Chemosphere, Vol. 38. No. 15. pp. 3463-3472, 1999
0 1999 Elsevier Science Ltd. All rights reserved
0045-6535/99/ $ - see front matter
PII: soa45-6535(98)00575-x
BIODEGRADATION
OF DIESEL OIL
BY COLD-ADAPTED MICROORGANISMS
IN PRESENCE
OF SODIUM DODECYL SULFATE
R. Margesin* and F. S&inner
Institute of Microbiology (N.F.), University of Innsbruck, Technikerstrasse 25,
A-6020 Innsbruck, Austria
* Corresponding author, e-mail: [email protected]
(Received in Germany 28 August 1998; accepted 15 October 1998)
ABSTRACT
The effect of different concentrations
of the anionic surtactant sodium dodecyl sulfate (SDS) on
biodegradation of diesel oil was assessed during 32 days at lO”C, under simulated environmental conditions,
in liquid culture and in an alpine soil. Low SDS concentrations (SO-100 mg 1-l) significantly enhanced oil
biodegradation
by a psychrotrophic inoculum in liquid culture, whereas higher SDS concentrations
(500-1000 mg 1-l) inhibited
hydrocarbon
biodegradation.
Oil biodegradation
by
the
indigenous
microorganisms in soil was inhibited at all SDS concentrations tested. The sm-factant itself was rapidly
biodegraded both in liquid culture and in soil. Q 1999Elsevier Science Ltd. ~11 rights reserved
Keywords: biodegradation, diesel oil, sodium dodecyl sulfate, cold-adapted microorganisms
1. INTRODUCTION
The environmental contamination with mineral oil hydrocarbons due to industrial wastes, transport and
storage accidents [l-3] is widespread. Diesel oil is one of the major contaminants of soil and groundwater
near petrol stations. The demand for the clean-up of contaminated sites increased with increasing public
attention towards the preservation of the environment. An efficient and ecologically acceptable treatment
method for the decontamination of many oil polluted areas is bioremediation which attempts to accelerate
the natural biodegradation rates by overcoming limiting factors [l-3].
The decontamination
of polluted cold environments has been recognized as an area of particular
importance since more than 80% of the biosphere show temperatures below 5°C. Evidence for oil
biodegradation at low temperatures in several cold marine and terrestrial ecosystems has been found,
3463
3464
successfbl bioremediation was described in arctic, subarctic, alpine and antarctic environments [for a review
see ref. 31.
However, there is an important limitation of bioremediation techniques: oil contaminations cannot be
reduced to zero, even after a prolonged treatment, Degradation rates below possible treshold concentrations
are slow or negligible [l]. In the course of biodegradation the bioavailability of the contaminants will
decrease and recalcitrant compounds accumulate [l-3]. It is not the degradability of the oil that is the
limiting step in the ultimate degradation rate of diesel oil; desorption or diffusion limitation of the oil
components are responsible [4]. Low aqueous solubility of highly hydrophobic hydrocarbons such as
mineral oil hydrocarbons and polycyclic aromatic hydrocarbons can negatively affect their bioavailability,
sorption characteristics and accessibility to microbial attack [5,6].
Surface-active agents reduce the interfacial tension between hydrocarbons and surfactant solutions [7]
and increase mobility and surface area available for microbial cell contact with hydrocarbons [8]; these
factors may promote biodegradation. The use of surfactants was proposed to enhance bioremediation of
hydrocarbon contaminated sites [6,7,9,10]. Sodium dodecyl sulfate (SDS) has been described as an effective
anionic surfactant for removing hydrophobic contaminants from contaminated sand [I l] and has been
utilized in soil-washing techniques [12]. The effect of exogenous addition of surfactants on enhanced
bioavailability of hydrophobic compounds has been studied by several authors, and the results are
contradictory. Both negative and positive effects have been reported in the literature ranging from inhibition
of biodegradation [6] to no effect [2] to stimulation of biodegradation [7,9,13]. No information is available
about the effect of surfactants on oil biodegradation at low temperatures.
