ENVIRONMENTAL ENGINEERING SCIENCE

Volume 21, Number 4, 2004

Mary Ann Liebert, Inc.

Biosurfactant Enhancement of Microbial Degradation

of Various Structural Classes of Hydrocarbon
in Mixed Waste Systems
Suresh Inakollu,1 Huang-Chin Hung,2 and Gina S. Shreve2,*
1Enviro

Matrix Inc.
Detroit, MI 48202
2Department of Chemical Engineering and Materials Science
Wayne State University
Detroit, MI 48202

ABSTRACT
This study examines the effect of two rhamnolipid biosurfactants on the first-order biodegradation rate
constant for a microbial consortium growing on a mixture of hydrocarbons representing four structural
classes of hydrocarbons. The microbial biodegradation rate of hexadecane, dodecane, benzene, toluene,
iso-octane, pristane (2,6,10,14 tetramethyl pentadecane), naphthalene, and phenanthrene in the presence
and absence of a mixture of rhamnolipid biosurfactant was determined. A first-order biodegradation model
was applied in these studies to better discern the differential solubilization and biodegradation rates for
specific structural classes of hydrocarbons in hydrocarbon mixtures. The biodegradation rate was enhanced
by the addition of biosurfactant levels above the critical micelle concentration for all hydrocarbon species
except phenanthrene and naphthalene. The time required for complete removal of each of hydrocarbons
from the culture was shortened due to the presence of surfactant, indicating a clear pattern in their order
of removal based upon their structural class. The rhamnolipid biosurfactants enhanced the rate of linear
alkane biodegradation more than the biodegradation rate of the monoaromatics. The rate constants for
hexadecane and dodecane increased by 111 and 76% to 4.7 and 0.3 mg/h, respectively, while those of
benzene and toluene increased by 34 and 65% to 3.1 and 4.0 mg/hr, respectively. The branched alkane
degradation rate constants were also increased by 71% for iso-octane and 39% for pristane. In contrast,
the biodegradation rate for the polycyclic aromatic hydrocarbons (PAHs), naphthalene and phenanthrene,
decreased by 25 and 27%, respectively. These were the only biodegradation rate decreases noted upon
addition of biosurfactant. Biodegradation rates of all other hydrocarbon species were characterized by
shorter times to total removal from the culture as well as increased removal rates. Hence, micellar solubilization affects the rate of hydrocarbon degradation for various hydrocarbons differently depending upon
their ability to partition into the micellar core. In mixed waste systems, PAHs must compete with other
substrates to partition into the micelle and a decreased rate of biodegradation may be observed due to ex-

*Corresponding author: Wayne State University, Department of Chemical Engineering & Material Science, 5050 Anthony
Wayne Drive, Detroit, MI 48202. Phone: 313-577-3774; Fax: 313-577-3810; E-mail: gshreve@eng.wayne.edu

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INAKOLLU ET AL.

clusion, or very low levels of solubilization within the micelle. Surfactant solubilization and enhancement
of biodegradation appears most important for insoluble hydrocarbons, and is significantly influenced by
the size and structure of the hydrocarbon contaminant.
Key words: biodegradation; hydrocarbon solubilization; microbial; rhamnolipid
INTRODUCTION

