Journalof

MethodsMicrobiological

Journal of Microbiological Methods 32 (1998) 155–164

Bioremediation of oil-contaminated soil: microbiological methodsfor feasibility assessment and field evaluation

*M.T. Balba , N. Al-Awadhi, R. Al-DaherKuwait 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. Thetechnology is very effective particularly in dealing with petroleum hydrocarbon contamination. However, bioremediation is asite-specific process and feasibility studies are required before full-scale remediation can be successfully applied. The typeand scale of the feasibility studies that will be needed are specific to the bioremediation approach to be employed duringfull-scale clean-up operation. In all cases however, these studies have the same goals: to accurately determine if specifichydrocarbon contaminants are amenable to biological treatment and to determine the time and cost required to treat thecontaminants of concern according to the regulated clean-up criteria. This contribution provides background information onthe chemistry and microbiology of hydrocarbon contamination, discusses the prospective of using biological methods foraddressing this problem and describes several microbiological methods which can be used for the feasibility assessment ofsoil bioremediation. The focus of this chapter is to highlight the needs for the integration of laboratory data to full-scalebioremediation. 1998 Elsevier Science B.V.

Keywords: Bioremediation; Petroleum hydrocarbons; Feasibility assessment

1. Introduction accidental spills that can be only minimized but noteliminated entirely. In recent years, leakage of

There is growing public concern as a wide variety gasoline from underground storage tanks primarily atof toxic organic chemicals are being introduced automobile service stations and from pipelines hasinadvertently or deliberately into the environment. been experienced at an alarming rate. Marine spillsPetroleum hydrocarbons are one common example of are also now becoming a frequent and major sourcethese chemicals, which enter the environment fre- of water and coastal contamination (US Environmen-quently and in large volumes through numerous tal Protection Agency, 1990). The Gulf War in 1991routes. The seepage from natural deposits is one of resulted in the worst man-made environmental disas-the major routes by which petroleum oil enters ter, with millions of gallons of crude oil releasedmarine environments (National Academy of Science, from the destroyed oil wells into the waters and1975). Human activities in the production, trans- surrounding land, forming more than 330 oil lakes,portation and storage of petroleum is another route covering an area of 49 square km (Salam, 1996).since such activities inevitably involve the risk of Such releases of large quantities of oil to marine and

terrestrial environments present a longterm threat to*Corresponding author. all forms of life. There is now an increasing need for

0167-7012/98/$19.00 1998 Elsevier Science B.V. All rights reserved.PI I : 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- and Gilbert, 1978; Bartha, 1986; Leahy and Colwell,carbon contamination. 1990; Haramaya et al., 1997; Colwell and Walker,

1977). It is now generally accepted by the scientificcommunity that no one species of microorganisms

2. Composition of petroleum hydrocarbons will completely degrade any particular oil (Colwelland Walker, 1977). The degradation of both crude

Petroleum hydrocarbons contain a complex mix- and refined oils seems to involve a consortium ofture of compounds which can be categorized for microorganisms, including both eukaryotic andsimplicity into four fractions: saturates, aromatics, prokaryotic forms. The most common genera knownresins (N, S, O) and asphaltene (Shell International to be responsible for oil degradation comprise mainlyLtd., 1983). The saturates fraction includes straight Nocardia, Pseudomonas, Acinetobacter, Flavobac-chain alkanes (normal alkanes), branched alkanes terium, Micrococcus, Arthrobacter, Corynebac-(isoalkanes), and cycloalkanes (naphthenes). The terium, Achromobacter, Rhodococcus, Alcaligenes,aromatic fraction contains volatile monoaromatic Mycobacterium, Bacillus, Aspergillus, Mucor,hydrocarbons such as benzene, toluene, xylenes etc., Fusarium, Penicillium, Rhodotorula, Candida andpolyaromatic hydrocarbons, naphthenoaromatics and Sporobolomyces, (Atlas, 1981; Bossert and Bartha,aromatic sulphur compounds such as thiophenes and 1984; Atlas and Bartha, 1992; Sarkhoh et al., 1990).dibenzothiophenes. It is noteworthy that the poly- Of the various petroleum fractions, n-alkanes ofaromatic hydrocarbon (PAHs) fraction which is intermediate length (C –C ) are the preferred10 20

associated with oil contamination, includes both substrates and tend to be most readily degradablesuspected and known carcinogens, the most toxic (Singer and Finnerty, 1984), whereas shorter chainbeing benzo(a)pyrene. The resins (N, S, O) and compounds are rather more toxic (Klug and Mar-asphaltene fractions consist of polar molecules con- kovetz, 1971). Longer chain alkanes known as waxestaining nitrogen, sulphur and oxygen. Resins are (C –C ) are hydrophobic solids and consequently20 40

