Colloids and Surfaces

A: Physicochemical and Engineering Aspects 162 (2000) 1 – 14

www.elsevier.nl/locate/colsurfa

Effect of additives on biodegradation of PAH in soils

T. Sobisch c,*, H. Heß a, H. Niebelschütz b, U. Schmidt a
a
AERES Angewandte Umweltforschung GmbH, Rudower Chaussee 29 (OWZ), 12484 Berlin, Germany
b
ARGUS Umweltbiotechnologie GmbH, Reuchlinstraße 11 – 13, 10553 Berlin, Germany
c
L.U.M. Gesellschaft für Labor-, Umweltdiagnostik & Medizintechnik mbH, Rudower Chaussee 5, 12489 Berlin, Germany

Received 22 September 1997; accepted 27 April 1999

Abstract

In a series of experiments with soil samples of contaminated sites we investigated the effect of special surfactant
combinations and other additives on the kinetics and extent of solubilization and degradation of PAH. Biodegrada-
tion tests showed a positive effect of adding surfactant combinations T10 and T15. The effect was more pronounced
when samples exhibited low degradation effectivity without surfactants. Addition of surfactants counterbalanced the
dependence of bioavailability of the individual PAH compounds on their solubility in pure water. Changing the
composition of surfactant combinations different tendencies were observed for the extent of solubilization and
degradation, i.e. very high concentrations of PAH in the liquid phase may have toxic effects on the soil microflora.
However, addition of a sorbent had the most remarkable effect on residual pyrene concentration in a sandy soil,
whereby the contamination was transferred to the sorbent phase. Sorption to the sorptive phase and solubilization are
substantially faster processes than degradation. © 2000 Elsevier Science B.V. All rights reserved.

Keywords: PAH; Pyrene; Biodegradation; Solubilization; Soil clean-up; Surfactant combinations

1. Introduction improve bioavailability seems a viable option but

Bioremediation, often a very economical option
for soil decontamination, has restricted applicabil-
ity for soils contaminated with polycyclic aro-
matic hydrocarbons (PAH’s).
Biodegradation of PAH’s in soil – water systems
of contaminated sites has been shown to be lim-
ited by certain factors. Addition of surfactants to
* Corresponding author. Fax: +49-306-7198189.
E-mail address: helpdesk@lum.fta-berlin.de (T. Sobisch)

This article was originally submitted to the IAP ’97 Spe-
cial Issue.
has been conversely discussed in literature [1,2].
In previous investigations we identified special
surfactant combinations which enhanced the bio-
logical activity (respiration rates) in tar–oil–water
systems without preferential degradation of sur-
factants added [3].
To evaluate the applicability of these effects for
remediation of contaminated soils and to support
commercialization we investigated the effect of
different surfactant combinations of this type on
the kinetics and extent of solubilization and
degradation in a series of experiments with soil
samples of contaminated sites.