ETROLEUM HYDROCARBONS are pollutants of concern

at many contaminated sites. While biodegradation of
petroleum hydrocarbon is a major mechanism of removal
in the subsurface, factors such as water solubility and hydrocarbon structure have been shown to greatly affect
natural attenuation times in the environment. Coupled
processes involving surfactant solubilization and microbial biodegradation have potential for enhancing both the
rate and total removal of the hydrocarbons. Surfactants
are known for their capability of enhancing the biodegradation of hydrocarbons from oil spills in surface waters
by reducing the surface tension and the droplet size,
which increases the dissolution rate (King and Martel,
1989). A few field experiments have indicated the potential for an enhanced biodegradation of subsurface hydrocarbons in the presence of surfactants (Rittman and
Johnson, 1989). Some laboratory studies indicated an enhanced rate of degradation of hydrocarbons in the presence of some nonionic surfactants below their critical micelle concentration (CMC) (Aronstein and Alexander,
1993; Aronstein et al., 1995), where as other investigators observed a strong inhibition of the biodegradation of
phenanthrene in the presence of some nonionic surfactants
which was presumed due to biocidal effects of the synthetic surfactant agent (Laha and Luthy, 1991). Biosurfactants possess the advantages that they may be produced
in situ, may be less biocidal, as well as being readily
degradable so as not to function as a pollutant themselves.
Rhamnolipid biosurfactants are produced by several
Pseudomonas species, and have been demonstrated to enhance the microbial biodegradation rate of alkane hydrocarbons in subsurface environments (Bury and Miller,
1993; Thangamani and Shreve, 1994; Sekelsky and
Shreve, 1999). While surfactants have been used as dispersants in the treatment of hydrocarbon contaminated
marine spills, their ability to enhance the solubilization
and subsequent biodegradation of specific classes of hydrocarbons typically found in complex mixtures in the
environment has not been systematically investigated.
This study examines the effect of two rhamnolipid biosurfactants on the first-order biodegradation rate constant
for a microbial consortia growing on a mixture of hydrocarbons including four structural classes of hydrocarbons. The biodegradation rate was examined for hexa-

decane, dodecane, benzene, toluene, iso-octane, pristane

(2,6,10,14 tetramethyl pentadecane), naphthalene, and
phenanthrene. A mechanistic solubilization and biodegradation model has been previously validated for hexadecane (Sekelsky and Shreve, 1999). In this study, apparent first-order degradation rates were compared for the
above classes of hydrocarbon to determine the influence
of rhamnolipid surfactant on observed biodegradation of
varying structural classes of hydrocarbon.
A well-designed surfactant-enhanced bioremediation
process should consider methods to mobilize hydrocarbon contaminants from the nonaqueous phase liquid
(NAPL) and make them available to the microbial population. However, no systematic study has been reported
that investigates surfactant enhanced biodegradation in a
mixture of hydrocarbons and how the rate of degradation
of the individual hydrocarbons is influenced by externally
added surfactants. The objective of this study is to further quantify the effect of biosurfactant solubilization of
four structural classes of hydrocarbons on the biodegradation kinetics of hydrocarbons present in complex hydrocarbon mixtures in the environment.

MATERIALS AND METHODS

Microbial media and hydrocarbon
mixture composition
The microbial culture medium was a slightly modified
version of the 4M medium used by Guerra-Santos et al.
(1986). KH2PO4 and K2HPO4 were used in the place of
H3PO4 at concentrations of 4 g/liter and 5 g/liter, respectively. The modified 4M medium used contained 2
g/liter NaNO3, 0.5 g/liter NaCl, 0.5 g/liter KCl, 0.025
g/liter CaCl2, 0.25 mg/liter FeSO4, 0.45 mg/liter H3BO3,
0.75 mg/liter ZnSO4  7H2O and 0.75 mg/liter MnSO4 
H2O. The hydrocarbon mixture was prepared as an organic liquid stock consisting of: 60 mL of benzene, 60
mL of toluene, 60 mL of iso-octanes, 60 mL of 2,6,10,14
tetramethyl pentadecane (pristane), 120 mL of hexadecane, 120 mL of dodecane, 3 g of naphthalene, 3 g of
phenanthrene, and 10 mL of trichloroethylene (TCE).
The mixture composition is 12% (v/v) for each monoaromatic and branched compound, 24% (v/v) for each linear alkane, 2% (w/v) for each polycyclic aromatic
hydrocarbon (PAH), and 2% (v/v) for TCE. In all ex-

BIOSURFACTANT ENHANCEMENT OF MICROBIAL DEGRADATION

periments, 1% (v/v) of super oil was added to the minimal medium. The biodegradation rate of TCE was not
measured, as no microbial strains were added that catabolized TCE under aerobic culture conditions.

