Journal

of
Microbiological
Journal of Microbiological Methods 32 (1998) 155–164
Methods

Bioremediation of oil-contaminated soil: microbiological methods

for feasibility assessment and field evaluation
M.T. Balba*, N. Al-Awadhi, R. Al-Daher
Kuwait Institute for Scientific Research, P.O. Box 24885, 13109 Safat, Kuwait

Abstract

Bioremediation is emerging as a promising technology for the treatment of soil and groundwater contamination. The
technology is very effective particularly in dealing with petroleum hydrocarbon contamination. However, bioremediation is a
site-specific process and feasibility studies are required before full-scale remediation can be successfully applied. The type
and scale of the feasibility studies that will be needed are specific to the bioremediation approach to be employed during
full-scale clean-up operation. In all cases however, these studies have the same goals: to accurately determine if specific
hydrocarbon contaminants are amenable to biological treatment and to determine the time and cost required to treat the
contaminants of concern according to the regulated clean-up criteria. This contribution provides background information on
the chemistry and microbiology of hydrocarbon contamination, discusses the prospective of using biological methods for
addressing this problem and describes several microbiological methods which can be used for the feasibility assessment of
soil bioremediation. The focus of this chapter is to highlight the needs for the integration of laboratory data to full-scale
bioremediation.  1998 Elsevier Science B.V.

Keywords: Bioremediation; Petroleum hydrocarbons; Feasibility assessment

1. Introduction accidental spills that can be only minimized but not

There is growing public concern as a wide variety
of toxic organic chemicals are being introduced
inadvertently or deliberately into the environment.
Petroleum hydrocarbons are one common example of
these chemicals, which enter the environment fre-
quently and in large volumes through numerous
routes. The seepage from natural deposits is one of
the major routes by which petroleum oil enters
marine environments (National Academy of Science,
1975). Human activities in the production, trans-
portation and storage of petroleum is another route
since such activities inevitably involve the risk of
*Corresponding author.
eliminated entirely. In recent years, leakage of
gasoline from underground storage tanks primarily at
automobile service stations and from pipelines has
been experienced at an alarming rate. Marine spills
are also now becoming a frequent and major source
of water and coastal contamination (US Environmen-
tal Protection Agency, 1990). The Gulf War in 1991
resulted in the worst man-made environmental disas-
ter, with millions of gallons of crude oil released
from the destroyed oil wells into the waters and
surrounding land, forming more than 330 oil lakes,
covering an area of 49 square km (Salam, 1996).
Such releases of large quantities of oil to marine and
terrestrial environments present a longterm threat to
all forms of life. There is now an increasing need for

0167-7012 / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved.

PII: S0167-7012( 98 )00020-7
156 M.T. Balba et al. / Journal of Microbiological Methods 32 (1998) 155 – 164
cost-effective remediation technologies for hydro-
carbon contamination.
2. Composition of petroleum hydrocarbons
Petroleum hydrocarbons contain a complex mix-
ture of compounds which can be categorized for
simplicity into four fractions: saturates, aromatics,
resins (N, S, O) and asphaltene (Shell International
Ltd., 1983). The saturates fraction includes straight
chain alkanes (normal alkanes), branched alkanes
(isoalkanes), and cycloalkanes (naphthenes). The
aromatic fraction contains volatile monoaromatic
hydrocarbons such as benzene, toluene, xylenes etc.,
polyaromatic hydrocarbons, naphthenoaromatics and
aromatic sulphur compounds such as thiophenes and
dibenzothiophenes. It is noteworthy that the poly-
aromatic hydrocarbon (PAHs) fraction which is
associated with oil contamination, includes both
suspected and known carcinogens, the most toxic
being benzo(a)pyrene. The resins (N, S, O) and
asphaltene fractions consist of polar molecules con-
taining nitrogen, sulphur and oxygen. Resins are
amorphous solids which are truly dissolved in oil,
whereas asphaltenes are large molecules colloidally
dispersed in oil. The relative proportions of these
fractions are dependent on many factors such as the
source, geological history, age, migration and altera-
tion of crude oil.
