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Biodegradation Kinetics of Hydrocarbons in Soil during Land Treatment of

Oily Sludge
Smadar Admon; Michal Green; Yoram Avnimelech

Online publication date: 03 June 2010

To cite this Article Admon, Smadar , Green, Michal and Avnimelech, Yoram(2001) 'Biodegradation Kinetics of
Hydrocarbons in Soil during Land Treatment of Oily Sludge', Bioremediation Journal, 5: 3, 193 — 209
To link to this Article: DOI: 10.1080/20018891079285
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 Biodegradation Kinetics of Hydrocarbons in Soil
during Land Treatment of Oily Sludge

Smadar Admon,* Michal Green, and Yoram Avnimelech

Department of Agricultural Engineering, Technion, Haifa 32000, Israel

Abstract: This study focuses on the processes influencing hydrocarbon residue persistence in soil, following land
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treatment of refinery oily sludge. Treating sludge applied to soil resulted in 70% to 90% degradation of total
petroleum hydrocarbon (TPH) during 2 months, regardless of their initial concentrations (9 to 60 g/kg soil). Kinetic
analyses performed on TPH degradation, in laboratory and field systems, revealed a degradation pattern charac-
terized by two consecutive first-order kinetics reactions in all experimental settings. The first stage lasted about
3 weeks and was characterized by a temperature dependent rate constant of 0.047 day–1 at 24°C. That value was
comparable to the rate constant obtained when combining the individual rate constants of the saturated, aromatic,
asphaltene and polar fractions. The subsequent slower stage rate constant was 0.012 day–1, insensitive to tempera-
ture and to hydrocarbon composition. The transition between the two stages (about 21 days) was independent of
the experimental temperature and the biodegradation extent during the first stage. It was concluded that the extent
of residual accumulation in the soil was determined by the biodegradation efficiency during the first three weeks
of treatment when biological processes dominated. During the following period, abiotic processes leading to
reduced bioavailability of the TPH were limiting the degradation rate. Practically, as the first few weeks of
treatment determine its efficiency, efforts to enhance the biological activity should be directed to that period.

Introduction Chaîneau et al., 1996; Genouw et al., 1994). In these

Petroleum refineries generate large quantities of oily
sludge as a waste byproduct. The sludge, containing a
complex mixture of hydrocarbons, water, and sedi-
ments, is defined as hazardous. In Israel, at the Haifa
Oil Refinery, the 600 tons of oily sludge produced
annually have been stored for years in concrete im-
poundments. Therefore, a need arose for a practical
and effective solution for the safe treatment of the
waste.
Bioremediation by land treatment, which has been
practiced successfully for decades for the treatment of
petroleum-derived substances by the oil refining in-
dustry (API, 1983; API, 1984; Sims and Sims, 1999),
is an attractive low-cost approach for the treatment of
large volumes of waste. Recently, land treatment tech-
nology has been used for the treatment of contami-
nated soils as well as for freshly applied waste to soils
in different climatic conditions and different types of
soils (Al-Awhadi et al., 1996; Bleckmann et al., 1997;
* Corresponding author. Tel: 972-4-8293334; Fax: 972-4-8221529; Email: agsmadar@tx.technion.ac.il
field studies, in which hydrocarbon concentration range
was from less than 1 mg to 80 mg per gram of soil,
nutrient amendment enhanced the biodegradation and
was essential for effective bioremediation. Yet, resi-
due persistence in the soil at the end of treatment has
been a concern.
The degradation pattern common to all land treat-
ment studies is characterized by a decrease in the
degradation rate with time, reaching an apparent pla-
teau associated with residue persistence in soil. How-
ever, the time scale for the degradation of the hydro-
carbons varied in the different cases probably as a
consequence of the environmental conditions and the
different hydrocarbon compositions. As an example,
after a year of active treatment, 80% of the hydrocar-
bons applied were degraded in Kuwait (Al-Awadhi et
al., 1996), while only 50% were degraded in Belgium
during the same period of time (Genouw et al., 1994).
In all cases, even after an extended period of treatment,
a fraction that consisted of 10% to 25% of the initial

