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Enzymes for Enhancing Bioremediation of

Petroleum-Contaminated Soils: A Brief Review
a a
Chi-Yuan Fan & S. Krishnamurthy
a
Releases Control Branch, Risk Reduction Engineering Laboratory , U.S.
Environmental Protection Agency , Edison , New Jersey , USA
Published online: 05 Mar 2012.

To cite this article: Chi-Yuan Fan & S. Krishnamurthy (1995) Enzymes for Enhancing Bioremediation of Petroleum-
Contaminated Soils: A Brief Review, Journal of the Air & Waste Management Association, 45:6, 453-460, DOI:
10.1080/10473289.1995.10467375

To link to this article: http://dx.doi.org/10.1080/10473289.1995.10467375

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 TECHNICAL PAPER
Enzymes for Enhancing Bioremediation of
Petroleum-Contaminated Soils: A Brief Review
Chi-Yuan Fan and S. Krishnamurthy
Releases Control Branch, Risk Reduction Engineering Laboratory,
U.S. Environmental Protection Agency, Edison, New Jersey
ABSTRACT
During the 1950s and 1960s, hundreds of thousands of
underground storage tanks (and above-ground storage
tanks) containing petroleum products and hazardous
chemicals were installed. Many of these tanks either have
been abandoned or have exceeded their useful lives and
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are leaking, thereby posing a serious threat to the nation's
surface and groundwater supplies, as well as to public
health. Cleaning up releases of petroleum hydrocarbons
or other organic chemicals in the subsurface environment
is a real-world problem,
Biological treatment of hydrocarbon-contaminated soil
is considered to be a relatively low-cost and safe technol-
ogy; however, its potential for effectively treating recalci-
trant wastes has not been fully explored. For millions of
years, microorganisms such as bacteria, fungi, actinomycete,
protozoa, and others have performed the function of recy-
cling organic matter from which new plant life can grow.
This paper examines the biological treatment technol-
ogy for cleaning up petroleum product-contaminated soils,
with special emphasis on microbial enzyme systems for en-
hancing the rate of biodegradation of petroleum hydrocar-
bons. Classifications and functions of enzymes, as well as
the microbes, in degrading the organic contaminants are
discussed. In addition, the weathering effect on biodegra-
dation, types of hydrocarbon degraders, advantages associ-
ated with enzyme use, methods of enzyme extraction, and
future research needs for development and evaluation of
enzyme-assisted bioremediation are examined.
IMPLICATIONS
Soil contaminated by petroleum products is a pervasive
problem in the United States. Regulations have mandated
the contaminant limits for various petroleum products. For
millions of years, microorganisms have performed the func-
tion of recycling organic matter from which new plant life
can grow. This natural process can be emulated, perhaps
even accelerated, and used for detoxification. Under cer-
tain conditions, intrinsic biodegradation could be enhanced
and considered as a cost-effective alternative for environ-
mental and health risk control.
Volume 45 June 199S
ISSN 1047-3289 /. Air & Waste Manage. Assoc. 45: 453-460
Copyright 1 DOS Air & Waste Management Association
INTRODUCTION
One of the major problems encountered as we approach a
new century is environmental pollution. Environmental
pollution cannot be accepted as inevitable for technologi-
cal society. Soil contaminated by petroleum products is a
pervasive problem in the United States. The cleanup of these
contaminated soils is a severe challenge. Regulations have
mandated the contaminant limits for various petroleum
products. Consideration of the current technology for re-
moval of pollutants clearly shows that physical and chemi-
cal methods are often uneconomical. However, biological
treatment technology may offer a solution to this problem.
Not all processes generating polluting waste can be replaced
with clean alternatives. However, pollution can be controlled
both at the source and in the contaminated plume after an
incident.
For millions of years, microorganisms such as bacteria,
fungi, actinomycete, protozoa, and others have performed
the function of recycling organic matter from which new
plant life can grow. This natural process can be emulated,
perhaps even accelerated, and used for detoxification. Mi-
croorganisms present in the environment can eventually
transform most of the toxic organics. The subject of biodeg-
radation has been treated critically.1 Environmental enhance-
ment, such as adjustment of pH, temperature, nutrient levels,
and aeration, often may be necessary to facilitate the
biotransformation process. Microbes generally metabolize
organic matter to produce energy for their synthesis, move-
ment, and respiration. Simple, naturally occurring organic
compounds are readily incorporated into the cells of the
microbes and oxidized under aerobic conditions. When mi-
crobes come into contact with complex organic materials,
extracellular enzymes are released to convert high molecu-
lar weight materials into diffusible fractions, which could
be transported through the cell wall for assimilation. Indig-
enous microbial populations, especially heterotrophic bac-
teria and fungi, are the chief agents causing biodegradation.
Microbes that transform specific compounds can be isolated,
cultured, adapted, and enriched under laboratory conditions.
This review will discuss limitations of petroleum hydro-
carbon biodegradation by microbes and the advantages of
enzyme systems for enhancing biodegradation.
Journal of the Air & Waste Management Association 453
 Fan and Krishnamurthy

