Environmental Pollution 136 (2005) 187e195

www.elsevier.com/locate/envpol

Bioremediation of petroleum hydrocarbons in contaminated

soils: Comparison of biosolids addition, carbon
supplementation, and monitored natural attenuation
Dibyendu Sarkar*, Michael Ferguson, Rupali Datta, Stuart Birnbaum
Department of Earth and Environmental Science, University of Texas San Antonio, 6900 North Loop,
1604 West, San Antonio, TX 78249-0663, USA
Received 17 May 2004; accepted 17 September 2004

Addition of biosolids is a potentially eective method of biostimulation for degradation

of petroleum hydrocarbons in soils.
Abstract
Two methods of biostimulation were compared in a laboratory incubation study with monitored natural attenuation (MNA) for
total petroleum hydrocarbon (TPH) degradation in diesel-contaminated Tarpley clay soil with low carbon content. One method
utilized rapid-release inorganic fertilizers rich in N and P, and the other used sterilized, slow-release biosolids, which added C in
addition to N and P. After 8 weeks of incubation, both biostimulation methods degraded approximately 96% of TPH compared to
MNA, which degraded 93.8%. However, in the rst week of incubation, biosolids-amended soils showed a linear two orders of
magnitude increase in microbial population compared to MNA, whereas, in the fertilizer-amended soils, only a one order of
magnitude increase was noted. In the following weeks, microbial population in the fertilizer-amended soils dropped appreciably,
suggesting a toxic eect owing to fertilizer-induced acidity and/or NH3 overdosing. Results suggest that biosolids addition is a more
eective soil amendment method for biostimulation than the commonly practiced inorganic fertilizer application, because of the
abilities of biosolids to supplement carbon. No statistically signicant dierence was observed between the biostimulation methods
and MNA, suggesting that MNA can be a viable remediation strategy in certain soils with high native microbial population.
2005 Elsevier Ltd. All rights reserved.
Keywords: Petroleum hydrocarbons; Biostimulation; Biosolids; Fertilizers; Natural attenuation

1. Introduction
Petroleum products are some of the most widely used
chemicals in society today. With the massive quantity of
fuel required to power automobiles and heat homes, and
the number of times each gallon of petroleum is stored,
transported, or transferred, accidents and leakages are
unavoidable. Petroleum contamination results from
leaking aboveground and underground storage tanks,
* Corresponding author. Tel.: C1 210 458 5453; fax: C1 210 458
4469.
E-mail address: dibyendu.sarkar@utsa.edu (D. Sarkar).
0269-7491/$ - see front matter 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2004.09.025

spillage during transport of petroleum products, abandoned manufactured gasoline sites, other unplanned
releases, and current industrial processes. As petroleum
contains hazardous chemicals such as benzene, toluene,
ethylbenzene, xylenes, and naphthalene, this contamination can be hazardous to the health of plants, animals,
and humans (Zhou and Crawford, 1995; Liebeg and
Cutright, 1999; Ting et al., 1999; Vasudevan and
Rajaram, 2001). Petroleum-contaminated soil is currently treated using three processes: physical, chemical,
and biological. The most common physical methods of
treatment of contaminated soils, such as disposal in
a landll, and incineration are expensive. Incineration is

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D. Sarkar et al. / Environmental Pollution 136 (2005) 187e195

also a source of air pollution (Ting et al., 1999).

Chemical treatment includes direct injection of chemical
oxidants into contaminated soil and groundwater
(Riser-Roberts, 1998), thereby altering native aquatic
chemistry. Biological treatment most commonly involves the breakdown of contamination into nontoxic
forms using microbiological processes (Riser-Roberts,
1998).
Bioremediation may be dened as the use of living
organisms to remove environmental pollutants from
soil, water, and gases (Collin, 2001). Organic compounds are metabolized under aerobic or anaerobic
conditions by the biochemical processes of microorganisms (Collin, 2001). Bioremediation of contaminants can
be accomplished by two methods, bioaugmentation and/
or biostimulation. The process of bioaugmentation, as it
applies to remediation of petroleum hydrocarbon
contaminated soil, involves the introduction of microorganisms that have been cultured to degrade various
chains of hydrocarbons into a contaminated system.
The cultures may be derived from the contaminated soil
or they may be obtained from a stock of microbes that
have been previously proven to degrade hydrocarbons.
Once introduced into the system, the cultured microorganisms selectively consume the hydrocarbons.
The process of biostimulation introduces additional
nutrients in the form of organic and/or inorganic
fertilizers into a contaminated system, which increases
the population of the indigenous microorganisms
(Pankrantz, 2001). The indigenous microorganisms
may or may not primarily target the hydrocarbons as
a food source. However, the hydrocarbons are assumed
to degrade more quickly in comparison to natural
attenuation due to the increased numbers of microorganisms caused by increased levels of nutrients.
The goal of bioremediation is to have microbes fully
degrade hydrocarbons to carbon dioxide and water.
Bioremediation has several benets over landll disposal
and incineration, such as the conversion of toxic wastes
to non-toxic end products, a lower cost of disposal (or
no disposal at all), reduced health and ecological eects
and long-term liabilities associated with non-destructive
treatment methods, and the ability to perform the
treatment in situ without unduly disturbing native
ecosystems.
Bioremediation of petroleum-contaminated soils has
been investigated since the late 1940s, but interest in the
eld did not become widespread until the Exxon Valdez
oil spill in 1989 (Margesin and Schinner, 1997; Jackson
and Pardue, 1998). Consequently, there have been
a large number of studies conducted, and bioremediation
has almost always been found to be an eective treatment
of hydrocarbon-contaminated sites (Huesemann and
Moore, 1993; Li et al., 1995; Zhou and Crawford, 1995;
Liebeg and Cutright, 1999). In the eld of biostimulation,
nutrient supplementation for hydrocarbon degradation