1058-8337/001/$.50
© 2001 by Battelle Memorial Institute
Biodegradtion
Bioremediation Kinetics of Hydrocarbons
Journal 5(3):193–209 in Soil during Land Treatment of Oily Sludge
(2001) 193
 hydrocarbons applied persisted in the soil. Since such
residues did not prevent the degradation of newly ap-
plied waste (API, 1984; Bleckmann et al., 1997;
Genouw et al., 1994), the process could be repeated on
the same piece of land with the resulting destruction of
large volumes of waste.
Analyzing the degradation patterns of the differ-
ent hydrocarbon classes during land treatment revealed
major differences in the extent to which they were
degraded. In most cases the saturated and aromatic
fractions were degraded more efficiently (54% to 80%)
than the polar and asphaltene compounds (0% to 20%)
(Bertrand et al., 1983; Cansfield and Racz, 1978;
Chaîneau et al., 1995; Dibble et al., 1979). Those
differences in degradation efficiency of the different
hydrocarbon classes were reflected in the composition
of the residues left in the soil, which consisted usually
of a larger fraction of the polar and asphaltene classes.
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As much as 300% increase in the asphaltene portion
and a decrease of 50% in the aromatic portion were
reported following land treatment of oily sludge
(Genouw et al., 1994).
An extensive evaluation on the performance of
land treatment demonstrated that slow biological pro-
cesses continued years after active remediation stopped,
leading to additional reduction in chemical concentra-
tion and mobility (Loehr and Webster, 1996). Recent
studies have demonstrated that the residues had no
adverse effect on ground water quality and toxicity to
soil organisms (Genouw et al., 1994; Wang et al.,
1990), but the yield of crops grown on treated land was
reduced (Chaîneau et al., 1996). Since a fraction of the
applied material persists as residues in the soil, the
extent and composition of those residues become a
concern when end point standards have to be reached.
More information on the composition of the residues,
the factors influencing their persistence and prediction
of the extent of their accumulation is needed.
Kinetic studies have been performed in order to
shed a light on hydrocarbons fate in soil and use the
knowledge acquired for predictive purposes. A first
order kinetics approximation is the most common used
model for the biodegradation of organic compounds.
Rate constants for the biodegradation of petroleum
hydrocarbon mixtures in soil have been derived in
laboratory and field scale applications. A wide range
of values, between 0.015 day–1 and 0.089 day–1, was
reported in different studies for diesel and crude oil
(Bleckmann et al., 1997; Bossert and Compeau, 1995;
Troy et al., 1993). A comprehensive comparison be-
tween published values for first-order kinetics con-
stants obtained in laboratory set-ups and values ob-
tained in field scale applications indicated that the
discrepancies between the two settings varied between
194
20% and 100% with laboratory values usually exceed-
ing field values (Blackburn, 1998). The conclusion
was that lab scale to field scale experimental linkage
was relatively poor. Some of the reasons could be the
different conditions used at the two scales and the lack
of sufficient data points in field applications for the
appropriate analyses. Comparisons between the degra-
dation rates of the different hydrocarbon classes agreed
with their persistence in the soil. The saturates had the
highest degradation rates, followed by the light aro-
matics, with high aromatic and polar compounds ex-
hibiting extremely low rates of degradation (Bossert et
al., 1984; Chaîneau, 1995; Dibble and Bartha, 1979;
Leahy and Colwell, 1990). Those results suggested
that the remaining residues were recalcitrant or very
slow degrading compounds. In this case, the persisting
refractory compounds are referred as potential exposed
hazards in the soil. Yet, when residuals were extracted
and reapplied to soil, further degradation occurred
(Weissenfels at al., 1992), suggesting that other pro-
cesses were involved.
The sequestration model (Alexander, 2000; Xing
and Pignatello, 1997) represents the more common
explanation to residue persistence. It is based on the
assumption that a certain fraction of hydrocarbons is
“locked up” in soil nanopores within soil aggregates,
making them inaccessible to microorganisms. More-
over, surface hydrophobicity in nanopores was directly
correlated to reduced microbial bioavailability of non-
polar compounds (Nam and Alexander, 1998). In that
case, the unavailable residues are not exposed and
might not represent a threat.
The initial high degradation rate, followed by the
very slow degradation rate during extended periods of
treatment can be interpreted by the two-compartment
model. The chemical in the “locked” compartment is
unavailable to microorganisms while in the second one
it is readily bio-transformed. After the supply of sub-
strate in the available compartment is biologically
depleted, the subsequent rate of biodegradation is gov-
erned by the rate of desorption or diffusion from the
inaccessible nanopores to sites containing active mi-
croorganisms. The rate at which the substrate is me-
tabolized or transferred to another compartment is
postulated to be proportional to the concentration of
the substrate in that compartment. The two-compart-
ment approach had been used to describe the biodeg-
radation in soil of the pesticide Triclopyr (Hamaker
and Goring, 1976) and dinitroanilines compounds
(Zimdahl and Gwynn, 1977). Aniline and phenol deg-
radation patterns gave a better fit to the two-compart-
ment model as well (Scow et al., 1986). All those cited
studies were performed on individual compounds in
laboratory-scale experiments. No attempts to use the
Admon et al.
 two-compartment approach for the analyses of mix-
tures of hydrocarbons in field studies or in laboratory
set-ups have been reported.
In spite of numerous studies conducted during the
years on land treatment, there are still open questions
concerning the residue persistence. What is the contri-
bution of biodegradation versus sequestration in con-
trolling residue persistence? How long does it take
until the compounds are sequestered to unavailable
sites? Is it affected by environmental conditions? Is it
possible to predict the amount of residues that will
persist in soil and the time it will take to reach that
“stable” situation? Is it possible to influence and con-
trol the level of residue remaining in the soil?
The present study focuses on the understanding of
the processes leading to the decrease in biodegradation
rate observed during land treatment in order to im-
prove the treatment efficiency. A kinetic approach was
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used for the study. Careful kinetic analyses of hydro-
carbon degradation in soil were performed on exten-
sive data collected from field and microcosms studies.
In addition, slurry systems were set as a means to
