International Journal of Marine Science 2015, Vol.5, No.27: 1-11
8
Indonesia). Gramafix is a granular type of SRF which
contain macro and micro nutrient (N:P:K:
Mg:C:S:Micro
element = 22:7:12:2:4:3:1) and used for agricultural
purpose. This was selected based on the result of
microcosm study in our laboratory previously
(Darmayati et al., 2014). Nutrient was added in the
appropriate amount to have a final concentration
equivalent to a C: N molar ratio of 100:15 for
biostimulation only treatment and 100:7.5 for
combination SRF plus single strain/consortium.
4.4 Bacterial culture preparation
Three selected strain cultures were obtained from the
Research Center for Oceanography Culture Collection,
Indonesian Institute of Sciences. These were
Alcanivora
x
sp. TE9,
Pseudomonas balearica
st 101
and RCO/B/08015, collected from Jakarta ports. They
have capability to degrade Poly Aromatic
Hydrocarbons (PAHs) and the first two have been
determined by 16sRNA partial sequencing (Darmayati,
2009; Hatmanti and Darmayati, 2009). According to
preliminary observation,
Alcanivorax
sp. TE9 and
consortium B (a mixture of the three strains) showed
the best performance (unpublished data).
Stock cultures from nutrient agar were transferred
onto marine agar plates, then transferred to the
adaptation media. Pre-acclimated bacteria conducted
using media contain sterilized Cilacap seawater which
was amended with NH
4
NO
3
(500 mg/L), KH
2
PO
4
(100 mg/L),
yeast
extract 0,01% (w/v) and Arabian
Light crude oil 10% (w/w). The cultures were
transferred in the log time to the fresh media with a
higher volume. The volume of the media was
increased in stages: 250 mL, 500 mL, 1.0 L and 12 L.
4.5 Experimental design
A controlled application of petroleum hydrocarbon on
a sandy beach was used to test the efficacy of
bioremediation strategies for enhancing the
oil-biodegradation process. The strategies were, the
addition of fertilizer only (F), single strain culture +
fertilizer (FSC) and consortium + fertilizer (FCC).
Intrinsic remediation was used as a control (C). Each
treatment was conducted in triplicate. Tilling was
done once a week to add oxygen and to homogenize
the sediment.
A randomized complete block design with three
blocks was established, with three plots assigned per
block. Plot elevation, and location relative to the
Donan River, served as criteria. Treatments were
assigned randomly to plots within a block such that
each block would have all the three potential
treatments. The plots assigned were presented in Fig.4.
Three blocks were established, the distance between
blocks was 6 meters and between plots (treatments)
was 2 meter (Xu et al., 2005)
Figure 4 Plot lay out on the inter-tidal foreshore of Donan river
bank on a randomized complete block design
4.6 Mesocosm design and construction
Mesocosms measuring 50 x 50 x 170 cm were
constructed with 4 bamboo walls coated in transparent
plastic (Figure 5). The top and bottom were open to
allow seawater in and out from the bottom, also
sunlight in for photo oxidation from above. The
enclosure structures (plots) were inserted 30 cm deep
into the ground. The upper parts of the constructions
were 140 cm in height above ground to prevent the oil
from spilling into the environment at high tides. There
was a 25 cm strip of clean sediment between the
treated
sediment and the inside wall of each enclosure, and
also a clean base of 40 cm was provided to eliminate
any impact from background oil in the surrounding
area. This was done by removing 100 x 100 x 55 cm
Figure 5 A) Diagram of Mesocosm B) Mesocosms at high tide