0927-7757/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.
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2 T. Sobisch et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 162 (2000) 1–14
2. Experimental
2.1. Soil samples
All soil samples, except sample A (fraction of
soil fines from a soil washing plant), were homog-
enized to avoid ill reproducibility of the initial
composition by passing through a 4 mm screen
and by mixing.
Sample A: fraction of soil fines (B 63 mm)
containing cationic flocculants from a soil wash-
ing plant with an initial PAH concentration of 88
mg/kg (16 individual PAH compounds according
to the EPA 610 priority pollutant list).
Sample B: sandy soil of a former gaswork site
with an initial PAH concentration of 646 mg/kg.
Sample C: sandy soil of a former gaswork site
with an initial PAH concentration of 1086 mg/kg,
having a lower content of 5 and 6 ring com-
pounds than sample B. Soil graines had a film of
mineral oils (total petroleum hydrocarbons ac-
cording to DEV H-18: 1000 mg/kg) on their
surface.
Sample D: subsample of C with an initial
pyrene concentration of 417 mg/kg. After sapon-
ification (see below) an additional concentration
of 3 mg/kg pyrene was determined, where from it
is supposed that the fraction of PAH fixed in the
organic soil matrix is low.
Sample D was further characterized by wet-
sieving into size fractions 4 – 2 mm, 2 mm–63 mm,
63–20 mm after mechanical disintegration in a
laboratory attrition cell (30 g soil, 30 g water, 10
min at 80 rpm). The dispersion of soil particlesB
20 mm was centrifuged at 3600 rpm for 5 min. The
size fractions had pyrene concentrations and dis-
tribution summarized in Table 1.
Table 1
Distribution of size fractions and pyrene contamination in
sample D
Size fraction Wt% Pyrene (mg/kg)
4–2 mm 1.7 360
2–0.063 mm 93.8 230
63–20 mm 3.4 2020
B20 mm 1.1 1440
Calculating the overall pyrene concentration
from the four fractions results in 306 mg/kg
pyrene. The difference between this and the initial
value seems to be due to the removal of PAH
with the wash water, mainly along with dispersed
oil (resulting from the initial oil film on the parti-
cles).
2.2. Soil microcosms
Results of soil microcosm studies are averaged
from two or three sets run in parallel. Deviations
were normally in the range of 5%.
Procedure A: The extent of the removal of
PAH was investigated in soil suspensions (20 g
soil–180 ml mineral salt solution). The mineral
salt solution (pH: 6.8) was prepared by adding 1 g
(NH4)2SO4, 0.3 g NaH2PO4, 0.5 g KH2PO4 and
0.05 g MgSO4 to 1 l of tapwater. The nonsteril-
ized soil samples were amended with different
surfactant combination. After shaking for 10 days
at a frequency of 125/min the soil was removed by
filtration and air dried. PAH’s were analyzed by
HPLC according to EPA 610.
Procedure B: The extent and kinetics of solubi-
lization and removal of pyrene from sample D
was investigated in soil slurries (10 or 20 ml
solution/5 g soil). Nonsterilized soil samples were
amended with nutrients (per g of soil: 2.38 mg
(NH4)2SO4; 1.67 mg NaH2PO4; 0.425 mg
KH2PO4; 1.09 mg K2HPO4) and different addi-
tives. Tap water was used. After shaking for a
distinct period of time at a frequency of 150/min
the soil was removed by centrifugation for 15 min
at 4500 rpm, air dried, extracted with methanol in
an ultrasonic bath at 40°C for 1 h. Extracts were
centrifuged at 3500 rpm for 5 min. Optionally the
extracted soil was rinsed with methanol, again
separated by centrifugation and treated with 80
ml boiling aqueous methanolic KOH (2N) to have
a measure for the fraction of pyrene not available
by simple extraction. After slurry tests pyrene
concentration in the liquid and solid phase was
determined by UV-derivative spectrometry [4].
The extent of PAH sorption to the container
walls was measured by equilibration with
methanol after removal of soil and aqueous
phase.
 T. Sobisch et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 162 (2000) 1–14
2.3. Additi6es
Surfactant combinations: T10, T15, T16, T35
and T36 are each mixtures of a nonionic hy-