follow the degradation under intensive mixing condi-
tions. The changes in the kinetic behavior with time
and the resulting residue composition were analyzed in
order to draw practical conclusions.
Materials and Methods
Sludge
The sludge was collected from Haifa Oil Refinery’s
open storage impoundments. Samples of sludge were
dried at 100°C and then analyzed for total hydrocarbon
content. The results gave an average of 15% hydrocar-
bons, 30% soil, and 55% water (w/w). As the sludge
has been stored for years in the open impoundments, it
was assumed that drying the samples at 100°C resulted
mostly in water loss. During the period of the experi-
Table 1. Soil characteristics.
Biodegradtion Kinetics of Hydrocarbons in Soil during Land Treatment of Oily Sludge
ments, different batches of sludge with slightly differ-
ent consistencies were used. These different batches
could have contributed to some variations in the re-
sults obtained. The sludge contained a range of heavy
metals: lead (0.27 g/kg dry-weight), nickel (0.48 g/kg
dry-weight), chromium (0.48 g/kg dry-weight), zinc
(0.69 g/kg dry-weight), copper (0.5 g/kg dry-weight)
and vanadium (2.4 g/kg dry-weight). Their concentra-
tions were determined by Inductively Coupled Plasma
(ICP) analysis following two hours of HNO3 (65%)
digestion at 85°C.
Soil
The soil was collected from an agricultural area in the
Izrael valley, which had never been exposed to hydro-
carbon contamination. The soil had a clay texture with
the characteristics described in Table 1.
Cation exchange capacity (CEC), field capacity,
calcium carbonate content (CaCO3) and mineral nitro-
gen content (NO3– and NH4+) were determined by stan-
dard soil analytical methods (Page et al., 1984; Sparks,
1996). Organic matter was estimated by measuring the
organic carbon, using a modified Walkley Black pro-
cedure (Raveh and Avnimelech, 1973). The organic
matter in the soil was calculated, assuming that the
total organic carbon comprised 58% of the organic
matter in the soil.
Nutrients
Nutrients were supplied in all experiments. For the
slurry system experiments, Bushnell Hass broth (Difco
0578-17-3) was used, containing 1 g/L KH2PO4, 1 g/L
K2HPO4, 1 g/L NH4NO3, 0.2 g/L MgSO4.7H2O, 0.05
g/L FeCl3, and 0.02 g/L CaCl2.2H2O. For the labora-
tory microcosms and the field scale applications, the
commercial fertilizers urea and superphosphate (25%
P) were added. The quantities supplied were targeted
for C:N:P 50:10:1. The assumption was that the hydro-
195
 carbons constituted 15% of the sludge weight from
which 90% was carbon. For each kg of sludge applied
a total of 60 g urea and 27 g of superphosphate were
planned to be applied. The phosphate was added in one
batch, while only a fifth of the urea was initially added.
During the experiments, urea was added only when N
concentrations were below 100 to 200 mg/g soil.
Monitoring nutrients levels revealed that usually only
one more addition was needed during a month of
treatment, suggesting that a ratio C:N of 10:1 was
adequate.
Total Petroleum Hydrocarbon (TPH)
Analysis
TPH analysis was performed according to a modified
U.S. EPA 418.1 method (U.S. EPA, 1983). Prior to the
extraction, soil samples were dried at 60°C for 48
hours, ground and passed through a 2-mm sieve. Five
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grams of soil were extracted three times each in 20 mL
carbon tetrachloride by quick and vigorous hand shak-
ing (one minute each time). The pooled extracts were
filtered through crystalline Na2SO4. Polar material was
excluded by adding 3 grams of silica gel. After set-
tling, the absorbance of the extracts was measured in
the infrared range at 3.4 µm (2930 cm–1). Concentra-
tions of hydrocarbons were determined as mg TPH /g
dry soil. The EPA recommended standard for TPH
was used and consisted of the following mixture: 15
hexadecane:15 isooctane:10 chlorobenzene (v/v) (U.S.
EPA, 1983).
Hydrocarbon Fractionation
Hydrocarbon fractionation was performed according
to standard method for the fractionation of asphalt
(ASTM, 1986). For each sample analyzed 10 to 20
grams of dry soil were soxhlet extracted in 200 mL
dichloromethane for 4 h (16 cycles) prior to fraction-
ation. As the procedure was laborious and large samples
were needed for analyses, three samples from the field
application or three flasks from the slurries were pooled
prior to fractionation and no replicate procedures were
performed.
Experimental Design
Sludge biodegradation was investigated in different
experimental systems. Soil microcosms were compared
to field scale application. In addition, slurry set-up was
studied as a more controlled system.
Microcosm
A series of 60-L vessels each were used (55 cm length/
35 cm width/30 cm depth). A layer of fine gravel was
196
placed on the bottom of each container to provide
drainage of excess water. An opening was drilled at
one corner from which a drain was connected for the
collection of leachate. The vessels were filled with soil
amended with urea and superphosphate. Sludge was
applied to the soil and mixed thoroughly in the upper
10 cm using a modified electrical hand drill that had
three blades soldered in 5-cm intervals perpendicular
to the axis of rotation. The amounts of sludge applied
to the soil were 5%, 10%, 20%, and 30% (v/v), with
resulting initial TPH concentrations of 9, 17, 36, and
61 mg TPH per gram of dry soil in the upper 10 cm
incorporation zone. Sludge loads above 30% had a
negative effect on the soil structure, giving it a sticky
texture that prevented any mechanical procedures for
proper aeration. Each treatment was applied to tripli-
cate vessels.
During the experimental period, the soil was mixed
twice a week for homogenization and oxygen provi-
sion. Once a week the soil was watered to maintain
20% water content (20 g H2O/100 g dry soil). Compos-
ite samples of 100 grams from five locations in the
upper 10 cm were collected periodically from each
vessel and analyzed for hydrocarbon concentrations.
The nutrient levels in the soil were estimated by ana-
lyzing ammonium and nitrate concentrations and their
levels in the soil were adjusted such that the nitrogen
concentration remained above 100 mg/g dry soil. The
presence of nitrates was an indication that nitrification
processes were taking place. As nitrification is an oxy-
gen sensitive process, monitoring nitrate levels in the
soil and the leachate was an indicator for the prevailing
aerobic conditions.
Field Studies
Five plots, 9 m × 1.5 m, lined by 0.6 mm high-density
polyethylene (HDPE) at a depth of 50 cm were pre-
pared. A drainage system was installed for leachate
collection. The volumes of sludge applied were 360 to
720 liters per plot of 13 m2, equivalent to 27 to 55 m3
of sludge per 1000 m2. The sludge was left on the soil
for 2 days until the water was drained. Then, 2 days
after sludge application, a rotor tiller was used to in-
corporate the sludge in the upper 20 cm of the soil,
resulting in initial hydrocarbon concentrations in the