be exposed to biodegradation. Therefore, the lighter hydro-

COMMON NAMI SYSTEMATIC NAME TYPICAL, MEMBER
Paraffin Alkane CH3-CH3
carbon fractions (e.g., short-chain aliphatics and single-ring
Olefin Alkene CH2=CH2 aromatic hydrocarbons) in a gasoline contaminated-soil site
will have a relatively higher biodegradation rate than a No.
H H H H H H H H 2 fuel oil-contaminated site because of relatively high solu-
I I I I I I I I bility in water of gasoline compositions.
_ _ _ _ _ . cCH
H c c c c c c
Weathering can affect the composition concentrations
I I I I I I I I of petroleum products. The constituents of a fresh petro-
H H H H H H H H
Paraffin leum product can differ from those of a weathered sample
that contains a depletion of some straight-chain alkanes.
H H H H H H
We know that straight-chain hydrocarbons are more easily
I I I I I I degraded than their branched-chain counterparts. Because
H _ C C C C = C C C CH

I I I I I I I I the branched-chain hydrocarbons are more resistant to bio-

H H H H H H H H degradation, weathered petroleum product-contaminated
Otefln
soils typically have low straight-chain to branched-chain
ratios, thus extending bioremediation duration.

BIODEGRADATION OF PETROLEUM

jQi,
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\ /
H-C-C-H
H H
Cyeloalkane
Figure 1 . Aliphatic, cyeloalkane, and aromatic structures.
CHEMICAL CONSTITUENTS OF
PETROLEUM PRODUCTS
Most petroleum products released from leaking underground
storage tanks are referred to as single bulk fluids. However,
petroleum products comprise a vast continuum of hydro-
carbonsfrom short-chain aliphatic and simple aromatic
hydrocarbons in gasoline to kerosene, diesel, and heavy oils
and to lubricating oils or Vaseline, each gradation with in-
creasing carbon chains and complexity. Figure 1 illustrates
the basic structure of petroleum hydrocarbons. Gasolines
are light distillates with a boiling-point range of -12 to
200 C, and their chemical composition varies significantly
with the sources of crude oil and the blending process to
ensure proper fuel volatility and car performance at each
locale and for each season. Theoretically, gasoline could con-
tain more than 1,200 different hydrocarbons, of which ap-
proximately 230 are in the carbon number range of C3 to
C12.2 The middle distillates, such as diesel and jet fuels with
a boiling-point range from 170 to 340 C, contain the car-
bon range of C10 to C28. Heavy petroleum products (e.g.,
lubricating oil, paraffin wax, and asphalt) have boiling points
higher than 350 C.
Table 1 indicates the major constituents of three com-
monly used petroleum products (e.g., automotive gasoline,
No. 2 fuel oil, and No. 4 jet fuel). In evaluating the effects of
biological treatment of these petroleum products contami-
nation on a particular site, a knowledge of the solubility in
water and the number of carbon atoms of the petroleum
products will be essential. For instance, the hydrocarbons
with high solubility in water and low carbon number will
HYDROCARBONS
Petroleum microbiology has been well documented.3 More
Aromatic
than 200 species of bacteria, yeasts, and fungi have been
identified, which are capable of degrading hydrocarbons.4
In order of importance, these are as follows: (1) heterotrophic
bacteria, (2) fungi, (3) aerobic bacteria, (4) actinomycete, (5)
phototrophic microbes, and (6) oligotrophic bacteria.
Table 2 lists common aerobic microorganisms that de-
grade petroleum hydrocarbons. As indicated, Corynebacte-
rium spp. are believed to be major agents causing
hydrocarbon degradation. Pseudomonas spp. can transform
many different compounds. They are adaptable to bring
about biotransformation through plasmid transfer of genetic
material. Fungi have considerable ability to degrade hydro-
carbons of complex structures and long chain length. Bac-
teria and yeasts, on the other hand, show decreasing ability
to degrade alkanes with increasing chain length. Organisms
in two orders of fungiMucorales (such as Cunningharriella)
and Monililiales (Fusarium, Aspergillus, and Penicillium)
show high biotransformation potential. Fungal metabolism
often results in incomplete metabolism; hence, subsequent
association with bacteria is necessary for complete mineral-
ization. In many cases, bioremediation may be combined
with a complementary physical or chemical process to im-
prove the reliability and effectiveness of both processes.
Figure 2 illustrates general oxidative pathways for eucary-
otic and procaryotic organisms. The first step in aerobic deg-
radation of aromatics is the activation of the ring for cleavage
by hydroxylation.
Both short- and long-chain alkanes are oxidized to the
corresponding alcohol, aldehyde, and then acid.5 The pri-
mary alcohol derived from the alkane is believed to be oxi-
dized to the aldehyde by an alcohol dehydrogenase, and
the aldehyde oxidized to the acid by an aldehyde dehydro-
genase. Fatty acids resulting from the alkanes are thought
to be oxidized by an inducible beta oxidation system to the

454 Journal of the Air & Waste Management Association Volume 45 June 1995
 Fan and Krishnamurthy

Table 1 . Unweathered composition of three common hydrocarbon products.

Selected Representative Concentrations (% w/w)