amorphous solids which are truly dissolved in oil, are difficult to degrade due their poor water solu-whereas asphaltenes are large molecules colloidally bility and bioavailability (Bartha, 1986); brancheddispersed in oil. The relative proportions of these chain alkanes are also degraded more slowly than thefractions are dependent on many factors such as the corresponding normal alkanes (Singer and Finnerty,source, geological history, age, migration and altera- 1984).tion of crude oil. Many microorganisms are also known to degrade

a wide range of aromatic compounds (Cerniglia,1984; Gibson and Subramanian, 1984; Weissenfels et

3. Biodegradation of petroleum hydrocarbons al., 1990). The degradation of polyaromatic hydro-carbons (PAH) by microorganisms depends to a

Current evidence suggests that in aquatic and large extent on their molecular weights among manyterrestrial environments microorganisms are the chief other factors (Weissenfels et al., 1990; Balba, 1993).agents for the biodegradation of molecules of en- One of the concerns which has risen out of a numbervironmental concern, including petroleum hydrocar- of in-vitro studies has been the possible productionbons (Alexander et al., 1982; Swanell and Head, of certain intermediates from PAH degradation,1994). Hydrocarbon-degrading bacteria, yeast and particularly dihydrodiols, which are of greater toxici-fungi are widely distributed in marine, fresh water ty than the parent compounds (Cerniglia, 1984).and soil habitats. Bacteria and yeast appear to be the However, studies on PAH degradation in sedimentsdominant degraders in aquatic ecosystems while suggested that accumulation of such compounds mayfungi and bacteria are the main degraders in soil not actually occur in the natural environment due toenvironments (Cooney and Summers, 1976; Hanson the rapidity with which they are further transformedet al., 1997). There is a vast amount of literature on (Herbes and Schwall, 1978). Most of the literaturethe subject of oil breakdown by microorganisms with concerning the microbial transformation of PAHs hasseveral major review papers (Atlas, 1977; Higgins centered on the lower-molecular-weight compounds

M.T. Balba et al. / Journal of Microbiological Methods 32 (1998) 155 –164 157

such as naphthalene, anthracene and phenanthrene, mentally sound, since it simulates natural processes,but more recent studies have described the capa- and since it can result in the complete destruction ofbilities of other microorganisms to metabolize higher hazardous compounds into innocuous products. Themolecular weight compounds, including the white rot use of bioremediation to remove pollutants is typical-fungus Phanerochaete chrysosporium (Bumpus, ly less expensive than the equivalent physical /1989; Field et al., 1992; Sack and Gunther, 1993; chemical methods (Russell, 1992). In situBarr and Aust, 1994; Yateem et al., 1998; Suga and bioremediation techniques also offer the potential toLindstrom, 1997). Recently, a soil Mycrobacterium remediate contaminated soil and groundwater with-strain was also shown to be able to metabolize out the need for excavation, which is a majorpyrene as the sole source of carbon and energy and advantage. By using this approach, bioremediationits rate of metabolism was doubled by the addition of can be implemented below existing buildings, with-a solvent such as paraffin oil (Jimenz and Bartha, out disturbing normal site operation.1996). While bioremediation has many advantages, it is a

Cycloalkane degradation rates are somewhat vari- site specific process and successful biological treat-able but tend to be much slower than alkanes and ment of contaminated soils presents a challenge tooften involve several microbial species (Perry, environmental scientists and engineers for reasons1981). Highly condensed aromatic and cycloparaf- including: (a) heterogeneity of the contaminants, forfinic structures, tars, bitumen and asphaltic materials example, the contaminants can be found as solids,have the highest boiling points and exhibit the liquid, gases, free or tightly bound to the particulategreatest resistance to biodegradation (Atlas, 1981; matter; (b) extreme concentrations of hydrocarbons,Blakebrough, 1978). for example, the presence of high concentrations of

Asphaltenes are the product of petroleum hydro- hydrocarbons can be inhibitory or toxic to thecarbons in soil that appear to be resistant to micro- microorganisms while extremely low concentrationsbial degradation (Bossert and Bartha, 1984). It has may not be adequate to support microbial activities;been proposed that such residual material from oil (c) variable site environmental conditions such asdegradation is analogous to, and could even be soil type and depth and soil microorganisms as wellregarded as, humic material (Jobson et al., 1972). as physical conditions such as pH, temperature,Due to its inert characteristics, insolubility and oxygen availability, redox potential, moisture contentsimilarity to humic materials it is unlikely to be and substrate bioavailability. These conditions canenvironmentally hazardous. substantially affect the microbial growth and

biodegradation of organic contaminants; and (d)bioremediation is also a slow process and subject to