3. Biodegradation of petroleum hydrocarbons
Current evidence suggests that in aquatic and
terrestrial environments microorganisms are the chief
agents for the biodegradation of molecules of en-
vironmental concern, including petroleum hydrocar-
bons (Alexander et al., 1982; Swanell and Head,
1994). Hydrocarbon-degrading bacteria, yeast and
fungi are widely distributed in marine, fresh water
and soil habitats. Bacteria and yeast appear to be the
dominant degraders in aquatic ecosystems while
fungi and bacteria are the main degraders in soil
environments (Cooney and Summers, 1976; Hanson
et al., 1997). There is a vast amount of literature on
the subject of oil breakdown by microorganisms with
several major review papers (Atlas, 1977; Higgins
and Gilbert, 1978; Bartha, 1986; Leahy and Colwell,
1990; Haramaya et al., 1997; Colwell and Walker,
1977). It is now generally accepted by the scientific
community that no one species of microorganisms
will completely degrade any particular oil (Colwell
and Walker, 1977). The degradation of both crude
and refined oils seems to involve a consortium of
microorganisms, including both eukaryotic and
prokaryotic forms. The most common genera known
to be responsible for oil degradation comprise mainly
Nocardia, Pseudomonas, Acinetobacter, Flavobac-
terium, Micrococcus, Arthrobacter, Corynebac-
terium, Achromobacter, Rhodococcus, Alcaligenes,
Mycobacterium, Bacillus, Aspergillus, Mucor,
Fusarium, Penicillium, Rhodotorula, Candida and
Sporobolomyces, (Atlas, 1981; Bossert and Bartha,
1984; Atlas and Bartha, 1992; Sarkhoh et al., 1990).
Of the various petroleum fractions, n-alkanes of
intermediate length (C 10 –C 20 ) are the preferred
substrates and tend to be most readily degradable
(Singer and Finnerty, 1984), whereas shorter chain
compounds are rather more toxic (Klug and Mar-
kovetz, 1971). Longer chain alkanes known as waxes
(C 20 –C 40 ) are hydrophobic solids and consequently
are difficult to degrade due their poor water solu-
bility and bioavailability (Bartha, 1986); branched
chain alkanes are also degraded more slowly than the
corresponding normal alkanes (Singer and Finnerty,
1984).
Many microorganisms are also known to degrade
a wide range of aromatic compounds (Cerniglia,
1984; Gibson and Subramanian, 1984; Weissenfels et
al., 1990). The degradation of polyaromatic hydro-
carbons (PAH) by microorganisms depends to a
large extent on their molecular weights among many
other factors (Weissenfels et al., 1990; Balba, 1993).
One of the concerns which has risen out of a number
of in-vitro studies has been the possible production
of certain intermediates from PAH degradation,
particularly dihydrodiols, which are of greater toxici-
ty than the parent compounds (Cerniglia, 1984).
However, studies on PAH degradation in sediments
suggested that accumulation of such compounds may
not actually occur in the natural environment due to
the rapidity with which they are further transformed
(Herbes and Schwall, 1978). Most of the literature
concerning the microbial transformation of PAHs has
centered on the lower-molecular-weight compounds
 M.T. Balba et al. / Journal of Microbiological Methods 32 (1998) 155 – 164
such as naphthalene, anthracene and phenanthrene,
but more recent studies have described the capa-
bilities of other microorganisms to metabolize higher
molecular weight compounds, including the white rot
fungus Phanerochaete chrysosporium (Bumpus,
1989; Field et al., 1992; Sack and Gunther, 1993;
Barr and Aust, 1994; Yateem et al., 1998; Suga and
Lindstrom, 1997). Recently, a soil Mycrobacterium
strain was also shown to be able to metabolize
pyrene as the sole source of carbon and energy and
its rate of metabolism was doubled by the addition of
a solvent such as paraffin oil (Jimenz and Bartha,
1996).
