Giraud
(1)
L., Durand
(2)
M.J., Andrès
(1)
Y., Merlin
(3)
F.X. and Raynaud. F
(4)
.
(1) GEPEA, UMR CNRS 6144, Ecole des Mines de Nantes, BP 20722, 44307 Nantes Cedex 3, tel. 02 51 85 82 84 or 02 51 85 81 40, fax. 02 51 85 82 99, e-mail. yves.andres@emn.fr
, laurence.giraud@emn.fr
(2) GEPEA, UMR CNRS 6144, Laboratoire CBAC-ERT 1052, IUT Génie Biologique, 18 Bd
Gaston Defferre, 85035 La Roche sur Yon, tel. 02 51 47 84 42, fax. 02 85 47 84 51, email. Marie-Jose.Durand-Thouand@univ-nantes.fr
(3) CEDRE, Rue Alain Colas, BP 20413, 29604 Brest Cedex, tel. 02 98 33 10 10, fax. 02 98
44 91 38, e-mail. francois.merlin@ifremer.fr
(4)
Vérité SA, Parc d’activités Vendée Atlantique, Chemin des Charmes, 85210 St Hermine.
Tel. 02 51 48 94 42, fax. 02 51 48 94 41, e-mail. fraynaud.verite1@libertysurf.fr
Abstract
Marine pollution by hydrocarbon has become a big problem with the development of the petrochemical industry, installation of numerous petrol stations and transport. Physical, chemical and biological technologies have been developed to remove hydrocarbon pollutants from soils and restore environmental quality. But there is not really specific methodology to evaluate remediation treatment. So, the aim of this study was to develop a methodology that permitted to economic actors to evaluate a degradation process. This methodology included toxicological evaluation, remediation evaluation and more specify biodegradation evaluation, consortium analysis and hydrocarbons analysis. Conception of the pilot was based on a exsitu process : a biopile treatment. Sandy sediments (67 Kg) were contaminated with 1.5 Kg of petroleum (DEPOL), and 6.7 L of refinery sludge The evaluation test of biodegradation was based on Sturm test (ISO 9439), the acute toxicity evaluation was based on LUMIStox test
(ISO 11348-2), the mutagenicity followed the Ames test (ISO 16240), analysis of the inoculum was performed with the Biolog ECOPlate (biochemical analysis) and the hydrocarbons were analysed by GC-MS.
Keywords : methodology, petroleum hydrocarbons, remediation evaluation, marine wastes
1. Introduction
A significant quantity of petroleum compounds has been released into the soil environment. These releases have been either from leakage of underground storage tanks or from accidental spills. A wide range of methods have been used to treat these contaminated sites and a lot of studies were performed to understand biodegradation of petroleum (Atlas,
1975 ; Perry, 1979 ; Atlas, 1981, 1984 ; Leahy et Colwell, 1990 ; Atlas et Bartha, 1992 ;
Swannell, 1996). Treatment processes have incorporated physical, chemical or biological phenomena, or a combination of these processes.
The aim of this study is to evaluate biological or chemical treatment processes for wastes polluted by petroleum hydrocarbons following an oil spill. The finality is to propose a methodology composed of simple and normalised evaluation tests of toxicity, biodegradation and bacteria consortium. This methodology could permit to economic actors to respond to a
1

