Environmental Pollution 136 (2005) 187e195

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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 effective 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 first 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 effect owing to fertilizer-induced acidity and/or NH3 overdosing. Results suggest that biosolids addition is a more
effective soil amendment method for biostimulation than the commonly practiced inorganic fertilizer application, because of the
abilities of biosolids to supplement carbon. No statistically significant difference 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.
Ó 2005 Elsevier Ltd. All rights reserved.

Keywords: Petroleum hydrocarbons; Biostimulation; Biosolids; Fertilizers; Natural attenuation

1. Introduction spillage during transport of petroleum products, aban-

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).
doned manufactured gasoline sites, other unplanned
releases, and current industrial processes. As petroleum
contains hazardous chemicals such as benzene, toluene,
ethylbenzene, xylenes, and naphthalene, this contami-
nation 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 cur-
rently treated using three processes: physical, chemical,
and biological. The most common physical methods of
treatment of contaminated soils, such as disposal in
a landfill, and incineration are expensive. Incineration is

0269-7491/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2004.09.025
188 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 in-
volves the breakdown of contamination into nontoxic
forms using microbiological processes (Riser-Roberts,
1998).
Bioremediation may be defined as the use of living
organisms to remove environmental pollutants from
soil, water, and gases (Collin, 2001). Organic com-
pounds are metabolized under aerobic or anaerobic
conditions by the biochemical processes of microorgan-
isms (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 micro-
organisms 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 micro-
organisms 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 micro-
organisms 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 benefits over landfill 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 effects
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
field 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 effective treatment
of hydrocarbon-contaminated sites (Huesemann and
Moore, 1993; Li et al., 1995; Zhou and Crawford, 1995;
Liebeg and Cutright, 1999). In the field 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 effects 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 hydrocar-
bon degradation (Ferguson, 2003). The primary benefits
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 difficulty
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 pro-
cesses 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 biostimu-
lation, monitored natural attenuation (MNA), if effec-
tive, provides significant benefits in terms of cost and
effort (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
difference 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
significant difference 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 diesel-
contaminated, carbon-poor soil. The secondary objec-
tive of the study was to determine if C supplementation
from biosolids application could be a factor of any
significance 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
differing approaches.
2. Materials and methods
2.1. Soil
Soil was collected from the Freeman Ranch in San
Marcos, Texas. It was dark brown in color and
consisted of 90% clay. Batte (1984) classified the soil
as Tarpley Clay that overlays the Edwards Limestone
Formation (Fisher, 1983). The soil was analyzed to
determine moisture content, pH and nutrient concen-
trations 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 molybdate-
ascorbic acid method (Sparks, 1996). Bioavailable P was
determined using the Mehlich-3 extraction technique
(Sparks, 1996).
2.2. Diesel
The diesel fuel used in this experiment is commer-
cially 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%
(O’Connor and Sarkar, 1999). The biosolids were
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-YieldÒ
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.
2.5. Microbial analysis
Microbial populations were determined using the 5-
tube 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).
2.6. Petroleum hydrocarbon analysis
A Varian CP 3800 Gas Chromatograph with flame
ionization detector (FID) and a Varian Chrompak
fused silica column was used to determine the total
petroleum hydrocarbon (TPH) concentration. Stand-
ards 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
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, commer-
cially available diesel fuel was added to each soil pan for
an initial total petroleum hydrocarbon (TPH) concen-
tration of approximately 4000 mg/kg (varied between
3350 and 4250 mg/kg). The soil, fertilizer/biosolids,
190 D. Sarkar et al. / Environmental Pollution 136 (2005) 187e195
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 concen-
tration, 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 five 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).
3. Results and discussion
3.1. Characterization of soil and amendments
The native soil had a neutral pH and low concen-
trations of C, N, total and bioavailable P (Table 1).
Largo biosolids had a pH of w6 and contained
significantly greater concentrations of total and bio-
available 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 first week in biosolids-amended soils (Fig. 1). The
Table 1
Characterization results for soils and amendments
Matrix Carbon (%) Nitrogen (%)
Largo biosolids 37.14G1.48 5.77G0.33
Inorganic fertilizer mix 0.00 35.0
Soil 1.53G0.12 0.15G0.02
4500
Control 1 - Soil Low Rate Biosolid High Rate Biosolid
4000
TPH Concentration (mg/kg)
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 biosolids-
amended 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 hydro-
carbons 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 first 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
profiles of diesel-contaminated soils (not shown) dem-
onstrated 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 degra-
dation (65.5e76.3%) was accomplished during the first
Total phosphorus (mg/kg) Bioavailable phosphorus (mg/kg) pH
34,800G1200 20400G1200 6.16G0.02
246,400G43,300 241900G16,800 3.24G0.02
275G92 9.7G1.8 7.14G0.11
 D. Sarkar et al. / Environmental Pollution 136 (2005) 187e195 191