has traditionally focused on addition of N and P, either

organically or inorganically. Because C is a major
constituent of petroleum fuels, its traditional role in
bioremediation research has typically been as an index to
determine the amount of N and P that need to be added
to reach the optimal C:N:P ratio (Riser-Roberts, 1998).
More recently, the role of C supplementation in
hydrocarbon biodegradation has been investigated with
the use of glucose, biosolids, and composts (Namkoong
et al., 2002). However, these experiments did not isolate
C as a nutrient supplement, but measured the eects of
a combination of nutrients from various sources, C being
one of them. Moreover, the role of C supplementation in
hydrocarbon bioremediation in low organic matter or
otherwise C-poor soils has never been investigated.
Various nutrient sources such as inorganic fertilizer,
urea, sawdust, compost, manure, and biosolids have
been used in biostimulation (Rosenberg et al., 1992;
Walworth and Reynolds, 1995; Cho et al., 1997;
Williams et al., 1999; Namkoong et al., 2002). Biosolids
seem to be a promising nutrient source for microbes in
bioremediation (Namkoong et al., 2002); however, very
few studies have been reported to date that explore the
potential of biosolids application to enhance hydrocarbon degradation (Ferguson, 2003). The primary benets
of biosolids include their low cost (or no cost), slow
release of the nutrients (similar to animal manures), and
easy availability (McBride, 2003). Moreover, because
the biosolids are slow-release nutrient sources, the
possibility of ecosystem contamination from excess
nutrients (as in the case of fertilizer application) is also
minimal. However, the typical problems encountered
with land application of biosolids include the diculty
of supplying the deeper soils with the nutrients and the
possibility of contaminating the soil with metals
(McBride, 2003).
Sanchez et al. (2000) describe natural attenuation as
a collection of biological, chemical and physical processes that occur naturally resulting in the containment,
transformation, or destruction of undesirable chemicals
in the environment. Processes include some combination
of sorption, volatilization, dilution, and dispersion
coupled with biodegradation. In contrast to biostimulation, monitored natural attenuation (MNA), if eective, provides signicant benets in terms of cost and
eort (Sanchez et al., 2000). Previous studies suggest
nutrient supplementation stimulates bioremediation by
increasing microbial biomass (Sanchez et al., 2000;
Margesin and Schinner, 2001; Duncan et al., 2003; Maki
et al., 2003). In all of these cited reports biostimulation
caused a rapid start to bioremediation. Duncan et al.
(2003) saw an initial rapid response associated with
fertilizer application, but there seemed to be no
dierence between the MNA and fertilizer-treated plot
after 2 years. Similarly Maki et al. (2003), in comparing
biostimulation with MNA in a marine setting, noted an

D. Sarkar et al. / Environmental Pollution 136 (2005) 187e195

initial increase in activity in the treated plot, but no

signicant dierence by the sixth week of data
collection. Only Margesin and Schinner (2001) saw
a permanent improvement of biostimulation over
MNA.
The primary objective of the reported study was to
determine if addition of biosolids enhances hydrocarbon
bioremediation by promoting biostimulation in a dieselcontaminated, carbon-poor soil. The secondary objective of the study was to determine if C supplementation
from biosolids application could be a factor of any
signicance in the petroleum hydrocarbon degradation
process in a soil with low organic matter content. These
two methods of biostimulation were compared to MNA
to develop a more complete understanding of the
diering approaches.

189

pulverized in order to accelerate distribution of nutrients

to the microbes. Powdered biosolids were autoclaved
prior to mixing with the diesel-contaminated soil to
eliminate the possibility of bioaugmentation; 50 g of
pulverized biosolids were autoclaved twice for 2 h each
time at 121  C.
2.4. Fertilizer
The inorganic fertilizer used was a mixture of reagent
grade NH4NO3, and commercially available Hi-Yield
Triple Superphosphate (TSP; 0-45-0). Bioavailability of
N in NH4NO3 was estimated as 100% and the
bioavailability of P in TSP was 90e100%. The fertilizers
were mixed with deionized water and autoclaved prior
to mixing with the contaminated soil; 50 mL of
inorganic fertilizer was autoclaved for 15 min at 121  C.

2. Materials and methods

2.5. Microbial analysis

2.1. Soil

Microbial populations were determined using the 5tube Most Probable Number (MPN) Method (Collins
et al., 1989). A 10% PTYG (Peptone, Tryptone, Yeast,
Glucose) solution was used as the growth medium
(Wilson et al., 1983; Balkwill and Ghiorse, 1985; Bone
and Balkwill, 1988; Colwell, 1989).

Soil was collected from the Freeman Ranch in San

Marcos, Texas. It was dark brown in color and
consisted of 90% clay. Batte (1984) classied the soil
as Tarpley Clay that overlays the Edwards Limestone
Formation (Fisher, 1983). The soil was analyzed to
determine moisture content, pH and nutrient concentrations following standard protocols (Klute, 1996;
Sparks, 1996). The amount of total C, H, and N was
determined using a dry combustion method with a PE
2400 Elemental Analyzer.
Total, organic, and inorganic P concentrations were
determined using the Ignition Method (Sparks, 1996).
Phosphorus was measured colorimetrically by a UV/
Visible light spectrophotometer using the molybdateascorbic acid method (Sparks, 1996). Bioavailable P was
determined using the Mehlich-3 extraction technique
(Sparks, 1996).

