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 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,
aban- 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 landll, and incineration are
expensive. Incineration is
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 dened 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 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 hydrocar- bon 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
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 eec- tive,
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
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 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.
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) 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 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%
(OConnor 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-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.
2.5. 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 ame
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 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).
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
signicantly 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 rst 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 - SoilLow Rate BiosolidHigh 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 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) 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 rst
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
3.3. Degradation rates of TPH
5000

4500 Kinetic modeling was performed to estimate the

4000 Control 2 - SoilLow Rate FertilizerHigh Rate Fertilizer rates of chemical reactions in the studied systems.
TPH Concentration (mg/kg)

Kinetics of a chemical reaction can be described in

3500
terms of its order (Snoeyink and Jenkins, 1980). While
3000 other authors (Nocentini et al., 2000; Namkoong et al.,
2002) adequately described their hydrocarbon
degradation data using a rst-order kinetic model, in
2500

2000 the present study, experimental results were better

1500 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
1000

500 diagram. For a rst-order reaction, the plot of ln[TPH]

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 fertilizer- versus time should be a straight line while for a
amended soils in comparison to control soils without amendments. second- order 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
week in the fertilizer-amended soils (Fig. 2). By week by curvilinear equations, whereas plots of 1/[TPH]
4, 92.3% of the original TPH was removed in the versus time were better described by linear models
2
MNA treatment (control 2), 91.5% in the low-rate (data not shown). The R value represents the
2
fertilizer treatment, and 93.8% in the high-rate coecient of correlation; the nearer the value of R to
fertilizer treat- ment. By the end of the experiment 1, the stronger the correlation of the data (Everitt,
2
period (week 8), 94.5% of the original TPH was 2002). The R values for the linear plots using the
removed in the MNA treatment (control 2), 95.5% in second-order model were higher than those obtained
the low-rate fertilizer treatment, and 96.8% in the using the rst-order model in ve of the six
high-rate fertilizer treat- ment. 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 second-order rate law is equal to 8k, where k is the
authors (Ting et al., 1999; Nocentini et al., 2000). rate constant (Snoeyink and Jenkins, 1980). Although
Namkoong the dierences between the calculated reaction rates
et al. (2002), however, did not show such a huge were statistically insignicant, TPH degradation in
reduction in TPH concentration in the control pan; the MNA soils were described by the lowest reaction
this can be attributed to the lack of aeration in their rate constants, the biosolids (both rates) and high-
study. Table 2 shows the initial and nal TPH rate fertilizer amended soils had the fastest reaction
concentration in each soil and the percent decrease in rate constants (Table 4). Rate constant of the low-rate
TPH concentration through 8 weeks of treatment. The fertilizer amended soil was intermediate. The rate
low-rate and high- rate biosolids-amended soils constants were reective of the relative eects of
degraded a similar percent- age of the original TPH various treatments on TPH degradation in diesel-
contamination. The low-rate and high-rate fertilizer- contaminated soils.
amended soils also degraded a similar percentage of
the original TPH contamination. The untreated soils
(MNA) degraded lesser percentages of the original 3.4. Microbial population
TPH contamination than the treated soils, although
the dierences were not signicant at the 95% The total viable microbial population of the
condence interval using the LSD method. biosolids treated soils and associated MNA soils are
presented in
Table 2 Table 3
Initial and nal TPH concentrations in control, biosolids and Comparison of correlation coecients for rst- and second-order
fertilizer amended soils

Matrix Initial TPH Final TPH Percent linear models

concentration concentration decrease Matrix First-order linear Second-order linear
2 2
(mg/kg) (mg/kg) in TPH model R model R
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
9
(MNA) soil (10 cfu/g soil dry weight) were much
7 al.
higher than the 10 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 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 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
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
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
ControlLow Rate BiosolidHigh 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
constant (per day) throughout the experimental period (Fig. 4); the
5 viable microbe count increased by only 124% and
1.00!10
1.25!10
5 140% (compared to a 57% increase in MNA) in the
1.25!10
5
low- and high-rate fertilizer treated soils, respectively
5
1.00!10 (Table 5).
5
1.13!10
5 Dramatic changes in microbial population have
1.25!10
been reported by other authors experimenting with
nutrient- supplemented, hydrocarbon-contaminated
soils (Ting
et al., 1999; Vasudevan and Rajaram, 2001). Microbial
population in the nutrient system studied by Ting et
(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 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
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 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; Riser- Roberts, 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).
 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 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
fertilizer- amended 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 biosolids- amended soils were within or near the
optimum pH range for hydrocarbon degradation of
6.5 to 8.5 (Riser- Roberts, 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
1.00E+10
Microbial Population (cfu)

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 hydro- carbon degradation was
observed in the biosolids- amended 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 fertilizer- treated soils
(124e140%) in comparison with that in biosolids-
treated soils (11 384e12 340%; Table 5). Ap- parently,
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 incuba- tion 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 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 tion.
identied such synergistic eects of biosolids
addition on TPH biodegradation. The eects of The reaction rates calculated for each treatment
chemical toxicity in the fertilizer-amended systems followed a similar pattern with biosolids-amended
were immediate and gradu- ally waned with soils and the high-rate fertilizer treated soil
incubation time, thereby resulting in a similar experiencing the fastest rate of degradation. The large
degradation pattern of residual TPH in fertilizer- increase in microbial population in the biosolids
amended and biosolids-amended soils. amended soils suggests that carbon supplementation
may enhance degradation of petroleum hydrocarbons
in organic- matter or otherwise C-poor soils. The TPH
degradation rate and the amount of TPH degraded in
4. Summary and conclusions the high-rate fertilizer-treated soil when compared to
those in the low- rate fertilizer-treated soil indicate
that certain levels of N and P supplementation may
Bioremediation can be a viable and eective be capable of stimulating hydrocarbon degradation
response to soil contamination by petroleum similar to that provided by sources that contribute C
hydrocarbons. This investigation compared to the system.
monitored natural attenuation with two methods of These results reveal that MNA performed
biostimulation: inorganic fertilizer amendment and extremely well paralleling the amended soils with
biosolids amendment, at two rates, low and high. only modest dierences in reaction rates and total
Results revealed that biodegradation of petroleum degradation. Soil types (e.g. texture, composition,
hydrocarbons was enhanced by the addition of microbial population) dier signicantly, even in
biosolids (and also fertilizers to a lesser extent) to geographically constrained regions. Some soils will be
diesel- contaminated soils; the biosolids-treated soils poor candidates for MNA while others will prove to
showed marked decrease in TPH concentrations and be excellent candidates. A greater understanding of
marked increase in microbial population during week the factors controlling MNA eciencies must be
1. After 8 weeks, the biosolids-amended soils and the investigated to maximize remedia- tion while
high-rate fertilizer-treated soils degraded more than reducing costs.
96% of the

1100 1200
Control 2 - SoilLow Rate FertilizerHigh Rate Fertilizer
Control 1 - SoilLow Rate BiosolidHigh Rate Biosolid
1000
1000
Bioavailable Phosphorus (mg/kg)

900

800 800
700
Bioavailable Phosphorus (mg/kg)

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- Fig. 6. Bioavailable phosphorus concentrations in low- and high-
rate biosolids-amended soils in comparison to control soils without rate fertilizer-amended soils in comparison to control soils without
amendments. amendments.
 D. Sarkar et al. / Environmental Pollution 136 (2005) 187e195 195
and biostimulation) of diesel-oil-contaminated soil in an alpine
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