4. Bioremediation of oil-contaminated soil regulatory constraints which influence its selection asthe clean-up technology, particularly with respect to

A variety of technologies are currently available to the required clean-up standards and the pressure fortreat soil contaminated with hazardous materials, immediate site spill or clean-up mandated by publicincluding excavation and containment in secured concern, which do not allow enough time for processlandfills, vapour extraction, stabilization and solidifi- optimization.cation, soil flushing, soil washing, solvent extraction, The two general approaches to bioremediation are:thermal desorption, vitrification and incineration (US (a) environmental biostimulation, such as throughEnvironmental Protection Agency, 1988; Russell, fertilizer addition, aeration and; (b) addition of1992). Many of these technologies, however, are adapted microbial hydrocarbon degraders by bioaug-either costly or do not result in complete destruction mentation. The first is the most commonly usedof contamination. On the other hand, biological approach for field application. The full claims of thetreatment ‘bioremediation’ appears to be among the effectiveness of soil seeding on enhancing oil degra-most promising methods for dealing with a wide dation has not yet been fully demonstrated in therange of organic contaminants, particularly petro- field.leum hydrocarbons. The technology is also environ- 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 bioremediation is a useful tool for following themicrobial growth and activities. Bioremediation changes and discerning for microbes active in hydro-methods fall in two major categories: (a) on-site or carbon degradation. A strong correlation betweenabove-ground treatment; such methods include land- microbial counts and hydrocarbon degradation hasfarming/solid-phase bioremediation, composting, been reported (Al-Awadhi et al., 1996; Song andbiologically enhanced soil washing and slurry bio- Bartha, 1990). During landfarming of oil-contami-reactors, with the first two being the most commonly nated soil, the total microbial counts in the form ofused and; (b) in situ bioremediation (in-place), for total colony forming units (TCFU) were increasedthe remediation of subsurface soil and groundwater. by four orders of magnitudes (Balba et al., 1998).This is usually achieved by the manipulation of the Bacterial count is usually determined in repre-groundwater constituents or the stimulation of air sentative soil composite samples, using the standardmovement, or both. serial dilution and nutrient agar plate-counting tech-

To demonstrate that a bioremediation technology niques (Lorch et al., 1995). The plate counts foris potentially useful, it is important that the ability to mesophilic bacteria are typically incubated at 308Cenhance the rate of hydrocarbon biodegradation be for 24 h before bacterial counts are conducted. Thedemonstrated under controlled conditions. For practi- counts are usually expressed in the form of CFU’s.cal reasons this cannot be easily accomplished in situ The hydrocarbon-utilizing bacteria (HUB) can beand thus must be accomplished in feasibility studies. assayed similarly, with the exception that solidSuch studies are also used to provide information on mineral basal media are used and a suitable hydro-the estimated cost and duration of treatment. These carbon compound such as n-hexadecane provided asstudies usually involve microbiological laboratory the sole source of energy and carbon. In this case,methods to measure the effectiveness of bioremedia- hexadecane is added on a disc of sterilized filtertion under predetermined conditions. The goal of a paper which is then placed in the lid of the invertedlaboratory feasibility study is to identify limiting plate. The plates are incubated at 308C for 72 hfactors and recommend ways to mitigate these before HUB are counted. The use of specific hydro-limitation in the field. carbon degrading bacterial counts provide additional

The following section describes several estab- information on the hydrocarbon biodegradation po-lished microbiological methods and approaches tential in a particular soil. The percentage of HUB towhich can be used for the feasibility assessment of the total heterotrophic bacterial counts usually re-soil bioremediation. flects the extent of microbial acclimation and hydro-

carbon degradation activities in an oil-contaminatedsites. The agar plate microbial-counts technique has

5. Microbiological methods for bioremediation several limitations particularly when dealing withassessment nonculturable microorganisms.