range of 15 to 80 mg TPH/g dry soil for each applica-
tion (Table 2). The initial load of each application was
calculated by subtracting the residue in the soil before
the new application from the measured TPH on the day
of the application. The cumulative load of TPH for
1000 m2 field was calculated assuming a 20 cm depth
of hydrocarbon incorporation zone and soil volumetric
density of 1.2 kg/m3.
Admon et al.
 Table 2. Sludge application in field studies: TPH loads
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Conditions for biodegradation in the soil were
achieved by using conventional agricultural practices.
The soil was amended with urea and superphosphate at
initial rates of 200 kg/1000 m2, a week after the first
sludge application, when treatment began. It should be
mentioned that hydrocarbon loss was observed only
after nutrients were amended, suggesting that no sig-
nificant evaporation was taking place. Tilling was per-
formed twice a week as a means to introduce oxygen
to the soil and to homogenize the mixture. Urea was
added during the treatment periods to maintain a nitro-
gen concentration above 100 mg/g dry soil (100 to 500
mg/g). The 20-cm depth of the tilling defined the zone
of sludge incorporation as well as the biodegradation
activity zone. A 20% moisture content (g/g-dry weight),
which was equivalent to about 40% of field capacity,
was maintained. It was achieved through weekly irri-
gation by sprinkling at a rate of approximately 30 m3
water/1000 m2 soil, attempting to cover a 5 to 6 mm
daily evaporation rate. Three composite samples of
100 g each were collected weekly from the top 20 cm
of predesignated locations on each plot. Each sample
was analyzed for TPH, NO3–, and NH4+ concentrations.
Slurry System
Slurries were prepared as 20% (w/w) solids in Bushnell
Hass medium, which provided the necessary mineral
nutrients for microbial growth. The solid phase com-
prised of a mixture of 20% sludge in soil, resulting in
concentrations of 15 to 20 mg TPH/g dry soil. Flasks
of 50 mL or 200 mL were used with a total sample
volume of 20 mL and 80 mL respectively. The flasks
were plugged with cotton balls, enabling the transfer
Biodegradtion Kinetics of Hydrocarbons in Soil during Land Treatment of Oily Sludge
of air. Aeration and mixing of the slurries were achieved
by rotating the flasks on an orbital shaker at 200 rpm.
All the experiments were conducted at 24°C. Sterile
controls, containing 1% HgCl2, were set to estimate
abiotic losses. Flasks were sacrificed periodically for
analysis. After centrifugation, the precipitate (solid
phase) was dried at 60°C for 48 h before hydrocarbon
analysis.
Results and Discussion
TPH Biodegradation
The pattern of TPH degradation in the microcosms
was found to be similar with all initial hydrocarbon
concentrations studied (9 to 61 mg TPH/g soil). The
process was characterized by a period of fast decrease
in hydrocarbon concentrations during the first three
weeks, followed by a period with slower activity (Fig-
ure 1). A direct relationship between the degradation
rate and the TPH concentration was observed. It was
manifested by the decrease in degradation rate with
time as the substrate concentration decreased and by
the higher degradation rate at higher sludge loads. The
degradation rate increased from 0.25 mg TPH/g soil/
day to 1.8 mg TPH/g soil/day during the first week, for
initial hydrocarbon concentrations of 9 mg TPH/g soil
to 61 mg TPH/g soil, respectively. The amount of TPH
that was removed during the first few weeks was equiva-
lent to 50% of the initial amount applied, regardless of
the initial concentration.
The field application data exhibited the same pat-
tern of biodegradation (Figure 2a), with a substantial
drop in TPH removal rate after the first three weeks.
197
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Figure 1. TPH degradation in microcosms at different initial concentrations. Initial TPH concentrations of 9, 17, 36, and 61 mg/g
soil correspond to sludge loads of 5%, 10%, 20%, and 30%.
The extent of degradation during those first weeks
reached approximately 80% with an additional 10%
loss of hydrocarbons in the following month. An ap-
parent plateau with only very slow degradation was
observed after the first 2 months, during a year of
treatment (Figure 2b). From a practical point of view,
the process seemed to be over. Thus, the efficiency of
the bioremediation treatment, that is, the extent of
degradation, was mostly determined by the first three
weeks of treatment.
The field set-up was further used to evaluate the
bioremediation efficiency with multiple applications
(Admon, 1999). The average degradation extent of the
sludge, which was applied to the plots at different time
intervals and loads during the summer (Table 2), was
found to be 85% to 90% regardless of the application
schedule or the application number (Table 3). The
extent and pattern of degradation of each freshly ap-
plied sludge load (up to eight applications; Admon,
1999) was similar, showing again that most of the
activity was occurring during the first three weeks of
treatment. It was concluded that there was no accumu-
lation of inhibitory metabolites in the soil. The transi-
tion to a slower activity stage does not seem to be due
to development of toxic conditions in the soil.
In the present work, a close analysis of those
degradation periods was performed in an attempt to
understand the transition to the slower activity stage.
198
TPH Kinetic Analyses
Kinetic analyses were performed on the data from the
different systems. As the analysis was actually performed
on the lumping effect of many simultaneous processes, a
pseudo first order equation could be derived. It was based
on the assumption that under the working conditions,
most factors were held constant except the substrate con-
centration. Whereas oxygen, moisture, and nutrients were
kept at non-rate-limiting concentrations and microorgan-
isms were in abundance, the substrate concentration was
changing continuously as the hydrocarbons disappeared.
Thus, first-order kinetic approximation was applied using
the linear integrated form ln(C/Co) = -kt and the rate
constant (k) was obtained by linear regression.
First-order kinetic analyses performed on the mi-
crocosm data are demonstrated in Figure 3. A rather
poor fit was obtained when all data were used as
illustrated by the dotted line in the framed graph for the
5% sludge (Figure 3: one stage k; R2 = 0.68). How-
ever, the points in the graph seemed to fall on two
straight lines with a transition point observed after
approximately three weeks, suggesting that two differ-
ent processes were taking place. Two separate regres-
sion analyses were done on the two apparent stages,
yielding a much better fit to the data. The first fast
stage, characterized by a rate constant of 0.037 day–1
(R2=0.92), was followed by a second slower stage with
a rate constant of 0.0086 day–1 (R2= 0.79).
Admon et al.
Downloaded By: [Romanian Ministry Consortium] At: 09:56 7 April 2011

