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International Journal of Marine Science, 2017, Vol.7, No.18, 176-182
177
The aim of this study was to isolate bacteria played important role in the process of bioremediation of
oil-contaminated marine environments.
1 Materials and Methods
1.1 Water sampling
Samples collected from five stations at two depths from Khor Al-Zubair channel, southern Iraq (Figure 1). Sterile
glass container (500 ml) used to collection for water samples and kept in ice boxes till reaching the laboratory.
Figure 1 Sampling sites
1.2 Isolation and identification of bacterial species
One ml of every sample was cultured in a conical flask containing 100 ml mineral salts medium (MSM), which
composed of 0.3 gm KCl, 1.0 gm K
2
HPO
4
, 0.5 gm KH
2
PO
4
, 0.01 gm FeSO
4
.7H
2
O, 30.0 gm NaCl, 0.5 gm
MnSO
4
.7H
2
O, 0.2 gm CaCl
2
and 1000 ml D.W. (Fujisawa and Murakami, 1980). Four concentrations of crude oil
added separately to the medium as 0.25 ml, 0.5 ml, 1 ml and 2.0 ml (Provided by Al-Shua’aba Refinery). Decimal
dilutions of 7 days grown culture was cultivated at 30ᵒC for 24 h. Nutrient agar (Hi media- India) with 30 ppt
sodium chloride and marine agar (Difco, USA) were used to isolate
Vibrio vulnificus; Brevundimonas
diminuta/vesicularis; Sphingomonas paucimobilis
and
Ochrobactrum anthropic
which identified by the Vitek II
system (VK2C8300, USA).
1.3 Degradability study of crude oil
One ml of broth culture of each bacteria was incubated separately in 250 ml Erlenmeyer flasks containing 50 ml
of MSM at 20 FID C for 7 days with shaking at 120 rpm using a cooling incubator shaker (Germany Sartorius
Stidem-Certomat) (Al-Sulami et al., 2014). All the experiments were carried out in two duplicates, and the
residual crude oil was estimated after 7 days.
1.4 Extraction of residual crude oil
Liquid-liquid extraction method was used for extraction of residual crude oil (Adebusoye et al., 2007),by using
separating funnel,the aqueous phase is removed while the residual oil, dried in the oven at 40ᵒC to eliminate CCl
4
.
The separation column (length 25 cm, 3 cm) was used to separate the aliphatic fraction as described by Farid
(2006), the remaining oil dissolved in n-hexane (25 ml), and poured in the separation column and drawn off the
aliphatic part in 50 ml beaker as well as the control vials similarly, GC technique (Agilent Chem Station) used to
estimate n-alkanes.
