Phylogenetic analysis and characterization of diesel oil degrading
bacteria isolated from oil-contaminated soils
92-EC-17-A-10-SI-0013
Fo-Ting Shen1, Ta-Chen Lin2, Mann-Jing Ho1,
Hui-Ling Lu1, Wei-Shuo Huang1 and Chiu-Chung Young1
1
Dept. Soil and Environmental Sciences, National Chung Hsing University, Taiwan
2
Dept. Environmental Engineering, National Chung Hsing University, Taiwan
ABSTRACT
Bioremediation is a low-cost treatment alternative for the cleanup of petroleumcontaminated soils and groundwater. Under selective pressure of environmental
pollution, a microbial capacity to degrade hydrocarbon might be harnessed for
pollutant removal in biotechnological processes. The present study envisages
phylogeny of diesel oil degrading bacteria from oil-contaminated sites with emphasis
also on the range of substrates degraded. Diverse soil samples were collected and
incubated in 50 ml sterile water supplemented with 1% diesel oil at 30oC, at 160 rpm
for 72 h. After enrichment twenty-two isolates that showed better performance are
subsequently transferred to the BH medium containing 1% diesel as sole source of
carbon. All the isolates were analyzed with 16S rDNA partial sequences (ca 500 bp)
and phylogenetic dendrogram was obtained from neighbor joining method using
multiple sequences alignment with CLUSTAL X software. Further six diesel oil
degraders were tested for their ability to grow on common aromatic hydrocarbons
(0.1% (v/v)) as sole carbon and energy source, that also included methyl tert-butyl
ether (MTBE), benzene, toluene, ethylbenzene, xylene and naphthalene (0.1% (w/v))
(Table 1). The growth was recorded and categorized as minimum (OD) ranging
between 0.1-0.3, moderate (OD 0.3-0.5) and optimum (OD 0.5-0.8). Benzene, toluene,
ethylbenzene and xylene (BTEX) served as growth substrate for at least three tested
isolates, while no organisms grew or least utilized MTBE. Three bacterial strains,
designated as CC-BC11, CC-CF5 and CC-JG39 were able to mineralize four kinds of
aromatic compounds and were identified as Rhodococcus, Acinetobacter and
Gordonia, respectively. Eleven distinctive bacterial species were represented from 7
genus with diverse phenotypic characteristics and 6 isolates were novel and had
98%-99% similarity compared to other described species from the GenBank, hence
might be an new representatives (Fig 1). Alcaligenes, Bacillus, Comamonas,
Gordonia, Pseudomonas, Rhodococcus and Sphingobium were common oil degrading
bacteria
while
Achromobacter
xylosoxidans,
Acinetobacter
calcoaceticus,
Pseudomonas aeruginosa and Serratia marcescens which are of clinical significance
and human pathogens were also recovered from the polluted soils. Detailed taxonomic
results showed that bacteria belonging to α, β, γ – Proteobacteria, Bacilli and
actinomycetes (Actinobacteria) are widespread in oil-contaminated soils. Such
isolates may have the potential to utilize hydrocarbon and with certain precautions can
be tried for the removal of pollutants.
Table 1. Growth of six isolates on various carbon sources
Strain
Carbon sources
MTBE
-1
Benzene
Toluene Ethylbenzene
-
Xylene
CC-BC04
++
+++
CC-BC11
+++
+
+
CC-CF3
++
+
CC-CF5
+
++
+
+
CC-JG30
++
+
+
CC-JG39
+
+
+
1
-, OD600 <0.1; +, OD600 0.1-0.3; ++, OD600 0.3-0.5; +++, OD600 0.5-0.8
Naphthalene
++
+
Fig 1. Phylogenetic analysis (16S rRNA gene sequences analysis) of diesel oil
degrading bacteria