5000 3.3. Degradation rates of TPH

Control 2 - Soil Low Rate Fertilizer High Rate Fertilizer
4500

4000 Kinetic modeling was performed to estimate the rates

TPH Concentration (mg/kg)

3500
of chemical reactions in the studied systems. Kinetics of
3000
a chemical reaction can be described in terms of its order
(Snoeyink and Jenkins, 1980). While other authors
2500
(Nocentini et al., 2000; Namkoong et al., 2002)
2000
adequately described their hydrocarbon degradation
1500 data using a first-order kinetic model, in the present
1000 study, experimental results were better described using
500 a second-order kinetic model.
0 To determine the order of the reactions in each of the
Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8
soil treatments, the data was plotted in a scatter
Time (week)
diagram. For a first-order reaction, the plot of ln[TPH]
Fig. 2. TPH degradation pattern in low- and high-rate fertilizer- versus time should be a straight line while for a second-
amended soils in comparison to control soils without amendments. order reaction, the plot of 1/[TPH] versus time should be
linear (Snoeyink and Jenkins, 1980). Data plots of
week in the fertilizer-amended soils (Fig. 2). By week 4, ln[TPH] versus time were better described by curvilinear
92.3% of the original TPH was removed in the MNA equations, whereas plots of 1/[TPH] versus time were
treatment (control 2), 91.5% in the low-rate fertilizer better described by linear models (data not shown). The
treatment, and 93.8% in the high-rate fertilizer treat- R2 value represents the coefficient of correlation; the
ment. By the end of the experiment period (week 8), nearer the value of R2 to 1, the stronger the correlation
94.5% of the original TPH was removed in the MNA of the data (Everitt, 2002). The R2 values for the linear
treatment (control 2), 95.5% in the low-rate fertilizer plots using the second-order model were higher than
treatment, and 96.8% in the high-rate fertilizer treat- those obtained using the first-order model in five of the
ment. six treatments (Table 3).
The degradation pattern in each treatment (Figs. 1 The slope of the line for reactions that follow the
and 2) is similar to the results obtained by other authors second-order rate law is equal to 8k, where k is the rate
(Ting et al., 1999; Nocentini et al., 2000). Namkoong constant (Snoeyink and Jenkins, 1980). Although the
et al. (2002), however, did not show such a huge differences between the calculated reaction rates were
reduction in TPH concentration in the control pan; this statistically insignificant, TPH degradation in the MNA
can be attributed to the lack of aeration in their study. soils were described by the lowest reaction rate
Table 2 shows the initial and final TPH concentration in constants, the biosolids (both rates) and high-
each soil and the percent decrease in TPH concentration rate fertilizer amended soils had the fastest reaction
through 8 weeks of treatment. The low-rate and high- rate constants (Table 4). Rate constant of the low-rate
rate biosolids-amended soils degraded a similar percent- fertilizer amended soil was intermediate. The rate
age of the original TPH contamination. The low-rate constants were reflective of the relative effects of various
and high-rate fertilizer-amended soils also degraded treatments on TPH degradation in diesel-contaminated
a similar percentage of the original TPH contamination. soils.
The untreated soils (MNA) degraded lesser percentages
of the original TPH contamination than the treated 3.4. Microbial population
soils, although the differences were not significant at the
95% confidence interval using the LSD method. The total viable microbial population of the biosolids
treated soils and associated MNA soils are presented in
Table 2
Initial and final TPH concentrations in control, biosolids and fertilizer Table 3
amended soils Comparison of correlation coefficients for first- and second-order
linear models
Matrix Initial TPH Final TPH Percent
concentration concentration decrease Matrix First-order linear Second-order linear
(mg/kg) (mg/kg) in TPH model R2 model R2
Control soil 1 3500G400 215G14 93.8G0.7 Control soil 1 0.8496 0.9532
Low-rate biosolids 3350G240 130G70 96.2G0.3 Low-rate biosolids 0.8867 0.7651
High-rate biosolids 3350G330 130G30 96.2G0.4 High-rate biosolids 0.751 0.8511
Control soil 2 4250G200 234G8 94.5G0.2 Control soil 2 0.8558 0.9373
Low-rate fertilizer 3950G150 175G87 95.5G0.2 Low-rate fertilizer 0.8867 0.9351
High-rate fertilizer 4050G300 130G47 96.8G0.3 High-rate fertilizer 0.7797 0.8998
192
Table 4
Second-order reaction rate constants for TPH degradation in control,
biosolids and fertilizer amended soils
Soil
Control soil 1
Low-rate biosolids
High-rate biosolids
Control soil 2
Low-rate fertilizer
High-rate fertilizer
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, respec-
tively (Table 5). The MNA soils also experienced an
increase in microbial population (57%; Table 5)
accompanied by significant 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 biosolids-
amended 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
significant differences 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
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
Control Low Rate Biosolid
1.00E+01
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.
D. Sarkar et al. / Environmental Pollution 136 (2005) 187e195
hydrocarbon degradation during the initial stages of
bioremediation. The microbial populations in the
fertilizer treated soils remained relatively invariable
Reaction rate throughout the experimental period (Fig. 4); the viable
constant (per day)
microbe count increased by only 124% and 140%
1.00!10ÿ5 (compared to a 57% increase in MNA) in the low- and
1.25!10ÿ5
1.25!10ÿ5
high-rate fertilizer treated soils, respectively (Table 5).
1.00!10ÿ5 Dramatic changes in microbial population have been
1.13!10ÿ5 reported by other authors experimenting with nutrient-
1.25!10ÿ5 supplemented, 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 micro-
bial growth, and/or (b) the potential toxic effect 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 degrada-
tion 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 effectiveness (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
High Rate Biosolid
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; Riser-
Roberts, 1998). However, using a fixed 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).
 D. Sarkar et al. / Environmental Pollution 136 (2005) 187e195 193