2.6. Petroleum hydrocarbon analysis

A Varian CP 3800 Gas Chromatograph with ame
ionization detector (FID) and a Varian Chrompak
fused silica column was used to determine the total
petroleum hydrocarbon (TPH) concentration. Standards in n-pentane were prepared from pure diesel. For
diesel-spiked soil samples, TPH was extracted using
n-pentane in a 1:4 soil/extractant ratio. The extract was
analyzed according to the Texas Commission on
Environmental Quality Method 1005 (TCEQ, 2001).
2.7. Remediation experimental design

2.2. Diesel
The diesel fuel used in this experiment is commercially available and was obtained from a gasoline pump
at a typical gasoline station.
2.3. Biosolids
Exceptional quality, Grade A biosolid pellets were
obtained from a sewage treatment plant in Largo,
Florida. In addition to grading, such biosolids have
various nutrient ratios with respect to C, N and P.
The nutrient availability for vegetative growth (i.e.
bioavailability) in these biosolids is mostly inorganic,
90e95%, with a small organic percentage, 5e10%
(OConnor and Sarkar, 1999). The biosolids were

Five hundred grams of soil and varying amounts of

sterile inorganic fertilizer and biosolids were placed in
each of six sterilized glass pans with a capacity of 1.4 L.
The pulverized and autoclaved Largo biosolids were
mixed thoroughly with the soil in each pan at two rates:
low (5%) and high (10%). Deionized water was then
added to the pans to achieve soil moisture content of
approximately 60% of the water holding capacity
(Moller et al., 1995; Zhou and Crawford, 1995; Cho
et al., 1997; Margesin and Schinner, 1997; Quinn and
Reinhart, 1997; Nocentini et al., 2000). Next, commercially available diesel fuel was added to each soil pan for
an initial total petroleum hydrocarbon (TPH) concentration of approximately 4000 mg/kg (varied between
3350 and 4250 mg/kg). The soil, fertilizer/biosolids,

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D. Sarkar et al. / Environmental Pollution 136 (2005) 187e195

3. Results and discussion

3.1. Characterization of soil and amendments
The native soil had a neutral pH and low concentrations of C, N, total and bioavailable P (Table 1).
Largo biosolids had a pH of w6 and contained
signicantly greater concentrations of total and bioavailable nutrients compared to the soil. The inorganic
fertilizer mix was highly acidic (pH w3) and contained
greater concentrations of N, total and bioavailable P
than either the biosolids or the native soil. The fertilizer
mix did not contain any C (Table 1).
3.2. Degradation pattern of TPH
TPH degradation was accomplished primarily during
the rst week in biosolids-amended soils (Fig. 1). The

4500
Control 1 - Soil

Low Rate Biosolid

High Rate Biosolid

4000

TPH Concentration (mg/kg)

water, and diesel were mixed thoroughly by hand to

achieve a homogeneous distribution of hydrocarbons,
water, and nutrients. Fertilizers were added such that N
and P contents of the fertilizer-treated soils roughly
mimicked the amount of those in the biosolids-amended
systems. The biosolids also supplied C in addition to P
and N.
The pans were covered with punctured plastic wrap
to allow gas exchange. The pans were placed under
constant light (Williams et al., 1999) and maintained at
a temperature of 22  CG2  C and a relative humidity of
65e80% (Ting et al., 1999). The pans were tilled three
times a week with a steel hand trowel sterilized with
alcohol (Margesin and Schinner, 1997).
Immediately after mixing the components, the soils
were analyzed to establish baselines for TPH concentration, pH, nutrient concentration, and microbial
population. The pH, TPH, and microbial population
of the pans were monitored weekly throughout the
experiment. Bioavailable P was measured in ve of the
eight weeks of experiment. During the experimental run,
the moisture content of each pan was adjusted by adding
water one day prior to sampling.
Precision of generated data was evaluated using
standard deviation of three replicates and the accuracy
was estimated by spike recovery. Correlation analysis
was performed using Sigma Plot version 4.01 (SPSS,
1997).

3500
3000
2500
2000
1500
1000
500
0
Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8

Time (week)

Fig. 1. TPH degradation pattern in low- and high-rate biosolidsamended soils in comparison to control soils without amendments.

soils showed reductions in TPH concentrations by

84.4% for MNA (control 1), 91.0% for the low-rate
biosolids treatment, and 90.4% for high-rate biosolids
treatment during week 1. The high-rate of TPH
degradation in the MNA soil demonstrates that the
native soil microbes were capable of degrading hydrocarbons to a large extent. Although a portion of this
TPH reduction is due to volatilization, abiotic loss of
diesel fuel has been reported to be generally below 10%
at 25  C in the rst 30 days (Margesin and Schinner,
1997). By week 4, 89.7% of the original TPH was
removed in the MNA treatment (control 1), 91.9% was
removed in the low-rate biosolids treatment, and 90.9%
in the high-rate biosolids treatment. By the end of the
experimental period (week 8), 93.8% of the original
TPH was removed in the MNA treatment (control 1),
96.2% in the low-rate biosolids treatment, and 96.2% in
the high-rate biosolids treatment. This residual TPH is
expected (Nocentini et al., 2000). Chromatography
proles of diesel-contaminated soils (not shown) demonstrated that only the heavier carbon compounds
remained in the soils at the completion of the
experiment. Nocentini et al. (2000) obtained similar
results and concluded that the heavier compounds in
diesel fuel were depleted at slower rates than the lighter
compounds. Overall, the biosolids treated soils (high
and low-rate of amendments) showed similar patterns of
TPH degradation (Fig. 1).
Although to a considerably lesser extent than the
biosolids-treated soils, the majority of the TPH degradation (65.5e76.3%) was accomplished during the rst