Hydrocarbon Representative Solubility in Automotive Jet Fuel
Group Hydrocarbon Water (mg/L) Gasoline #2 Fuel Oil JP-4
n-Alkanes 10.8-29.6
04 n-butane 61.4 4.8-7.0 0.12
C5 n-pentane 38.5 1.9-4.5 1.06
C6 n-hexane 13.3 2.0- 12.9 2.21
C7 n-heptane 2.2 0.2-2.3 3.67
C8 n-octane 0.43 1.3 3.80
09 n-nonane 0.12 0.4 - 0.8 2.25
C10-C14 n-decane 0.05 0.2-0.8 8.73
Branched Alkanes 18.8-59.5
04 Isobutane 49 0.7-2.2 0.66
C5 Isopentane 48 8.6- 17.3
C6 2-methylpentane 78 4.6-9.7 2.27
C7 2-methylhexane 2.54 1.4-8.3 5.48
C8 2,4-dimethylhexane 1.29 1.8- 16.7 8.82
09 2,2,4-trimethylhexane 0.53 1.2-2.7 3.36
Others 0.5-2.6 1.35
Cycloalkanes 3.2- 13.7
C6 Cyclohexane 55 0.2 2.40
C7 Methylcyclohexane 14 1.0-3.9 3.77
C8 1,2,4-trimethylcyclopentane 0.2- 1.4 1.35
C9 1,1,3-trimethylcyclohexane 1.77 0.2-0.7 3.21
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Others 1.6-7.5
Olefins 5.5- 13.5
C4 1-butene 222 0.9
C5 1-pentene 148 1.3-3.3
C6 1-hexene 69.7 0.8- 1.8
Others 2.5-7.5
Monoaromatics 18.6-40.2
Benzene Benzene 1,760 0.9-4.4 0.50
Toluene Toluene 470 4.0-6.5 1.33
Xylenes m-xylene 172 5.6-8.8 0.07 2.32
Ethyl benzene Ethyl benzene 140 1.2-1.4 0.03 0.37
C3-benzenes 1,3,4-trimethylbenzene 48.2 3.2-11.3 0.67 3.59
C4-benzenes 1,4-diethylbenzene 15 2.1 -2.6 0.88 3.98
Others 1.6-5.2
Phenols
Phenol Phenol 82,000 0.001
C1-phenols o-cresol 31,000 0.01
C2-phenols 2,4-dimethylphenol 4,600 0.02
C3-phenols 2,4,6-trimethylphenol 14,000 0.02
C4-phenols m-ethylphenol 0.01
Indanol Indanol 0.001
Polyaromatics
Fluorene 1.69 0.57
Aromatic amines
C1-anilines 0.003
C2-anilines 0.004
Heterocyclic base Quinoline 60,000 0.002
Di-aromatics Naphthalene 30 0.7 3.43 1.59
Carboxylic acids Benzoic acid 2.7 0
Saturated hydrocarbons
08 n-octane 0.43 0.05
09 n-nonane 0.12 0.20
010 n-decane 0.05 0.58
011 n-undecane 0.003 0.98
012 n-dodecane 0.008 1.14