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 1.15EC09G1.31EC09 1.81EC09G1.36EC09 57
Low-rate biosolids 1.05EC09G3.61EC08 1.30EC11G1.00EC00 12300
High-rate biosolids 6.40EC08G2.12EC08 7.35EC10G7.78EC09 11400
Control soil 2 1.15EC09G1.3EC09 1.81EC09G1.36EC09 57
Low-rate fertilizer 5.43EC08G1.70EC08 1.21EC09G1.81EC09 124
High-rate fertilizer 1.91EC09G2.62EC09 4.58EC09G8.3EC09 140

Nutrient levels in the diesel-contaminated native and in the fertilizer-treated soils (Fig. 6). The TSP started
biosolids/fertilizer-amended soils are presented in Table releasing P immediately, almost 100% of which was
6. The MNA soils (controls 1 and 2) had less C, N, and available to the microbes. The w20% decrease in
P than the biosolids amended soils. The MNA soils had bioavailable P between week 0 and week 1 indicates
similar C percentage as the fertilizer-amended soils, but that the released P was being utilized by the microbes. It
significantly less N and P. The biosolids- and fertilizer- is also possible that a portion of the soluble/bioavailable
amended soils had similar concentrations of N and P, P leached to the bottom of the pan and was not sampled
but, expectedly, the biosolids-treated soils had much during analysis of surface soils. The die-off cycle for the
greater C content compared to the fertilizer-treated microbial population began in week 5 after which
soils. The pH of the MNA soils and the biosolids- the bioavailable P concentration became constant until
amended soils were within or near the optimum pH the end of the experiment (Fig. 6). Bioavailable P in the
range for hydrocarbon degradation of 6.5 to 8.5 (Riser- control soils (MNA) remained low throughout the
Roberts, 1998). The pH of the fertilizer-amended soils experiment (Figs. 5 and 6). Although N bioavailability
was below this optimum range (Table 6). was not monitored in the reported study, given the
The biosolids-treated soils experienced an increase of typical mineralization pattern of N in clay soils, it is not
bioavailable P during the first week of the experiment expected to be considerably different from the P profile.
(Fig. 5). This is due to the time-release nature of Despite the fact that the fertilizer-treated soils had
nutrients in the Largo biosolids; P was released slowly more bioavailable P and N, greater percentage hydro-
and did not become available to the microbes until after carbon degradation was observed in the biosolids-
one week of biosolids amendment. Between week 1 and amended soils during week 1 (Figs. 1 and 2). This is in
week 5, there was a steady decrease in bioavailable P as agreement with the much smaller increase in microbial
the microbes continued to metabolize the nutrients. population (normalized to MNA) noted in the fertilizer-
After week 5, as the microbes started to die off, the treated soils (124e140%) in comparison with that in
bioavailable P concentration in the biosolids treated biosolids-treated soils (11 384e12 340%; Table 5). Ap-
soils reached a constant level and remained that way parently, nutrient bioavailability was not the only factor
until the end of the experiment (Fig. 5). Bioavailable P governing TPH degradation. In a C-poor soil such as
decreased sharply during the first week of the experiment 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
1.00E+12 C:N:P ratio of 33:5:1 (Riser-Roberts, 1998). On the
1.00E+11 other hand, the C:N:P ratio in the biosolids-treated soils
1.00E+10 ranged between 27:4:1 and 24:4:1 (Table 6), which were
Microbial Population (cfu)