Table 1
Characterization results for soils and amendments
Matrix

Carbon (%)

Nitrogen (%)

Total phosphorus (mg/kg)

Bioavailable phosphorus (mg/kg)

pH

Largo biosolids
Inorganic fertilizer mix
Soil

37.14G1.48
0.00
1.53G0.12

5.77G0.33
35.0
0.15G0.02

34,800G1200
246,400G43,300
275G92

20400G1200
241900G16,800
9.7G1.8

6.16G0.02
3.24G0.02
7.14G0.11

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D. Sarkar et al. / Environmental Pollution 136 (2005) 187e195

3.3. Degradation rates of TPH

5000

TPH Concentration (mg/kg)

4500

Control 2 - Soil

Low Rate Fertilizer

High Rate Fertilizer

4000
3500
3000
2500
2000
1500
1000
500
0
Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8

Time (week)

Fig. 2. TPH degradation pattern in low- and high-rate fertilizeramended soils in comparison to control soils without amendments.

week in the fertilizer-amended soils (Fig. 2). By week 4,

92.3% of the original TPH was removed in the MNA
treatment (control 2), 91.5% in the low-rate fertilizer
treatment, and 93.8% in the high-rate fertilizer treatment. By the end of the experiment period (week 8),
94.5% of the original TPH was removed in the MNA
treatment (control 2), 95.5% in the low-rate fertilizer
treatment, and 96.8% in the high-rate fertilizer treatment.
The degradation pattern in each treatment (Figs. 1
and 2) is similar to the results obtained by other authors
(Ting et al., 1999; Nocentini et al., 2000). Namkoong
et al. (2002), however, did not show such a huge
reduction in TPH concentration in the control pan; this
can be attributed to the lack of aeration in their study.
Table 2 shows the initial and nal TPH concentration in
each soil and the percent decrease in TPH concentration
through 8 weeks of treatment. The low-rate and highrate biosolids-amended soils degraded a similar percentage of the original TPH contamination. The low-rate
and high-rate fertilizer-amended soils also degraded
a similar percentage of the original TPH contamination.
The untreated soils (MNA) degraded lesser percentages
of the original TPH contamination than the treated
soils, although the dierences were not signicant at the
95% condence interval using the LSD method.
Table 2
Initial and nal TPH concentrations in control, biosolids and fertilizer
amended soils
Matrix

Control soil 1
Low-rate biosolids
High-rate biosolids
Control soil 2
Low-rate fertilizer
High-rate fertilizer

Initial TPH
concentration
(mg/kg)

Final TPH
concentration
(mg/kg)

Percent
decrease
in TPH

3500G400
3350G240
3350G330
4250G200
3950G150
4050G300

215G14
130G70
130G30
234G8
175G87
130G47

93.8G0.7
96.2G0.3
96.2G0.4
94.5G0.2
95.5G0.2
96.8G0.3

Kinetic modeling was performed to estimate the rates

of chemical reactions in the studied systems. Kinetics of
a chemical reaction can be described in terms of its order
(Snoeyink and Jenkins, 1980). While other authors
(Nocentini et al., 2000; Namkoong et al., 2002)
adequately described their hydrocarbon degradation
data using a rst-order kinetic model, in the present
study, experimental results were better described using
a second-order kinetic model.
To determine the order of the reactions in each of the
soil treatments, the data was plotted in a scatter
diagram. For a rst-order reaction, the plot of ln[TPH]
versus time should be a straight line while for a secondorder reaction, the plot of 1/[TPH] versus time should be
linear (Snoeyink and Jenkins, 1980). Data plots of
ln[TPH] versus time were better described by curvilinear
equations, whereas plots of 1/[TPH] versus time were
better described by linear models (data not shown). The
R2 value represents the coecient of correlation; the
nearer the value of R2 to 1, the stronger the correlation
of the data (Everitt, 2002). The R2 values for the linear
plots using the second-order model were higher than
those obtained using the rst-order model in ve of the
six treatments (Table 3).
The slope of the line for reactions that follow the
second-order rate law is equal to 8k, where k is the rate
constant (Snoeyink and Jenkins, 1980). Although the
dierences between the calculated reaction rates were
statistically insignicant, TPH degradation in the MNA
soils were described by the lowest reaction rate
constants, the biosolids (both rates) and highrate fertilizer amended soils had the fastest reaction
rate constants (Table 4). Rate constant of the low-rate
fertilizer amended soil was intermediate. The rate
constants were reective of the relative eects of various
treatments on TPH degradation in diesel-contaminated
soils.
3.4. Microbial population
The total viable microbial population of the biosolids
treated soils and associated MNA soils are presented in
Table 3
Comparison of correlation coecients for rst- and second-order
linear models
Matrix

First-order linear
model R2

Second-order linear
model R2

Control soil 1
Low-rate biosolids
High-rate biosolids
Control soil 2
Low-rate fertilizer
High-rate fertilizer

