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Professor Irena
B.Ivshina, PhD, Head of the Alkanotrophic Microorganism
Laboratory
Dr. Maria S.
Kuyukina, PhD, Senior Scientist of the Alkanotrophic
Microorganism Laboratory
Ms. Marina I.
Ritchkova, Research Scientist of the Alkanotrophic
Microorganism Laboratory
Institute of
Ecology and Genetics of Microorganisms, Ural Branch of Russian
Academy of Sciences, Perm, Russia Email:
ivshina@ecology.psu.ru
kyukina@ecology.psu.ru
Professor Nick
Christofi, PhD, Email:
N.Christofi@napier.ac.uk
Dr. James C. Philp,
PhD, Lecturer. Email:
J.Philp@napier.ac.uk
School of Life
Sciences, Napier University, Edinburgh,
Scotland, UK
Mr. Colin J.
Cunningham, Research Co-ordinator
Contaminated Land Assessment and
Remediation Research Centre (CLARRC), Edinburgh, Scotland, UK
Email: c.cunningham@ed.ac.uk
In
recent years methods of biological remediation of
oil-contaminated soils have become more and more significant
due to their ecological safety. There are two principal
approaches to the biological degradation of hydrocarbons in
water and soil: (1) introduction of specifically selected
associations of microorganisms, which degrade various classes
of hydrocarbon pollutants (bioaugmentation); (2) activation of
indigenous oil-oxidizing microflora by providing optimal
conditions for its growth (biostimulation). The most effective
are biotechnologies including both approaches e.g. use of
biofertilizer containing microbial cultures and inorganic
nutrients.
It
should be noted, that in the Ural climatic zone with its short
warm period (120-125 days) natural attenuation of
oil-contaminated soils is slow. In the Perm region, therefore,
biotechnologies that involve stimulation of hydrocarbon
oxidation by natural oil-oxidizing microorganisms are
essential. Several agrotechnical methods, namely, tilling and
loosening; watering and drainage systems; addition of organic
materials (straw, compost etc) and mineral fertilizers could
decrease the contamination level up to 30-40 % due to the
oxidation of easily degradable petroleum components (Christofi
et al., 1998). However, high molecular paraffins,
aromatic and polycyclic compounds, with concentrations in
crude oil from 20 to 65 %, are not degraded for years. The
above compounds bind to the soil particles, producing
hydrophobic residues, and become non-bioavailable to
microorganisms. To increase the bioavailability of hydrocarbon
pollutants, surface-active substances (surfactants) are used
which allow desorption and solubilization of petroleum
hydrocarbons and thus facilitate their assimilation by
microbial cells (Deschenes et al., 1995; Volkering et al.,
1995; Thibault et al., 1996). However, synthetic surfactants
commonly used for this purpose can be highly toxic and
non-biodegradable (Chin et al., 1996). Application of
synthetic surfactants leads to the accumulation of
ecologically harmful compounds in soil.
Effective and ecologically safe biosurfactants produced by
Rhodococcus bacteria isolated from oil-extraction areas in
Perm region were developed in the
alkanotrophic
microorganism laboratory
of the Institute of Ecology and Genetics of Microorganisms,
and the Pollution Research Unit of Napier University within
the frame of Collaboration Project 638072 P750/LJH supported
by the Royal Society (1994-1997). The biosurfactants produced
possess significant advantages over synthetic ones,
particularly biodegradability, stable activity under extreme
environment conditions, a wide range of functional
characteristics and the possibility to be produced from
non-traditional and relatively cheap raw materials. Laboratory
study data on recovery of heavy oils (density 0.93 to 0.97
g/cm3) from oil-contaminated sand using
Rhodococcus biosurfactant complexes showed that the rate
of oil removal exceeds the control data 15-20 fold (Ivshina et
al., 1998).
In
this paper we discuss the application of oleophilic
biofertilizer based on bacterial surfactants for
decontamination of soil heavily polluted (200 g/kg of total
recoverable petroleum hydrocarbons – TRPH) with crude oil.
Materials and
methods
Biofertilizer production.