marine pollution. For this purpose, a pilot for treatment evaluation is designed in order to evaluate the degradation processes.
Therefore, laboratory or field-scale investigations dealing with the transformation of pollutants are required to optimise the remediation process. Data from laboratory scale experiments provide information on the metabolic pathways involved in the biodegradation process, the degradation rate, the bioavailability, and the ecotoxicity of pollutants. A better understanding of chemical and biological processes involved in the transformation of contaminated wastes helps in the design of more efficient processes.
Biodegradation rate determination, study of consortium diversity and chemical analysis of hydrocarbons permit the evaluation of a process and to perform biological or chemical treatment of pollutants. In order to evaluate toxicity of biodegraded wastes we used a battery of ecotoxicity bioassays. These data could be used to adapt the good treatment to the pollution type.
2.
Experimental methodology
2.1.
Properties of petroleum, sandy sediment and bacteria inoculum
Petroleum (DEPOL, CEDRE : Centre of Documentation, Research and
Experimentation, Brest, in France) is composed of : Draugen (41%), Forties (33%), Saharan
(13%) et Statfjord (13%). Most of the volatile compounds have been eliminated by evaporation. Table 1 give some characteristics of petroleum before evaporation.
Density (15°C)
Viscosity (10°C)
Viscosity (20°C)
% Asphaltics
Pour point
% Evaporation in mass (150°C)
% Evaporation in mass (200°C)
0.844
9 mm2/s
7 mm2/s
1.5%
< -20°C
19%
29%
Table 1 : Characteristics of DEPOL (provided by CEDRE, France).
Sand was collected from the sea side of La Turballe (in France) and was used directly raw for contamination with petroleum, to simulate a poorly polluted waste (< 5%, in mass).
The initial oil contents was 22.4 g.kg
-1 of sand.
This polluted sandy sediment was polluted with refinery activated sludge (Donges in
France) at 0.55 gMES (suspension).kg
-1 . Heaviest particles have been eliminated by simple decantation and the sludge was washed with deionised water.
2.2.
Bioremediation treatment
Experiments were performed in a steel reactor (50*94*80, l*L*h) covered by a polyvinyl chloride (PVC) lid (figure 1). Firstly, 67 kg of sand was mixed with 1.5 kg of
DEPOL and disposed in the reactor. Then, 6.7 L of sludge were inoculated with the polluted sand. This bioreactor simulated biopile treatment. The contaminated sand was humidified with water that percolated from the reactor. Periodically, water was added to balance
2

evaporation.. Weekly manual tilling with gloves and nutrients (C/N/P = 100/10/1, ammonium chlorate and sodium phosphate) addition in reactor ensured a homogenised biodegradation of polluted waste. A control of temperature and moisture rate was realised in order to control the biodegradation conditions. A system of CO
2
trapping was placed before the inlet of air and was composed of 3 sodium hydroxide (NaOH, 4 M) flasks and 1 barium hydroxide (Ba(OH)
2
,
0.0125 M) flask. Percolated water containing some microorganisms and non-degraded hydrocarbons was continuously circulated at a flow rate of 3 L hour -1 , and passed in the
VOCs sorbed on glass cartridges
Control of moisture rate activated sludge reactor which was supplemented by air, and in the clarification system, and finally in the reactor.
CO
2
trapped in Ba(OH)
2
(0.0125M)
S1 gloves
S2 S3
Clarification system
Control of temperature
Air without CO production system
2
Hydrocarbon contaminated beach sand water
L1/
L2
Activated sludge
S1, S2, S3, L1 and L2 : sample points
Figure 1 : Scheme of pilot treatment plant.
2.3.
Samplings
Samples were taken from 3 equidistant points of the reactor (S1, S2, S3) and from the percolated water (L1, L2). air
3