0.8496
0.8867
0.751
0.8558
0.8867
0.7797

0.9532
0.7651
0.8511
0.9373
0.9351
0.8998

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D. Sarkar et al. / Environmental Pollution 136 (2005) 187e195

Table 4
Second-order reaction rate constants for TPH degradation in control,
biosolids and fertilizer amended soils
Soil

Reaction rate
constant (per day)

Control soil 1
Low-rate biosolids
High-rate biosolids
Control soil 2
Low-rate fertilizer
High-rate fertilizer

1.00!105
1.25!105
1.25!105
1.00!105
1.13!105
1.25!105

Fig. 3. Total viable counts of the uncontaminated

(MNA) soil (109 cfu/g soil dry weight) were much higher
than the 107 cfu/g soil dry weight typically encountered
in soils (Ting et al., 1999), indicating that the Tarpley
Clay soil had high microbial population natively. After
adding biosolids to the diesel-contaminated soil, the
microbial population was immediately stimulated,
resulting in an increase of 12 430% and 11 384% for
the low- and high-rate biosolids treated soils, respectively (Table 5). The MNA soils also experienced an
increase in microbial population (57%; Table 5)
accompanied by signicant TPH degradation (Figs. 1
and 2), indicating that the indigenous soil microbes
utilized a portion of the C supplied by the diesel fuel as
a potential nutrient source.
Increases in microbial population corresponded to
decreases in TPH concentration during the same time
period in the biosolids-treated soils (Figs. 1 and 3). The
increase in microbial population in the biosolidsamended soils is likely due to availability of all three
major nutrients, namely P, N, and C (Table 1).
However, this increase did not necessarily translate to
signicant dierences in TPH degradation, which
suggest existence of a certain threshold level to nutrient
supplementation that might be able to accelerate

1.00E+13
1.00E+12

Microbial Population (cfu)

1.00E+11
1.00E+10
1.00E+09
1.00E+08
1.00E+07
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01

Control

Low Rate Biosolid

High Rate Biosolid

1.00E+00
Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8

Time (week)

Fig. 3. Total viable microbial population in low- and high-rate

biosolids-amended soils in comparison to control soils without
amendments.

hydrocarbon degradation during the initial stages of

bioremediation. The microbial populations in the
fertilizer treated soils remained relatively invariable
throughout the experimental period (Fig. 4); the viable
microbe count increased by only 124% and 140%
(compared to a 57% increase in MNA) in the low- and
high-rate fertilizer treated soils, respectively (Table 5).
Dramatic changes in microbial population have been
reported by other authors experimenting with nutrientsupplemented, hydrocarbon-contaminated soils (Ting
et al., 1999; Vasudevan and Rajaram, 2001). Microbial
population in the nutrient system studied by Ting et al.
(1999) decreased after the initial increase, and then
increased again. A similar pattern was observed in the
high-rate fertilizer treatment of this study (Fig. 4).
The microbial population changes for the remainder of
the experiment could be due to interactions between
various microbial populations and the resulting changes
to the environment. The fact that the increases in
microbial population in fertilizer-treated soils were
negligible compared to the increases in biosolids-treated
soils may be indicative of either of the following: (a)
carbon supplementation by biosolids stimulated microbial growth, and/or (b) the potential toxic eect of
fertilizer-induced acidity (pH of the fertilizer mix w3,
Table 1) and other chemical factors on indigenous soil
microbes. Zhou and Crawford (1995) found that
excessive bioavailable N supplied by fertilizer inhibited
hydrocarbon degradation due to NH3 toxicity that
retarded microbial growth. Characterization of the
microbial community within the system could give
further insight to the overall microbial population
changes.
3.5. Nutrient status
The primary limiting nutrients in microbial degradation of petroleum hydrocarbons have been historically
understood to be N and P (Zhou and Crawford, 1995;
Ting et al., 1999). These nutrients are important to
cellular production and their supplementation increases
the hydrocarbon degradation eectiveness (Walworth
and Reynolds, 1995; Zhou and Crawford, 1995; Ting
et al., 1999). The quantity of N and P required to
convert 100% of the petroleum C to biomass may be
calculated from the C:N and C:P ratios found in cellular
material (Dibble and Bartha, 1979). Various authors
provide ratios for these nutrients. There is some
disagreement on the exact ratios, but they are not too
dissimilar (Riser-Roberts, 1998); the optimal C:N:P
ratio reported in the literature is 100:15:3 (33:5:1) for
hydrocarbon biodegradation (Zitrides, 1983; RiserRoberts, 1998). However, using a xed ratio of total
nutrients as a biostimulation index may be misleading,
as the entire amount of nutrients may not be readily
bioavailable (Sims and Bass, 1984).

