i~mwwio~rui Bi~~~kre~k~~ntiorr & Bi,~rl~~~~o~krri~~n.Vol. 40. No.

2 4 ( I YY 7) IO I IO6
1 1997 Elsevier Science Limited
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ELSEVIER

Effect of Organic Matter and Clays on the

Biodegradation of Phenanthrene in Soils

J. J. Ortega-Calvo, M. Lahlou & C. Saiz-Jimenez

Imtituto de Rrrw.w.s Nuturdes J Agrohiologiu de Sevillu. Cormjo Superior de Investigaciones Cicnt(fic,us. Apartado 1052,
4/080-Scvillu, Spin

Soil organic matter and clays represent an important hinderance for

bioremediation technologies, as they may cause a retardation in the biological
removal of hydrophobic pollutants. To overcome this limitation it is necessary to
understand how these two soil constituents influence biodegradation. The results
herein reported suggest that the bioavailability of phenanthrene in soils is
affected not only by sorption to the organic matter or clays. but also by the
interaction of phenanthrene with humic fractions4ay complexes.
Los coloides del suelo representan un importante inconveniente para las
tecnologias de biorecuperacion. ya que retrasan la elimination biologica de 10s
contaminantes hidrofobos. Los resultados obtenidos sugieren que la
biodisponibilidad del fenantreno en suelos se ve afectada mo solo por la
adsorcion a la materia orginica o ascillas, sino tambien por la interaction entre
fenantreno y el complejo arcillo-hlmico. icl 1998 Elsevier Science Ltd. All rights
reserved

INTRODUCTION the humic and non-humic fractions on the

sorption of organic compounds has been studied
Polycyclic aromatic hydrocarbons (PAHs) are previously (Chiou et al., 1986; Amy et al., 1991).
environmental pollutants of widespread The affinity of hydrophobic organic compounds
occurrence in soils. One of the most important to soil clays is generally considered of less
ways of removal of such pollutants is degradation importance due to the latter’s hydrophilic surface.
by soil microorganisms, whose activity can be However, the contribution of clay surfaces to
enhanced through bioremediation technologies. hydrophobic sorption in soils can be significant
However, microbial degradation of PAHs in soil when the OM content is below 68% (Hassett and
is limited by different factors that, in practice, Banwart, 1989).
often cause bioremediation not to be as efficient There are studies on the binding of some organic
as expected. One of these factors is bioavailability, compounds to humic acids (HA) and the influence
which has been pointed out as a research priority on their biodegradation (Amador and Alexander,
in bioremediation (Alexander, 199 1). Biological 1988; Amador et al., 1989; Robinson and Novak,
soil cleaning often results in residual 1994). Clay may also affect the biodegradation of
concentrations (still above legal standards) of organic pollutants (Cracker et al., 1995;
chemicals that are not accessible to Magdaliniuk et al., 1995; Miller and Alexander,
microorganisms. One of the most important 1991; Knaebel et al., 1994). However, fulvic acids
residual fractions is constituted by pollutants (FA), HA and clays often coexist in soil not as
sorbed to soil colloids, organic matter (OM) and separate components, but forming complexes.
clays having high sorptive properties. Although their interactions have been studied in
Previous studies have shown that soil organic detail (Cornejo and Hermosin, 1996) there is very
matter (SOM) is involved in the sorption of little information on how OM and clay
hydrophobic organic pollutants in water-saturated interactions affect the biodegradation of
soils, and a strong correlation exists between the hydrophobic organic pollutants. Hence, a study
extent of sorption and its content (Chiou, 1989). was conducted to compare rates of biodegradation
The influence of the source and composition of of phenanthrene in soils with different physico-

101
102 J. J. Ortega-Calvo et al

chemical properties, and to assess the

biodegradability of phenanthrene sorbed to soil
components.