3. Material and methods
3.1. Biodegradation evaluation
Evaluation of biodegradation was carried out measuring CO
2
production (according to the Sturm test, ISO 9439; Sturm, 1973). The gas produced in reactor was trapped in a solution of barium hydroxide (Ba(OH)
2
, 0.0125 M). The quantity of CO
2
produced was determined by titration of the excess of Ba(OH)
2
by a standard solution of hydrochloric acid (HCl, 0.05 M) using phenolphthalein as indicator. The theoretical carbon dioxide content (mg ThCO
2
) produced by total oxidation of the material was calculated from :
ThCO
2
 mgC
44
12
3.2. Extraction and purification of hydrocarbons
Hydrocarbons were extracted from the liquid phase by decantation with dichloromethane (75-09-2, 99.5 %, Carlo Erba).
30 g of sand sample were placed in a 250 mL volumetric flask and 50 mL of dichloromethane was added. The sample was ultrasonicated in the sonication bath for 15 min, filtered with a
0.5 mm grille and recovered in a specific glass vial. The extracts were concentrated by evaporation system (rotavapor) at 800 mbar and 40°C. Samples were stocked at 4°C for chemical analysis and at –20°C for ecotoxicological analysis.
3.3. Adsorption of volatile hydrocarbons
Volatile hydrocarbons were sorbed on glass cartridges (150*4 mm ID), which are made of Carbosieve SIII (very volatile compounds, C2-C6), Chromosorb 106 (wide range of
VOCs including hydrocarbons from C5-C12, and volatile oxygenated compounds) and Tenax
TA (aromatics except benzene, apolar compounds and less volatile compounds, C7-C26).
Cartridges were conditioned with helium (20-40 mL.min-1) initially and before each sampling
(320°C, 10 min). Cartridges are placed in a desorption oven (Turbo Matrix TD, Perkin Elmer) and hydrocarbons are separated in a gas chromatograph and analysed by a mass spectrometer.
3.4. Hydrocarbons analysis
Chemical analysis were realised on hydrocarbons extracted each week from sandy samples (30 g S1, 30 g S2, 30 g S3) and hydrocarbons extracted each week from 20 mL of percolated water. Internal standard PAHs and n -alkanes, used for quantification, were added to the matrix prior purification. Extracts were purificated with a silica column
(10mm*300mm, Labosi) by elution with 40mL of a mixture of pentane (109-66-0, 99.7 %,
Carlo Erba)/dichloromethane (80/20, v/v) in order to retain asphaltics compounds and to collect only aliphatic and aromatics compounds. The saturated and aromatic fractions were finally reconcentrated to a few mL by evaporation (800 mbar, 40°C), and analysed by gas chromatography –mass spectrometry (GC-MS).
4

The extracts were analysed with a Perkin Elmer GC-MS system, using a split less injection for 1 min and split at 50 mL.min
-1
at 270°C, and using helium as carrier gas (1mL.min
-1
).
Electron impact (EI) ionisation at 70 eV was used. The GC separation was performed on a column VF1MS (Varian; 30 m, 0.25 mm i.d., film thickness 0.25 µm). The ion source temperature and interface temperature were adjusted to 220 and 320°C, respectively. The temperature program was held at 50°C for 1 min and increased from 50 to 300°C at 5°C.min
-1 and finally was stabilised at 300°C for 30 min. The sample volume injected was 1µL. The mass spectrometer was operated in selected ion monitoring mode (SIM), detecting the following ion masses : 57, 128, 134, 136, 142, 148, 152, 154, 156, 162, 164, 166, 170, 176,
178, 180, 184, 188, 191, 192, 194, 206, 208, 212, 216, 217, 218, 220, 226, 228, 230, 231, 240,
242, 244, 252, 256, 264, 270, 276, 278. Internal standard calibration was used for quantification of the extracts : naphtalene-d
8
, biphenyl-d
10
, phenanthrene-d
10
, chrysene-d
12
, benzo[a]pyrene-d
2
for PAHs (Carlo Erba-SDS, Val-de-Reuil, France) and 1-eicosene for n alkanes (Sigma Aldrich, St Quentin Fallavier, France). The n -alkanes standard solution used for calculation of response factors, was purchased from Promochem (Wesel, Germany) and the Standard Reference Materiel, SRM 1647d “Priority Pollutant Polycyclic Aromatic
Hydrocarbons (in acetonitrile), was provided by the National Institute of Standards and
Technology (NIST, Gaithersburg, MD, USA).
3.5. Toxicity evaluation
Toxicity tests were realised every 3 weeks with the extracted fraction from 30 g of sandy samples (10 g S1 + 10 g S2 + 10 g S3), on hydrocarbons extracted from 20 mL of percolated water, and with 20 mL of raw percolated water. Extraction procedure was the same as for chemical analysis.
Solvent exchange
Dichloromethane is shown to be not compatible with the Ames test due to it intrinsic toxicity. A solvent exchange was then performed by replacing the extraction solvent by dimethyl sulfoxide (DMSO) that is compatible with the Ames test. To do so, dichloromethane was evaporated at room temperature. DMSO was added juste before complete evaporation. The procedure was continued until only DMSO was left in the sample.
Acute toxicity
A LUMIStox 300 luminometer, a LUMIStherm incubator, and the non-pathogenic bacteria Vibrio fischeri NRRL-B-11177, all obtained from Dr. Lange, GmbH, Germany, were used for toxicity measurements. The LUMIStox® (ISO 11348-2) acute toxicity test was performed according to the standard procedure outlined in Lange (1998). Briefly, the procedure included reconstitution of liquid-dried bacteria, preparation of homogenous bacterial suspension, and measurement of the initial bioluminescence. Control solutions (8.0 mg of potassium dichromate.L
-1
, 50 mg of zinc sulphate heptahydrate.L
-1
, 12 mg of 3,5-dichlorophenol.L
-1 and 7%
NaCl) were used with each batch of bacteria to verify the bacteria and reagent quality. Bacterial suspensions were then exposed to 9 dilutions of each DMSO (dimethyl sulfoxyde) extract, osmotically adjusted to 2% NaCl, and were incubated for 5, 15, 30 minutes under a constant temperature (15°C). After each incubation time the change in bioluminescence was recorded.
5