Figure 2. TPH degradation in field first application. a: Data from four different plots that received
sludge on the same day. Day 0 is the day of nutrient application. b: Long-term active
bioremediation treatment performed in one of the plots (plot 4).

The kinetic analyses for the 10%, 20%, and 30%
sludge loads yielded similar results (Figure 3). The
two consecutive stage model gave a better fit than the
single stage model for all sludge loads (Table 4). The
rate constants for the first and second stages were
similar for all initial hydrocarbon concentrations tested,
indicating that the first-order kinetics approximation
was indeed appropriate. The average rate constant for
the first-stage was 0.036 day–1 (SD = 0.0022) while the
rate constant for the second-stage was three times as
low, with a value of 0.011 day–1 (SD = 0.0015) (Table
4). The transition time was similar as well, except for
earlier one in the case of the 30% sludge load.
Kinetic analyses of field data demonstrated simi-
larities in the overall process with a distinction in the
value of the first stage rate constant. The data from the

Biodegradtion Kinetics of Hydrocarbons in Soil during Land Treatment of Oily Sludge 199
 Table 3. Cumulative TPH degradation extents in field studies.
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Figure 3. Two stages kinetic analyses at different initial TPH concentrations in microcosms. Framed figure 5% sludge compares
between one-stage and two-stage kinetic analyses. 5% 10%, 20%, and 30% sludge loads correspond to initial TPH concentrations
of 9, 17, 36, and 61 mg/g soil. k1 and k2 represent the first and second stage rate constants respectively. Correlation coefficients
(R2) for the regressions are listed in Table 4.

200 Admon et al.

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Figure 4. Kinetic analyses for TPH degradation in field first sludge application. k1 and k2 represent the first and
second stages, respectively.

Table 4. Degradation rate constants in microcosm set-up. k1 and k2 are

the first order rate constant derived for the first and second degradation
stages respectively. One stage k represents the rate constant for a single
stage analysis. R2 is the correlation coefficient obtained for the regression
analyses