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D. Sarkar et al. / Environmental Pollution 136 (2005) 187e195

Table 5
Increase in microbial population after week 1 in control, biosolids, and fertilizer amended soils
Matrix

Week 0 (cfu)

Week 1 (cfu)

% Change

Control soil 1
Low-rate biosolids
High-rate biosolids
Control soil 2
Low-rate fertilizer
High-rate fertilizer

1.15EC09G1.31EC09
1.05EC09G3.61EC08
6.40EC08G2.12EC08
1.15EC09G1.3EC09
5.43EC08G1.70EC08
1.91EC09G2.62EC09

1.81EC09G1.36EC09
1.30EC11G1.00EC00
7.35EC10G7.78EC09
1.81EC09G1.36EC09
1.21EC09G1.81EC09
4.58EC09G8.3EC09

57
12300
11400
57
124
140

Nutrient levels in the diesel-contaminated native and

biosolids/fertilizer-amended soils are presented in Table
6. The MNA soils (controls 1 and 2) had less C, N, and
P than the biosolids amended soils. The MNA soils had
similar C percentage as the fertilizer-amended soils, but
signicantly less N and P. The biosolids- and fertilizeramended soils had similar concentrations of N and P,
but, expectedly, the biosolids-treated soils had much
greater C content compared to the fertilizer-treated
soils. The pH of the MNA soils and the biosolidsamended soils were within or near the optimum pH
range for hydrocarbon degradation of 6.5 to 8.5 (RiserRoberts, 1998). The pH of the fertilizer-amended soils
was below this optimum range (Table 6).
The biosolids-treated soils experienced an increase of
bioavailable P during the rst week of the experiment
(Fig. 5). This is due to the time-release nature of
nutrients in the Largo biosolids; P was released slowly
and did not become available to the microbes until after
one week of biosolids amendment. Between week 1 and
week 5, there was a steady decrease in bioavailable P as
the microbes continued to metabolize the nutrients.
After week 5, as the microbes started to die o, the
bioavailable P concentration in the biosolids treated
soils reached a constant level and remained that way
until the end of the experiment (Fig. 5). Bioavailable P
decreased sharply during the rst week of the experiment

1.00E+12
1.00E+11

Microbial Population (cfu)

1.00E+10
1.00E+09
1.00E+08
1.00E+07
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01

Control

Low Rate Fertilizer

High Rate Fertilizer

1.00E+00
Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8

Time (week)

Fig. 4. Total viable microbial population in low- and high-rate

fertilizer-amended soils in comparison to control soils without
amendments.

in the fertilizer-treated soils (Fig. 6). The TSP started

releasing P immediately, almost 100% of which was
available to the microbes. The w20% decrease in
bioavailable P between week 0 and week 1 indicates
that the released P was being utilized by the microbes. It
is also possible that a portion of the soluble/bioavailable
P leached to the bottom of the pan and was not sampled
during analysis of surface soils. The die-o cycle for the
microbial population began in week 5 after which
the bioavailable P concentration became constant until
the end of the experiment (Fig. 6). Bioavailable P in the
control soils (MNA) remained low throughout the
experiment (Figs. 5 and 6). Although N bioavailability
was not monitored in the reported study, given the
typical mineralization pattern of N in clay soils, it is not
expected to be considerably dierent from the P prole.
Despite the fact that the fertilizer-treated soils had
more bioavailable P and N, greater percentage hydrocarbon degradation was observed in the biosolidsamended soils during week 1 (Figs. 1 and 2). This is in
agreement with the much smaller increase in microbial
population (normalized to MNA) noted in the fertilizertreated soils (124e140%) in comparison with that in
biosolids-treated soils (11 384e12 340%; Table 5). Apparently, nutrient bioavailability was not the only factor
governing TPH degradation. In a C-poor soil such as
Tarpley Clay, even after diesel contamination, the
C:N:P ratio varied between 7:4:1 and 10:3:1 in the
fertilizer-treated soils, which was far from the optimal
C:N:P ratio of 33:5:1 (Riser-Roberts, 1998). On the
other hand, the C:N:P ratio in the biosolids-treated soils
ranged between 27:4:1 and 24:4:1 (Table 6), which were
much closer to the optimal C:N:P ratio for hydrocarbon
degradation. Zhou and Crawford (1995) reported that
excess N (C:NZ2:1) can impair biodegradation due to
ammonia toxicity, although it does not completely
inhibit biodegradation. In the fertilizer-treated soils,
there is a possibility that ammonia toxicity (C:N ratio of
2:1 and 3:1), coupled with the low pH (5.2e5.5)
impacted TPH degradation in the very critical rst
week of incubation. The toxic eect on the microbial
population gradually decreased with increasing incubation time possibly because of an increase in the rate of
nitrication with increased tillage and aeration; NO3eN
is typically not toxic to microbes. Because the biosolids
added C to the system, it helped optimize the C:N:P

194

D. Sarkar et al. / Environmental Pollution 136 (2005) 187e195

Table 6
Total nutrients in control, biosolids, and fertilizer amended soils
Soil

Carbon (%)

Nitrogen (%)

Phosphorus (%)

C:N:P

pH

Control soil 1
Low-rate biosolids
High-rate biosolids
Control soil 2
Low-rate fertilizer
High-rate fertilizer

1.53G0.12
3.22G0.11
4.15G0.48
1.54G0.22
1.49G0.16
1.62G0.05

0.15G0.02
0.48G0.02
0.66G0.05
0.15G0.01
0.48G0.05
0.91G0.03

0.03G0.01
0.12G0.01
0.17G0.02
0.03G0.01
0.15G0.01
0.24G0.01

56:5:1
27:4:1
24:4:1
50:5:1
10:3:1
7:4:1

7.14G0.16
6.56G0.03
6.22G0.04
7.14G0.09
5.54G0.03
5.23G0.00

ratio in the C-poor Tarpley Clay, further aided by the

biosolids slow-release nature of bioavailable nutrient
supplementation. Namkoong et al. (2002) also identied
such synergistic eects of biosolids addition on TPH
biodegradation. The eects of chemical toxicity in the
fertilizer-amended systems were immediate and gradually waned with incubation time, thereby resulting in
a similar degradation pattern of residual TPH in
fertilizer-amended and biosolids-amended soils.