MATERIALS AND METHODS

Five soils from continental ecosystems of central

Spain were selected on the basis of their content in
organic matter and clays (Table 1). Soil samples,
collected from the A horizon, were air-dried and
sieved to pass a 2-mm screen. To measure
phenanthrene mineralization, duplicate 25 g-
samples of soil were placed in 250-ml Erlenmeyer
flasks, and sterile, distilled water was added to
yield a moisture level of approximately 70% of
the field capacity of the soils. The samples were
supplemented with approximately 80 000 dpm of
[9J4C] phenanthrene (8.3 mCi mM_‘;
radiochemical purity > 98%; Sigma, Deisenhofen,
Germany) and enough unlabelled compound to
give 1 mg kg-i soil. The substrate was added in a
dichloromethane solution, which was allowed to
evaporate. The flasks, maintained at laboratory
temperature (20-22°C) were closed with teflon-
lined stoppers to prevent drying out of the
samples during the measurements, and 14C02
production was measured as radioactivity
appearing in an alkali trap suspended from the
stopper (Ortega-Calvo et al., 1995). Radioactivity
was measured with a Beckman model LS5000TD
liquid scintillation counter. Results are given as
means of duplicate measurements. An F-test was
used to analyse statistically the difference between
means at p = 0.05.
The sorption of phenanthrene to soil particles
was determined by the batch method. Briefly,
duplicated 1 g samples of soil were introduced in
10ml screw-cap glass tubes, and 10ml of distilled
water was added. Labelled and unlabelled
phenanthrene were added to give a final
concentration of 1 pgg-’ soil, and the soil
suspensions were equilibrated overnight on an
orbital shaker at 150g. After centrifugation,
dissolved phenanthrene was determined in the
supernatant by liquid scintillation counting.
Colour in the supernatant did not interfere with
the measurements.
Experiments with inoculated soils were
performed with a phenanthrene-degrading
bacterium isolated from a Cambisol soil from
Guadalajara, Spain (soil no. 3 in Table 1).
Isolation was performed through the enrichment
 Biodegradation qf’phenanthrenr in .roils 103

procedure. using liquid medium with mineral salts 1 jlgrnll’. The suspensions were again equilibrated
and phenanthrene as the sole source of carbon overnight, and centrifuged. Clay suspensions were
and energy (Efroymson and Alexander, 1994). The treated in the same way. After centrifugation, the
bacterium was characterized by standard supernatant was used to measure the non-sorbed
microbiological methods (Holt ef al., 1994). The fraction of phenanthrene, and then discarded. The
phenanthrene-utilizing isolate formed opaque, pellet, containing the sorbed phenanthrene, was
mucous, yellowish colonies of regular shape when mixed thoroughly with the soil. This was
grown on TSA plates. This bacterium is a slightly performed first inside the tubes by mixing the
curved rod. arranged in short chains. The pellet and portions of the soil samples using a
bacterium was Gram-negative, oxidase-positive, glass stick, and then mixing these portions with
and catalase positive. The bacterium was the rest of the soil samples inside 250ml flasks.
identified as Pseudomornas jluorescens and The soils were then inoculated and mineralization
designated strain Bl . It was able to use was measured as already described. The total
phenanthrene as a sole carbon and energy source amount of phenanthrene adsorbed, and therefore
for growth. The bacterium was maintained in added to the soil, was 3.76f0.5pg for clay alone
phenanthrene-containing liquid medium identical and 3.96fO. 15, 3.47f0.04 and 4.00f0.08pg for
to that used for enrichment culture. Inoculation of complexes containing, respectively, HA from the
soils with phenanthrene-conditioned bacteria was Typic Xerorthent soil, and FA and HA from the
performed as described previously, after Typic Xerochrept soil.
elimination of phenanthrene crystals from the
inoculum (Ortega-Calvo et al., 1995). The final
inoculum density used for the experiments was RESULTS AND DISCUSSION
3.5x lo6 cells”-’ soil.
Humic fractions were isolated from soils: HA In the five soils tested, phenanthrene was
and FA were extracted from a Typic Xerochrept mineralized to CO9 by the autochthonous
soil and HA from a Typic Xerorthent soil. A microbial population (Fig. 1). However, the
detailed description of methods and chemical kinetics of transformation was different according
characteristics of the humic fractions was to the content of OM and clay of the soils. Figure
published elsewhere (Saiz-Jimenez et al., 1986). la shows mineralization of phenanthrene in three
Sodium-montmorillonite from Crook County, soils with a marked difference in OM content (soil
WY (Source Clay Minerals Repository, University nos. l-3 from Table 1). In these soils the
of Missouri-Columbia, MO) was purified by maximum rates of mineralization were achieved
gravity settling in deionized water to remove after an acclimation period of several weeks. The
particles larger than 2pm. The clay was saturated subsequent rates and extents of mineralization
with 1 M CaCl, dialyzed to remove excess anions, were statistically lower in the soils with a higher
and lyophilized. OM content. Differences in phenanthrene
Sorption of humic fractions to clay was mineralization were also observed among soils
performed in 10 ml screw-cap, glass tubes into with similar OM, but differing in clay contents
which tom1 suspensions containing 5OOpg of (Fig. lb). Whereas in the soil with a lower clay
dissolved humic fractions and 100 mg of content the mineralization curve was S-shaped
montmorillonite were introduced. The caps were and included an acclimation period of several
lined with teflon to prevent losses due to weeks, mineralization in the clay-rich soils was
adsorption to the inner surface of the caps. After linear and occurred at statistically lower rates and
overnight equilibration at 21°C on a rotary shaker extents.
operating at 15Og, the non-sorbed humic fraction To investigate further the effect of OM and clays
was eliminated by centrifugation. In preliminary on the mineralization of phenanthrene, a series of
tests. equilibrated suspensions showed upon experiments was performed with soils inoculated
centrifugation a marked decrease in coloration of with the phenanthrene-degrading bacterium
the supernatant, suggesting that most of the isolated from soil. Mineralization of phenanthrene
humic fractions were adsorbed onto clay. The by this bacterium started without an acclimation
resulting pellet was resuspended in sterile distilled phase, both in liquid medium with phenanthrene
water, and labelled and unlabelled phenanthrene as the sole source of carbon and in the soils. The
were added to give a final concentration of mineralization rate in the soils was lower than in
104 J. J. Ortega-Calvo et al.