1.00E+09 much closer to the optimal C:N:P ratio for hydrocarbon

1.00E+08
degradation. Zhou and Crawford (1995) reported that
1.00E+07
excess N (C:NZ2:1) can impair biodegradation due to
1.00E+06
ammonia toxicity, although it does not completely
1.00E+05
inhibit biodegradation. In the fertilizer-treated soils,
1.00E+04
there is a possibility that ammonia toxicity (C:N ratio of
1.00E+03
2:1 and 3:1), coupled with the low pH (5.2e5.5)
1.00E+02
impacted TPH degradation in the very critical first
1.00E+01 Control Low Rate Fertilizer High Rate Fertilizer
week of incubation. The toxic effect on the microbial
1.00E+00
Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 population gradually decreased with increasing incuba-
Time (week) tion time possibly because of an increase in the rate of
Fig. 4. Total viable microbial population in low- and high-rate nitrification with increased tillage and aeration; NO3eN
fertilizer-amended soils in comparison to control soils without is typically not toxic to microbes. Because the biosolids
amendments. 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 1.53G0.12 0.15G0.02 0.03G0.01 56:5:1 7.14G0.16
Low-rate biosolids 3.22G0.11 0.48G0.02 0.12G0.01 27:4:1 6.56G0.03
High-rate biosolids 4.15G0.48 0.66G0.05 0.17G0.02 24:4:1 6.22G0.04
Control soil 2 1.54G0.22 0.15G0.01 0.03G0.01 50:5:1 7.14G0.09
Low-rate fertilizer 1.49G0.16 0.48G0.05 0.15G0.01 10:3:1 5.54G0.03
High-rate fertilizer 1.62G0.05 0.91G0.03 0.24G0.01 7:4:1 5.23G0.00

ratio in the C-poor Tarpley Clay, further aided by the original TPH contamination whereas the control soils
biosolids slow-release nature of bioavailable nutrient (MNA) degraded 94% of the original TPH contamina-
supplementation. Namkoong et al. (2002) also identified tion.
such synergistic effects of biosolids addition on TPH The reaction rates calculated for each treatment
biodegradation. The effects of chemical toxicity in the followed a similar pattern with biosolids-amended soils
fertilizer-amended systems were immediate and gradu- and the high-rate fertilizer treated soil experiencing the
ally waned with incubation time, thereby resulting in fastest rate of degradation. The large increase in
a similar degradation pattern of residual TPH in microbial population in the biosolids amended soils
fertilizer-amended and biosolids-amended soils. suggests that carbon supplementation may enhance
degradation of petroleum hydrocarbons in organic-
matter or otherwise C-poor soils. The TPH degradation
4. Summary and conclusions rate and the amount of TPH degraded in the high-rate
fertilizer-treated soil when compared to those in the low-
Bioremediation can be a viable and effective response rate fertilizer-treated soil indicate that certain levels of N
to soil contamination by petroleum hydrocarbons. This and P supplementation may be capable of stimulating
investigation compared monitored natural attenuation hydrocarbon degradation similar to that provided by
with two methods of biostimulation: inorganic fertilizer sources that contribute C to the system.
amendment and biosolids amendment, at two rates, low These results reveal that MNA performed extremely
and high. well paralleling the amended soils with only modest
Results revealed that biodegradation of petroleum differences in reaction rates and total degradation. Soil
hydrocarbons was enhanced by the addition of biosolids types (e.g. texture, composition, microbial population)
(and also fertilizers to a lesser extent) to diesel- differ significantly, even in geographically constrained
contaminated soils; the biosolids-treated soils showed regions. Some soils will be poor candidates for MNA
marked decrease in TPH concentrations and marked while others will prove to be excellent candidates. A
increase in microbial population during week 1. After 8 greater understanding of the factors controlling MNA
weeks, the biosolids-amended soils and the high-rate efficiencies must be investigated to maximize remedia-
fertilizer-treated soils degraded more than 96% of the tion while reducing costs.