Microbial strains and experimental procedure

The micro-organisms used in this study were a mixed
consortia of Pseudomonas aeriginosa strain PG201,
Rhodococcus H131A, and a Pseudomonas strain which
produced the rhamnolipid Dyna270. The microbial consortia inocula was grown together in 50 mL 4M medium
containing 12% of the hydrocarbon mixture in shaken
Erlhenmeyer flasks at 3035C. The culture flasks were
shaken in a Forma Scientific model 4580 rotary shaker
operating at 120 rpm at room temperature. Purified Dyna
270, and a mixture of rhamnolipids R1 and R3 was obtained from Drs. William Finnerty and Urs Ochsner, respectively. The surface active properties of these rhamnolipid biosurfactants are typical of rhamnolipids, and
have been described in detail elsewhere (Thangamani and
Shreve, 1994; Hung and Shreve, 2001).
Experiments were conducted with and without added
biosurfactant to determine the influence of externally
added biosurfactants on hydrocarbon degradation. While
the micro-organisms used for biodegradation have the
ability to produce biosurfactants, exogenous biosurfactants were added to avoid lags and kinetic changes due to
nonconstant biosurfactant concentration. Thus, the experiments were conducted over a relatively short time duration. The kinetic data used for determination of biodegradation rates was obtained within the initial 72 h of each
experiment. Exogenous biosurfactant was added to a concentration six times the CMC for each of the biosurfactants. The CMC of the mixed surfactant micelles against
the hydrocarbon mixture was determined to be 6.9 mg/L
using a Kruss K12 interfacial tensiometer and the DuNouy
ring push method. Interfacial tensions in the cultures were
monitored periodically to determine the presence and effectiveness of the biosurfactants. The interfacial tension
in the culture to which biosurfactant was added remained
approximately constant at approximately 12.75 mN/m, indicating no apparent degradation of biosurfactant over the
course of the experiment. Cultures where no biosurfactant
was added remained above 68 mN/m.
Ten milliliters of the mineral medium was added in
50- mL vials to maintain enough headspace. The vials
were selected with Teflon-coated caps and sterilized.
One milliliter of the hydrocarbon mixture was injected
into the vials. All experimental flasks contained the same
amount of biomass as 0.5 mL of inoculum culture was
added from one actively growing culture flask to experimental treatments with and without surfactant. The inoculum was grown under identical rpm, temperature, and

465

nutrient conditions as the experimental cultures. At approximately 24 h of growth the inoculum was applied to
the experimental flasks. Sterile air was injected every 6
h through a syringe to prevent oxygen deficiency in experimental flasks and abiotic controls. The experiments
were conducted over 7 days and sampled at 6-h intervals.
The entire solution of the vial was extracted with hexane
for every sampling point. Extraction efficiencies were
measured separately in samples containing known quantities of each hydrocarbon and biosurfactant and were determined to be typically greater than 60%. All concentrations reported were corrected as necessary to account
for the extraction efficiency of the specific hydrocarbon.
Triplicates of the samples were analyzed to calculate
standard deviations associated with each time point concentration. These standard deviations reflected error inherent in extraction and instrument, and were consistently
less than 7% of the experimental value. A control vial
was autoclaved to heat-kill micro-organisms prior to hydrocarbon addition, and was sampled every 12 h as an
abiotic control. Maximum losses observed in the abiotic
bottles did not exceed 5% for the most volatile components benzene and toluene, and were negligible for other
hydrocarbons. The entire solution of each bottle was extracted by hexane for gas chromatography (GC) analysis
to avoid sampling errors. Samples were diluted as necessary to maintain concentrations within the calibration
standard range. The following first-order rate law was
used to simply describe the combined solubilization and
biodegradation rate (dCtot)/(dt)  kdCtot. In this rate
law the first-order constant (kd) represents the coupled
processes of hydrocarbon solubilization in the micelle,
transport to the micro-organism, and microbial catabolism of hydrocarbon substrate.