Two Rhodococcus strains isolated from Perm
oil-extracting area (West Urals) were used for biofertilizer
production. R. erythrolopis IEGM 708 was isolated from
the bottom of crude oil waste storage pit, and the R. ruber
IEGM 327 was isolated from oil-contaminated soil. The ability
of strains to use petroleum hydrocarbons as sole carbon source
was tested on mineral agar containing 0.5 % of individual
compound (naphthalene was tested in a vapor phase). The growth
of strains on crude oil was tested in shake flask experiments
(Mueller et al., 1992). Freeze-dried strains were stored at
the Regional Specialized Alkanotrophic Microorganism
Collection IEGM (Catalogue of strains, 2001).
Strains were grown in a
mineral salts medium contained (per liter of distilled water)
KH2PO4, 1.0 g; K2HPO4,
1.0 g; KNO3, 1.0 g; NaCl; 1.0 g; MgSO4·7H2O,
0.2 g; CaCl2·2H2O, 0.02 g; FeCl3·7H2O,
0.01 g; trace element solution, 1.0 ml; yeast extract, 0.1 g.
Diesel used as carbon source was added at a concentration of
3% (v/v). Strains pregrown in the above mineral medium with 3%
v/v of n-hexadecane were used as inocula at a
concentration of 3.5 x 105 bacterial cells/ml.
Cultivation was performed in a bioreactor with agitation (300
rpm) at 28oC for 3 days. Broth culture was settled
for 2 hours and most of the water fraction was removed. The
remaining oleophilic material containing rhodococcal cells and
residual hydrocarbons was amended by the addition of NPK
mineral salts in ratio 70:5:1 and Rhodococcus
biosurfactant complex in concentration of 10 g/l. The growth
conditions for biosurfactant producing strain and surfactant
complex isolation procedure are described elsewhere (Ivshina
et al., 1998; Kuyukina et al., 2001).
The biofertilizer was stored
at the temperature below 10oC and was diluted with
water before use.
Microbiological analyses.
To achieve maximum desorption of microorganisms from the
surface of soil particles, the soil samples, with a small
amount of water, were subjected to ultrasonic treatment (22
kHz, 0.3 A, 1-2 min) (Ivshina and Kuyukina, 1997).
Heterotrophic bacteria enumeration was performed routinely by
inoculation of nutrient agar plates. Enrichment cultures of
hydrocarbon-oxidizing microorganisms were obtained using a
liquid mineral medium supplemented with diesel as a carbon
source. Enumeration and isolation of hydrocarbon-degrading
microorganisms was performed by plate count on mineral agar. A
mixture of C12-C17 n-alkanes was
used as the carbon source. Cultures were incubated at 28oC
for a week. All the experiments were performed in triplicate.
The identification of the
oil-degrading strains isolated was performed by polyphasic
taxonomy methods, including phenotypic, chemotaxonomic and
immunochemical, using specific polyclonal antisera, analyses (Ivshina
et al., 1986; Ivshina et al., 1995).
Analytical methods.
The oil content in soil and
slurry samples was determined gravimetrically as the amount of
total recoverable petroleum hydrocarbons (TRPH) extracted by
chloroform. Oil fraction analysis was performed using
Iatroscan TLC-FID Analyzer MK-5 (Iatron Laboratories Inc.,
Japan). Soil and slurry samples were extracted with
dichloromethane-pentane (3:1), the pentane-soluble fractions
were applied onto chromarods (type S III) and consecutively
eluted with n-hexane to separate saturated
hydrocarbons, dichloromethane-pentane (55:45) to separate
aromatics, and dichloromethane-methanol (98:2) to separate
heterocyclics. The rods were scanned by FID, the area for each
peak was calculated, and the composition (i.e. the percentage
of saturated hydrocarbons, aromatic hydrocarbons and
heterocyclics) was determined. Tars/asphaltenes content was
determined gravimetrically.
Bioremediation study. Soil
experiments.
Experiments were carried out
in 8-litre vacuum desiccators. Oil-contaminated soil (208
g/kg of TRPH) taken from the oil waste storage pit was
homogenized and added to clean agricultural soil at different
concentrations. To improve mechanical structure and to enhance
aeration of the soil, different bulking materials were added
(plastic crumbs, wood-chips and wood shavings) in the ratio of
1:8, bulking agent : soil. The surfactant-based biofertilizer
in concentration 10 g/l was added weekly for one month. During
the experiment the soil was tilled and watered daily to
maintain the moisture content of 20%.