4. Summary and conclusions

Bioremediation can be a viable and eective response
to soil contamination by petroleum hydrocarbons. This
investigation compared monitored natural attenuation
with two methods of biostimulation: inorganic fertilizer
amendment and biosolids amendment, at two rates, low
and high.
Results revealed that biodegradation of petroleum
hydrocarbons was enhanced by the addition of biosolids
(and also fertilizers to a lesser extent) to dieselcontaminated soils; the biosolids-treated soils showed
marked decrease in TPH concentrations and marked
increase in microbial population during week 1. After 8
weeks, the biosolids-amended soils and the high-rate
fertilizer-treated soils degraded more than 96% of the

original TPH contamination whereas the control soils

(MNA) degraded 94% of the original TPH contamination.
The reaction rates calculated for each treatment
followed a similar pattern with biosolids-amended soils
and the high-rate fertilizer treated soil experiencing the
fastest rate of degradation. The large increase in
microbial population in the biosolids amended soils
suggests that carbon supplementation may enhance
degradation of petroleum hydrocarbons in organicmatter or otherwise C-poor soils. The TPH degradation
rate and the amount of TPH degraded in the high-rate
fertilizer-treated soil when compared to those in the lowrate fertilizer-treated soil indicate that certain levels of N
and P supplementation may be capable of stimulating
hydrocarbon degradation similar to that provided by
sources that contribute C to the system.
These results reveal that MNA performed extremely
well paralleling the amended soils with only modest
dierences in reaction rates and total degradation. Soil
types (e.g. texture, composition, microbial population)
dier signicantly, even in geographically constrained
regions. Some soils will be poor candidates for MNA
while others will prove to be excellent candidates. A
greater understanding of the factors controlling MNA
eciencies must be investigated to maximize remediation while reducing costs.

1200

1100
Control 1 - Soil

Control 2 - Soil

High Rate Biosolid

Bioavailable Phosphorus (mg/kg)

Bioavailable Phosphorus (mg/kg)

1000

Low Rate Biosolid

900
800
700
600
500
400
300
200

Low Rate Fertilizer

High Rate Fertilizer

1000

800

600

400

200

100
0

0
Week 0

Week 1

Week 3

Week 5

Week 8

Time (week)

Fig. 5. Bioavailable phosphorus concentrations in low- and high-rate

biosolids-amended soils in comparison to control soils without
amendments.

Week 0

Week 1

Week 3

Week 5

Week 8

Time (week)

Fig. 6. Bioavailable phosphorus concentrations in low- and high-rate

fertilizer-amended soils in comparison to control soils without
amendments.

D. Sarkar et al. / Environmental Pollution 136 (2005) 187e195

References
Balkwill, D.L., Ghiorse, W.C., 1985. Characterization of subsurface
bacteria associated with two shallow aquifers in Oklahoma.
Applied and Environmental Microbiology 50, 580e588.
Batte, C.D., 1984. Soil Survey of Comal and Hays Counties, Texas.
United States Government Printing Oce, Washington, DC.
Bone, T.L., Balkwill, D.L., 1988. Morphological and cultural comparison of microorganisms in surface soil and subsurface sediments at
a pristine study site in Oklahoma. Microbial Ecology 16, 49e64.
Cho, B-H., Chino, H., Tsuji, H., Kunito, T., Nagaoka, K., Otsuka, S.,
Yamashita, K., Matsumoto, S., Oyaiz, H., 1997. Laboratory-scale
bioremediation of oil-contaminated soil of Kuwait with soil
amendment materials. Chemosphere 35 (7), 1599e1611.
Collin, P.H., 2001. Dictionary of Ecology and the Environment, fourth
ed. Peter Collin Publishing, London.
Collins, C.H., Lyne, P.M., Grange, J.M., 1989. Microbiological
Methods, 6th Edition. Butterworth-Heinemann, London.
Colwell, F.S., 1989. Microbiological comparison of surface soil and
unsaturated subsurface soil from a semiarid high desert. Applied
and Environmental Microbiology 55, 2420e2423.
Dibble, J.T., Bartha, R., 1979. Eect of environmental parameters on
the biodegradation of oil sludge. Applied and Environmental
Microbiology 37, 729e739.
Duncan, K., Jennings, E., Buck, P., Wells, H., Kolhatkar, R.,
Sublette, K., Potter, W.T., Todd, T., 2003. Multi-species ecotoxicity assessment of petroleum-contaminated soil. Soil and Sediment
Contamination 12, 181e206.
Everitt, B., 2002. The Cambridge Dictionary of Statistics. Cambridge
University Press, Cambridge, UK.
Ferguson, M.C., 2003. Biodegradation of petroleum hydrocarbons in
contaminated soils: Eects of biosolids addition and role of
carbon. M.S. Thesis, University of Texas at San Antonio, Texas.
Fisher, W.L., 1983. Geologic Atlas of Texas, San Antonio Sheet.
Bureau of Economic Geology, Austin, TX.
Huesemann, M.H., Moore, K.O., 1993. Compositional changes during
landfarming of weathered Michigan crude oil-contaminated soil.
Journal of Soil Contamination 2 (3), 245e264.
Jackson, W.A., Pardue, J.H., 1998. Potential for enhancement of
biodegradation of crude oil in Louisiana salt marshes using
nutrient amendments. Water, Air, and Soil Pollution 109, 343e355.
Klute, A. (Ed.), 1996. Methods of Soil Analysis: Part 1: Physical and
Mineralogical Methods. SSSA Publications, Madison, WI.
Li, K.Y., Zhang, Y., Xu, T., 1995. Bioremediation of oil-contaminated
soil d a rate model. Waste Management 15, 335e338.
Liebeg, E.W., Cutright, T.J., 1999. The investigation of enhanced
bioremediation through the addition of macro and micronutrients
in a PAH contaminated soil. International Biodeterioration and
Biodegradation 44, 55e64.
Maki, H., Hirayama, N., Hiwatari, T., Kohata, K., Uchiyama, H.,
Watanabe, M., Yamasaki, F., Furuki, M., 2003. Crude oil
bioremediation eld experiment in the Sea of Japan. Marine
Pollution Bulletin 47, 74e77.
Margesin, R., Schinner, F., 1997. Laboratory bioremediation experiments with soil from a diesel-oil contaminated site e signicant
role of cold-adapted microorganisms and fertilizers. Journal of
Chemical and Biotechnology 70, 92e98.
Margesin, R., Schinner, F., 2001. Bioremediation (natural attenuation
and biostimulation) of diesel-oil-contaminated soil in an alpine
glacier skiing area. Applied and Environmental Microbiology 67,
3127e3133.