Light readings were normalized using blank solutions containing the same NaCl and DMSO concentration as test sample. The effective concentration percentages of tested solutions resulting in a 50 % decrease in bioluminescence (EC
50
) were calculated following the procedures outlined in Lange (1998).
Mutagenicity
The test followed the ISO 16240. This test was realised on the hydrocarbons extracted from sand and percolated effluent, and on the raw petrol. The Ames assay was a measure of soil mutagenicity, which is a potential indicator of chronic toxicity. Extract were tested with and without the addition of S9 (metabolic activator mix), the supernatant of rat liver homogenate.
Exposure to S9 simulates mammalian metabolism of the toxicant.
The table 2 show the sample preparation used in our work.
Sample sterilite control S9mix
Concentration
DMSO
4-Nitro-o-phenylenediamine 2,5 µg/plate
Benzo(a)pyrene extract "sand"
10 µg/plate
0,5mg/plate extract "sand" 0,1mg/plate extract "sand" 10 µg/plate extract "sand" 1µg/plate extract "L1" extract "L1"
50 µg/plate extract "L1"
5 µg/plate
0,5 µg/plate extract "L2" 100µl/plate
DEPOL 25µg/plate
DEPOL 2,5µg/plate
DEPOL 250ng/plate
DEPOL 25ng/plate
Results were expressed as reversion factor : reversion factor
DEPOL 2,5ng/plate
Table 2 : Concentration of the test samples.
 number of revertant / plate for sample number of spontaneou s revertant / plate in control
3.6. Consortium analysis
Analysis of the bacteria consortium were realised on 30 g sand (10 g S1 + 10 g S2 + 10 g
S3), 15 mL of sludge in the percolated water and 15 mL of sludge in the activated sludge reactor, every 3 weeks. Samples were collected in sterile flasks. Bacteria from sandy samples were extracted in sterile phosphate buffer during 30 min.
Biolog ECO microplates are used (BIOLOG, Inc.). The ECO plate contains three replicate well of 31 carbon substrates. These substrates are predominantly amino acids (n = 6), carbohydrates
(n = 10), and carboxylic acids (n = 7) (table 3). A control well contain no sole-carbon source,
6