Cycloalkane degradation rates are somewhat vari-
able but tend to be much slower than alkanes and
often involve several microbial species (Perry,
1981). Highly condensed aromatic and cycloparaf-
finic structures, tars, bitumen and asphaltic materials
have the highest boiling points and exhibit the
greatest resistance to biodegradation (Atlas, 1981;
Blakebrough, 1978).
Asphaltenes are the product of petroleum hydro-
carbons in soil that appear to be resistant to micro-
bial degradation (Bossert and Bartha, 1984). It has
been proposed that such residual material from oil
degradation is analogous to, and could even be
regarded as, humic material (Jobson et al., 1972).
Due to its inert characteristics, insolubility and
similarity to humic materials it is unlikely to be
environmentally hazardous.
4. Bioremediation of oil-contaminated soil
A variety of technologies are currently available to
treat soil contaminated with hazardous materials,
including excavation and containment in secured
landfills, vapour extraction, stabilization and solidifi-
cation, soil flushing, soil washing, solvent extraction,
thermal desorption, vitrification and incineration (US
Environmental Protection Agency, 1988; Russell,
1992). Many of these technologies, however, are
either costly or do not result in complete destruction
of contamination. On the other hand, biological
treatment ‘bioremediation’ appears to be among the
most promising methods for dealing with a wide
range of organic contaminants, particularly petro-
leum hydrocarbons. The technology is also environ-
157
mentally sound, since it simulates natural processes,
and since it can result in the complete destruction of
hazardous compounds into innocuous products. The
use of bioremediation to remove pollutants is typical-
ly less expensive than the equivalent physical /
chemical methods (Russell, 1992). In situ
bioremediation techniques also offer the potential to
remediate contaminated soil and groundwater with-
out the need for excavation, which is a major
advantage. By using this approach, bioremediation
can be implemented below existing buildings, with-
out disturbing normal site operation.
While bioremediation has many advantages, it is a
site specific process and successful biological treat-
ment of contaminated soils presents a challenge to
environmental scientists and engineers for reasons
including: (a) heterogeneity of the contaminants, for
example, the contaminants can be found as solids,
liquid, gases, free or tightly bound to the particulate
matter; (b) extreme concentrations of hydrocarbons,
for example, the presence of high concentrations of
hydrocarbons can be inhibitory or toxic to the
microorganisms while extremely low concentrations
may not be adequate to support microbial activities;
(c) variable site environmental conditions such as
soil type and depth and soil microorganisms as well
as physical conditions such as pH, temperature,
oxygen availability, redox potential, moisture content
and substrate bioavailability. These conditions can
substantially affect the microbial growth and
biodegradation of organic contaminants; and (d)
bioremediation is also a slow process and subject to
regulatory constraints which influence its selection as
the clean-up technology, particularly with respect to
the required clean-up standards and the pressure for
immediate site spill or clean-up mandated by public
concern, which do not allow enough time for process
optimization.
The two general approaches to bioremediation are:
(a) environmental biostimulation, such as through
fertilizer addition, aeration and; (b) addition of
adapted microbial hydrocarbon degraders by bioaug-
mentation. The first is the most commonly used
approach for field application. The full claims of the
effectiveness of soil seeding on enhancing oil degra-
dation has not yet been fully demonstrated in the
field.
The main goal of the bioremediation design should
158 M.T. Balba et al. / Journal of Microbiological Methods 32 (1998) 155 – 164
be the creation of the most favourable conditions for
microbial growth and activities. Bioremediation
methods fall in two major categories: (a) on-site or
above-ground treatment; such methods include land-
farming / solid-phase bioremediation, composting,
biologically enhanced soil washing and slurry bio-
reactors, with the first two being the most commonly
used and; (b) in situ bioremediation (in-place), for
the remediation of subsurface soil and groundwater.