5.1. Microbial enumeration5.2. Dehydrogenase activity

Initial soil analyses of the total heterotrophicmicrobial counts and specific hydrocarbon degrading Biological oxidation of organic compounds ismicrobial counts in the contaminated soil can pro- generally a dehydrogenation process, which is cata-vide useful information on soil biological activities, lyzed by dehydrogenase enzymes (Lenhard, 1956;and the extent to which the indigenous microbial Paul and Clark, 1989; Page et al., 1982). Therefore,population has acclimated to the site conditions. The these enzymes play an essential role in the oxidationresults will also indicate whether the soil contains a of organic matter by transferring hydrogen from thehealthy indigenous microbial population capable of organic substrates to the electron acceptor. Manysupporting bioremediation. In addition to the initial different specific enzyme systems are involved in themicrobial assessment of the contaminated soil, moni- dehydrogenase activity of the soils. These systemstoring microbial populations during the soil are an integral part of the soil microorganisms and

M.T. Balba et al. / Journal of Microbiological Methods 32 (1998) 155 –164 159

reflect to a great extent the soil biochemical ac- using either expensive automated equipment whichtivities. can handle a large number of samples simultaneous-

The assay of dehydrogenase in contaminated soil ly, or by simple respirometric-flask methods, whichcan be used as a simple method to examine the are commonly used (Bartha and Pramer, 1965;possible inhibitory effect of the contaminants on the Pritchard et al., 1992). In the latter case, measure-soil microbial activities (Bartha and Pramer, 1965). ment of oxygen consumption can be carried out byFor example, toluene and chloroform, if present at the use of a U-tube manometer and barometricelevated concentration, can strongly inhibit soil control, so when oxygen is consumed by aerobicdehydrogenase, but have little effect at low con- metabolism, a measurable negative pressure developscentrations (Page et al., 1982). However, because the within the respirometric flask which is directlydehydrogenase activity depends on the total metabol- related to the oxygen partial pressure. Carbon diox-ic activities of soil microorganisms, its values in ide which is evolved during the respiration process isdifferent soils do not always reflect the total number simultaneously trapped in a potassium hydroxideof viable microorganisms isolated on a particular (KOH) solution located in a central well or in themedium (Page et al., 1982). side arm attached to the respirometric flasks. The

The most widely used method for the determi- amount of carbon dioxide absorbed, is then measurednation of soil dehydrogenase activities is the by titrating the residual KOH with a standardcolorimetric method, involving the use of 2,3,5- solution of hydrochloric acid, after barium chloridetriphenyl tetrazolium chloride (TTC) which acts as is added to precipitate the carbonate ions. The levelsan electron acceptor for many dehydrogenase en- of cumulative oxygen consumed and carbon dioxidezymes (Page et al., 1982). When this compound is evolved can then be calculated and plotted in mmol /reduced by the catalytic effect of the soil dehydro- kg of dry soil as a function of incubation time. Ingenase, it forms triphenyl formazan (TPH) which has addition to the assessment of the degradation po-a characteristic reddish colour which can be assayed tential of petroleum hydrocarbons, the respirometricat 485 nm (Page et al., 1982). The intensity of red tests can also be applied to assess the possiblecolour produced from the dehydrogenase assay is a inhibitory effects of heavy metals, toxic compounds,good index for microbial activities within the tested and pH on the soil microbial activities.soil. However, several factors may affect the ac- The Biometer flask, involves the use of smalltivities of soil dehydrogenase. Also nitrate, nitrite amounts of soil and can be easily adapted to assessand ferric ions seem to inhibit dehydrogenase activi- mineralization rates, particularly when a large num-ty due to the ions acting as alternative electron ber of samples need to be tested. Examples of theacceptors. data generated from such a mineralization test are

shown in Figs. 1 and 2 which present the respiromet-5.3. Soil respirometric tests ric results of routine monitoring of field trials,

involving the remediation of oil-contaminated desertMineralization studies involving measurements of soil by the windrow composting method, in Kuwait

total CO production can provide excellent infor- (Al-Daher et al., 1995). The soil treatment continued2

mation on the biodegradability potential of hydro- for a period of ten months during which compositecarbons in contaminated soils. The approach, which soil samples were collected monthly for chemicalis considered a preliminary step in the feasibility analyses and respirometric assessment, using biome-study, provides rapid, relatively unequivocal time- ter flasks. The respirometric tests were used to assesscourse data suitable for testing different biological the soil microbial activities during the bioremedia-treatment options, such as the effect of nutrient tion program, by measurement of carbon dioxidesupplementation, microbial inoculation, etc. The test production. The results presented in Fig. 1 show thecan be useful also for confirming active hydrocarbon relationship between the water content of the treateddegradation during full scale bioremediation. During soil and microbial activities, measured in the form ofthe respiration tests, oxygen consumption and/or carbon dioxide. Maximum respiration rate correlatedcarbon dioxide evolution rates can be monitored by well with the level moisture content of the soil. The

