BIODEGRADATION OF DIESEL OIL IN SOIL

BY A MICROBIAL CONSORTIUM

FACUNDO J. MRQUEZ-ROCHA , VANESSA HERNNDEZ-RODRGUEZ and

MA. TERESA LAMELA
Centro de Investigacin Cientfica y de Educacin Superior de Ensenada, Ensenada, Mxico
( author for correspondence, e-mail: fmarquez@cicese.mx; address: Aquaculture Department,
CICESE, P.O. Box 434844, San Diego, CA 92143, U.S.A.)

(Received 11 October 1999; accepted 1 June 2000)

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.

Keywords: bacterial consortium, biodegradation, diesel, inoculum

1. Introduction

The extent of hydrocarbon biodegradation in contaminated soils is critically de-

pendent upon four factors, namely the creation of optimal environmental conditions
to stimulate biodegradative activity, the predominant petroleum hydrocarbon types
in the contaminated matrix and the bioavailavility of the contaminants to microor-
ganisms. The petroleum hydrocarbon degradation is also affected by the molecular
composition of the hydrocarbons, characteristic which is directly related with the
bioavailability of these compounds, and as a consequence, the biodegradation rate
may be altered (Huesemann, 1995).
Bioremediation is being used or proposed as a treatment option at many hydro-
carbon-contaminated sites (Braddock et al., 1997). The effectiveness of bioremedi-
ation is often a function of the microbial population or consortium and how it can
be enriched and maintained in an environment. Microorganisms with the ability
to degrade crude oil are ubiquitously distributed in soil and marine environments,
(Venkateswaran and Harayama, 1995). However, when few or no indigenous de-

Water, Air, and Soil Pollution 128: 313320, 2001.

2001 Kluwer Academic Publishers. Printed in the Netherlands.
314 F. J. MARQUEZ-ROCHA ET AL.

gradative microorganisms exist in a contaminated area or when there is not time

for the natural enrichment of a suitable population, inoculation (bioaugmentation)
can be a realistic option. Inoculation of bacteria with hydrocarbon biodegradation
capabilities shorten the time of the treatment. The same ecological principles that
influence biodegradation with the native microorganism population, in general, will
also govern the effectiveness of the inoculation, regardless of whether they are
natural isolates or genetically engineered microorganisms (Liu and Suflita, 1993).
Some researchers have reported that inoculation had no positive, or only mar-
ginal effects on oil biodegradation rates. Microorganisms able to degrade organic
pollutants in cultures may fail to function when inoculated into natural environ-
ments, because they may be susceptible to toxins or predators in the environment.
They may use other organic compounds in preference to the pollutant, or they may
be unable to move through the soil to the contaminated site. The successful use of
microbial inocula in soils requires that the microorganisms contact the contamin-
ant. Physical adsorption to soil particles or filtration through small pores may limit
the transport of organisms, (Margesin and Schinner, 1997).
Most physical-chemical technologies have the ability to be scaled up with pre-
dictive reliability from laboratory or pilot experiments to full-scale, a normally ex
situ operation. This is true for the processing industries as well as for nonbiological
environmental remediation applications. Bioremediation, especially, in situ biore-
mediation, has been identified as one technology in which accurate scale-up can be
problematic, and the prediction of how long a remediation treatment will require
and how well that treatment will work can at times be uncertain (Blackburn, 1998).
In this work, we describe a procedure to improve the ability of a microbial consor-
tium to degrade diesel from a contaminated soil, a procedure based on inoculum
preparation in two steps.

2. Materials and Methods

2.1. BACTERIAL COMMUNITY AND FUNGAL STRAIN

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

2.2. S OIL PREPARATION

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%.

2.3. B IODEGRADATION CONDITIONS

2.3.1. Laboratory Scale

Five grams of soil with diesel adsorbed were placed in a flask, it was inoculated
with the bacterial consortium (6.11.6 105 heterotrophic bacteria g (dry soil)1 ),
at room temperature. The control test was carried out without addition of BC
(BC). All the experiments were carried out by triplicate and the sampling was
programmed once a week during six weeks. Three flasks were inoculated with P.
ostreatus (P) by using 1 g of wheat seed (in each cylindrical flask) covered with the
fungus and other flasks were treated with both bacterial consortium and P. ostreatus
(BCP).