013 n-tridecane 0.013 1.20
014 n-tetradecane 0.0022 1.31
015 n-pentadecane 1.42
016 n-hexadecane 0.0009 1.53
017 n-heptadecane 1.51
018 n-octadecane 1.31
019 n-nonadecane 1.16
020 n-eicosane 0.99
021 n-heneicosane 0.51
022 n-docosane 0.29
023 n-tricosane 0.15
024 n-tetracosane 0.05
Pristane 0.52
Phytane 0.46
Waxes
Base compounds
Unknowns 6.6- 13.8

NOTE: Blanks indicate the unavailability of data and do not indicate the absence of a particular compound from the hydrocarbon product.

Volume 45 June 1995 Journal of the Air & Waste Management Association 455
 Fan and Krishnamurthy

Table 2. Aerobic microbial alkanes, cycloalkanes, and aromatic degraders.

ALKANES AROMATICS AROMATICS

Bacteria Bacteria Fungi

Pseudomonas Pseudomonas Phyktochytrium
Acinetobacter Aeromonas Phizophlyctis
Alcaligenes Moraxelia Phytophthora
Torulopsis Beijerinckia Thraustochytrium
Bacillus Flavobacterium Hyphochytrium
Arthrobacter Achromobacter Cunningharriella
Chlorella Nocardia Syncephalastrum
Brevibacterium Corynebacterium Mucor
Corynebacterium Gilbertella
Mycobacterium Algae Absidia
Oscillatoria Zygorrhynchus
Fungi Microcoleus Cokeromyces
Candida Nortoc Choanephora
Saccharomyces Anabaena Phycomyces
Streptomyces Agmenellum Circinella
Coccochloris Thamnidium
Aphanocapsa Rhizopus
CYCLOALKANES Porphyridium Basidiobolus
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Petalonia Conidiobolus
Bacteria Cylindrotheca Smittium
Bacterium aliphaticum Amphora Saproiegnia
Achromobacter Nitzschia Saccharomyces
Pseudomonas Navidula Emericellopsis
Corynebacterium Chorella Neuropora
Micrococcus Dunaliella Sordana
Nocardia Chlamydomonas Claviceps
Sarcina Ulva Candida
Mycobacterium Debarydmyces
Acetobacter Psilocybe
Alcaligenes Panaeolus
Aspergillus
Fungi Penicillium
Penicillium Curvuiarea
Guocladium
Epicocurn
Epicocum
Pestalotia
Helicortylum