160 M.T. Balba et al. / Journal of Microbiological Methods 32 (1998) 155 –164

1984). Microcosms can vary in complexity fromsimple static soil jars of contaminated soil to highlysophisticated systems designed to enable variationsin various environmental parameters encountered onsite to be more accurately simulated in the labora-tory. The microcosm design that closely models realenvironmental conditions is most likely to producerelevant results. In such experiments, it is importantto include appropriate controls, such as sterile treat-ments, to separate the effects of abiotic weatheringof oil from actual biodegradation. Soil microcosmexperiments can be a useful tool to assess thebiodegradation potential of hydrocarbon contamina-tion and the development of models for predictingthe fate of these pollutants. Mathematical equations

Fig. 1. Correlation between respiration rate and moisture contentcan then be formulated to describe the kinetics ofduring soil bioremediation of petroleum hydrocarbons.each of the processes involving transformation ofspecific hydrocarbon constituents under considera-

relationship between respiration rate versus total tion. Concentrations of the hydrocarbon constituentshydrocarbon concentration are presented in Fig. 2. and their degradation products can subsequently beThe decrease in the carbon dioxide production rate, monitored in various components of the microcosm,towards the end of the treatment, is possibly caused to obtain useful kinetic information in relation toby the exhaustion of the readily degradable organic their, equilibrium partitioning, biodegradation trans-fraction. formation behaviour, under predetermined environ-

mental conditions. Additionally, the test can be used5.4. Biodegradation microcosm test for screening bioremediation treatments to establish

the most appropriate bioremediation strategy forThere are many definitions of ‘microcosm’. A large scale application (Balba et al., 1992; Compeau

typical one is that of an intact, minimally disturbed et al., 1991).piece of an ecosystem brought into the laboratory for Similarly, the biodegradation potential of hydro-study in its natural state (Prichard and Bourquin, carbons can be assessed by using slurry reactors

(10–15% soil: water w/v), which offer severaladvantages over the soil microcosms. Due to moreefficient mixing, aeration and improved substratebioavailabilty, the duration of a treatability study canbe significantly reduced.

During the treatability study, microcosms tests areusually 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 microbialcounts, pH, nutrient concentration and moisturecontent.

To determine the rate of hydrocarbon biodegrada-tion, accurate and reliable analyses are critical. Oneof the recommended standard analyses for totalFig. 2. Correlation between respiration rates and hydrocarbon

content during soil bioremediation. petroleum hydrocarbons (TPH) is based on the use

M.T. Balba et al. / Journal of Microbiological Methods 32 (1998) 155 –164 161

of infrared (IR) absorption (Standard Methods for nature so that the initial analytical data vary fromthe Examination of Water and Wastewater, 1985; very low to very high concentrations over relativelyPotter, 1993). The method involves the extraction of small areas. In addition, large volumes, generally inthe soil or the soil slurry with Freon, and the IR the order of thousands of cubic meters of soil, are

21absorption of the extract is measured at 2930 cm . involved. Under such circumstances, it is veryThis absorption band reflects the C-H stretching difficult to obtain statistically meaningful data with-vibrations of the hydrocarbons and, consequently, the out recourse to analyzing a massive number ofmeasurement has to be performed in solvents that are samples which would be prohibitively expensive.free of C-H bonds. The IR method is therefore more Because of the difficulties in the quantification ofsensitive to saturated hydrocarbons than to aromat- hydrocarbons in large scale bioremediation, theics. For the best quantitative results, a standard has to ratios of hydrocarbons compounds within the com-be prepared from a balanced mixture of hydro- plex hydrocarbon mixture can be used to assesscarbons, with a composition that approximates the hydrocarbon biodegradation. Hydrocarbon degradinghydrocarbon contamination in the samples to be microorganisms usually degrade branched alkanesanalyzed. The method, however cannot provide and isoprenoid compounds such as pristane, phytaneinformation on the fate of individual hydrocarbon and hopane compounds at much slower rates thanconstituents. To obtain specific information on the straight-chain alkanes. Therefore, the ratio ofbiodegradation of oil constituents, the extract has to straight-chain alkanes to these highly branched bio-be first fractionated by appropriate chromatographic marker compounds can reflect the extent to whichtechniques and the obtained fractions need to be then microorganisms have degraded the hydrocarbons in aanalyzed by gas chromatography fitted with a capil- petroleum mixture (Wang et al., 1994; Prichard andlary column and flame ionization detector (GC–FID) Costa, 1991; Kennicutt, 1988). This ratio concept isor mass spectrometry (GC–MS). Such methods based on the assumption that nonbiodegradationallow detailed information to be obtained on the processes such as weathering, volatilization andresidual concentration of aliphatics, aromatics and leaching, will not produce differential losses ofbiomarker constituents (Wang et al., 1994). How- normal and branched hydrocarbons that have similarever, the quantification of individual compounds is gas chromatographic and correspondingly, chemicalrestricted to those which can be resolved by the gas behaviour (Kennicutt, 1988). An example of the datachromatographic technique. generated from such analyses is shown, in Table 1.

