SCREENING, ISOLATION AND CHARACTERIZATION OF HYDROCARBONOCLASTIC BACTERIA FROM OIL CONTAMINATED SOILS
Loading...
Date
2017-05
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Petroleum and its products continue to serve as a principle source of energy for
industries and daily life. However, their release into the environment is a worldwide
concern since some products are acutely toxic or possess mutagenic, teratogenic and
carcinogenic properties. Several oil disposal methods have been applied over time
with bioremediation emerging as the most promising technology. It takes advantage
of the versatility of soil microbes to degrade hydrocarbon contaminants. Unlike
conventional disposal methods, bioremediation is an environmentally friendly and
cost effective method that simulates natural processes for complete degradation of
hydrocarbons into innocuous compounds. This study focused on isolation,
morphological and biochemical characterization as well as molecular identification of
bacteria possessing hydrocarbon-degrading properties. The study also aimed at
optimizing appropriate culture conditions for the isolates as well as screening for
alkane hydroxylase enzyme. Isolation of hydrocarbon degrading microbes from soils
polluted with used motor oil around Ngara, Nairobi-Kenya was carried out using
Bushnell Haas media supplemented with used engine oil. The isolates were screened
for ability to utilize heating oil, hexane, octane, toluene and diesel oil hydrocarbons.
Characterization of the isolates was carried out by performing Gram’s iodine and
potassium hydroxide as well as starch, catalase and carbohydrate fermentation tests.
The isolates were also identified through PCR amplification and sequencing of 16S
rDNA gene and comparison of obtained sequences with those retrieved from Genbank
database. Optimization of culture conditions of three efficient degraders was
performed using diesel oil and cellular growth monitored through biomass
determination. Hydrocarbon analysis was performed using GC-MS following
culturing in diesel oil. Alkane hydroxylase (alkB) gene was amplified using alk-3F
and alk-3R primer pair. Among 21 microbes isolated, nine were selected based on
their ability to utilize the hydrocarbons and characterized. The isolates were observed
to mineralize heating oil, hexane, octane and toluene as well as diesel oil. PCR
amplification of 16S rDNA gene revealed that the nine isolates belong to six different
genera; Pseudomonas, Acinetobacter, Klebsiella, Enterobacter, Salmonella and
Ochrobactrum. Based on their ability to degrade the hydrocarbons, three isolates were
selected and their growth conditions optimized. Optimum degradation of diesel oil
was recorded at <1 % substrate concentration, pH 7, temperature of 37 0C and using
yeast extract as a nitrogen source. GC-MS analysis of diesel oil degradation
demonstrated that the isolates were capable of readily degrading linear, branched,
cyclic and isoprenoid alkanes as well as aromatic hydrocarbons with fatty acids,
aldehydes and alcohols produced as intermediate metabolites. Isolate 1C was
identified as the most efficient hydrocarbon degrader based on utilization of the
different hydrocarbons tested. Its alkane hydroxylase gene was successfully amplified
indicating the isolate’s potential catabolic capability in degrading alkanes. Overall, the
characterized bacterial isolates may constitute potential candidates for
biotechnological application in environmental cleanup of petroleum contaminants