level of acetate for even-chain alkanes and propionate for
odd-chain alkanes. Acetate is subsequently oxidized in the
Krebs cycle. Although the mechanism of the initial step in
alkane oxidation is not clear, oxygen has been determined
to be an obligatory reactant in alkane oxidation catalyzed
by microbial systems. Diterminal and subterminal oxida-
tion pathways are also known.6-7
BIOLOGICAL TREATMENT OF
PETROLEUM-CONTAMINATED SOIL
A recent paper8 sets forth in detail the attractiveness of
bioremediation of petroleum-contaminated soils.
Bioremediation of petroleum-contaminated soil offers a
cost-competitive treatment for many sites that are currently
faced with costly incineration or the extended liability of
land disposal. In the field, under conditions of full-scale
site remediation, this technology has been shown to be
cost effective.910
A key factor that all biotechnology applications share is
that the application design must effectively compensate for
the rate-limiting effects of various parameters, both micro-
biological and environmental. Some of these rate-limiting
factors, which may apply at a specific site, are as follows:
The complete absence or low density of a bacterial
population or species that is capable of degrading
hydrocarbons,
Temperature and moisture,
Oxygen supply,
Available nutrient supply,
Contaminant bio-availability and soil chemistry, and
Population ecology.
Temperature affects both the specific growth rate of the
degrading microbes and the activity of the enzyme respon-
sible for the oxidation of the contaminant. For most petro-
leum-contaminated soils, the temperature that provided
favorable growth was 27 C.

4 5 6 Journal of the Air & Waste Management Association Volume 45 June 1995
 Fan and Krishnamurthy

the cells themselves.12 Of these, the glycolipids produced

OH
H have the additional advantage of nontoxidty.
trans-Dihydrodiol
cit-Dihydrocliol
by fermentation are the most important. Many of the gly-
colipids are extremely effective surfactants, lowering the in-
terfacial tension to below 0.0001 dynes/cm.13 Such materials
The contaminant's chemical structure may also limit the
rate. The chain length and degree of branching influence
the rate of biodegradation. The time required to degrade
petroleum hydrocarbons substantially increases with chain
length. Usually, the order of persistence to biodegradation
is Bunker C, diesel, No. 2 heating oil, jet fuel, and gaso-
line,14 because of a higher boiling point and lower solubil-
ity of hydrocarbon constituents contained in heavier
products.

Figure 2. General oxidative pathways for eucaryotic and procaryotic ADVANTAGES OF USING ENZYME SYSTEMS IN
organisms. SOIL BIOREMEDIATION
The use of microbes for bioremediation is beset with many
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rate-limiting factors. Several limitations apply to the use of