bioremediation companies (Piotrowski and

a 0.8%
Cunningham, 1996).

k
Phenanthrene is a highly hydrophobic
17.0%
compound, and this property causes its rapid
association to solid surfaces within the soil.
36.9% Assuming that soil microorganisms transform
compounds dissolved in water more easily than
those sorbed to matrices, removal of the substrate
from the aqueous phase due to sorption could
b 5.6%
cause a decrease in the rate and extent of its
microbial transformation. To assess whether the
differential biodegradability observed in our study
13.4% could be attributed to sorption to soil particles,
f
the sorptive properties of the five soils were tested
by determining the aqueous concentration of
phenanthrene in soil suspensions. The results are
0 20 40 60 80 100 120 included in Table 1, where the observed
DAYS
maximum rates and extents of mineralization by
Fig. 1. Mineralization of phenanthrene (1 mg kg-’ soil) by the autochthonous microorganisms are also
the autochthonous microbial flora in soils with different compared. Dissolved phenanthrene in the presence
organic matter (a) and clay contents (b). Contents are
expressed as percentages. of soil particles decreased with the SOM content.
The clay content seemed to have no influence. The
data agree with previous studies on the sorption
liquid culture (Fig. 2). A different phenanthrene
of hydrophobic compounds by soils in aqueous
biodegradability was also observed among the
systems (Chiou, 1989).
soils under these conditions. Higher OM content
The different mineralization data in soils 1, 2
of the soils led to a reduced mineralization. In this
and 3 may be explained in terms of sorption to
experiment, the long acclimation periods for the
SOM, since low dissolved phenanthrene
biological destruction of phenanthrene in non-
concentrations were related to low rates and
inoculated soils were eliminated by the addition of
extents of mineralization. These results are
a large number of degrading microorganisms, but
consistent with those obtained by Weissenfels rt
the data suggest that exogenous bacteria have
ul. (1992) who observed a strong sorption of
similar limitations to the autochthonous soil
PAHs, including phenanthrene, to SOM and
microbial population. This is remarkable since
concomitantly, a decrease in biodegradation. They
large-scale cultivation of degrading
found that in a contaminated soil from a coking
microorganisms, for their introduction into
plant, containing a high percentage of OM,
contaminated soils, is a common practice of
biodegradation did not occur even after addition
of nutrients and PAH-degrading bacteria.
However, PAH extraction and addition to the
extracted soil material led to a rapid
biodegradation by introduced bacteria. The
authors concluded that physical binding or
sorption of the PAHs to the soil was the cause for
the initial lack of mineralization. Manila1 and
Alexander ( 199 1) compared mineralization rates
of phenanthrene in four soils, and found a clear
5 reduction in a soil with high OM content, when
n compared to the other three soils with lower OM
- 0 5 10 15 20 25
DAYS
content. It was suggested that this reduction was
caused by sorption to SOM. However, sorption
Fig. 2. Mineralization of 25pg phenanthrene by a soil and mineralization data in the latter three soils,
Pseudomomas fluorescens in lOOmI of mineral medium
and in inoculated soils with different content in organic with similar contents of OM, did not correlate
matter. well. Sorption of other organic pollutants in the
 !05