Hydrocarbon analysis
All hydrocarbons except TCE were analyzed on a
Perkin-Elmer Autosystem GC using a flame ionization
detector. A Restek Rtx-1, 0.53-mm i.d. megabore column
with a stationary phase thickness of 7 m was used for
the hydrocarbon analysis. Helium was used as the carrier
gas at a flow rate of 8 mL/min. The oven temperature
program started at an initial temperature of 125C, held
at this temperature for 0.5 min, then proceeded with a
single temperature ramp from 125 to 167C at a rate of
10C/min. The flow rates of hydrogen and air were 40
and 400 mL/min, respectively. The injector temperature
was held at 280C, and the detector temperature was held
at 290C. The total hydrocarbon concentration in each
experimental bottle was measured by extracting the
serum bottle contents with 20 mL of pure HPLC-grade
hexane. Triplicate 1-L samples of the hexane phase
were then analyzed on the GC to determine the average
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hydrocarbon concentration. The calibration standards of

each hydrocarbon were used to develop linear calibration
curves for the instrument over an approximate range of
0.1 to 3 ppm for each hydrocarbon. External standards
for each hydrocarbon were interspersed throughout the
GC sample analysis runs to ensure GC precision between
samples was generally within 1%.

RESULTS
Biodegradation rates in the absence
of exogenous biosurfactants
Biodegradation rates for each of the hydrocarbons
were measured over the 57-day experimental time period with the linear alkane and monoaromatic hydrocarbons, dodecane, and hexadecane, being largely degraded
within 72 h (Fig. 1). Since linear alkane hydrocarbon concentrations were twofold higher in the hydrocarbon mixture the biodegradation rate for dodecane (3.6 mg/h) was
higher than that of benzene (2.33 mg/h) and toluene (2.43
mg/h), while that of hexadecane was similar at a rate of
2.2 mg/h. Branched alkanes, iso-octanes, and pristane
were degraded within 5 days. Phenanthrene and naphthalene were detectable at concentrations below 60 ppm

Figure 1.

at the conclusion of the experimental period. Over the

course of this same experimental period the stoppered
abiotic experimental hydrocarbon concentrations remained approximately constant. Hence, the order of disappearance of the hydrocarbons from the culture mixture
demonstrated biodegradation of monoaromatic and linear alkanes followed by branched alkanes followed by
PAHs. Apparent first-order rate constants were calculated
by linear regression analysis over the approximately linear portion of the degradation rate curve, typically 12 h
and 3, 5, or 7 days, depending on the hydrocarbon as discussed above, for each hydrocarbon species (Table 1).
These apparent biodegradation rate constants confirm
that the monoaromatic hydrocarbons, benzene and
toluene, and hexadecane were degraded at similar rates
with dodecane being degraded at a slightly higher rate by
microbial consortia in the presence of added biosurfactant. Also, both branched alkanes, iso-octane and pristane, were degraded at a higher rate than either naphthalene or phenanthrene.

Biodegradation rates in the presence of

exogenous biosurfactant
The biodegradation rate was enhanced for all hydrocarbon species except phenanthrene and naphthalene

Biodegradation kinetics of hydrocarbon species in the absence of a surfactant.

BIOSURFACTANT ENHANCEMENT OF MICROBIAL DEGRADATION

Table 1.

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Biodegradation kinetics in the absence and presence of an exogenous surfactant.

Hydrocarbon
Benzene
Toluene
Iso-octanes
Pristane (2,6,10,14 tetramethyl pentadecane)
Hexadecane
Dodecane
Naphthalene
Phenanthene

Rate without
surfactant
(mg per hour)

Rate with
surfactant
(mg per hour)

2.33
2.43
0.87
2.01

3.13
4.01
1.49
2.79

34.3
65
71.3
38.8

2.20
3.60
0.36
0.73

4.65
6.34
0.27
0.53

111
76.1
25
27.4

(Fig. 2). Hence, the time required for removal of the hydrocarbon from the culture was shortened to roughly 2
days for monoaromatics, 3 days for alkanes, and 4 days
for branched alkanes, indicating a slight shift in their removal order to that which is reflected by the effect of exogenous surfactant on the apparent first-order rate constant. The rhamnolipid mixture enhanced the rate of linear
alkane biodegradation more than that of the monoaromatics. The biodegradation rate constants for hexadecane
and dodecane increased by 111 and 76%, respectively,
while those of benzene and toluene increased by 34 and

Figure 2.