thus any colour development there presumably indicates utilisation of carbon sources inherent in the inoculated water.
150µL of sample was inoculated into each well of the microplates. Plates were incubated at room temperature (20°C) in the dark. The optical density (λ = 590 nm and 750 nm) of each well was determinate after 24h thereafter up to 120h with a Biolog microstation reader, model
(BioTek Inc.).
Polymers Carbohydrates Carboxylic acids Amino acids Amines Phenolic compounds
Tween 40
Tween 80
D-galactonic acid γ -l act one pyruvatic acid methyl ester
β-methyl-D-glucoside
D-galacturonic acid
L-arginine
L-asparagine phenyl ethylamine 2-hydroxybenzoic acid putrescine 4-hydroxybenzoic acid
α-cyclodextrine glycogen
D-xylose i-erythritol
γ- hydr oxybut yr ic aci d itaconic acid
L-phenylalanine
L-serine
D-mannitol
N-acetyl-D glucosamine
D-cellobiose glucose-1-phosphate
α-ketobutyric acid
D-malic acid
D-glucosaminic acid
L-threonine glycyl-L-glutamic acid
D,L-α -glycerol phosphat e
α-D-lactose
Table 3 : Carbon substrates in Biolog ECO microplates
4. Results and discussion
3.1. Biodegradation evaluation
Respiration rates seemed to reflect a specific degradation of the petroleum. The degradation pattern followed a typical first-order degradation. A similar pattern was reported by most authors independently on the starting concentration. However, no lag time appeared at the beginning of the experiment probably due to the origin of the microorganisms. They were already adapted to hydrocarbons degradation. Thouand and Block (1993), Thouand et al .
(1996), Thouand et al . (1999) reported that the origin of inoculum is very important in the biodegradation process. Thouand et al . (1996) found that the probability of para -nitrophenol biodegradability tests versus adapted P.
putida density was a simple sigmoid. So, the use of adapted inocula appears very interesting in biodegradability assays because biodegrader number are increased. The risk of error in biodegradability tests decreased in comparison with non-adapted inocula.
An exponential phase appeared between the 0-day and the 6-day (figure 2). A plateau was obtained between the 6-day and the 12-day. After this period, a slow increase occurred to reach a new stationary phase. Petroleum contained approximately 80 % of aliphatic and 20 % of aromatic, after asphaltic elimination. Microorganisms degrade n-aliphatic compounds before cyclic-aliphatic compounds and before aromatic compounds (Atlas et Bartha, 1992 ;
Massias et al ., 2003). Some compounds could be necessary to enhance degradation of other hydrocarbons (cometabolism). Bouchez et al . (1995) explain this case for fluorene and phenanthrene. Each of these compounds were not degraded when they were individually present in the experiment, but they were degraded when they were present in pair. Otherwise some hydrocarbons should have an inhibitory effect on the degradation of other compounds :
7

naphtalene inhibited the degradation of phenanthrene because of its high water-solubility
(30ppm) (Bouchez et al ., 1995). A lot of PAH interactions occurred during a biological process. These interactions happen near the active site of enzymes, particularly the initial oxygenase. It is possible, like this, to have some stages in the biodegradation treatment.
Therefore, aromatic hydrocarbons were more slowly degraded than alkanes. This fact can explain the results.
125 mg CO
2
/kg sand were produced in 70 days. This value was small compare to the theoretical carbone dioxide content (70.4 g/Kg sandy sediments). Biodegradation evaluation could be underestimated due to diffusion and adsoption of petroleum (Gogoi et al ., 2003 ;
Jonker et al ., 2003 ; Johnsen et al ., 2005). Furthermore, hydrocarbon fraction was washed by the recirculated water and was not evaluated for CO
2
production. In addition, volatile hydrocarbons after stripping must be taken into account.
Respirometry evaluation following the Sturm test is not enough precise to evaluate a biodegradation process. Not all the CO
2
will be trapped leading to errors cumulate from daily determination. The test is also time consuming and require a considerable equipment. Some authors have criticised Sturm test and have studied other methods to determine respirometric activity (Larson et al ., 1996 ; Itävaara and Vikman, 1996 ; Van der Zee et al ., 1997 ; Calmon et al ., 2000 ; Srinivasan et Viraraghavan, 2000). For example, Calmon et al . (2000) proposed a CO
2
determination by IR spectroscopy and compared the results with the results obtained with Sturm test procedure. They found that the automated test was more reproducible, less costly and more compact.
150,00
100,00
50,00
0,00
0 10 20 30 40 50 60 70
Time (days)
Figure 2 : CO
2
production (mg/Kg sandy sediments) with degradation time.
Further analysis by GC-MS (Gas chromatography) and HPLC (High Pressure Liquid
Chromatography) will be realised in order to give more information about the percentage of each hydrocarbons family biodegradation and their biodegradation.
3.2. Chemical analysis
First results were agreed with literature (Del’Arco and de França, 2001). Number of compounds detected along biodegradation treatment decreased and some others appeared.
8