Most hydrocarbon degradation is caused by aerobic bac-
microbes for detoxification. Costly and time-consuming
teria. This is largely because of the great energy-yielding ca-
methods may be necessary to produce microbial cultures.
pacity of aerobic respiration relative to both anaerobic
Furthermore, severe conditions such as chemical shock, ex-
respiration and fermentation. Soil oxygen content is fre-
tremes of pH and temperature, toxins, predators, and high
quently a rate-limiting factor in bioremediation.11 To miti-
concentrations of the pollutants or their products may irre-
gate the potential for oxygen limitation, delivery of oxygen
versibly damage or metabolically inactivate microbial cells.
is included, either by air injection or by the addition of hy-
Difficulty in maintaining active cells during transportation
drogen peroxide. Usually the nutrient deficiency of soils is
to the polluted site also limits the use of whole-cell detoxifi-
corrected by adding nitrogen and phosphorus.
cation technologies. Other factors that could restrict the use
The biological availability of hydrophobic contaminants
of microbes include limited mobility of the cells within the
is often the rate-limiting factor in biodegradation. This fac-
soil, alternate carbon sources, and weakness of the inocu-
tor is a function of soil chemistry. In many soils, the con-
lated microorganisms in competition with the indigenous
taminant may be unavailable because of contaminant
population.15
hydrophobicity, sorption onto soil colloids, or dissolution
into soil organic matter. Hence, a surfactant may have to be Biotransformation involves a series of enzyme-catalyzed
incorporated in the treatment. Some bacteria secrete their reactions. Thus, most of these adverse factors can either be
own surfactants. During the growth of microbes, an emulsi- eliminated or mitigated if the enzymes (preferably immobi-
fication of water-insoluble hydrocarbons often occurs, which lized) are used instead of the microbes. As previously indi-
is generally mediated either by extracellular products or by cated, biodegradation is mediated by microbial enzymes.16
Enzymes are complex proteins functioning as bio-oxidation
catalysts that cause a host of reactions. They exhibit specific-
ity and are characterized by showing an optimum tempera-
/
,'T\
i \ y. Activation energy of a
ture and pH for their actions. Similar to other catalysts, they
/ i ' nonanzymatlc reaction accelerate the chemical reaction rate by lowering the activa-
/ i
tion energy for a particular reaction. As shown in Figure 3,17
\\ the activation energy for the enzyme-catalyzed reaction is
\
SS Energy

/ i
/ i ^ x - Activation energy of an
^ - ^ ^ enEymatlcally catalyzed
much smaller than that for the nonenzyme-catalyzed reac-
reaction
tion. This results in a much faster reaction rate. Furthermore,
A \
enzymatic catalysis involves the formation of an intermedi-
Educts
Enzym:
educts
\ ^ Energy ol
rsactlon
ate complex on binding sites on the enzyme. The tremen-
complex Enzyme:
products dous increase in the rate of a reaction under enzyme catalysis
complex
Proc ucts
can be illustrated with a simple example: the hydrolysis of
sucrose by yeast invertase at 37 C is a trillion times faster
Progress of Reaction
than that caused by hydrogen ions.
Figure 3. The activation energy of an enzymatically catalyzed re- According to the Commission on Enzymes of the Inter-
action as compared to a noncatalyzed chemical reaction. national Union of Biochemistry, enzymes are classified into