This is usually achieved by the manipulation of the
groundwater constituents or the stimulation of air
movement, or both.
To demonstrate that a bioremediation technology
is potentially useful, it is important that the ability to
enhance the rate of hydrocarbon biodegradation be
demonstrated under controlled conditions. For practi-
cal reasons this cannot be easily accomplished in situ
and thus must be accomplished in feasibility studies.
Such studies are also used to provide information on
the estimated cost and duration of treatment. These
studies usually involve microbiological laboratory
methods to measure the effectiveness of bioremedia-
tion under predetermined conditions. The goal of a
laboratory feasibility study is to identify limiting
factors and recommend ways to mitigate these
limitation in the field.
The following section describes several estab-
lished microbiological methods and approaches
which can be used for the feasibility assessment of
soil bioremediation.
5. Microbiological methods for bioremediation
assessment
5.1. Microbial enumeration
Initial soil analyses of the total heterotrophic
microbial counts and specific hydrocarbon degrading
microbial counts in the contaminated soil can pro-
vide useful information on soil biological activities,
and the extent to which the indigenous microbial
population has acclimated to the site conditions. The
results will also indicate whether the soil contains a
healthy indigenous microbial population capable of
supporting bioremediation. In addition to the initial
microbial assessment of the contaminated soil, moni-
toring microbial populations during the soil
bioremediation is a useful tool for following the
changes and discerning for microbes active in hydro-
carbon degradation. A strong correlation between
microbial counts and hydrocarbon degradation has
been reported (Al-Awadhi et al., 1996; Song and
Bartha, 1990). During landfarming of oil-contami-
nated soil, the total microbial counts in the form of
total colony forming units (TCFU) were increased
by four orders of magnitudes (Balba et al., 1998).
Bacterial count is usually determined in repre-
sentative soil composite samples, using the standard
serial dilution and nutrient agar plate-counting tech-
niques (Lorch et al., 1995). The plate counts for
mesophilic bacteria are typically incubated at 308C
for 24 h before bacterial counts are conducted. The
counts are usually expressed in the form of CFU’s.
The hydrocarbon-utilizing bacteria (HUB) can be
assayed similarly, with the exception that solid
mineral basal media are used and a suitable hydro-
carbon compound such as n-hexadecane provided as
the sole source of energy and carbon. In this case,
hexadecane is added on a disc of sterilized filter
paper which is then placed in the lid of the inverted
plate. The plates are incubated at 308C for 72 h
before HUB are counted. The use of specific hydro-
carbon degrading bacterial counts provide additional
information on the hydrocarbon biodegradation po-
tential in a particular soil. The percentage of HUB to
the total heterotrophic bacterial counts usually re-
flects the extent of microbial acclimation and hydro-
carbon degradation activities in an oil-contaminated
sites. The agar plate microbial-counts technique has
several limitations particularly when dealing with
nonculturable microorganisms.
5.2. Dehydrogenase activity
Biological oxidation of organic compounds is
generally a dehydrogenation process, which is cata-
lyzed by dehydrogenase enzymes (Lenhard, 1956;
Paul and Clark, 1989; Page et al., 1982). Therefore,
these enzymes play an essential role in the oxidation
of organic matter by transferring hydrogen from the
organic substrates to the electron acceptor. Many
different specific enzyme systems are involved in the
dehydrogenase activity of the soils. These systems
are an integral part of the soil microorganisms and
 M.T. Balba et al. / Journal of Microbiological Methods 32 (1998) 155 – 164
reflect to a great extent the soil biochemical ac-
tivities.
The assay of dehydrogenase in contaminated soil
can be used as a simple method to examine the
possible inhibitory effect of the contaminants on the
soil microbial activities (Bartha and Pramer, 1965).