drophilic and a nonionic hydrophobic component.
Combinations had low water solubility but could
be easily dispersed in water. The components are
biodegradable and are available at the market at
reasonable prices. Different ratios between hy-
drophilic and hydrophobic compounds have been
used, marked with 1,/2,/3 in order of increasing
content of the hydrophobic compound, i.e. T10/1,
T10/2 and T10/3.
Other additives: polymeric compounds and sor-
bents (activated carbon, wood, S1) were also tested.
All additives (incl. surfactant combinations)
mentioned in this paper are available from L.U.M.
GmbH, Berlin.
3. Results and discussion
3.1. Relation between reduction of PAH
concentrations measured and biodegradation
In this paper microcosm studies are only investi-
gated by the variation of PAH concentrations in
soil and solution. Besides biodegradation of PAH
there are several biotic and abiotic factors which
may contribute to the variations measured.
In principle the following processes may play a
role in the reduction of the initial PAH content, i.e.
sorption to container walls, volatilization, photo-
chemical degradation, oxidation and physical,
chemical or biological bound residue formation.
However, PAH initially not available for extraction
may be released by physical, chemical and biolog-
ical processes too.
Sorption of PAH to container walls during the
microcosm studies varied between 0.5 and 3% of
the initial PAH content and was taken into account
by a correction.
Volatilization should only have a measurable
effect for two and three ring PAH, but will be
relatively small during 1 or 2 weeks. Variations
caused by photochemical and oxidative degrada-
tion are expected to be of minor importance in case
of the present investigations.
However, ‘bound residue formation’ or even
‘bound residue destruction’ could interfere seri-
3
ously with experimental results. Only a fraction of
the overall PAH content in contaminated soils is
available by solvent extraction. This fraction can be
increased by solvent extraction with additional
saponification, however, the fraction of non-ex-
tractable PAH may amount up to more than 60%
[5]. On the other hand, bound residues or non-ex-
tractable PAH are connected with soil organic
matter and clays, but the content of organic matter
and clays is very low for soil samples B, C and D.
The low percentage of pyrene additionally ex-
tractable after saponification may indicate that the
overall content of non-extractables is also low. In
the case of soil fines (sample A) no distinction
between ‘real’ biodegradation and bound residue
formation can be made.
3.2. Effect of special surfactant combinations on
slurry biodegradation tests with different
contaminated soils
Fig. 1 illustrates some typical results of biodegra-
dation tests in suspension (procedure A). The
variation of the content of selected PAH (phenan-
threne as three ring, pyrene as four ring, ben-
zo(a)pyrene as five ring, benzo(g,h,i)perylene as six
ring compound and the total of the 16 EPA
compounds) before and after biological action (10
days) demonstrates a positive effect of adding
T10/1 (1%) to different soils (sample A: fraction of
soil fines from a soil washing plant with relatively
low contamination, sample B and C: both sandy
soil of a former gaswork site, sample B with a
higher content of five and six ring compounds).
The soil microflora, when amended only with the
mineral salt medium, exhibited no PAH-degrading
activity in the case of soil fines (sample A), had only
a moderate effect on the PAH content of sample
B but reduced the amount of lower molecular
weight PAH compounds substantially in the case
of sample C.
The addition of T10/1 to sample A, having an
overall low bioavailability of PAH, caused a reduc-
tion of the 16 EPA compounds by 36%. The PAH
content of sample B was reduced by 44% and of
sample C by 90%.
Addition of surfactants may be especially useful
if the entire degradation effectivity of the soil
4 T. Sobisch et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 162 (2000) 1–14