In case of exogenous surfactant addition, the stimulation of hydrocarbon biodegradation without
residual surfactant pollution is desirable. Alkyl sulfates such as SDS are readily broken down by bacteria and
constitute the most readily biodegradable surfactant in common use [14]. Biodegradation of SDS at low
temperatures by cold-adapted diesel oil degraders [IS] and by microbial populations from a polluted river
[ 161 was reported.
We wanted to know whether the addition of SDS will enhance hydrocarbon bioavailability at low
temperatures in both aqueous and terrestrial systems. It was &ther of interest to know if and to what extent
the time course of oil biodegradation is influenced in the presence of SDS. Therefore we investigated the
effect of SDS at different concentrations on biodegradation of diesel oil in liquid culture and in an alpine
soil. We measured biodegradation of both compounds during 32 days at 10°C to simulate environmental
conditions. A comparable temperature prevails in ground and surface water, in non-heated underground
water treatment plants, in subsoils of temperate climates and in alpine soils where temperatures above
8-1O'Care reached only during high solar irradiation [ 171.
2. MATERIALS
AND METHODS
2.1. Mineral medium
A phosphate-buffered pa-neutral
mineral medium [ 181, containing filter-sterilized SDS (dodecyl sulfate
sodium salt tryst., research grade, min. 99%) and/or diesel oil (85.7% C, density 820 g l-1, added as a
liquid) as the sources of carbon and energy was used.
3465
2.2. Microorganisms
The investigated psychrotrophic inoculum RM8/11 was isolated from a high-moor soil (8”C, pH 6.2) in the
Tyrolean Alps at 2000 m a.s.1. [ 191 and degraded both diesel oil and the anionic surfactant SDS efficiently at
10°C [20]. Growth in mineral medium containing SDS and/or diesel oil was visible at 0-30°C, diminished at
3YC and absent at 4O“C. The inoculum was a mixture of two bacteria that were assigned to the genera
Pseudomonas sp. and Arthrobucter sp. [20]. The inoculum was cultivated for 96 h at 10°C and 180 ‘pm in
mineral medium supplemented with SDS (500 mg 1-1)and diesel oil (5000 mg 1”).
2.3. Soil
The soil selected for this study was an uncontaminated subsoil (C-horizon) from Hahntennjoch in the
Tyrolean Alps at 1714 m a.s.1. Soil properties are described elsewhere in detail [18]; briefly, it was a
pH-neutral, carbonate-rich, nutrient-deficient, sandy soil.
2.4. Biodegradation studies in liquid culture
One hundred and forty IOO-ml Erlenmeyer flasks were prepared, each containing 20 ml of sterilized mineral
medium and 5000 mg diesel oil I-t. In order to test the effect of five SDS-concentrations, 28 flasks each
received 0, 50, 100, 500 or 1000 mg SDS 1-l. Thirty five flasks (7 flasks for each SDS concen-tration) were
not inoculated (sterile controls); the residual 105 flasks received 0.5 ml of inoculum.
The flasks were closed with cotton wool stoppers and incubated in the dark at 10°C and 180 rpm.
Water losses during incubation were compensated for regularly by the addition of sterile water. After 2, 4,
7, 10, 15, 21 and 32 days of incubation, three inoculated flasks and one control flask of each SDSconcentration were removed to measure the residual contents of diesel oil and SDS.
2.5. Biodegradation studies in soil
One hundred and forty 100~ml Erlenmeyer flasks were prepared, each containing 10 g of soil, 5000 mg
diesel oil kg-* soil dry weight (dw) and a water-soluble inorganic fertilizer at a C:N:P ratio of 100: 10:2 [ 171.
To test the effect of five SDS-concentrations, 28 flasks each received 0, 50, 100, 500 or 1000 mg SDS kg-l
soil dw. To determine abiotic losses, poisoned controls (35 flasks; 7 flasks for each SDS concentration)
received AgNO3 at a final concentration of 0.3% (w/w) [ 181. The water content was adjusted with sterile
water to 50-60% of the soil’s maximum water-holding capacity in all flasks.
