International Journal of Marine Science, 2017, Vol.7, No.42, 399-410
400
1 Materials and Methods
1.1 Collection of samples
Samples were collected from Khor Al-Zubair channel, southern Iraq (Figure 1)
from five stations. Water
samples (500 ml) were collected at two depths in sterile glass bottle, all samples transferred to an ice box and
transported to the laboratory.
Figure 1 Sampling locations
1.2 Isolation and identification of bacteria
One milliliter of each sample was cultured in a conical flask containing 100 ml mineral salts medium (MSM),
the composition of the MSM was 0.3 gm of KCl, 1.0 gm of K
2
HPO
4
, 0.5 gm of KH
2
PO
4
, 0.01 gm of
FeSO
4
.7H
2
O, 30.0 gm of NaCl, 0.5 gm of MnSO
4
.7H
2
O, 0.2 gm of CaCl
2
and 1000 ml DW (Fujisawa and
Murakami, 1980). Four concentrations of crude oil as 0.25 ml, 0.5 ml, 1 ml and 2.0 ml (Provided by
Al-Shua’aba Refinery) were added separately to the medium. Decimal dilutions of 7 days grown culture was
cultivated at 30°C.
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
and identified by the Vitek II system (VK2C8300, USA).
1.3 Degradation of PAH
One millilitre of broth culture of each bacterial isolate was incubated separately in 250 ml Erlenmeyer flask
containing 50 ml of MSM at 20ᵒC for 7 days at 120 rpm using a cooling incubator shaker (Al-Sulami et al.,
2014). All the experiments were carried out in two duplicates, and the residual crude oil was performed after 7
days.
1.4 Gas chromatographic analysis of residual crude oil
The residual crude oil was extracted by liquid-liquid extraction, as described by Adebusoye et al. (2007). The
aqueous phase was removed by separating funnel and the residual oil dried in the oven at 40ᵒC to remove CCl
4
.
The aromatic fraction was separated using separation column (25 cm length, 3 cm diameter) containing 8 gm of
silica gel over a little amount of glass cotton (Farid, 2006). The residual oil dissolved in 25 ml of benzene and
poured into the separation column and drawn off the aromatic fraction in 50 ml beaker. Control flasks were also
extracted similarly, aromatic fraction hydrocarbons estimated by FID gas chromatography technique (Agilent
Chem Station).