Fig. 1. Effect of surfactant addition on slurry degradation tests (10 days).

 T. Sobisch et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 162 (2000) 1–14 5

Fig. 2. Removal of individual PAH compounds as function of their original aqueous solubilities.
6 T. Sobisch et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 162 (2000) 1–14
Fig. 3. Effect of composition of surfactant combinations on solubilization and removal of pyrene (sample D, 5 g/20 ml, 2%
surfactant per soil).
microflora is low, as in the case of sample A. As
could be expected with sample C, in the presence
of mineral oils with appropriate bioavailability,
surface active bioproducts may be released (see
below), which would have a positive effect on the
physico-chemical and biological removal of PAH
without addition of technical surfactants.
Bioavailability of individual PAH compounds is
related to their solubility in the aqueous phase. This
trend is also evident for the degradation tests with
no surfactant added (Fig. 2). The percentage of the
individual compounds remaining in soil after treat-
ment is depicted against their aqueous solubility
(S). The literature data for the solubilities are
compiled from refs. [6,7]. Unreasonably high values
were excluded, which was also the case for the only
available data for indeno(1,2,3-ed)pyrene (0.062
mg/l [7]). The addition of 1% T10/1 or T15/1 to the
soil caused the fraction of the individual com-
pounds remaining in soil to be nearly independent
on their original aqueous solubilities for sample B.
In the case of sample C with higher biological
activity the dependence on the solubility values is
more pronounced. The addition of T10/1 and
T15/1 weakens this tendency.
The solubilizing action of the surfactant combi-
nations used is not solubilization in terms of
‘preparation of thermodynamically stable isotropic
solutions of substances otherwise only slightly
soluble’ [8], i.e. micellar solubilization. Because of
these surfactant combinations are only dispersed in
the water phase, the PAH will partition between the
soil and the dispersed surfactant phase. Therefore,
the critical volume necessary for solubilization of
PAH of high molecular weight [9] may not be a
limiting factor, as in the case of micellar solubiliza-
tion. It is obvious from Fig. 2 that addition of 1%
T15/1 caused a reduced removal of PAH, whereas
addition of 0.1% was almost as effective as the
amendment with 1% T10/1.
3.3. Effect of surfactant composition on the
extent of solubilization and degradation of pyrene
Fig. 3 shows the influence of the composition of
surfactant combinations on solubilization and
degradation for sample D, a subsample of sample
C with an initial pyrene concentration of 417
mg/kg. The residual pyrene concentration in soil
and in the water phase was analyzed after one week
shaking of the soil suspension (5 g/20 ml) amended
 T. Sobisch et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 162 (2000) 1–14
Fig. 4. Effect of surfactant concentration on solubilization and removal of pyrene (sample D, 7 days).
with nutrients and 2% surfactant per soil. Chang-
ing the quantity of the hydrophobic compound
between 37 and 70% only a slight variation in
residual and solubilized amounts of pyrene was
observable. With T15, were only the hydrophobic
component in T10 was changed (same hydrophilic
compound), the solubilizing power decreased by
more than 50%. Replacing the hydrophilic compo-
nent in T10 and T15 by a slightly more hydrophilic
surfactant, which differed only in the degree of
ethoxylation (HLB: 0.5), solubilization was en-
hanced (T10“ T36) or slightly reduced (T15“
T35). On the other hand, if the hydrophilic
compound in T10 was substituted by a surfactant
of the same HLB value ( “ T16), the degree of
solubilization was reduced by an order of magni-
tude.
Therefrom it is evident that the structure of the
hydrophilic compound has a marked influence on
the solubilization effect of the formulation.
Interestingly, the extent of biodegradation, de-
duced from the total of pyrene determined in soil
and solution, is the highest for the surfactant
combinations with low solubilizing power and vice
versa. This may be caused by the very high concen-
tration of PAH in the liquid phase, which may
have had a toxic or inhibiting effect on the soil
microflora.
7
During the slurry tests the fraction of pyrene
extractable after additional saponification re-
mained on the low initial level. Hence biological
fixation of PAH might not have been occurred.
3.4. Influence of surfactant concentration on
solubilization and residual concentration of pyrene
The degree of solubilization and concentration
of pyrene in soil (sample D) after one week shaking
was measured as function of concentration of
T15/2 (Fig. 4).
As could be expected, the degree of solubiliza-
tion is increased with increasing surfactant concen-
tration. On the other hand the total residual pyrene
content in the soil slurry (soil+ liquid phase) is
lowest at a surfactant concentration of 0.5%.
Lower surfactant concentrations (0.1–0.3%) ex-
hibited a negative effect on residual concentration.
Increasing the soil–water ratio to 5 g/10 ml the
degree of degradation as well as the fraction of
pyrene solubilized from soil is enhanced. Enhanced
solubilization corresponds to higher ratios of PAH
in the liquid phase per surfactant added at
concentrations of 0.5% T15/2. This might be due
to the formation of active bioproducts (biosurfac-
tants or biopolymers) released by the soil mi-
croflora, maybe preferentially during degradation
8 T. Sobisch et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 162 (2000) 1–14
Fig. 5. Effect of different additives on solubilization and removal of pyrene (sample D, 5 g/10 ml, 7 days).
of the mineral oil contaminants. Release of such
bioproducts is reflected by increased foaming,
which had been observed after a distinct period of
shaking. It seems that dilution of the soil microflora
has an adverse effect on its overall activity, i.e.
biodegradation and production of active metabo-
lites. Another indication of the production of active
metabolites is the enhanced solubility of pyrene in
the soil slurries amended only with nutrients. In the
case of 5 g soil/20 ml the pyrene concentration in
the liquid phase reaches a value of 0.18 mg/l after
one week, which is only slightly above the value for
its solubility in pure water (0.13 mg/l), but a value
of 3.1 mg/l for 5 g soil/10 ml. After 2 weeks the
concentration of pyrene increases to a value of 0.9
mg/l (5 g soil/20 ml). Enhanced mutual attrition of
soil grains may play also an important role in
increasing the extent of solubilization if (technical
or biological) surface active substances are present
to stabilize mineral oils in suspension which will
carry PAH into the liquid phase.
3.5. Screening of effecti6ity of different additi6es
The performance of different surfactant combi-
nations and other additives (polymeric compounds
and sorbents) was further tested under standard
conditions, i.e. shaking over a period of 1 week, 5
g soil (sample D)/10 ml. Selected results are com-
piled in Fig. 5.
Compared with the sample not amended with
additives (only with nutrients) addition of various
surfactant combinations caused only a further
reduction of the residual pyrene concentration in
soil of about 10% (5 g/10 ml values). In the case
of the formulation with high solubilization power
T36/2 this could be reduced further by 10%, but
degradation was completely inhibited. Addition of
0.1% of the polymeric compound Z18 reduced the
amount of pyrene remaining in soil to 58% of the
initial value. When 0.1% Z18 and 0.5% T15/2 were
added simultaneously a reduction to 52% was
reached. In these cases the relatively high concen-
trations of pyrene in the liquid phase did not
inhibited degradation.
 T. Sobisch et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 162 (2000) 1–14