Biodegradtion Kinetics of Hydrocarbons in Soil during Land Treatment of Oily Sludge 201
 four plots gave a good fit to the two consecutive first
order kinetics processes as well. The rate constants
derived for the two stages were similar in all plots
(Figure 4). The average first-stage rate constant (k1)
for the four plots was 0.075 day–1 (SD = 0.004), while
the second-stage rate constant (k2) was 0.013 day–1 (SD
= 0.005). The first rate constant (k1) was more than
twice the value obtained in the microcosms, while the
(k2) value was similar to the one in the microcosms. As
there were more variations in the field data than in the
laboratory experiments, the average correlation coeffi-
cients R2 were not as high: 0.85 for (k1) and 0.45 for
(k2).
In addition to the solid systems, the pattern of
biodegradation in slurries was followed as well in
order to assess the influence of the intensive mixing. If
stirring provides better contact between microorgan-
isms and substrates, an improvement in the degrada-
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tion efficiency is expected. The biodegradation pattern
in the slurries was similar to the solid systems with two
consecutive first-order kinetics reactions as well (Figure
5). The rate constant for the first stage was 0.047day –1
(R2 = 0.91), higher than the average rate constant
derived for the microcosm (0.036 day–1) but lower than
the rate constant derived for the field application (0.075
day–1), implying that the stirring had no effect on the
degradation rate. On the other hand, the second slower
stage rate constant 0.012 day–1 (R2 = 0.99) was similar
to the rate constants obtained for the microcosm and
the field (0.011 day–1, 0.013 day–1).
Figure 5. Kinetic analysis for TPH degradation in slurries. k1 and k2 represent
the first and second stage rate constants respectively. Initial TPH concentration
was 18.5 mg TPH/ g soil.
202
These differences in the first-stage rate constants
found for the three systems can be attributed to differ-
ent experimental conditions. In all the different sys-
tems, oxygen and nutrient concentrations were not rate
limiting. Transport and diffusion conditions could not
explain the differences in degradation rates because
the degradation rates in the slurries were lower than in
the field application. However, a correlation between
the rate constants and the experimental temperatures
could be made.
The microcosm experiments were conducted at an
ambient average temperature of 21°C, the slurry ex-
periments were conducted in the controlled tempera-
ture room at 24°C, and the field experiments were
conducted during the summer with an average ambient
temperature of 31°C. An estimation of the different
rate constants normalized to 21°C was performed us-
ing the Arrhenius equation: k2=k1.θ(T2–T1) with a tem-
perature coefficient θ = 1.088 as reported for hydro-
carbon degradation in soil (Troy et al., 1993). The first
stage rate constants normalized for 21°C were similar
for the three systems: 0.037 day–1 for the slurry, 0.036
day–1 for the microcosm and 0.032 day–1 for the field
application (Table 5). These results imply that the
differences in the degradation rate found in the three
systems during the first three weeks were mainly due
to the differences in the experimental temperature con-
ditions. The similarity between the slurry system and
the microcosm was surprising and unexpected (Manilal
and Alexander, 1991; Zhang et al., 1995), suggesting
Admon et al.
 Table 5. Comparison between degradation rate constants at different
temperatures.
that under the working conditions mass transfer stimu-
lation in the slurries was not a rate determining factor.
In contrast to the first-stage rate constant, the
values of the second-stage rate constants did not vary
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with the different temperatures, suggesting that the
controlling processes during that stage were not bio-
logical. Those processes could involve sorption and
aging of hydrocarbons in the soil, making them un-
available to microorganisms (Hatzinger and Alexander,
1995; Tang et al., 1998; Weissenfels et al., 1992).
Another possibility could be that the transition to the
slower stage was a reflection of the degradability of
different hydrocarbon fractions. Assuming that the more
labile chemicals would be biodegraded first and faster,
molecules that were more refractory due to their chemi-
cal structure would tend to be degraded slower and
accumulate in the soil along with refractory metabo-
lites.
The realization that not only the degradation rate
was higher with the rise in temperature but also the
extent of degradation increased, suggested that the
transition to the slower stage was not due to the deple-
tion of the more biodegradable substrates. Analyses on
the hydrocarbon fraction degradation were performed
in order to clarify these issues.
TPH Fractions Degradation
An extensive analysis of the four major hydrocarbon
classes — saturates, aromatics, asphaltenes, and polars
— was performed on samples collected from the field
experiment. The composition profile on the day of
sludge application was compared to the profile 4 weeks
later and the cumulative load of each one of the frac-
tions was compared to the accumulated residues with
each sludge application (Figure 6). At the beginning of
a given degradation cycle, on the day of sludge appli-
cation, the saturated and aromatic classes constituted
the major hydrocarbon fractions with an average of
50% and 26% from the total hydrocarbons respec-
tively. The rest was composed of an average of 8%
Biodegradtion Kinetics of Hydrocarbons in Soil during Land Treatment of Oily Sludge
asphaltene and 15% polar compounds. The biodegra-
dation process induced a change in the hydrocarbon
composition. It was characterized by a sharp decrease
in the saturates and aromatics masses that was re-
flected by the reduction in their proportional mass
(from 76% to 50%) and an increase in the proportional
mass of the asphaltenes and polars (from 23% to 50%).
This pattern of composition change repeated itself with
subsequent sludge applications regardless of the num-
ber of applications. At the end of the treatment, follow-
ing 4 to 8 applications, the hydrocarbon composition
in the soil was similar to the composition found at the
end of the first application treatment. The saturates and
aromatics were reduced to 50% of the total mass of
hydrocarbons left in the soil and the asphaltenes and
polars increased to about 50%, demonstrating the en-
richment of molecules more resistant to biodegrada-
tion. The calculated degradation extents were 87% and
78% for the saturates and aromatics respectively, while
the asphaltenes and polars degradations were 57% and
48% (Table 6).
Those results were in agreement with other stud-
ies that demonstrated that the saturates and the aromat-
ics as a group were more susceptible to biodegradation
than the asphaltenes and some of the polar constituents
(Chaîneau et al., 1995; Genouw et al., 1994; Leahy and
Colwell, 1990). Moreover, the heterogeneous polar
fraction could also include biodegradation metabolites
that could contribute to an increase in their mass fol-
lowing the biodegradation processes (Huesemann and
Moore, 1993). The observation that there was no in-
crease in the degradation level of any of the fractions
following the repetitive and longer exposure of the
microorganisms to the substrates implied that there
was no selective enrichment of organisms able to trans-
form those compounds.
Rates of TPH Fractions Degradation
The contribution of the individual degradation rates of the
hydrocarbon classes to the apparent TPH degradation rate
203
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Figure 6. Hydrocarbon composition changes during repetitive field applications. a: Cumulative

values on day of application. b: Values 4 weeks later. Data shown are from one representative plot
(plot 2).