These results were obtained from field demonstration5.5. Biomarker compounds involving the bioremediation of oil-contaminated soil

3in Kuwait (2160 m ), using three different methods,3The evaluation of hydrocarbon degradation in the namely landfarming, composting piles (480 m ), and

3field is much more difficult than in the laboratory static bioventing piles (240 m ). The results summa-due to the heterogeneity of contamination. Polluted rizes the progressive changes in C :phytane ratio18

sites are often remarkably heterogeneous in their during the course of soil remediation. Hydrocarbon

Table 1Correlation of C :phytane ratio with TPH degradation18

Treatment C :phytane ratio TPH concentration (mg/kg) TPH reduction18

T T T T T T (%)0 6 12 0 6 12

Landfarming 2.4 0.3 ND 39 400 14 000 7200 81.7Control test 2.4 2.3 2.2 39 400 35 500 31 700 19.5Windrow piles 2.4 0.4 ND 34 700 19 400 9500 72.6Control test 2.4 2.4 2.2 35 900 39 800 30 600 14.8Static piles 1.7 0.5 ND 14 400 8500 4600 68.1Control Test 1.7 1.6 1.4 14 100 13 600 12 200 13.5

ND5Less than 0.3.

162 M.T. Balba et al. / Journal of Microbiological Methods 32 (1998) 155 –164

degradation in the treated soil was accompanied by crotox test assesses the toxicity of the soil extracts bysignificant reduction in the ratio, compared to little measuring the reduction in light emission by Photo-or no change in the control tests (Balba et al., 1998). bacterium phosphoreum, while the Ames Test ex-However, the method has some limitations, due to amines the mutagenic effect of the contaminated soilthe fact that branched alkanes including phytane on Salmonella typhimurium (Maron and Ames,biodegrade slowly, which means that the 1983).C :phytane ratio underestimates hydrocarbon18

biodegradation. Also, octadecane (C ) usually de- 5.7. Microbial survival test and tracking of GEM18

grades rapidly, making the ratio technique usefulonly during the early stages of oil degradation. There is a great deal of interest in the use ofHopane compounds are also molecular fossils that genetically modified or engineered microorganismsare derived from the biomass that give rise to the (GEM) to enhance oil degradation, particularly thecrude oil. These molecules are slowly biodegradable degradation of high molecular weight polyaromaticand thus become enriched within the residual oil as compounds and alkane hydrocarbons. Microorga-the oil weathers by evaporation and biodegradation. nisms with enhanced capabilities to degrade par-Some of these compounds have been successfully ticularly aromatic hydrocarbons and their derivatives,used as biomarkers for oil degradation assessment have already been developed (Thomas and Ward,(Prince et al., 1994). 1994; Krume et al., 1994). Although technologies

based on these concepts hold promise for improved5.6. Ecological impact and toxicity assessment bioreactor performance, experience gained from

bioaugmentation tests suggest that the use of GEMsIn addition to the demonstration of the treatment will be ineffective without development of tech-

efficacy, it is necessary to demonstrate that niques to improve their survival in the face ofbioremediation does not produce any toxic inter- competition from indigenous microbial populationmediate products and to avoid undesired environ- (Atlas, 1992). Convenient, economical and effectivemental and ecological effects (Cerniglia, 1984; Pr- methods of tracking engineered microorganisms haveince et al., 1994). Fertilizers should not be applied at been developed to enable the examination of theirexcessive rates and the use of sodium nitrate is survival, transport and ecological impact, whendiscouraged because of the problem of introducing a released in new environments (Veal and Stokes,potential contaminant into groundwater (Russell, 1992; O’Donnell and Hopkins, 1993). This topic has1992). Nitrate has been known to cause the ‘blue been addressed in detail by other authors in thisbaby’ syndrome in small infants and the maximum special issue of the Journal of Microbiologicalconcentration of nitrate in drinking water supply is Methods.10 mg/ l. Necessary engineering measures shouldalso be considered to ensure the containment of theremediation zone and prevent leachate migration 6. Concluding remarksoutside the treatment zone (Ellis et al., 1990).