1100 1200
Control 1 - Soil Low Rate Biosolid High Rate Biosolid Control 2 - Soil Low Rate Fertilizer High Rate Fertilizer
1000
Bioavailable Phosphorus (mg/kg)
Bioavailable Phosphorus (mg/kg)

900 1000

800
800
700

600
600
500

400
400
300

200 200
100

0 0
Week 0 Week 1 Week 3 Week 5 Week 8 Week 0 Week 1 Week 3 Week 5 Week 8
Time (week) Time (week)

Fig. 5. Bioavailable phosphorus concentrations in low- and high-rate Fig. 6. Bioavailable phosphorus concentrations in low- and high-rate
biosolids-amended soils in comparison to control soils without fertilizer-amended soils in comparison to control soils without
amendments. amendments.
 D. Sarkar et al. / Environmental Pollution 136 (2005) 187e195 195

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

has promotion of beneficial use discounted the risks? Advances in
Environmental Research 8, 5e19.
Balkwill, D.L., Ghiorse, W.C., 1985. Characterization of subsurface
Moller, J., Gaarn, H., Steckel, T., Wedbye, E.B., Westermann, P.,
bacteria associated with two shallow aquifers in Oklahoma.
1995. Inhibitory effects on degradation of diesel oil in soil-
Applied and Environmental Microbiology 50, 580e588.
microcosms by a commercial bioaugmentation product. Bulletin
Batte, C.D., 1984. Soil Survey of Comal and Hays Counties, Texas.
of Environmental Contamination Toxicology 54, 913e918.
United States Government Printing Office, Washington, DC.
Namkoong, W., Hwang, E., Park, J., Choi, J., 2002. Bioremediation of
Bone, T.L., Balkwill, D.L., 1988. Morphological and cultural compar-
diesel-contaminated soil with composting. Environmental Pollu-
ison of microorganisms in surface soil and subsurface sediments at
tion 119, 23e31.
a pristine study site in Oklahoma. Microbial Ecology 16, 49e64.
Nocentini, M., Pinelli, D., Fava, F., 2000. Bioremediation of a soil
Cho, B-H., Chino, H., Tsuji, H., Kunito, T., Nagaoka, K., Otsuka, S.,
contaminated by hydrocarbon mixtures: the residual concentration
Yamashita, K., Matsumoto, S., Oyaiz, H., 1997. Laboratory-scale
problem. Chemosphere 41, 1115e1123.
bioremediation of oil-contaminated soil of Kuwait with soil
O’Connor, G.A, Sarkar, D., 1999. Fate of land applied residuals
amendment materials. Chemosphere 35 (7), 1599e1611.
bound phosphorus. Final Project Report; Florida Department of
Collin, P.H., 2001. Dictionary of Ecology and the Environment, fourth
Environmental Protection, Contract #WM661.
ed. Peter Collin Publishing, London.
Pankrantz, T.M., 2001. Environmental Engineering Dictionary and
Collins, C.H., Lyne, P.M., Grange, J.M., 1989. Microbiological
Directory. CRC Press, Boca Raton, FL.
Methods, 6th Edition. Butterworth-Heinemann, London.
Quinn, J.W., Reinhart, D.R., 1997. Bioremediation of diesel contam-
Colwell, F.S., 1989. Microbiological comparison of surface soil and
inated soil-using biopiles. Practice Periodical of Hazardous, Toxic,
unsaturated subsurface soil from a semiarid high desert. Applied
and Radioactive Waste Management 1, 18e25.
and Environmental Microbiology 55, 2420e2423.
Riser-Roberts, E., 1998. Remediation of Petroleum Contaminated
Dibble, J.T., Bartha, R., 1979. Effect of environmental parameters on
Soil: Biological, Physical, and Chemical Processes. CRC Press
the biodegradation of oil sludge. Applied and Environmental
LLC, Boca Raton, FL.
Microbiology 37, 729e739.
Rosenberg, E., Legmann, R., Kushmaro, A., Taube, R., Adler, E.,
Duncan, K., Jennings, E., Buck, P., Wells, H., Kolhatkar, R.,
Ron, E.Z., 1992. Petroleum bioremediation d a multiphase
Sublette, K., Potter, W.T., Todd, T., 2003. Multi-species ecotox-
problem. Biodegradation 3, 337e350.
icity assessment of petroleum-contaminated soil. Soil and Sediment
Sanchez, M.A., Campbell, L.M., Brinker, F.A., Owens, D., 2000.