Slurry bioreactor. A bench-scale slurry bioreactor was designed using the following
operation parameters: reactor volume 2 liter; working volume 1.5
liter; mixing speed 300 rpm; air supply at the pressure 1.5
kg/cm2 and temperature 25oC. The
bioreactor was filled with 500 g of oil-contaminated soil and
900
ml tap water. During the experiment (2.5 months), water was
added periodically to maintain the total volume of 1.2
liter and solid phase content not more than 40%. The 5 g of
biofertilizer was added on the 1, 15, and 43 days of the
experiment.
Soil
and slurry physical parameters (temperature, pH, oxygen
dissolved in slurry, soil moisture content) were monitored
daily. Samples for microbiological and chemical analyses were
taken weekly.
Results and
Discussion
Physiological and catabolic
properties of Rhodococcus bacteria used in
biofertilizer are shown below.
Strain R.
erythropolis IEGM 708 assimilates n-alkanes (C10-C20),
aromatic hydrocarbons (benzene, toluene, xylene, naphthalene,
acenaphthalenes, e.g. methyl-, dimethyl- and
trimethylnaphthalene); grows at up to 7% NaCl, pH 5.7-8.0,
temperature 10-40oC; survives after 15-min heating
at 75oC; tolerates heavy metals (grows in 30 mM of
cadmium and chromium) and xenobiotics (0.03% basic fuchsin,
0.0001% crystal violet, 5% propylene glycol).
Strain R.
ruber IEGM 327 assimilates gaseous (ethane, propane,
butane) and liquid (C10-C20) n-alkanes,
aromatic hydrocarbons (benzene, toluene, xylene, phenol,
naphthalene, acenaphthalenes, e.g. methyl-, dimethyl- and
trimethylnaphthalene); grows at up to 7% NaCl, pH 5.7-8.0,
temperature 10-40oC; survives after 15-min heating
at 75oC; tolerates heavy metals (grows in 30 mM of
cadmium and chromium) and xenobiotics (0.003% basic fuchsin,
0.0001% crystal violet, 5% propylene glycol); fixes
atmospheric nitrogen (N2-fixation reaches 62 mg N per
liter
of medium).
Growth of R.
erythropolis and R. ruber in mineral medium with 3
% (v/v) of diesel resulted in biomass yield of 2.5 and 3.3 g/l
(dry weight) respectively. Both strains
grew to high cell density (106-108
bacterial cells/ml) in medium containing 0.5 and 1.0 % (v/v)
of crude oil from the Kokuyskoye oilfield.
Results of
field experiments on introduction of
Rhodococcus
bacteria into oil-contaminated soil showed different
ecological behavior of
R. erythropolis
and R. ruber (Ivshina et al., 1998a). Specifically,
under optimal environmental conditions (excess hydrocarbon
substrate, water and mineral nutrients in soil) the number of
R. erythropolis rose rapidly (10-fold within two weeks
after introduction), demonstraiting the successful ecological
competitiveness of this organism in the autochthonous
microbial community. In contrast, R. ruber introduced
into oil-contaminated soil showed insignificant (2.4-fold)
increase in population, and the number of bacteria was stable
through the experiment, indicating the capability of this
organism to survive under limiting environmental conditions
(i.e. low rainfall,
nutrient deficiency and
lack of readily
biodegradable hydrocarbons). Thus, the combination of two
Rhodococcus
species ensured effective and stable operation of
biofertilizer under variable environmental conditions.
It is noteworthy, that
rhodococcal cells, due to their hydrophobic cell surfaces,
have high affinity for oleophilic substances, and therefore
cells remain viable for long periods in the oleophilic matrix
of the biofertilizer. This explains the high viability of
oil-degrading bacteria presented in biofertilizer under
different storage conditions (Fig. 1). Thus biofertilizer in
oleophilic form is suitable for prolonged storage and
transportation. Moreover, since rhodococcal cells maintain
high activity in the oleophilic matrix, the biofertilizer,
unlike lyophilized bacterial preparations, does not need
preliminary activation before use.
This laboratory studies,
including soil and slurry bioreactor experiments, were
conducted to examine the effectiveness of oleophilic
biofertilizer for bioremediation of crude oil-contaminated
soil.
