Environmental Engineering Reference
In-Depth Information
2
Materials and Methods
Light crude oil (API gravity = 40) was obtained from an oil processing factory of
Sarkan in the west of Iran and added to the dry soil with concentrations of 1-5 %
(w/w). The soil and the oil were mixed to make homogenized contaminated soil
and transferred to 1 l pots. Each sample consisted of 500 g of dry soil. Animal
manure was added to the soil as fertilizer before seeding. Fifty seeds of Tall Fescue
were planted in the soil. The control sample was also prepared as above but did not
receive oil. After 2 months, the plants were removed from the soil and the roots
washed with water to dislodge excess soil adhering to the roots. Roots and shoots
were separated and kept frozen at − 10 °C for further experiments. One gram of
roots or shoots was homogenized in 10 ml of NaCl (0.15 M) by a homogenizer to
destroy the cells. The homogenized solution was centrifuged (3000 ×
g
for 10 min)
to remove unbroken cells. The supernatant was used as cell free extract. The en-
zyme assay was performed in 0.1 M phosphate buffer (pH 7.0). The reaction was
started by adding 100 µl of guaiacol solution to the test tubes containing 100 µl of
enzyme solution (cell free extract) and 100 µl of different concentrations of H
2
O
2
as substrate. The final volume was always 2 ml. The colorimetric change of guaia-
col to tetraguaicol was detected during 10 min using Perkin-Elmer visible spec-
trophotometer. Results were the average of at least two separate experiments and
expressed as mean ± Standard deviation (± SD).
3 Results
Figure
13.1
shows the number of seeds germinated in the vegetated samples 30 af-
ter planting. In 0 % sample, the number of germinated seeds was higher than in the
other samples while it was lower in 5 % sample. However the difference number of
germinated seeds between the control and 5 % sample was not significant. The dry
biomass of roots and shoots was measured at the end of the experiment (Fig.
13.1
).
The separation of roots from the soil showed that the distribution of roots in the soil
has decreased by increasing the crude oil concentration. The higher root biomass
was observed in 0 % sample, in which the roots were well distributed in the soil.
The lower root biomass was observed in 5 % sample. The total dry biomass (roots
+ shoots) was also high in 0 %, while it was low in 3 and 5 % samples. A sudden
decrease in total dry biomass was observed in 1 % sample in comparison with that
in the control (0 %) sample and the difference was significant between control and
all of contaminated samples (Fig.
13.1
). The length of leaves decreased by increas-
ing crude oil concentration; the shorter leaves were observed in 5 % sample, while
the tallest in 0 % sample (Fig.
13.2
). The number of green plants at the end of ex-
periment was lower in 5 %, while the higher was observed in 0 % followed by 1 %
samples (Fig.
13.2
).