Volume 45 June 1995 Journal of the Air & Waste Management Association 457
 Fan and Krishnamurthy
six general groups*8: (1) oxidoreductases, catalyzing oxi-
dation-reduction reactions; (2) transferases, catalyzing a
chemical group from one molecule to another; (3) hydro-
lases, catalyzing hydrolytic enzymes; (4) lyases, catalyzing
the addition of groups to double bonds or vice versa; (5)
isomerases, catalyzing intramolecular rearrangements; and
(6) ligases (or synthetases), catalyzing the condensation
of two molecules coupled with the cleavage of a pyrophos-
phate bond of ATP or similar triphosphate.
The enzymes responsible for hydroxylation of aromatic
rings are the oxygenases, both of the mono-oxygenase type
that inserts one oxygen atom of an oxygen molecule into
their substrates or the dioxygenase type that inserts two
oxygen atoms into their substrates. Subsequently, the ring
is cleaved and, by a series of reactions, is converted to
2-ketoadipate or another compound that can be utilized
by the organism. The biodegradation process of naphtha-
lene is shown in Figure 4.
Bollag19 discussed the decontamination of soils using
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enzymes. The use of horseradish peroxidase in the detoxi-
fication of industrial wastewater has been studied by
Maloney et al.20 The enzyme laccase oxidizes phenolic
compounds to form their corresponding anionic free radi-
cals. The ability of the laccase from the fungus R. pmticola
to transform and thereby detoxify phenolic compounds
has also been described by Bollag et al.21
Enzymes bound to solid supports show increased effi-
ciency. The immobilization of laccase on soil supports in-
creases the enzyme's thermostability, its resistance to
degradation by protease, and its half-life.22 Compared to
the free enzyme, the immobilized enzyme retains its ac-
tivity for a much longer period. Furthermore, the immo-
LOJ - \g
NAPHTHALENE CIS-NAPHTHALENE DIHYDRODIOL SALICYLIC ACID
CH 3 - CO - SCoA
"C COOH
COOH
ACETYL-CO-ENZYMEA 3-OXO-ADIPICACID CATECHOL
CH3-COOH CO2 + H2O
Figure 4. Biodegradation process of naphthalene.
4 5 8 Journal of the Air & Waste Management Association
bilized enzyme could be recovered from the reaction solu-
tion and reused to transform substrate, with minimal loss
of its activity.23
Immobilized enzymes may prove more economical be-
cause they are biochemically stable and can be used repeat-
edly to detoxify xenobiotics. Laccase can be purified from a
commercial strain of Agaricus biisporus.24 The use of an im-
mobilized bacterial enzyme (a hydrolase) for detoxification
of pesticides has been described by Munnecke.25 The crude
enzyme was immobilized on porous glass. A laccase of the
basidiomycetes Trametes versicolor was immobilized on po-
rous glass beads. The enzyme laccase has the advantage that
it does not require the addition of hydrogen peroxide such
as peroxidase.26 The glass beads support immobilized 100%
of the enzyme, and 90% of the original activity was retained.
After immobilization, the enzyme was active in a wider pH
and temperature range, and its heat stability and reuse were
greatly improved, compared to those of the free laccase. The
immobilized enzyme was reusable in treating different sub-
strates. The use of enzymes to detoxify pesticide-contami-
nated soils and waters has also been reviewed by Nannipieri
and Bollag.27
For the reasons previously stated, the use of cell-free en-
zymes (preferably immobilized) offers great promise. Intra-
cellular enzyme preparations are often unstable under
cell-free conditions. Enzyme stability can be increased by
immobilizing it on a solid support. Immobilized enzymes
can be easily recovered and reused, can display enhanced
stability to extremes of temperature and pH, and are more
resistant to proteolytic degradation. Thus, their life in soil is
prolonged. More than 100 enzyme immobilization proce-
dures have been elaborated, including covalent attachment
to solid supports, adsorption on solid supports, entrapment
in polymeric gels, cross-linking with biofunctional reagents,
and encapsulation within a solid support.28 Immobilization
techniques have been refined to the point at which almost
every enzyme can now be immobilized, with full retention
of activity.
The foregoing considerations underline the high poten-
tial for the use of immobilized enzymes in detoxification of
varied xenobiotics, and, most certainly, appropriate immo-
bilized enzymes could be used to accelerate remediation of
petroleum-contaminated soils.
CONCLUSIONS AND RECOMMENDATIONS FOR
FUTURE WORK
This article has attempted to emphasize the great promise
and potential shown by the technology that can be described
as enzyme-accelerated remediation of petroleum-contami-
nated sites. Profitable lines of future work that may aid in
realizing the technology's full potential are delineated as
follows:
1. The mechanism of action should be studied for the en-
zymes associated with the petroleum-degrading microbes.
Volume 45 June 1995