Contamination 12, 181e206.
Attenuation the natural way. A former wood-preserving site offers
Everitt, B., 2002. The Cambridge Dictionary of Statistics. Cambridge
a case study for evaluating the potential of monitored natural
University Press, Cambridge, UK.
attenuation. Industrial Wastewater 5, 37e42.
Ferguson, M.C., 2003. Biodegradation of petroleum hydrocarbons in
Sims, R., Bass, J., 1984. Review of in-place treatment techniques for
contaminated soils: Effects of biosolids addition and role of
contaminated surface soils, vol. 1, Technical Evaluation. EPA
carbon. M.S. Thesis, University of Texas at San Antonio, Texas.
Report 540/2-84-003a, USEPA.
Fisher, W.L., 1983. Geologic Atlas of Texas, San Antonio Sheet.
Snoeyink, V.L., Jenkins, D., 1980. Water Chemistry. Wiley, New
Bureau of Economic Geology, Austin, TX.
York.
Huesemann, M.H., Moore, K.O., 1993. Compositional changes during
Sparks, D.L. (Ed.), 1996. Methods of Soil Analysis: Part 1: Physical
landfarming of weathered Michigan crude oil-contaminated soil.
and Mineralogical Methods. SSSA Publications, Madison, WI.
Journal of Soil Contamination 2 (3), 245e264.
SPSS, 1997. Sigma Plot version 4.01. SPSS, Chicago, IL.
Jackson, W.A., Pardue, J.H., 1998. Potential for enhancement of
TCEQ, 2001. Total Petroleum Hydrocarbons. Method 1005, Revision
biodegradation of crude oil in Louisiana salt marshes using
03. http://www.tnrcc.state.tx.us/enforcement/csd/qa/method_1005.
nutrient amendments. Water, Air, and Soil Pollution 109, 343e355.
Ting, Y.P., Hu, H.L., Tan, H.M., 1999. Bioremediation of petroleum
Klute, A. (Ed.), 1996. Methods of Soil Analysis: Part 1: Physical and
hydrocarbons in soil microcosms. Resource and Environmental
Mineralogical Methods. SSSA Publications, Madison, WI.
Biotechnology 2, 197e218.
Li, K.Y., Zhang, Y., Xu, T., 1995. Bioremediation of oil-contaminated
Vasudevan, N., Rajaram, P., 2001. Bioremediation of oil sludge-
soil d a rate model. Waste Management 15, 335e338.
contaminated soil. Environment International 26, 409e411.
Liebeg, E.W., Cutright, T.J., 1999. The investigation of enhanced
Walworth, J.L., Reynolds, C.M., 1995. Bioremediation of a petro-
bioremediation through the addition of macro and micronutrients
leum-contaminated cryic soil: effects of phosphorus, nitrogen, and
in a PAH contaminated soil. International Biodeterioration and
temperature. Journal of Soil Contamination 4 (3), 299e310.
Biodegradation 44, 55e64.
Williams, C.M., Grimes, J.L., Mikkelsen, R.L., 1999. The use of
Maki, H., Hirayama, N., Hiwatari, T., Kohata, K., Uchiyama, H.,
poultry litter as co-substrate and source of inorganic nutrients and
Watanabe, M., Yamasaki, F., Furuki, M., 2003. Crude oil
microorganisms for the ex situ biodegradation of petroleum
bioremediation field experiment in the Sea of Japan. Marine
compounds. Poultry Litter 78, 956e964.
Pollution Bulletin 47, 74e77.
Wilson, J.T., McNabb, J.F., Balkwill, D.L., Ghiorse, W.C., 1983.
Margesin, R., Schinner, F., 1997. Laboratory bioremediation experi-
Enumeration and characterization of bacteria indigenous to
ments with soil from a diesel-oil contaminated site e significant
a shallow water-table aquifer. Groundwater 21, 135e141.
role of cold-adapted microorganisms and fertilizers. Journal of
Zhou, E., Crawford, R., 1995. Effects of oxygen, nitrogen, and
Chemical and Biotechnology 70, 92e98.
temperature on gasoline biodegradation in soil. Biodegradation 6,
Margesin, R., Schinner, F., 2001. Bioremediation (natural attenuation
127e140.
and biostimulation) of diesel-oil-contaminated soil in an alpine
Zitrides, T., 1983. Biodecontamination of spill site. Pollution
glacier skiing area. Applied and Environmental Microbiology 67,
Engineering 15, 25e27.
3127e3133.