For example, toluene and chloroform, if present at
elevated concentration, can strongly inhibit soil
dehydrogenase, but have little effect at low con-
centrations (Page et al., 1982). However, because the
dehydrogenase activity depends on the total metabol-
ic activities of soil microorganisms, its values in
different soils do not always reflect the total number
of viable microorganisms isolated on a particular
medium (Page et al., 1982).
The most widely used method for the determi-
nation of soil dehydrogenase activities is the
colorimetric method, involving the use of 2,3,5-
triphenyl tetrazolium chloride (TTC) which acts as
an electron acceptor for many dehydrogenase en-
zymes (Page et al., 1982). When this compound is
reduced by the catalytic effect of the soil dehydro-
genase, it forms triphenyl formazan (TPH) which has
a characteristic reddish colour which can be assayed
at 485 nm (Page et al., 1982). The intensity of red
colour produced from the dehydrogenase assay is a
good index for microbial activities within the tested
soil. However, several factors may affect the ac-
tivities of soil dehydrogenase. Also nitrate, nitrite
and ferric ions seem to inhibit dehydrogenase activi-
ty due to the ions acting as alternative electron
acceptors.
5.3. Soil respirometric tests
Mineralization studies involving measurements of
total CO 2 production can provide excellent infor-
mation on the biodegradability potential of hydro-
carbons in contaminated soils. The approach, which
is considered a preliminary step in the feasibility
study, provides rapid, relatively unequivocal time-
course data suitable for testing different biological
treatment options, such as the effect of nutrient
supplementation, microbial inoculation, etc. The test
can be useful also for confirming active hydrocarbon
degradation during full scale bioremediation. During
the respiration tests, oxygen consumption and / or
carbon dioxide evolution rates can be monitored by
159
using either expensive automated equipment which
can handle a large number of samples simultaneous-
ly, or by simple respirometric-flask methods, which
are commonly used (Bartha and Pramer, 1965;
Pritchard et al., 1992). In the latter case, measure-
ment of oxygen consumption can be carried out by
the use of a U-tube manometer and barometric
control, so when oxygen is consumed by aerobic
metabolism, a measurable negative pressure develops
within the respirometric flask which is directly
related to the oxygen partial pressure. Carbon diox-
ide which is evolved during the respiration process is
simultaneously trapped in a potassium hydroxide
(KOH) solution located in a central well or in the
side arm attached to the respirometric flasks. The
amount of carbon dioxide absorbed, is then measured
by titrating the residual KOH with a standard
solution of hydrochloric acid, after barium chloride
is added to precipitate the carbonate ions. The levels
of cumulative oxygen consumed and carbon dioxide
evolved can then be calculated and plotted in mmol /
kg of dry soil as a function of incubation time. In
addition to the assessment of the degradation po-
tential of petroleum hydrocarbons, the respirometric
tests can also be applied to assess the possible
inhibitory effects of heavy metals, toxic compounds,
and pH on the soil microbial activities.
The Biometer flask, involves the use of small
amounts of soil and can be easily adapted to assess
mineralization rates, particularly when a large num-
ber of samples need to be tested. Examples of the
data generated from such a mineralization test are
shown in Figs. 1 and 2 which present the respiromet-
ric results of routine monitoring of field trials,
involving the remediation of oil-contaminated desert
soil by the windrow composting method, in Kuwait
(Al-Daher et al., 1995). The soil treatment continued
for a period of ten months during which composite
soil samples were collected monthly for chemical
analyses and respirometric assessment, using biome-
ter flasks. The respirometric tests were used to assess
the soil microbial activities during the bioremedia-
tion program, by measurement of carbon dioxide
production. The results presented in Fig. 1 show the
relationship between the water content of the treated
soil and microbial activities, measured in the form of
carbon dioxide. Maximum respiration rate correlated
well with the level moisture content of the soil. The
160 M.T. Balba et al. / Journal of Microbiological Methods 32 (1998) 155 – 164
Fig. 1. Correlation between respiration rate and moisture content
during soil bioremediation of petroleum hydrocarbons.
relationship between respiration rate versus total
hydrocarbon concentration are presented in Fig. 2.