In this connection, mention can be made of the power-
ful tool of whole cells of bacteria.29
This recommendation is based on the unique capa-
bilities and the appropriateness of resonance Raman spec-
troscopy for investigating structural and mechanistic
features of delicate systems. The following outlines the
features of this method for use in the present context:
Raman spectroscopy is based on the scattering of light
with an accompanying shift in frequency. Raman spec-
troscopy, a scattering technique, provides detailed infor-
mation on the vibrations of molecules and hence direct
structural information on those molecules. Raman scat-
tering is a relatively weak effect. The advent of lasers with
high-powered monochromatic light sources has en-
hanced the capability for Raman spectroscopy. Resonance
Raman spectroscopy is a special case of the Raman effect
that occurs when the frequency of the laser excitation
source matches the frequency of an absorption band of
the molecule.30
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This matching of excitation and absorption frequen-
cies enhances by a thousandfold to a millionfold the in-
tensities of vibration. Hence, sensitivity increases, and a
degree of sensitivity is imparted to the method. The in-
vestigation of the heme protein cytochrome oxidase is
an example of the power of this method. When the heme
iron atoms are in their ferrous forms, the heme absorp-
tion from this protein occurs at 442 nm. Using a laser
frequency of 441.6 nm, Raman spectra of this protein
within whole cells of bacteria have been obtained. This
type of whole-cell study opens the way for investigating
the enzymatic mechanism of this and other enzymes in
their natural biological venues.31
2. The possible uses of thermophilic enzymes for hydrocar-
bon mineralization should be examined. Aldolases from
thermophilic bacteria are known to accept a broad range
of compounds at catalytic rates more than 100 times
greater than previously known aldolases. Thermophylic
enzymes are able to catalyze reactions in harsh environ-
ments, and this exciting possibility should be explored.
3. The study of the microbiology of subsurface environment
has brought to light some new knowledge that can be
useful in petroleum detoxification. Highly diverse mi-
crobial populations with a broad array of metabolic ac-
tivities exist in an environment that, until the past few
decades, was considered devoid of life.32 The enzymes
associated with the subsurface should be isolated, im-
mobilized, and studied for their ability to degrade petro-
leum hydrocarbons.
4. Because the availability of the contaminant to the en-
zyme is crucial, the addition of suitable surfactants dur-
ing bioremediation should be studied. As previously
mentioned,12-13 surface active compounds are produced
by some bacteria. A glycolipid was found to lower dra-
matically the interfacial tension. These glycolipids should
Volume 45 June 19S5
Fan and Krishnamurthy
be studied, along with other possible natural surfactants,
with a view to use them in conjunction with the immobi-
lized enzymes.
5. Some stable enzymes have been isolated from soil. A stable
peroxidase was extracted from soil using 50 mM phos-
phate buffer at pH 6.0.33 The enzyme was believed to be a
glycoprotein, and its stability in soil presumably arose from
the protective action afforded by the polysaccharide. The
possibility of the occurrence of other stable enzymes in
the soil, such as lipoproteins, should be examined with a
view to their potential use. It is intriguing to speculate on
the possible effects of adding a polysaccharide to the en-
zyme to be immobilizedthis combination may work
well. A cell-free esterase was isolated from soil by 0.2 M
alkali extraction and 560-fold purification by manganese
chloride treatment, protamine sulfate treatment, and
QAE-sephadex A-50 chromatography.34 The esterase was
not subject to proteolysis nor was it inactivated metal ions.
A search should be made for the possible occurrence of
other cell-free enzymes, and a study of these may be
helpful.
Finally, the concept of using enzyme systems for en-
hancing bioremediation of petroleum hydrocarbon-
contaminated soils is relatively new; reported data mainly
are limited to laboratory experiments. Future research
should emphasize large-scale pilot studies to evaluate the
effectiveness of cell-free enzymes in contrast to microbial
inoculation, analyze the cost of cell-free enzymes and cul-
turing organism systems, and develop engineering pro-
cess design parameters and system optimization models
coupled with enzyme kinetics.
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About the Authors
Mr. C. Fan is an environmental engineer at the Re-
leases Control Branch, Risk Reduction Engineering
Laboratory, U.S. Environmental Protection Agency,
2890 Woodbridge Avenue, Edison, NJ 08837. He is a
diplomate of the American Academy of Environmen-
tal Engineers and has specialized in water pollution
control and remediation of soils at leaking underground
storage tank sites.
Dr. S. Krishnamurthy is a senior chemist from the
American Association of Retired Persons working at
the Releases Control Branch, Risk Reduction Engi-
neering Laboratory, U.S. Environmental Protection
Agency, Edison, NJ 08837. He is a member of a re-
search group investigating new and innovative tech-
nologies for remediating contaminated soil.
Volume 45 June 1995