The flasks were closed with cotton wool stoppers and incubated in the dark at 10°C. To avoid
anaerobic conditions, the contents of the flasks were mixed thoroughly every second day. Water losses
during incubation were compensated for regularly by the addition of sterile water. After 2, 4, 7, 10, 15, 21
and 32 days of incubation, three non-poisoned flasks and one control flask of each SDS-concentration were
removed to measure the residual contents of diesel oil and SDS.
2.6. Hydrocarbon content
The residual total petroleum hydrocarbon concentration was measured in liquid culture and in soil according
to the German standard method [21] after extraction with 1,1,2-trichloro-trifluoro-ethane
by infrared spectroscopy as described [ 181.
and quantification
3466
2.7. SDS content
SDS in liquid culture was quantified using a modification [ 151 of the methylene-blue-active-substances
assay
for anionic surfactants of Hayashi [22]. SDS in soil was extracted by shaking 10 g soil with 40 ml of
distilled water for 30 min at 150 rpm and room temperature. After 5 min of settling down, 1.5 ml of the
“clear” phase was centrifuged; SDS quantification was performed in the clear supematant as described
3. RESULTS
Both diesel oil and SDS could be recovered completely from the mineral medium and from soil immediately
after their addition. Statistical analysis of variance (95% confidence level) showed that the presence of SDS
did not influence efIiciency of hydrocarbon extraction.
3.1. Biodegradation
in liquid culture
3.1.1. SDS
Independent of the SDS concentration, no abiotic elimination of SDS was observed in sterile mineral
medium. The inoculum degraded all SDS concentrations tested in presence of diesel oil at 1O“C: 50 and
100 mg SDS l-1 were fUly degraded after 4 and 7 days, respectively; 500 and 1000 mg SDS l-1 were fully
degraded after 15 days (Fig. 1).
1000
800
L
600
E
$
400
SDS concentration
0
5
10
15
Time
(mg I-‘)
A1000
~500
0100
20
25
30
*50
35
(days]
Fig. 1: Biodegradation of SDS in presence of diesel oil (5000 mg 1-l) in liquid culture by a psychrotrophic
inoculum at 1O’C. Data are means of replicates (n=3) f standard deviation (n-l).
3467
3.1.2. Diesel oil
In the absence of SDS, 15% of the initial diesel oil contamination were lost in sterile mineral medium after
32 days at 10°C. This loss could be attributed to abiotic processes such as chemical transformations,
sorption mechanisms and evaporation of volatile compounds [19]. In inoculated flasks, total hydrocarbon
loss in the absence of SDS was 58%, thus 43% of the hydrocarbon loss could be attributed to degradation
by the inoculum (Fig. 2). Biodegradation of diesel oil was highest during the first lo-15 days; thereafter no
Ii&her hydrocarbon loss was detected.
The presence of SDS had no effect on hydrocarbon degradation by the inoculum during the first
15 days. Within the following days, the hydrocarbon content decreased markedly in inoculated flasks
containing 50 and 100 mg SDS l-l, although these favourable SDS concentrations were already filly
biodegraded afler 4-7 days. After 32 days, the residual hydrocarbon content in flasks without SDS was
2070 mg 1-l which was remarkably higher than in flasks with 50 and 100 mg SDS I-1 (residual hydrocarbon
contents of 960 and 850 mg l-1, respectively). On the other hand, higher concentrations of SDS inhibited
hydrocarbon biodegradation: residual hydrocarbon contents in flasks containing 500 and 1000 mg SDS 1-l
were 3360 and 3500 mg l-l, respectively (Fig. 2).
Abiotic hydrocarbon loss in sterile mineral medium was not influenced in the presence of 50-100 mg
SDS l-l, whereas higher SDS concentrations were able to bind more hydrocarbons and increased abiotic
losses significantly (Fig. 2).
5000
z
4000
‘-
E
3000
r
:
5 2000
0
_;;
f
1000
00
0
100
1000
500
SDS concentration
(mg I-‘)
Fig. 2: Effect of SDS on hydrocarbon loss (initial concentration 5000 mg diesel oil 1-l) in sterile mineral
medium and in mineral medium inoculated with a psychrotrophic inoculum after 32 days at 10°C. Data are
means of replicates (n=3) * standard deviation (n-l).
