomonas acidaminiphila, Acinetobacter calcoaceticus, Achromobacter xylosoxidans and Achromobacter piechaudii. The reference taxa of the six genotypes from Gloria Norte were: Pandoraea pnomenusa, Acinetobacter calcoaceticus, Bacillus cereus, Stenotrophomonas acidaminiphila, Achromobacter xylosoxidans and Achromobacter ruhlandii. The twelve obtained sequences, the eight reference taxa (partial 16 S sequences extracted from GenBank) and two out-groups (Deinococcus, Deinococci; Holorubrum, Archaea, partial 16S sequences also extracted from GenBank) were aligned using Muscle version 3.8.31 to build an unsmoothed Maximum Likelihood phylogeny using RAxML version 7.2.8 (). Gene sequences of the twelve isolated strains were deposited into GenBank (see or Supplementary Material for accession numbers). […]

Pipeline specifications

Software tools MUSCLE, RAxML
Applications Phylogenetics, RNA-seq analysis, Nucleotide sequence alignment
Organisms Bacteria, Escherichia coli