The decrease in the carbon dioxide production rate,
towards the end of the treatment, is possibly caused
by the exhaustion of the readily degradable organic
fraction.
5.4. Biodegradation microcosm test
There are many definitions of ‘microcosm’. A
typical one is that of an intact, minimally disturbed
piece of an ecosystem brought into the laboratory for
study in its natural state (Prichard and Bourquin,
Fig. 2. Correlation between respiration rates and hydrocarbon
content during soil bioremediation.
1984). Microcosms can vary in complexity from
simple static soil jars of contaminated soil to highly
sophisticated systems designed to enable variations
in various environmental parameters encountered on
site to be more accurately simulated in the labora-
tory. The microcosm design that closely models real
environmental conditions is most likely to produce
relevant results. In such experiments, it is important
to include appropriate controls, such as sterile treat-
ments, to separate the effects of abiotic weathering
of oil from actual biodegradation. Soil microcosm
experiments can be a useful tool to assess the
biodegradation potential of hydrocarbon contamina-
tion and the development of models for predicting
the fate of these pollutants. Mathematical equations
can then be formulated to describe the kinetics of
each of the processes involving transformation of
specific hydrocarbon constituents under considera-
tion. Concentrations of the hydrocarbon constituents
and their degradation products can subsequently be
monitored in various components of the microcosm,
to obtain useful kinetic information in relation to
their, equilibrium partitioning, biodegradation trans-
formation behaviour, under predetermined environ-
mental conditions. Additionally, the test can be used
for screening bioremediation treatments to establish
the most appropriate bioremediation strategy for
large scale application (Balba et al., 1992; Compeau
et al., 1991).
Similarly, the biodegradation potential of hydro-
carbons can be assessed by using slurry reactors
(10–15% soil: water w / v), which offer several
advantages over the soil microcosms. Due to more
efficient mixing, aeration and improved substrate
bioavailabilty, the duration of a treatability study can
be significantly reduced.
During the treatability study, microcosms tests are
usually monitored regularly for petroleum hydro-
carbon degradation, by either sacrificing whole mi-
crocosm systems or by subsampling techniques.
Other parameters which may be monitored, in addi-
tion to petroleum hydrocarbons, include microbial
counts, pH, nutrient concentration and moisture
content.
To determine the rate of hydrocarbon biodegrada-
tion, accurate and reliable analyses are critical. One
of the recommended standard analyses for total
petroleum hydrocarbons (TPH) is based on the use
 M.T. Balba et al. / Journal of Microbiological Methods 32 (1998) 155 – 164
of infrared (IR) absorption (Standard Methods for
the Examination of Water and Wastewater, 1985;
Potter, 1993). The method involves the extraction of
the soil or the soil slurry with Freon, and the IR
absorption of the extract is measured at 2930 cm 21 .
This absorption band reflects the C-H stretching
vibrations of the hydrocarbons and, consequently, the
measurement has to be performed in solvents that are
free of C-H bonds. The IR method is therefore more
sensitive to saturated hydrocarbons than to aromat-
ics. For the best quantitative results, a standard has to
be prepared from a balanced mixture of hydro-
carbons, with a composition that approximates the
hydrocarbon contamination in the samples to be
analyzed. The method, however cannot provide
information on the fate of individual hydrocarbon
constituents. To obtain specific information on the
biodegradation of oil constituents, the extract has to
be first fractionated by appropriate chromatographic
techniques and the obtained fractions need to be then
analyzed by gas chromatography fitted with a capil-
lary column and flame ionization detector (GC–FID)
or mass spectrometry (GC–MS). Such methods
allow detailed information to be obtained on the
residual concentration of aliphatics, aromatics and
biomarker constituents (Wang et al., 1994). How-
ever, the quantification of individual compounds is
restricted to those which can be resolved by the gas
chromatographic technique.