3468
3.2. Biodegradation
in soil
3.2.1. SDS
As already observed with sterile mineral medium, no abiotic loss of SDS was measured in poisoned soil
controls where microbial growth was completely excluded [ 181. Biodegradation of low SDS concentrations
by the indigenous soil microorganisms was comparable to that in liquid culture; 50 and 100 mg SDS kg-1
soil were degraded after 4 and 7 days, respectively. Higher SDS concentrations (500 and 1000 mg kg-l)
were already fidly degraded after 10 days in soil (Fig. 3), but only after 15 days in liquid culture (Fig. 1).
1000
800
z
0
”
,”
600
g
400
0
5
10
15
Time
Fig. 3: Biodegradation
20
25
30
35
(days)
of SDS in presence of diesel oil (5000 mg kg-1 soil dw) by indigenous soil
microorganisms at 10°C. Data are means of replicates (n=3) f standard deviation (n-1).
3.2.2. Diesel oil
In the absence of SDS, 19% of the initial diesel oil content was lost in poisoned soil controls after 32 days at
10°C. These data corresponded to previous observations. Abiotic hydrocarbon losses are higher in soil than
in aqueous solution because of more irreversible sorption mechanisms and interactions with soil colloids
[18]. Abiotic hydrocarbon
loss was not influenced in the presence of low SDS concentrations
(So-100 mg kg-1 soil), but higher SDS concentrations enhanced the release of hydrocarbons from soil
colloids and their bioavailability: the initial diesel oil contamination could be recovered almost completely
after 32 days. This is contrary to results obtained with sterile mineral medium where high SDS
concentrations bind more hydrocarbons.
Nevertheless, total hydrocarbon loss in soil was significantly inhibited in presence of SDS; the higher
the SDS concentration, the higher was the inhibition. Atter 32 days at lO”C, the residual hydrocarbon
content in the absence of SDS was 2155 mg kg-1 soil. The presence of 50, 100, 500 and 1000 mg SDS kg-1
3469
soil resulted in residual hydrocarbon contents of 2735, 2830, 3329 and 3843 mg kg-l soil, respectively
(Fig. 4). Hydrocarbon degradation by the indigenous soil microrganisms could be calculated from the
difference between hydrocarbon losses in poisoned and non-poisoned soil. While 38% biodegradation was
noticed in the absence of SDS, biodegradation in presence
SDS
concentration
DO
Al000
I
I
0
5
(mg
~500
kg-’
of SDS was only 20-30%.
soil)
0100
*50
1’5
2’0
Time
(days.1
I
10
2’5
3’0
3’5
Fig. 4: Effect of SDS on hydrocarbon loss (initial concentration 5000 mg kg-l soil dw) in an alpine soil at
1O’C. Data are means of replicates (~3) f standard deviation (n-l).
4. DISCUSSION
Especially in subsoils and in aqueous systems, biodegradation of contaminants may be limited by available
nutrients such as nitrogen and phosphorus [l-3]. Therefore nutrients have to be added to the nutrientdeficient environments. In the described experiments, the presence of SDS did not markedly affect the C:N
ratio which was 1O:l without SDS and 12.4:1 at the highest SDS concentration tested.
The biodegradability and toxicity of surfactants depend on the temperature which changes the effects of
surfactants on microorganisms [23]. All SDS concentrations tested in this study were ti~lly degraded within
IO-15 days at 10°C in liquid culture and in soil. No abiotic loss of SDS was observed. Adsorption of
surfactants plays a rather insignificant role compared to biodegradation [7]. Biodegradation of high SDS
concentrations was faster by the indigenous soil microorganisms than by the psychrotrophic inoculum in
liquid culture. This may be explained by the fact that the soil system contained more microorganisms
have rapidly adapted
reported
their metabolism
in a creosote-contaminated
degraded within 10 days at 20°C 161.
to the contamination.
Complete
mineralization
of SDS was
that
also
soil where 60% of the added SDS (10, 100 and 500 mg kg-‘) were
3470
Contrary to the complete removal of SDS, complete diesel oil decontamination was not achieved in
liquid culture or in soil. A content of ca. lo-30% of the initial oil pollution remains at 1O’C in soil [ 171 and
in liquid culture [ 18,191, even after a prolonged treatment. Similar values were reported at mesophilic
temperatures [24]. The residual fraction of diesel oil consists mainly of multiple condensed cycloaliphatics
P51.