Background toxicity prior to soil bioremediation Bioremediation is a cost-effective and environ-and after treatment can also be measured by using mentally sound remediation technology, particularlyappropriate toxicity tests. The tests used for this for dealing with petroleum hydrocarbon contamina-purpose may include plant, microtox and Ames tests. tion. However, the degradation rates of hydrocarbonsIn the plant tests, the effect of the contaminated soils are site specific and are limited by the metabolicon the growth and germination of selected mono- capabilities of the hydrocarbon-degrading microbialcotyledonous and dicotyledonous plant species and populations and also by a wide range of environmen-ability of soil to support sustainable growth are tal factors. The effectiveness of the bioremediationassessed (El-Nawawy et al., 1995). In addition to depends therefore on the success in identifying thethese phytotoxicity tests, acute toxicity of contami- rate-limiting factors and optimizing them in thenated soil, can be determined by microtox assay and feasibility studies. In these studies, microbially basedAmes test (Mathew and Hastings, 1987). The mi- methods are usually used to determine site feasibili-

M.T. Balba et al. / Journal of Microbiological Methods 32 (1998) 155 –164 163

Balba, M.T., Ying, A.C., McNeice, T.G., 1992. Bioremediation ofty, the rate and extent of contaminants biodegrada-contaminated soil: bench-scale to field application. In: Thetion that might be attained during remediation, and toProceedings of National Research and Development Conference

provide design criteria. Feasibility studies are there- on the Control of Hazardous Materials, HMCRI, Washington,fore essential and can have an enormous impact on D.C., pp. 145–151.the cost of full-scale remediation. Depending on the Barr, D.P., Aust, S.D., 1994. Mechanisms white rot fungi use to

degrade pollutants. Environ. Sci. Technol. 28 (2), 79A.circumstances, screening tests such as microbialBartha, R., 1986. Biotechnology of petroleum pollutant biodegra-plate counts and enzyme assessment may be used to

dation. Microbiol Ecol. 12, 155–172.determine if existing conditions are favourable forBartha, R., Pramer, D., 1965. Features of a flask and method for

microbial growth and respirometer tests provide measuring the persistence of and biological effects of pesticidesconfirmation that the microbial population is in soil. Soil Sci. 100, 86–170.metabolically active. Treatability studies conducted Blakebrough, N., 1978. Interactions of oil and microorganisms in

soil. In: Chater, K.W., Somerville, J.H. (Eds.), The Crude Oilwith soil or slurries can be tested under severalIndustry and Microbial Ecosystems, Heydon and Son Ltd.,conditions, including unmodified microcosm, nutri-London, pp. 28–40.

ent-amended and biologically inhibited conditions, toBossert, I., Bartha, R., 1984. The fate of petroleum in soil

provide useful data on the rate and extent of conver- ecosystems. In: Atlas, R.M. (Ed.), Petroleum Microbiology,sion of contaminants. During bioremediation of Macmillan, New York, pp. 435–473.

Bumpus, J.A., 1989. Biodegradation of polyaromatic hydrocar-hydrocarbons in the field, the rate of degradation isbons by Phanerochaete chrysosporium. Appl. Environ. Mi-largely controlled by the rate of supply of nutrientscrobiol. 55, 154–155.and oxygen, which makes it difficult sometimes, to

Cerniglia, C.E., 1984. Microbial metabolism of polycyclic aro-extrapolate directly the results from laboratory to matic hydrocarbons. Adv. Appl. Microbiol. 30, 31–37.bioremediation in the field. Colwell, R.E., Walker, J.D., 1977. Ecological aspects of microbial

degradation of petroleum in the marine environment. Crit. Rev.Microbiol. 5, 423–445.

References Compeau, G.C., Mahaffey, W.D., Patras, L., 1991. Full-scalebioremediation of contaminated soil and water. In: Sayler, G.S.Al-Awadhi, N., Al-Daher, R., ElNawawy, A., Balba, M.T., 1996.(Ed.), Environmental Biotechnology for Waste Treatment,Bioremediation of oil-contaminated soil in Kuwait. I. Land-Plenum Press, New York, pp. 91–109.farming to remediate oil-contaminated soil. J. Soil Contamin. 5

Cooney, J.J., Summers, R.J., 1976. Hydrocarbon-using micro-(3), 243–260.organisms in three fresh water ecosystems. In: Sharpley, J.M. etAl-Daher, R., El-Nawawy, A., Al-Awadhi, N., 1995. Evaluational. (Eds.), Proceedings of the Third International Biodegrada-of on-site bioremediation of oil-contaminated soil throughtion Symposium, Applied Sciences, London, pp. 141–156.composting soil piles. Final Report, KISR4625, Kuwait Institute