Table 6. Comparison between degradation extents of individual hydrocarbon classes. Cumulative

degradation was estimated by comparing the cumulative load applied to the residues left
following a specific treatment cycle

204 Admon et al.

 was studied. The analysis was performed on the slurry
experimental data. A first order kinetics approximation
was performed on the data for each one of the four
fractions separately. The analysis included only the first
stage of degradation (~ 21 days), as the small concentra-
tion changes during the second-stage could not be fol-
lowed with accuracy by the methods used to determine
the concentration of the individual fractions. The results
of the regression analyses performed on the individual
fractions as well as TPH are given in Figure 7. The
different fractions exhibited distinct degradation rates
indicative of their degradability and related to their accu-
mulation in the soil during bioremediation (Figure 6).
The saturated fraction had the highest rate constant 0.057
day–1, followed by 0.038 day–1 for the aromatic. The more
refractory fractions, the asphaltene and the polar had rate
constants of 0.01 day–1 and 0.005 day–1, respectively. It
should be noted that the rate constant values for the
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asphaltenes and polars could be underestimated, due to
the small quantities of those fractions in the samples. In
any case, as the saturates and aromatics were the major
constituents of the sludge (75%) with rate constants four
to ten times higher than the rate constants for the more
polar compounds, the overall degradation rate was ex-
pected to be predominantly affected by them.
Using those rate constants and the initial proportional
concentrations of each one of the fractions in the experi-
ment (55%, 26%, 7%, 12% for the saturates, aromatics,
asphaltenes, and polars), the expected overall rate constant
k(TPH) can be calculated according to the equation:
k(TPH) = Σ kiCip (1)
where ki is the rate constant of a specific hydrocarbon
class and Cip is its initial proportional concentration.
When introducing the appropriate rates and fractional
concentrations, the overall rate constant obtained was:
k(TPH) = (0.55*0.057 day–1) + (0.26*0.038 day–1) +
(0.007*0.01 day–1) + (0.12*0.005 day–1) = 0.042 day–1
(2)
This value 0.042 day–1 was in agreement with the
TPH rate constant of 0.047 day–1 obtained from the
kinetic analysis based on measured TPH concentration
values (Figure 5). The rate constant observed was
indeed a reflection of the combined degradation rates
of the mixture of substrates present in the sludge. The
similarity in the values was even more conclusive
considering the different methods used to determine
the hydrocarbon concentrations: Soxhlet extraction
followed by chromatography and gravimetric analysis
for the fractions, and shake extraction followed by
infrared detection for TPH.
Biodegradtion Kinetics of Hydrocarbons in Soil during Land Treatment of Oily Sludge
The Transition from the Fast to the
Slow Degradation Rate
The transition between the fast degradation process
and the subsequent slow process was abrupt and took
place following approximately 3 weeks in all treat-
ments. This observation is important with regard to
both management of the process as well as to under-
standing the reasons for the incomplete degradation.
The following analysis was performed in order to have
a more conclusive interpretation to the processes af-
fecting the decrease in degradation rate.
The continuous changes in the hydrocarbon com-
position with time imply that the apparent TPH rate
constant (kTPH) is actually changing with time as the
proportional contribution of each one of the hydrocar-
bon classes is changing continuously. Since the satu-
rates and aromatics have higher degradation rates than
the asphaltenes and polars, the contribution of the
slower degrading compounds would be expected to be
more prominent when the relative masses of the faster
degrading compounds drop. This should be reflected
in a lower apparent rate constant as the degradation
proceeds and could have explained the transition to the
second slower degradation stage. In order to test this
hypothesis, the apparent rate constant at any time kt
can be calculated as the sum of the proportional rate
constants of the hydrocarbon fractions such that
kt = Σ {kiCi(t) / Ctotal(t)} (3)
where Ci(t) is a given hydrocarbon fraction concentra-
tion at time t and Ctotal(t) is the total concentration of
hydrocarbons at time t. It can be assumed that the rate
constant of the slow second stage (k2) is affected by the
changes in hydrocarbon composition and its value is
given as kt at the transition point (kt=k21). As the sec-
ond-stage rate constants for the individual fractions
could not be directly determined, the TPH- k2 rate
constant was calculated based on the individual frac-
tions using Equation 3. The expected apparent rate
constant at the transition point which occurred after
approximately 21 days was calculated as the sum of
the rate constants of each one of the fractions multi-
plied by their proportional mass. Ci(t) was calculated by
the following first-order equations:
Saturates: Csat = 55.exp(-0.057t) (4)
Aromatics: Caro = 26.exp(-0.038t) (5)
Asphaltenes: Casp = 7.exp(-0.010t) (6)
Polars: Cpol = 12.exp(-0.005t) (7)
205
Downloaded By: [Romanian Ministry Consortium] At: 09:56 7 April 2011
Figure 7. Kinetic analyses of hydrocarbon fractions degradation. The data was obtained in the slurry experiment. ksat, karo, kasp,
and kpol represent the rate constant for the different fractions, saturates, aromatics, asphaltenes and polars respectively. Ktotal is
the value obtained for the TPH analysis (Figure 5).
Using the calculated values for t = 21 days and intro-
ducing theses values into Equation 3, the apparent rate
constant k21 obtained was 0.033 day–1 (Table 7). That
rate constant was almost three times the value obtained
from the data analysis of the total hydrocarbons at the
beginning of the second stage where k2=0.012 day–1.
Thus, the second stage rate constant could not be ex-
plained by the direct kinetics of the relative contribu-
tions of the fractions during the degradation process.
The transition to a slower degradation stage was not
induced solely by the changes in the hydrocarbon com-
position.
A second explanation could be that the drop in
degradation rate at the end of the first stage of degra-
dation was a reflection of the accumulation of recalci-
trant compounds. If this was the case, the transition
from the fast to the slow stage should be a function of
the amount of hydrocarbons degraded, since the hy-
drocarbon composition was the same in all treatments.
However, it was shown that the transition took place at
the same time when the extent of degradation was
different.
Another possibility was that the change in the
kinetic behavior was caused by interactions of the
hydrocarbons with the soil, limiting their availability
to microorganisms. The different chemical properties
of the hydrocarbons might have influenced their dif-
ferential sorption and diffusion characteristics as well.
206
The similarities between the kinetics in the slurry sys-
tem and the microcosm suggested that the diffusion
properties and contact between the substrate and the
microorganisms were similar. Thus, if limiting condi-
tions were created under the working conditions, they
occurred in the micro-environment that was not af-
fected by the liquid media and intensive mixing in the
slurries.
The observation that the transition from the first
stage to the second one was independent of the degra-
dation rate, that is, the extent of degradation during the
first-stage, and was happening at the same time at all
temperatures was intriguing. An attempt to get more
insight into that process was made by the following
analysis. As mentioned previously, the rate constant
for the total hydrocarbon degradation was expected to
change continuously as the relative masses of the dif-
ferent substrates changed. At higher temperatures,
where the more labile fractions would be degraded
faster and earlier, one would expect that the rate con-
stant for the total hydrocarbons would decrease faster
than at lower temperatures. Theoretically, at a certain
time the rate constant at the different temperatures
might be close enough and thus explain the similarities
in the values of the second rate constants. A simulation
was done, in which temperature corrections were made
for the rate constants of the different fractions, where
the sums kt = Σ {kiCi(t) / Ctotal(t)} at 21°C, 24°C and
Admon et al.
 Table 7. Calculated k2 rate constant as predicted from the proportional contributions of
the individual hydrocarbon classes. Calculated k2 is k21 obtained using the Equation 3
k21 = Σ {kiCI(21) / Ctotal (21)}
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Figure 8. Estimated changes in the values of TPH rate constant during biodeg-
radation at different temperatures. kt at each time point was calculated using
Equation 3 for the different temperatures.