Fig. 6. Effect of different additives on the kinetics of pyrene removal (sample D, 5 g/10 ml).
9
10 T. Sobisch et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 162 (2000) 1–14

Fig. 7A. Kinetics of pyrene removal from sample D (without additives).

T. Sobisch et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 162 (2000) 1–14 11

Fig. 7B. Kinetics of pyrene removal from sample D (0.5% T15/2).

12 T. Sobisch et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 162 (2000) 1–14

Fig. 7C. Kinetics of pyrene removal from sample D (0.1% Z18).

T. Sobisch et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 162 (2000) 1–14 13

Fig. 7D. Kinetics of pyrene removal from sample D (5% SI + 0.1% Z18).
14 T. Sobisch et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 162 (2000) 1–14
Addition of 5% of the sorbent S1 reduced con-
tamination of soil substantially. S1 was far more
effective than other sorbents tested, i.e. activated
carbon or wood. Without further additives
present pyrene content of soil was lowered to
44%, when 41% of the initial amount of pyrene
was concentrated in the sorbent phase. With addi-
tional 0.5% T15/2 or 0.1% Z18 these levels
reached 36/44% (T15/2) and 34/43% (Z18).
3.6. Kinetics of pyrene remo6al and solubilization
Fig. 6 shows the kinetics of pyrene removal
from sample D. Figs. 7A, 7B, 7C and 7D summa-
rize the variation of the pyrene fraction in soil and
the liquid phase or bound to the sorbent.
In the presence of 5% of sorbent S1 + 0.1% Z18
a fast removal of pyrene was observed. After a
few days the residual pyrene concentration ap-
proached 34% of the initial value. Correspond-
ingly, 43% of pyrene was concentrated in S1 (Fig.
7D). In all other cases the pyrene concentration
reduced only slowly with only a moderate positive
effect of adding 0.5% T15/2 or 0.1% Z18.
The fraction of pyrene solubilized approaches a
maximum during the first week of shaking, re-
maining nearly constant after a rapid decline.
Sorption to the sorbent and solubilization are
substantially faster processes than degradation.
Degradation, measured as difference between
initial values of pyrene and the total amount
determined in the soil slurries, is small in all cases
after 3–5 weeks.
4. Conclusions
Biodegradation tests showed a positive effect of
adding surfactant combinations T10 and T15. The
effect was more pronounced when samples exhib-
ited low degradation effectivity without surfac-
tants. Addition of surfactants counterbalanced the
dependence of bioavailability of the individual
PAH compounds on their solubility in pure water.
By variation of composition and concentration
of surfactant combinations different tendencies
were observed for the extent of solubilization and
degradation, i.e. very high concentrations of PAH
in the liquid phase may have toxic effects on the
soil microflora.
Addition of the sorbent S1 had the most re-
markable effect on residual pyrene concentration
in a sandy soil, whereby the contamination was
transferred to the sorbent phase. Sorption to the
sorbent and solubilization are substantially faster
processes than degradation.
However, under no circumstances removal rates
and residual concentrations were in the order of
values which would be necessary for a competitive
decontamination process. Therefore a new two
stage bioreactor process was developed (patent
pending, described elsewhere [10,11]), which en-
hances removal rates drastically.
Acknowledgements
We gratefully acknowledge the support of the
Bundesministerium für Bildung, Forschung und
Technologie under grant number FKV 0211001J6
and FKV 0150402J6.
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