3.3. Toxicity evaluation
First results of the Ames assay are presented in table 4 for strains TA98 and TA100 at
T = 0 . No specific mutagenicity was showed in hydrocarbons extract of sandy sediment and of percolated water. No genotoxicity was detected in initial petroleum sample. These results were agreed with Sayles et al.
(1999).
No specific toxicity were determined in the sandy samples or in the percolated water samples at t=0, 21, 42, 63 days.
Sample Concentration S9 addition
Reversion factor for TA 98
Reversion factor for TA 100 extract "sand" extract "sand" extract "sand" extract "sand" extract "L1" extract "L1" extract "L1" extract "L2"
DEPOL
DEPOL
DEPOL
DEPOL
0,5mg/plate
0,1mg/plate
10 µg/plate
1µg/plate
50 µg/plate
5 µg/plate
0,5 µg/plate
100µl/plate
25µg/plate
2,5µg/plate
250ng/plate
25ng/plate
+
-
+
-
+
-
+
-
+
-
+
-
+
-
+
-
-
+
-
-
+
-
+
-
+
0,80 (± 0,01)
1,05 (± 0,02)
0,93 (± 0,02)
0,83 (± 0,01)
0,76 (± 0,06)
0,87 (± 0,24)
0,96 (± 0,05)
0,82 (± 0,08) nd* nd*
0,75 (± 0,1)
0,94 (± 0,03)
0,69 (± 0,04)
0,83 (± 0,01)
0,80 (± 0,1)
0,80 (± 0,13)
1,05 (± 0,12)
1,03 (± 0,12)
0,90 (± 0,02)
0,76
0,92 (± 0,07)
1,0 (± 0,03)
0,98 (± 0,26)
1,21 (± 0,16) nd* nd*
0,86 (± 0,03)
1,13 (± 0,05)
1,10 (± 0,05)
0,87 (± 0,14)
1,12 (± 0,18)
1,04 (± 0,18)
0,86 (0,14)
0,95 (± 0,07)
0,99 (± 0,04) DEPOL 2,5ng/plate
+ 1,00 (± 0,03) nd* : not determined
Table 4 : Results for Ames test on hydrocarbons extracts at T=0 of the treatment.
Some hypothesis can explain this results :
toxicological compounds were not present in extracts, so there were some problems with the extraction procedure;
Problems were occurred during the dilution of hydrocarbons extract in DMSO solvent and in NaCl, for the LUMIStox test; Jennings et al . (2001) have discussed about this limiting factor;
perhaps some hydrocarbons were not accessible to bacteria and so no toxicity was detected;
an other hypothesis should be that DEPOL petroleum was not toxic because a first chemical analysis showed that it was composed of 80% of alkanes and only 20% of polycyclic aromatic hydrocarbons principally composed of 70% of naphthalene and its derivative (figure 3).
9