soil system has also previously been shown to

affect microbial transformation negatively, such as
naphthalene (Guerin and Boyd, 1992) toluene
(Robinson rt al., 1990) styrene (Fu of al.. 1994)
and (2.4-dichlorophenoxy)-acetic acid (Ogram et
a/., 1985).
The differences in mineralization of
phenanthrene between soils 2, 4 and 5 (with
similar OM but different clay contents) cannot be
explained solely on the basis of sorption. taking
into account the phenanthrene concentrations
found in sorption experiments (Table 1). On the
other hand. clay seems not to be the sole factor
far reduced mineralization in these soils, given the
active biodegradation observed in soil no. 1, with
a clay content very close to soil no. 4. The data
suggest that a certain amount of SOM is needed 0 5 10 15 20 25
DAYS
for observation of this effect. These results can be
explained by the increase in tortuosity caused by a Fig. 3. Mineralization of phenanthrene in soils. initially
higher clay content. This concept has been sorbed to clay or Typic Xerorthent HA-clay complex (a), and
to complexes containing clay and FA or HA from a Typic
postulated by Alexander (1994) as affecting
Xerochrept soil (b).
bioavailability in particulated microbial
ecosystems (soil. subsoils and sediments). It refers
to the increase in diffusion paths of molecules due substrate carbon was mineralized when initially
to the presence of particles. The combination of present in the HA-clay complex. This suggests
sorption-desorption, diffusion and tortuosity may that the interaction between phenanthrene and the
affect the acquisition of biodegradable pollutants added particles is stronger when they contain HA.
by soil bacteria, which are fixed on the surface of This is remarkable because of the low proportion
particulate matter and depend on the flow of ( < 0.5% w/w) of HA in relation to the clay, which
nutrients through the soil structure. This increase did not affect the amount of phenanthrene
in tortuosity may have been the cause of the adsorbed in vitro (and therefore added to the soil)
reduced mineralization in soils no. 4 and 5, where but did affect its mineralization. This could be
the combination of OM and clay reduces the explained by a faster rate of desorption from the
accessibility of phenanthrene to the bacteria due clay than from the HAxlay complexes. The cause
to an increase in the surfaces where the compound for this difference would have been the limitations
can be transiently retained before its to intraparticle mass transfer caused by the
biodegradation. presence of HA. These synthetic complexes would
In other experiments, phenanthrene was added have functioned as the natural soil complexes,
to a soil with low OM - 0.8% (soil no. l), not as from which the rate of desorption of hydrophobic
dichloromethane solution but sorbed to clays or organic compounds is retarded by their microscale
to humic fractions-clay complexes. The complexes partitioning between intra-agreggate pore fluids
were formed by sorbing dissolved humic fractions and the solids making up the aggregate grains
onto clays. The colloids containing the sorbed (Wu and Gschwend, 1986).
substrate were homogenized with the soil, and The nature of the humic fraction present in the
then inoculated with the bacterium. The bacterium complex also affected the transformation. Figure
readily mineralized sorbed phenanthrene, 3b compares the biodegradation of phenanthrene
although differences were observed according to sorbed to a complex containing clay and HA or
the nature of the sorbent. Phenanthrene sorbed to FA from a Typic Xerochrept soil. Phenanthrene
the clay was more bioavailable and easily sorbed to the FAxlay complex was more easily
degraded than that sorbed to the HAslay degraded than that sorbed to the HA-clay
complex (Fig. 3a). More than 40% of the initial complex. FA are less hydrophobic than HA
carbon was converted to CO1 when the substrate (Piccolo, 1996) and therefore their interaction
was introduced with the clay alone, while 26% of with phenanthrene would have been weaker.
106 J. J. Ortega-Calve et al.

Although the influence of sorption to soil Fu. M.-H.. Mayton, H. & Alexander, M. (1994) Deso-
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Madison.
Holt, J. G., Krieg, N. R., Sneath, P. H. A., Staley, J. T. and
The authors greatly acknowledge Dr G. Williams, S. T. (1994) Bergeyk Manual of Determinative
Almendros, from the Centro de Ciencias Bacteriology. Williams and Wilkins, Baltimore.
Knaebel, D. B., Federle, T. W., McAvoy, D. C. & Vestal,
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