A bioflo 110 (3l volume) was used with 8

3rd European Bioremediation Conference P 124 BIOREMEDIATION AND TOXICITY DETERMINATION OF
M. Ruiz1, N. Pasadakis2, N. Kalogerakis1
1Department of Environmental Engineering, 2Dept of Mineral Resources Engineering, Technical University of Crete, 73100 Chania, Greece
The aim of this research was to compare the bioremediation treatment of seawater polluted
with two different concentrations of weathered crude oil (0.2 for experiment (a) and 1.14%
for experiment (b), v/v) by salt-tolerant consortia enriched from the sludge of a refinery
wastewater treatment facility, in a SBR. The use of a commercial bioremediation stimulant
(S200®) was also evaluated as an alternative to the traditional nitrogen and phosphorus

Oil spills are currently considered a major problem on the environment [13, 24].
Bioremediation, defined as the use of microorganisms to degrade environmental
contaminants [3, 4] has proved to be a useful tool in the clearing of this pollution. Since
crude oils contain such a wide range of molecular structures, it was postulated that mixed
cultures capable of rapidly degrading crude oil might have broader applications in the
general biotransformation of hazardous hydrophobic environmental contaminants. The use
of these mixed cultures eliminates the high cost implications of pure-culture installations,
provides greater metabolic diversity and can provide a process able to degrade a variety of
oily waste sludge [23].


2.1 Inoculum preparation

Seawater (3.5%) for culturing and experiments was periodically collected from Chania
beaches, stored at 4ºC and filtered (0.45µm) prior to use [16].
The same crude oil was used for both experiments and submitted to the same natural
weathering process, being the resulting weight loss: 28% (ρ=0.8202 g/ml) and 27.1%
(ρ=0.8166 g/ml), for crude oil A and B, respectively. This weight loss, similar to the
reported by other researchers [7, 24] was achieved through continuous shaking (200 rpm) at
constant temperature (30ºC).
The bacterial consortia used came from sludge from a refinery wastewater treatment facility
in Athens (Greece). It was enriched in seawater with 1% (v/v) weathered crude oil as the
only source of carbon for four weeks.
The following solutions were used as inoculum for the bioreactor:
a) 9 ml of the enriched culture, 180 ml filtered sterilised natural seawater, 0.44 ml (0.2%, v/v) weathered crude oil A, and NH4NO3 and K2HPO4 in such a concentration to keep a C:N:P ratio of 100:10:1 [12], assuming that 85% of the crude oil is carbon. b) 9.5 ml of the enriched culture, 190 ml filtered sterilised natural seawater, 1.9 ml (1%, v/v) weathered crude oil B and 0.95 ml of the microbial stimulant S200®. The latter
was not sterilised due to its thermal decomposition when exposed to temperatures above
In both cases, solutions were kept at 270C on a shaker incubator (150 rpm) for four days,
and then inoculated to the bioreactor: 148 ml of mixture (a) for the first experiment
(3.7x106 MPN/ml) and 190 ml (OD660=0.14725) of mixture (b) for the second experiment.
Adding these volumes of inoculum, and considering the volume of seawater present in the
bioreactor in each experiment (1.8 and 1.9L for experiment 1 and 2, respectively), the
inoculum was close to 10% of the total volume in the bioreactor [20, 8].

2.2 Bioreactor set-up

Both experiments were carried out in a batch mode in an autoclavable 3 L, BioFlo 110
bioreactor (New Brunswick Scientific) It is equipped with two six-bladed impellers, a
sparger, a thermowell RTD, a harvest tube, an exhaust condenser, a sampler, and dissolved
oxygen and pH electrodes, all of which are mounted through the head plate.
The initial conditions in the bioreactor for both experiments were:
a) 1800 ml sterilised natural seawater supplemented with C:N:P in a 100:10:1 ratio (Guerin,
2002), 270C, 300 rpm, aeration flow rate of 1.8 vvm, pH 7.71 and 100% DO. b) 1900 ml natural seawater, 27ºC, 400 rpm, aeration flow rate of 2 vvm, pH 7.64, and When the conditions in the bioreactor were stable, first the preculture, and afterwards the weathered crude oil were added near the rotating impeller shaft [11]. The bioreactor in experiment (a) received 4 ml weathered crude oil, while 21.6 ml were added on experiment (b). Foam production in the reactor was controlled by the addition of a single aliquot of 1.8 ml and 1.9 ml Dow Corning® 2210 for experiments (a) and (b) respectively. On the first case it was added after the first 17 h, due to the appearance of foam, therefore it was initially added when the second experiment was carried out. Finally, in experiment (a) the microbial stimulant S200® (0.95 ml, 0.05% v/v) was introduced into the bioreactor. The bioreactor was run as a closed system after inoculation, with the exception of regular sampling for crude oil concentration analysis (6 ml, two replicates), bacterial growth estimation (2 ml) and acute toxicity determination (3 ml). Once the stationary phase of the bacterial growth was reached (88.5 in experiment (a) and 93.5 h in experiments (b)) 900 ml of the culture were removed and 900 ml fresh media were added. The second batch was maintained for 82.5 and 92 h in both experiments, and 900 ml of broth were exchanged. The third run of the experiments were considered to be concluded after another 63 and 95 h, respectively. 2.3 Analytical methods
Microbial population in the bioreactor was determined by three methods. First, the bacterial
population density was estimated by the most-probable number (MPN) method for
hydrocarbon degraders [25], where results for each plate were registered visually following
the incubation period and calculations performed using MS-Excel® [6]. Second, cell
densities were estimated based on optical density at 660 nm (OD660) measurements [18].
Third, aqueous samples were transferred to 2 mL weighed Eppendorf tubes and centrifuged
immediately at 13.000 rpm for 15 min. and the cell pellets dried to constant mass at 800C

Samples for crude oil concentration were stored at 40C until time of analysis. They were
extracted by liquid-liquid extraction, with a 87.6% extraction efficiency. The IR spectra of
the extracts were obtained by FT-IR on a Perkin Elmer Spectrum 1000 with variable path
length liquid cell (Graseby Speac ZnSe 7009). The path length used was 0.05 cm. For
experiment (a), the maximum corrected height was recorded between 3100 and 2700 cm-1.
For experiment (b), the corrected height at 2855 cm-1 was recorded between 3200 and 2600
cm-1, due to the high concentration of the samples.