5.5. Biomarker compounds
The evaluation of hydrocarbon degradation in the
field is much more difficult than in the laboratory
due to the heterogeneity of contamination. Polluted
sites are often remarkably heterogeneous in their
Table 1
Correlation of C 18 :phytane ratio with TPH degradation
Treatment C 18 :phytane ratio
T0 T6 T 12
Landfarming 2.4 0.3 ND
Control test 2.4 2.3 2.2
Windrow piles 2.4 0.4 ND
Control test 2.4 2.4 2.2
Static piles 1.7 0.5 ND
Control Test 1.7 1.6 1.4
ND5Less than 0.3.
161
nature so that the initial analytical data vary from
very low to very high concentrations over relatively
small areas. In addition, large volumes, generally in
the order of thousands of cubic meters of soil, are
involved. Under such circumstances, it is very
difficult to obtain statistically meaningful data with-
out recourse to analyzing a massive number of
samples which would be prohibitively expensive.
Because of the difficulties in the quantification of
hydrocarbons in large scale bioremediation, the
ratios of hydrocarbons compounds within the com-
plex hydrocarbon mixture can be used to assess
hydrocarbon biodegradation. Hydrocarbon degrading
microorganisms usually degrade branched alkanes
and isoprenoid compounds such as pristane, phytane
and hopane compounds at much slower rates than
straight-chain alkanes. Therefore, the ratio of
straight-chain alkanes to these highly branched bio-
marker compounds can reflect the extent to which
microorganisms have degraded the hydrocarbons in a
petroleum mixture (Wang et al., 1994; Prichard and
Costa, 1991; Kennicutt, 1988). This ratio concept is
based on the assumption that nonbiodegradation
processes such as weathering, volatilization and
leaching, will not produce differential losses of
normal and branched hydrocarbons that have similar
gas chromatographic and correspondingly, chemical
behaviour (Kennicutt, 1988). An example of the data
generated from such analyses is shown, in Table 1.
These results were obtained from field demonstration
involving the bioremediation of oil-contaminated soil
in Kuwait (2160 m 3 ), using three different methods,
namely landfarming, composting piles (480 m 3 ), and
static bioventing piles (240 m 3 ). The results summa-
rizes the progressive changes in C 18 :phytane ratio
during the course of soil remediation. Hydrocarbon
TPH concentration (mg / kg) TPH reduction
T0 T6 T 12 (%)
39 400 14 000 7200 81.7
39 400 35 500 31 700 19.5
34 700 19 400 9500 72.6
35 900 39 800 30 600 14.8
14 400 8500 4600 68.1
14 100 13 600 12 200 13.5
162 M.T. Balba et al. / Journal of Microbiological Methods 32 (1998) 155 – 164
degradation in the treated soil was accompanied by
significant reduction in the ratio, compared to little
or no change in the control tests (Balba et al., 1998).
However, the method has some limitations, due to
the fact that branched alkanes including phytane
biodegrade slowly, which means that the
C 18 :phytane ratio underestimates hydrocarbon
biodegradation. Also, octadecane (C 18 ) usually de-
grades rapidly, making the ratio technique useful
only during the early stages of oil degradation.
Hopane compounds are also molecular fossils that
are derived from the biomass that give rise to the
crude oil. These molecules are slowly biodegradable
and thus become enriched within the residual oil as
the oil weathers by evaporation and biodegradation.
Some of these compounds have been successfully
used as biomarkers for oil degradation assessment
(Prince et al., 1994).