Soil experiments showed that
addition of the biofertilizer increased significantly the
number of soil hydrocarbon-oxidizing and heterotrophic
microorganisms. The number of heterotrophs increased 4-fold
and the number of hydrocarbon-oxidizers increased almost
100-fold after one week (correspondent numbers in control
untreated soil increased 2-10– fold). Each of the subsequent
additions of biofertilizer resulted in a sharp increase of
hydrocarbon-oxidizing microorganisms and further stabilization
at the level 5 x 107 - 5 x 108 bacterial
cells/g soil. The mean values of bacteria numbers in treated
soil systems with the initial contamination levels of 12 and
45 g/kg of TRPH did not significantly differ.
Rapid formation of an
oil-degrading bacteriocenosis correlated with the high rates
of oil biodegradation in the soil. In soil systems treated
with the biofertilizer, oil removal was 57-60 % after four
weeks, while that of the control one was only 37-47 %.
Therefore, the use of
oleophilic biofertilizer had a stimulating effect on the
growth of hydrocarbon-oxidizing microflora of oil-contaminated
soil, leading to 1.3-1.6 fold increase in the oil
biodegradation rate.
Microbiological studies of
the laboratory slurry bioreactor content shows that it
initially contained heterotrophic and hydrocarbon-oxidizing
bacteria in numbers of 3.4 x 105 and 9.2 x 104
bacterial cells/ml. Following biofertilizer treatment, the
number of heterotrophic bacteria increased 100-fold, while
that of hydrocarbon oxidizers increased 500-fold. During the
subsequent two weeks, the populations of these groups of
microorganisms decreased insignificantly. However, upon the
second biofertilizer treatment, their numbers increased again
and reached 108 bacterial cells/ml. It should be
noted, that each subsequent treatment with biofertilizer led
to a 10-fold increase in various microbial groups within the
reactor. Thereafter, the numbers of both groups of bacteria in
the bioreactor remained constant and were 109 and
108 bacterial cells/ml, respectively, within the
experiment.
The data showed that to
maintain a sufficient amount of hydrocarbon-oxidizing bacteria
in the bioreactor, it was necessary to add the biofertilizer
once a month, in contrast to soil systems where the
biofertilizer was added weekly. Taxonomic structure of the
oil-degrading microbiocenosis in the bioreactor was similar to
that of the soil microbial communities under study. A common
characteristic was that representatives of several bacterial
genera, e.g. Rhodococcus erythropolis and Gordonia
terrae predominated. Gram-negative microorganisms were
represented almost by various Pseudomonas species.
An improvement to the mixing
at the end of first week achieved complete homogeneity of the
contaminated soil slurry and oil degradation rate increased,
reaching 9.1-9.5 g/l/day within a month. Visual comparisons of
solid phase samples obtained from the bioreactor showed that
initially heavily contaminated soil contained much less
hydrocarbons, and the greater amount of the oil was present in
the liquid phase and was biodegraded effectively. Therefore,
use of biofertilizer provided effective desorption of
hydrocarbon contamination from the surface of clay particles,
thus, making it available to bacterial oxidation. The amount
of oil degraded was 82 % of the initial concentration by the
end of the second month. At the final stage of the experiment,
the extent of oil biodegradation reached 85 %.
Aliphatics and aromatics
decreased at a higher rate (Fig. 2). The initial portions of
these components in oil were 66 and 24 %, and they decreased
to 53 and 8 % by the end of the experiment. Bacterial
destruction of heterocyclics and tars/asphaltenes was
significantly slower, and the portion of these components in
residual oil increased by 12.3 and 16.5 %, respectively, at
the final experimental stage.
The studies performed using
laboratory soil systems and slurry bioreactor demonstrate the
efficacy of the oleophilic biofertilizer developed for
microbiological treatment of soil contaminated heavily with
oil, providing effective elimination of contamination.
The
field scale bioremediation of soil polluted with crude oil (up
to 200 g/kg of TRPH) was carried out using the oleophilic
biofertilizer on the territory of the Kokuyskoye oilfield.
After 3.5 months of bioremediation using field slurry
bioreactor and land farming cells the contamination was
reduced to 1.0-1.5 g/kg of TRPH. The oleophilic biofertilizer
was shown to enhance decontamination of heavily
oil-contaminated soils in cold climate regions.
Acknowledgements
This work was
supported by the Regional Complex Science-Technical Programme
“Ural”, grant from the Ministry for Industry, Science and
Technology of the Russian Federation and travel grant from the
British Council.
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