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BY A MICROBIAL CONSORTIUM
Abstract. Biodegradation of diesel oil was performed using a diesel oil-degrading bacterial consor-
tium, in both laboratory and pilot scale experiments. The bacterial consortium was prepared in liquid
for laboratory tests and for pilot scale experiments, it was prepared in two steps, liquid and then in
soil. The concentration of diesel in soil treated with the bacterial consortium was reduced to <15%
of the initial concentration, within a period of five weeks in both laboratory (135 to 19.32 g diesel
kg (soil dry weight)1 ) and pilot scale (118 to 17.5 g diesel kg (soil dry weight)1 ) experiments, in
comparison with controls (without bacterial consortium), in which initial concentration of diesel was
reduced by only 5 and 15%, respectively. Diesel biodegradation rate with the bacterial consortium
was 2.13 g diesel kg (soil dry weight)1 d1 , it was slightly enhanced by the addition of NH4 NO3
in the presence of bacterial consortium 2.78 g diesel kg (soil dry weight)1 d1 . The enhancement
of the microbial activity in hydrocarbon-contaminated soil can be achieved with the combination of
stepwise soil inoculation and nutrient additions.
1. Introduction
A microbial consortium grown on crude oil was obtained from the Mexican In-
stitute of Petroleum (IMP, Mexico City). It was acclimated to grow on a mineral
medium using diesel hydrocarbons as sole carbon source. The mineral medium
was prepared as follows (g L1 ): K2 HPO4 1.71, KH2 PO4 1.32, NH4 Cl 1.26, MgCl2
6H2 O 0.011, CaCl2 0.02, and 1 mL of trace mineral solution, 7 mL of diesel (85.7%
as C, 0.886 g mL1 of density) were added per 100 mL of culture medium. A
dense culture was lyophilized to preserve the bacterial activity. A sample of the
consortium was activated and used to prepare the inoculum for soil treatment. A
strain of the white rot fungus Pleurotus ostreatus ECS-110 (IE-8, Colegio de la
Frontera Sur (ECOSUR), Chiapas., Mxico) was used for laboratory experiments.
BIODEGRADATION OF DIESEL IN SOIL 315
The soil for laboratory experiments was dried at 80 C overnight (this may serve
to eliminate indigenous bacteria from the soil). Once dried, 0.177 g diesel g (dry
soil)1 was uniformly placed on the soil and it was allowed to be adsorbed during
30 min. After adsorption of diesel, the humidity was adjusted to approximately
43%. Diesel-contaminated soil was used for pilot scale experiments. The soil has
an organic matter content of 5.9%, nitrogen content of 0.21% and an available
phosphate content of 24.8%.
Three liters of dense culture (107 CFU as heterotrophic bacteria mL1 ) were in-
oculated on 200 kg of diesel-contaminated soil (1.5 105 CFU g1 soil), mixing
was provided mechanically during one week. These two hundred kg were placed in
each experimental unit (5 m2 ), except control (BC). Initial heterotrophic bacterial
count was 0.50.9 105 CFU g1 soil. Moisture was keeped at 435% and mixed
manually, temperature range was 1525 C.
The bacterial consortium was classified to their generic level, on the base of Gram
stain, catalase reaction, oxidase activity, and the ability to utilize glucose and lactose
under aerobic and anaerobic conditions.
316 F. J. MARQUEZ-ROCHA ET AL.
Figure 1. Time course of the bacterial consortium (BC) grown on diesel as sole carbon source in
liquid medium. Bacterial growth in control was not detected.
2.6. A NALYSIS
For liquid-liquid extraction of diesel, dichloromethane (1:1) was used. For solid-
liquid extraction, 5 g of soil were extracted with twice 50 mL of dichloromethane.
The organic phase was passed through NaSO4 and concentrated to 0.2 mL. It was
analyzed by gas chromatography, using a Varian model 3700, equipped with a
flame ionization detector and capillary column (30 m long, I.D. 0.25 mm, SPB-
20, Supelco). Peaks from 636 min were used for total diesel determination in
all experiments. At the end of the experiments, more than 95% of the diesel was
recovered in the control trial.
Figure 2. GC analyses of the remainder diesel when BC was grown in liquid culture: A) time zero of
the cultivation and B) after 13 days of cultivation.
The bacterial consortium was used to remove diesel adsorbed into soil. Soil with
diesel adsorbed was inoculated with the BC (11.5 105 CFU g1 soil), P. os-
treatus (P) and both BC + P (BCP). Figure 3 shows the diesel concentration in
soil when treated with the bacterial consortium (BC), P. ostreatus (P) and BCP.
318 F. J. MARQUEZ-ROCHA ET AL.
Figure 3. Time course of diesel biodegradation in soil at laboratory experiments; BC, treated with
the bacterial consortium; BC, control (without BC); P, Pleurotus ostreatus; BCP, treated with BC
and P. ostreatus.
Degradation effectiveness was 85, 91 and 96% for BC, P and BCP, respectively.
The biodegradation rates were 2.51, 3.11 and 4.28 g kg (soil dry weight)1 d1 ,
respectively. Diesel lost in controls (BC) was <5.4%. There is not statistical sig-
nificance between BC and P, but both were slightly different to BCP treatments, all
were significantly different to BC (controls). Theses results indicated BC and P.
ostreatus effectively remove diesel from soil. Four bacterial genera were identified
in the bacterial consortium (BC) named, Pseudomona, Serratia, Acinetobacter and
Flavobacterium, most of them have been reported as hydrocarbon utilizers (Atlas,
1981).
A simple inoculation procedure was established for pilot scale experiments. The
bacterial consortium was prepared in liquid (see material and methods), then it
was added to diesel-contaminated soil (200 kg). One week after BC addition, these
200 kg of soil were placed in the 5 m2 units containing diesel-contaminated soil,
keeping a relationship of 10%. The unit inoculated with the bacterial consortium
(BC) was able to remove more than 85% of the diesel (118.3 to 17.5 g diesel
kg (soil dry weight)1 ); the unit supplemented with NH4 NO3 and inoculated with
BC (BC + NH4 NO3 ), the diesel biodegradation was 90% of the initial concen-
tration (123 to 11.72 g diesel kg (soil dry weight)1 ) and the control unit lost
only 13% of the initial diesel concentration (Figure 4). Biodegradation rates were
2.13 and 2.78 g diesel kg (soil dry weight)1 d1 for BC and BC + NH4 NO3 ,
BIODEGRADATION OF DIESEL IN SOIL 319
Figure 4. Diesel biodegradation in soil at pilot experiments: BC, control; BC, with the bacterial
consortium; BC + NH4 NO3 , with the bacterial consortium and NH4 NO3 (0.5 g N kg1 dry soil)
addition.
burn, 1998). Our study might be established a simple scale up procedure with good
predictability by using a bacterial preparation in liquid and then inoculation of the
contaminated-soil. This procedure might be enhance the capabilities of a bacterial
consortia to degrade and mineralize hydrocarbons.
References