Percent increase

65%, respectively (Table 1). The branched alkane degradation rate constants were also increased by 71 for isooctane and 38% for pristane (Table 1). The abiotic negative control showed little physical losses over the
experimental period. In contrast, the biodegradation rate
constant for the PAHs, naphthalene and phenanthrene, decreased by 25 and 27%, respectively. These were the only
biodegradation rate decreases noted upon addition of biosurfactant. Biodegradation rates of all other hydrocarbon
species were characterized by shorter times to total removal from the culture as well as increased removal rates.

Biodegradation kinetics of hydrocarbon species in the presence of a rhamnolipid biosurfactant.

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DISCUSSION
The objective of these studies is to determine the effect of presence of biosurfactant on the observed
biodegradation rate of hydrocarbons from four structural
classes in a mixed waste system. Previous work with
structurally similar nonionic surfactants indicates that anionic surfactant micelles have definite solubilization preferences and accompanying interfacial effectiveness for
varying classes of hydrocarbons (Hung and Shreve,
2001). These interfacial packing properties preferences
roughly follow the biodegradation preferences observed
here, indicating what has been confirmed in further studies that competition in the micelle among various structural classes of hydrocarbons controls the solubilization
preferences observed in mixed waste systems (Hung and
Shreve, 2002; Inakollu and Shreve, 2002). While it is
likely that the solubilization step is rate limiting for most
of the substrates (Goswami and Singh, 1991; Sekelsky
and Shreve, 1999), it is difficult to measure this step independently for NAPL solubilization of a hydrocarbon
contaminant. The hydrocarbon specificity of these rhamnolipid biosurfactants for these substrates has been recently determined (Inakollu and Shreve, unpublished
data). These hydrocarbon solubilization studies for pure
hydrocarbons and mixed hydrocarbon systems have indicated that the biosurfactants hydrocarbon specificity,
or packing of hydrocarbon substrate within the micelle
core, is influenced by the size and shape of the hydrocarbon substrate in the mixed waste systems.
In a mixed waste system hydrocarbon substrates compete for a position in the micelle as well as for the catabolic resources of the micro-organisms. Hydrocarbon
solubilization studies for pure hydrocarbons and mixed
hydrocarbon systems have indicated that in the presence
of mixed wastes the biosurfactant hydrocarbon specificity, or packing of hydrocarbon substrate within the micelle core, is influenced by the size and shape of the hydrocarbon substrate. In the mixed waste systems the
micellar solubilization of phenanthrene decreases, presumably due to both size and shape constraints. Naphthalene micellar solubilization increased very slightly in
the mixture. This decreased micellar solubilization of
PAH constituents in hydrophobic organic contaminant
mixtures would directly explain the biodegradation rates
determined in this study in that phenanthrene biodegradation rate was decreased and the pristane biodegradation
rate was increased by only 38.8%, whereas iso-octane was
increased by 71%, indicating that the larger sized pristane
molecule may have been less favorably solubilized into
the micelle than the smaller branched iso-octanes. One
important effect of adding exogenous biosurfactant was
to change the observed order of biodegradation to that

of linear alkane  monoaromatic  branched alkane 

PAH, which is consistent with the catabolic preferences
of the aerobic organisms used in this study.
In this study, biosurfactant addition enhanced hydrocarbon biodegradation for each hydrocarbon substrate except the PAHs, naphthalene, and phenanthrene. Hence,
micellar solubilization appears to effect the rate of hydrocarbon degradation for these four hydrocarbon differently depending upon their ability to partition into the micellar core. In mixed waste systems, where PAHs must
compete with other substrates within the micelle, a decreased rate of biodegradation may be observed, potentially due to exclusion or very low levels of solubilization
within the micelle and transport to the micro-organism.
This mechanism of micellar enhancement of biodegradation will be most important for insoluble hydrocarbons,
but may be significantly influenced by the size and structure of both the surfactant and the hydrocarbon.

ACKNOWLEDGMENTS
The authors would like to thank Leland Rosenberger,
MS, for his help in developing of the GC analytical
methodology, and Dr. William Finnerty of Dynazyme,
Inc. for provision of microbial strains and Dyna-270
rhamnolipid biosurfactant. This work was supported by
the Environmental Protection Agencys Office of Exploratory Research under Research Grant R820 399-01-0
to Dr. G.S. Shreve.

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