31°C were plotted against time. The results showed
that the lines meet at a defined point where k = 0.02
day–1 on the 45th day (Figure 8). As this calculated rate
constant was still twice the value of the observed one
and the transition in the experiments occurred much
earlier (~21 days), that concept could not explain the
transition. It emphasized the suggestion that abiotic
interactions were interfering with the biological pro-
cesses, that is, the biodegradation capability of the
microorganisms was not the only factor affecting the
extent of biodegradation in the soil.
It seems that time-dependent decline in microbial
bioavailability was affecting the hydrocarbons biodeg-
radation. The bi-phasic behavior of the hydrocarbons
in the soil can be explained by the sequestration (two-
compartment) model, that is, a proportion of the ini-
tially “free” pollutants introduced into the soil is se-
questrated into soil nanopores, making them
inaccessible to the microorganisms. As those processes
are not as sensitive to temperature changes, the time
that was required for them to limit the biodegradation
in the different systems was similar. The present study
indicates that sequestration processes are only mini-
mal during the first few weeks and become dominant
after the first short period of rapid degradation. Thus,
enhancement of biological activity during the first
weeks of treatment could influence the residual levels
in the soil, avoiding sequestration of compounds that

Biodegradtion Kinetics of Hydrocarbons in Soil during Land Treatment of Oily Sludge 207
 could be readily degraded. As the soil structure and
composition affect the sorption characteristics and
behavior, the kinetics of the whole biodegradation pro-
cess would be different in different type of soils
(Alexander, 2000; Chung and Alexander, 1998; Manilal
and Alexander, 1991).
The practical conclusion from this study is that it is
important to create and maintain favorable conditions
for biodegradation during the initial treatment period for
maximal degradation capacity to be achieved. More-
over, bioremediation under unfavorable conditions
should be avoided to prevent the persistence of higher
levels of residuals in the soil as the degradation rate
during the first few weeks determines the treatment
efficiency. The higher the degradation rate during that
period, the fewer residues would be left in the soil.
Downloaded By: [Romanian Ministry Consortium] At: 09:56 7 April 2011
Acknowledgments
We thank Malik Kohva and Horacio Bach for techni-
cal assistance.
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