2.3.2. Pilot Scale Experiments

Three square units of 5 m2 and 40 cm of depth were used. One unit was used as
control (BC; not inoculated), another unit was inoculated and supplemented with
0.5 g NH4 NO3 kg1 soil (BC + NH4 NO3 ) previous to inoculation, and the third
unit was only inoculated with the bacterial consortium (BC).

2.4. I NOCULUM PREPARATION FOR PILOT SCALE EXPERIMENTS

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.

2.5. BACTERIAL ANALYSES

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.

3. Results and Discussion

3.1. BACTERIAL GROWTH ON DIESEL AS SOLE CARBON SOURCE

Figure 1 shows the time-course of a liquid culture of the bacterial consortium

(BC) using diesel as sole carbon source. A typical Monod-type growth kinetics
was observed, hydrocarbons were used for bacterial growth. Diesel was consumed
 BIODEGRADATION OF DIESEL IN SOIL 317

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.

by BC to an extent close to 90% (1S = 54 g L1 ) after 13 days. The lost of diesel

in controls (BC) was <5% (1S = 2.1 g L1 ). These results indicate that diesel
was used by the bacterial consortium, since the bacterial population increased from
2 104 to 107 CFU mL1 . The diesel biodegradation rate in liquid culture was
close to 4.0 g L1 d1 . Figure 2 shows the gas chromatography analyses, before
inoculation (Figure 2A) and after 13 days of treatment with BC (Figure 2B), long
chain alkanes (>C12 ) were not detected after the bacterial growth ceased (200 h
after inoculation, Figure 1). Diesel remainder in controls (BC) was identical to
Figure 2A (chromatogram not shown) after 13 days.

3.2. D IESEL BIODEGRADATION IN SOIL ( LABORATORY EXPERIMENTS )

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).

3.3. P ILOT SCALE EXPERIMENTS

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.

respectively. There is not statistical significant difference between BC and BC

+ NH4 NO3 treatments, but these two treatments are significantly different to
BC (control). Analysing kinetic parameter such as biodegradation rate, there is
a difference between BC and BC + NH4 NO3 treatments, it may depends of the
hydrocarbon concentration following a first kinetic order reaction with respect to
high concentration of substrate and microorganisms ds/dt = kX, (S >> Ks ).
Diesel oil consists mostly of linear and branched alkanes with different chain
lengths and contains a variety of aromatic compounds. Many of these compounds,
especially linear alkanes, are known to be easily biodegradable. However, due to
their low water solubility, the biodegradation of these compounds is often limited
by slow rates of dissolution, desorption, or transport. In general, the bioavailabil-
ity of hydrophobic compounds is determined by their sorption characteristics and
dissolution or partitioning rates and by transport process to microbial cell, (Sticher
et al., 1997). Diesel biodegradation does not appear to be limited by the metabolic
capabilities of the bacterial consortium, since a significant fraction of high mo-
lecular weight hydrocarbons (>C12 ) were biodegraded at the same rate. However,
bacterial inability for total hydrocarbon utilization, seems to be due to a mass
transfer limitation, in which hydrocarbons are not available to microorganisms.
P. ostreatus achieved more efficiently the removal of diesel, because this white rot
fungus grows well through the soil.
Bioremediation identified as a technology in which accurate scale-up can be
problematic, in which the prediction of how long a remediation treatment will
require and how well that treatment will work can at times be uncertain (Black-
320 F. J. MARQUEZ-ROCHA ET AL.

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

Atlas, R. M.: 1981, Microbiol. Rev. 45(1), 180.

Blackburn, J. W.: 1998, Bioremediation J. 1(3), 265.
Braddock, J. F., Ruth, M. L., Catterall, P. H., Walworth, J. L. and McCarthy, K. A.: 1997, Environ.
Sci. Technol. 31(7), 2078.
Huesemann, M. H.: 1995, Environ. Sci. Technol. 29(1), 7.
Liu, S. and Suflita, J. M.: 1993, TIBTECH 11(8), 344.
Margensin, R. and Schinner, F.: 1997, Appl. Environ. Biotechnol. 63(7), 2660.
Sticher, P., Jaspers, M. C. M., Stemmler, K., Harms, H., Zehnder, A. J. B. and van der Meer, J. R.:
1997, Appl. Environ. Microbiol. 63(10), 4053.
Venkateswaran, K. and Harayama, S.: 1995, Cand. J. Microbiol. 41, 767.