We observed that the effect of SDS on diesel oil decontamination was completely different in liquid
culture and in soil; biodegradation and abiotic loss (the total hydrocarbon loss is a sum of these two factors)
were intluenced in both systems to a different extent.
Low concentrations of SDS (SO-100 mg I-1) stimulated hydrocarbon biodegradation in liquid culture
significantly, without affecting the abiotic hydrocarbon loss. Remarkably, this stimulation occurred only very
late when the SDS was already lily degraded and when hydrocarbon loss had already reached a “saturation
plateau”. Possibly SDS is able to enhance hydrocarbon bioavailability on a long-term-basis. Surfactants at
low concentrations (So-100 mg l-1) were found to be useful for bioremediation of sites contaminated with
sorbed polycyclic aromatic hydrocarbons such as phenanthrene and biphenyl; the extent of hydrocarbon
desorption and mineralization was enhanced in nutrient-amended aquifer sand and in soil slurries [9].
On the other hand, we observed that hydrocarbon loss in soil was inhibited in presence of SDS: the
higher the SDS concentration, the higher was the inhibition. More interaction mechanisms in soil than in
liquid culture and/or the accumulation of inhibiting metabolites in the course of SDS biodegradation might
be responsible. Similar results were described [6]: the addition of 100 and 500 mg SDS kg-1 soil slowed the
biodegradation of three-ring polycyclic aromatic compounds and significantly inhibited biodegradation of
four-ring aromatic compounds in a weathered contaminated soil. The preferential utilization of surfactants
by degraders of high-molecular-mass compounds was assumed to be responsible for the inhibition of
hydrocarbon biodegradation [6]. The presence of anionic surfactants enhanced kerosene biodegradation in a
soil-water system at 2O”C, but the rate of biodegradation of surfactant and kerosene was higher in clean
water system [7].
Various explanations have been proposed for the inhibitory effects of surfactants on biodegradation
[ 10,141. One proposed effect is the partitioning of hydrophobic substances into surfactant micelles with the
assumption that microorganisms do not have direct access to compounds inside micelles and that the mass
of hydrophobic substances exiting micelles limits biodegradation [14]. An overdosing of surfactants could
have a negative effect on the biodegradation
rate because of a resulting decline in aqueous-phase
hydrocarbon concentration; the amount of surfactant at which such inhibition would occur depends on the
specific system [lo]. There may also be possible substrate competition between surfactant and contaminant,
and surfactants could be toxic to the soil microorganisms [6]. In this study, respirometric measurements
(data not shown) showed that the presence of SDS at all concentrations tested did not adversely affect the
microbial activity in soil and in liquid culture.
Our experiments have demonstrated that low concentrations of SDS enhanced significantly bioavailability and consequently biodegradation of diesel oil at low temperatures in an aqueous system, without
residual surfactant pollution. However, the application of SDS in soil was not successful. Thus, the
suitability of surfactants has to be tested for each system. The results of our study could be of particular
importance for in-situ bioremediation precesses in cold temperature environments,
Microbial degraders of both mineral oil hydrocarbons and anionic stufactants at low temperatures are of
considerable biotechnological interest for an improved low-temperature bioremediation of oil contaminated
3471
waters. The inoculum investigated in this study is able to degrade both high concentrations of SDS [ 151 and
diesel oil (unpublished results) over a broad temperature range (4-3O’C) in an aqueous system, this is of
special importance for in-situ applications when considering environmental temperature fluctuations. The
usefulness of such microorganisms for the low-temperature biological decontamination of waste waters
from garages and car-washs where anionic surfactants are extensively used to clean oil contaminated
vehicles, oil tanks etc. has already been demonstrated [20].
5. ACKNOWLEDGEMENTS
This study was supported by the Austrian Federal Ministry of Science and Traffic, we like to express our
special thanks to Dr. C. Fialla. We thank P. Thurnbichler for technical assistance.
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