To estimate the toxicity of the treated seawater of experiment (b), 24 h acute toxicity tests
with the rotifer Brachionus plicatilis were carried out with samples directly taken from the
bioreactor and stored at 4ºC until analyses were performed. A second scoring of the results
was taken after 48 h incubation. For that purpose, cysts of the mentioned rotifer were
hatched for 28 h at 25ºC and 4000 lux. Then, the multiwell test plates were filled with
samples collected not earlier than 36 h [9] and tests carried out following the ASTM
Standard Guide E1440-91 [2]. Results were analysed according to the procedure suggested
by EPA [10].

During the three sequential batches of each experiment, the bioreactor was operated under
controlled constant operational conditions (data not shown). The DO was maintained above
a non-limiting level of 30% air saturation during the whole experiment [17, 20].
As observed in figure 1, the growth curves for each batch run of both experiments are
shown. In the first run, the lag phase was only visible in experiment (a) (19 h) whereas in
experiment (b) it directly exhibited exponential growth phase, until 25 h when the
stationary phase was achieved. In experiment (a), the consortia grew from 19.5 to 66.5 h,
when it reached the stationary growth phase. During the second batch, the bacteria showed
lag-phase in none of the experiments, but rapid growth after the exchange of media: 89.5 h
and 93.5 h for experiments (a) and (b), respectively. In the third batch, the consortia grew
more in experiment (a) than in (b), possibly due to the high concentration of weathered
crude oil (1%, v/v) at which they were exposed in this last one, which could have inhibited
their higher growth.
Figure 1. OD660 vs crude oil concentration in both experiments. Experiment (a): OD660 rhomboidal
markers, HC (g/l) square markers. Experiment (b): OD660 linear markers, HC (g/l) circular markers.
The isolated consortia has proved to be considerably effective on the removal of
hydrocarbons (HC) from seawater polluted with weathered crude oil in a SBR, especially at
0.2% (v/v), although partial degradation was also achieved at 1% (v/v). The specific HC
degradation rates of the consortia during the three batches were: 0.063, 0.038 and 0.054 for
experiment (a) and 1.13, 2.28 and 1.08 mg HC/l.h.g dry biomass for experiment (b).
No partial mortalities were recorded when, in experiment (b), samples were analysed for
their acute toxicity to the marine rotifer Brachionus plicatilis.


Although the bioreactor was run under control of its operational conditions, on experiment
(b), the pH decreased during certain phases of the experiment. This fact has also been
reported by Kleijntjens and Luyben [14] who stated that during bath processing inhibiting
side products of the microbial breakdown may increasingly be released into the medium.
They pointed out the drop in pH due to humification processes as an example of this
The inoculum size chosen, ranging from 8.2 to 10% (OD660=0.14725), is in accordance
with Song et al. [21] who reported an OD540=0.167 as the appropriate size of the inoculum
for their degradation experiments. It has shown its adequacy for the treatment of natural
seawater with up to 1% (v/v) weathered crude oil in a SBR.
Three different methodologies have been employed in these experiments for the
enumeration of the bacteria present in the bioreactor. The use of the MPN procedure for the
enumeration of bacteria, both aerobic and anaerobic is widely spread [15, 1, 5]. However,
the need of incubating the microtiter wells for two weeks to obtain results makes it a
difficult procedure to apply when immediate bacterial enumeration is required. On the
contrary, the measurement of the optical density of the sample provides immediate values
of cell density, indicating the phase of the bacteria growth at which the culture in the
bioreactor is. Finally, the existence of a relationship between the optical density and the dry
biomass measurements was investigated on experiment (b). Wubbolts et al. [26] established
a good agreement for both methods at low cell densities (bellow 10 g dry mass) using a
constant conversion factor of 0.16 g cdw (cell dry weight) per litre per unit of OD440.
Reardon et al. [19] have found an OD-mass correlation linear over the cell concentration
range (up to 300 mg dry cell weight/L) with 1.00 OD=1000 mg/L. On this research, no
clear relationship between these two measurements could be established.
Relatively high removal of TPH was obtained after a short period of SBR operation
indicating that the effectiveness of the isolated consortia as weathered crude oil degraders.
Nevertheless, it might be appropriate, to increase the efficiency of the process, the addition
of a supplementary medium containing glucose or sucrose after the depletion of crude oil
[8]. It can also be useful to increase the temperature in the bioreactor to 30ºC, as it has
proved to be effective for the treatment of oily sludge in stirred aerated bioreactors [22].
The treated effluent appears to be quite toxic to the microorganism tested as can be
concluded from the mortalities recorded for the samples analysed from experiment (b). This
is due to the combined effect of the toxicity of the weathered crude oil itself, and to the
microbial stimulant used in this study, as shown from its LC50=20.520 ppm, and the
presence of some toxic compounds in its composition ((2-2-butoxyethoxy)ethanol).
This work was founded through Project HPMD-CT-2001-00060 from the Marie Curie
Development Host Fellowship.


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