5.6. Ecological impact and toxicity assessment
In addition to the demonstration of the treatment
efficacy, it is necessary to demonstrate that
bioremediation does not produce any toxic inter-
mediate products and to avoid undesired environ-
mental and ecological effects (Cerniglia, 1984; Pr-
ince et al., 1994). Fertilizers should not be applied at
excessive rates and the use of sodium nitrate is
discouraged because of the problem of introducing a
potential contaminant into groundwater (Russell,
1992). Nitrate has been known to cause the ‘blue
baby’ syndrome in small infants and the maximum
concentration of nitrate in drinking water supply is
10 mg / l. Necessary engineering measures should
also be considered to ensure the containment of the
remediation zone and prevent leachate migration
outside the treatment zone (Ellis et al., 1990).
Background toxicity prior to soil bioremediation
and after treatment can also be measured by using
appropriate toxicity tests. The tests used for this
purpose may include plant, microtox and Ames tests.
In the plant tests, the effect of the contaminated soils
on the growth and germination of selected mono-
cotyledonous and dicotyledonous plant species and
ability of soil to support sustainable growth are
assessed (El-Nawawy et al., 1995). In addition to
these phytotoxicity tests, acute toxicity of contami-
nated soil, can be determined by microtox assay and
Ames test (Mathew and Hastings, 1987). The mi-
crotox test assesses the toxicity of the soil extracts by
measuring the reduction in light emission by Photo-
bacterium phosphoreum, while the Ames Test ex-
amines the mutagenic effect of the contaminated soil
on Salmonella typhimurium (Maron and Ames,
1983).
5.7. Microbial survival test and tracking of GEM
There is a great deal of interest in the use of
genetically modified or engineered microorganisms
(GEM) to enhance oil degradation, particularly the
degradation of high molecular weight polyaromatic
compounds and alkane hydrocarbons. Microorga-
nisms with enhanced capabilities to degrade par-
ticularly aromatic hydrocarbons and their derivatives,
have already been developed (Thomas and Ward,
1994; Krume et al., 1994). Although technologies
based on these concepts hold promise for improved
bioreactor performance, experience gained from
bioaugmentation tests suggest that the use of GEMs
will be ineffective without development of tech-
niques to improve their survival in the face of
competition from indigenous microbial population
(Atlas, 1992). Convenient, economical and effective
methods of tracking engineered microorganisms have
been developed to enable the examination of their
survival, transport and ecological impact, when
released in new environments (Veal and Stokes,
1992; O’Donnell and Hopkins, 1993). This topic has
been addressed in detail by other authors in this
special issue of the Journal of Microbiological
Methods.
6. Concluding remarks
Bioremediation is a cost-effective and environ-
mentally sound remediation technology, particularly
for dealing with petroleum hydrocarbon contamina-
tion. However, the degradation rates of hydrocarbons
are site specific and are limited by the metabolic
capabilities of the hydrocarbon-degrading microbial
populations and also by a wide range of environmen-
tal factors. The effectiveness of the bioremediation
depends therefore on the success in identifying the
rate-limiting factors and optimizing them in the
feasibility studies. In these studies, microbially based
methods are usually used to determine site feasibili-
 M.T. Balba et al. / Journal of Microbiological Methods 32 (1998) 155 – 164
ty, the rate and extent of contaminants biodegrada-
tion that might be attained during remediation, and to
provide design criteria. Feasibility studies are there-
fore essential and can have an enormous impact on
the cost of full-scale remediation. Depending on the
circumstances, screening tests such as microbial
plate counts and enzyme assessment may be used to
determine if existing conditions are favourable for
microbial growth and respirometer tests provide
confirmation that the microbial population is
metabolically active. Treatability studies conducted
with soil or slurries can be tested under several
conditions, including unmodified microcosm, nutri-
ent-amended and biologically inhibited conditions, to
provide useful data on the rate and extent of conver-
sion of contaminants. During bioremediation of
hydrocarbons in the field, the rate of degradation is
largely controlled by the rate of supply of nutrients
and oxygen, which makes it difficult sometimes, to
extrapolate directly the results from laboratory to
bioremediation in the field.
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