The most probable hypothesis was the problem of PAH solubility in water. Loibner et al . (2004) discussed about the relation between the aqueous solubility of PAH and the inhibition of the bioluminescence. They found that only two to four-ring PAH showed a toxicity effect. These compounds are effectively the most soluble in water than the other compounds, so they are most accessible to Vibrio fischeri cells than others. They found that toxicity of PAHs in soil were difficult to evaluate due to accessibility of compounds adsorbed on soil particle.
Other experiments will be realised in order to understand if there is some problems in the experimental procedure or if the results can be explain by the chemical composition or distribution in the samples :
LUMIStox test and Ames assay will be performed in presence of a surfactant that will permit to ameliorate the hydrocarbons accessibility to the bacteria cells;
LUMIStox test and Ames assay will be performed with LCO (Light Cracking Oil,
CEDRE), the most petroleum toxic party, in order to observe if toxicological tests really respond and are adapted to this type of contaminant;
An other test will be realised on a micro crustacean Artemia salina (ARTOKIT M,
MicroBioTests Inc), for marine environment samples, which is a very simple and rapid
(24h).
80,00%
70,00%
60,00%
50,00%
40,00%
Chemical analysis of DEPOL (CEDRE)
Chemical analysis on sandy sediment in Greece (Papadopoulou and Samara, 2002)
Chemical analysis on sandy sediment in Nigeria (Olajire et al., 2002)
+ Toxicity
(Study on Daphnia magna )
-
30,00%
20,00%
10,00%
0,00%
)pe ryl ene
B enz o( g,h,i o[ b+k] fluor
B enz ant he ne o[ a] pyr ene o[ e] pyr
B enz
B enz ene
A nt hr ac ene cd) pyr
Inde no(
1,2,3ene
Fl uor ene
A ce na pht yl ene
Phe na nt hr ene
N apht al
ène
Pyr ene
Fl uor ant hè ne
C hr ys ene
D ibe nz
(a
,h) ant
B hr ac enz o[ ene a] ant
Fl hr ac ene ant he ne
,P yr ene uor D ibe nz ot hi ophe ne
Polycyclic aromatic hydrocarbons
B iphe nyl
Figure 3 : Comparison of chemical analysis of 3 polycyclic aromatic hydrocarbons family with their toxicity on
Daphnia magna (Kaiser, 1998)
10

3.4. Consortium analysis
Consortium profile of sandy sediment, percolated water and activated sludge reactor were different. Profiles of inoculum were difficult to determine because the initial cell density in each well of the microplate was not the same (Garland, 1996 ; Choi and Dobbs, 1999).
Table 5 show the carbon substrates in BIOLOG microplate which were not degraded by different consortia at the end of analyse (120 h). Results are difficult to interpret because almost all of the well of the microplate responded and very rapidly. So BIOLOG ECOPlate is not adapted to this type of consortium which is very full of microorganisms. Moreover, in the sand extract, some very fine particles modified the spectrophotometric measurement. Other experiments should be more precise like molecular analysis of the bacteria consortium.
Activated sludge reactor putrescine glycogen
Consortium profile
Percolated water
D,L-α -glycerol phosphat e D,L-α -glycerol phosphat e
Sandy sediment
α-D-lactose
Table 5 : Consortium profile with non-degraded compounds of samples of activated sludge reactor, percolated water and sandy sediment.
Some photographs with the optical microscopy

100 with immersion (Leica DM LB) have been taken to observe the consortium evolution. After two months of experiment, some protozoa were observed, and especially Vorticella and Aspidisca (figure 4a and 4b). Aspidisca
( Ciliata , Hypotrichida ) is a little ciliate that move at the surface of flocs. He is often observed in fully-grown activated sludge. This is a bacteriophage exclusively. Vorticella ( Ciliata ,
Peritrichida ) is a sign of a good oxygenation. All of them taken part in the consortium equilibrium.
Figure 4a : Vorticella Figure 4b : Aspidisca
11

4. Conclusion
The evaluation of a degradation process, and especially the biodegradation was not so easy because of
the relative difficulties to solubilise hydrocarbons in water for toxicity evaluation,
the imprecision on the microbial activity of the Sturm test,
the difficulties to have homogenate sample and cell density for consortium analysis by the
BIOLOG ECOPlate.
Only GC-MS and HPLC analysis could give precise analysis of the degradation of each hydrocarbons compounds and family hydrocarbons.
Further analysis must be realised in order to confirm first results and to have more information about remediation processes.
The study show the complexity to evaluate the efficiency of a waste treatment.
12

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