195

McBride, M.B., 2003. Toxic metals in sewage sludge-amended soils:

has promotion of benecial use discounted the risks? Advances in
Environmental Research 8, 5e19.
Moller, J., Gaarn, H., Steckel, T., Wedbye, E.B., Westermann, P.,
1995. Inhibitory eects on degradation of diesel oil in soilmicrocosms by a commercial bioaugmentation product. Bulletin
of Environmental Contamination Toxicology 54, 913e918.
Namkoong, W., Hwang, E., Park, J., Choi, J., 2002. Bioremediation of
diesel-contaminated soil with composting. Environmental Pollution 119, 23e31.
Nocentini, M., Pinelli, D., Fava, F., 2000. Bioremediation of a soil
contaminated by hydrocarbon mixtures: the residual concentration
problem. Chemosphere 41, 1115e1123.
OConnor, G.A, Sarkar, D., 1999. Fate of land applied residuals
bound phosphorus. Final Project Report; Florida Department of
Environmental Protection, Contract #WM661.
Pankrantz, T.M., 2001. Environmental Engineering Dictionary and
Directory. CRC Press, Boca Raton, FL.
Quinn, J.W., Reinhart, D.R., 1997. Bioremediation of diesel contaminated soil-using biopiles. Practice Periodical of Hazardous, Toxic,
and Radioactive Waste Management 1, 18e25.
Riser-Roberts, E., 1998. Remediation of Petroleum Contaminated
Soil: Biological, Physical, and Chemical Processes. CRC Press
LLC, Boca Raton, FL.
Rosenberg, E., Legmann, R., Kushmaro, A., Taube, R., Adler, E.,
Ron, E.Z., 1992. Petroleum bioremediation d a multiphase
problem. Biodegradation 3, 337e350.
Sanchez, M.A., Campbell, L.M., Brinker, F.A., Owens, D., 2000.
Attenuation the natural way. A former wood-preserving site oers
a case study for evaluating the potential of monitored natural
attenuation. Industrial Wastewater 5, 37e42.
Sims, R., Bass, J., 1984. Review of in-place treatment techniques for
contaminated surface soils, vol. 1, Technical Evaluation. EPA
Report 540/2-84-003a, USEPA.
Snoeyink, V.L., Jenkins, D., 1980. Water Chemistry. Wiley, New
York.
Sparks, D.L. (Ed.), 1996. Methods of Soil Analysis: Part 1: Physical
and Mineralogical Methods. SSSA Publications, Madison, WI.
SPSS, 1997. Sigma Plot version 4.01. SPSS, Chicago, IL.
TCEQ, 2001. Total Petroleum Hydrocarbons. Method 1005, Revision
03. http://www.tnrcc.state.tx.us/enforcement/csd/qa/method_1005.
Ting, Y.P., Hu, H.L., Tan, H.M., 1999. Bioremediation of petroleum
hydrocarbons in soil microcosms. Resource and Environmental
Biotechnology 2, 197e218.
Vasudevan, N., Rajaram, P., 2001. Bioremediation of oil sludgecontaminated soil. Environment International 26, 409e411.
Walworth, J.L., Reynolds, C.M., 1995. Bioremediation of a petroleum-contaminated cryic soil: eects of phosphorus, nitrogen, and
temperature. Journal of Soil Contamination 4 (3), 299e310.
Williams, C.M., Grimes, J.L., Mikkelsen, R.L., 1999. The use of
poultry litter as co-substrate and source of inorganic nutrients and
microorganisms for the ex situ biodegradation of petroleum
compounds. Poultry Litter 78, 956e964.
Wilson, J.T., McNabb, J.F., Balkwill, D.L., Ghiorse, W.C., 1983.
Enumeration and characterization of bacteria indigenous to
a shallow water-table aquifer. Groundwater 21, 135e141.
Zhou, E., Crawford, R., 1995. Eects of oxygen, nitrogen, and
temperature on gasoline biodegradation in soil. Biodegradation 6,
127e140.
Zitrides, T., 1983. Biodecontamination of spill site. Pollution
Engineering 15, 25e27.