Ellis, B., Balba, M.T., Theile, P., 1990. Bioremediation of oil-of Scientific Research, Kuwait, 175–180.contaminated land. Environ. Technol. 11, 443–455.Alexander, S.K., Schropp, S.J., Schwarz, J.R., 1982. Spatial and

El-Nawawy, A., Al-Daher, R., Yateem, A., Al-Awadhi, N., 1995.seasonal distribution of hydrocarbon-utilizing bacteria of sedi-Bioremediation of oil-contaminated soil in Kuwait by landfarm-ment from the northwestern Gulf of Mexico. Contrib. Mar. Sci.ing technology. Final Report (KISR) 4596), Kuwait Institute for25, 13–19.Scientific Research, Kuwait.Atlas, R.M., 1977. Stimulated petroleum biodegradation, CRC

Field, J.A., Jong, E.D., Costa, G.F., Bont, J.A., 1992. Biodegrada-Crit. Rev. Microbiol. 5, 371–386.tion of polyaromatic hydrocarbons by new isolates of white rotAtlas, R.M., 1981. Microbial degradation of petroleum hydro-fungi. Applied Environ. Microbiol. 58 (7), 2219–2226.carbons: An environmental perspective. Microbiol. Rev. 45,

Gibson, D.T., Subramanian, V., 1984. Microbial degradation of180–209.aromatic hydrocarbons. In: Gibson, D.T. (Ed.), MicrobialAtlas, R.M., 1992. Molecular methods for environmental moni-Degradation of Organic Compounds, Dekker, New York, pp.toring and containment of genetically engineered microorga-181–282.nisms. Biodegradation 3, 137–146.

Hanson, K.G., Nigam, A., Kapadia, M., Desai, A., 1997.Atlas, R.M., Bartha, R., 1992. Hydrocarbon biodegradation andBioremediation of crude oil contamination with Acinetobacteroil-spill bioremediation. In: Marshall, K.C. (Ed.), Advances insp. A3. Current Microbiol. 35, 191–193.Microbial Ecology, vol. 12, Plenum Press, New York, pp.

Haramaya, S., Venkateswaran, K., Toki, H., Komukai, S., Goto,287–338.M., Tanaka, H., Ishihara, M., 1997. Degradation of crude oil byBalba, M.T., 1993. Microorganisms and detoxification of in-marine bacteria. J. Mar. Biotechnol. 3, 239–243.dustrial waste. In: Gareth Jones, D. (Ed.), Exploitation of

Herbes, S.E., Schwall, L.R., 1978. Microbial transformation ofMicroorganisms, Chapman and Hall, London, pp. 411–409.polyaromatic hydrocarbons in pristine and petroleum-contami-Balba, M.T., Al-Daher, R., Al-Awadhi, N., 1998. Bioremediationnated sediments. Appl. Environ. Microbiol. 35, 306–316.of oil-contaminated soil in Kuwait. Environ. Int. 24 (1), 163–

173. Higgins, I.J., Gilbert, P.D., 1978. The biodegradation of hydro-

164 M.T. Balba et al. / Journal of Microbiological Methods 32 (1998) 155 –164

carbons. In: Chater, K.W., Somerville, H.J. (Eds.), The Oil Miller, R.N. (Eds.), Hydrocarbon Bioremediation, Lewis Pub-lishers, London, pp. 107–124.Industry and Microbial Ecosystems, Hedon and Son Ltd.,

Pritchard, P.H., Mueller, J.H., Rogers, J.C., Kremer, F.V., Glaser,London, pp. 80–117.J.A., 1992. Oil spill bioremediation:experience, lessons andJimenz, I., Bartha, R., 1996. Solvent-augmented mineralization ofresults from the Exxon Valdez oil spill in Alaska. Biodegrada-pyrene by a Mycobacterium sp. Appl. Environ. Microbiol. 62tion 3, 315–335.(7), 2311–2316.

Russell, D.L., 1992. Remediation Manual for Petroleum-Contami-Jobson, A., Cook, F.D., Wastelake, D.W.S., 1972. Microbialnated Sites, Technomic Publishing Co., Lancaster, PA., USA.utilization of crude oil. Appl. Microbiol. 32, 1082–1089.

Sack, U., Gunther, T., 1993. Metabolism of PAH by fungi andKennicutt, M.C., 1988. The effect of biodegradation on crude oilcorrelation with extracellular enzymatic activities. J. Basicbulk and molecular composition. Oil Chem. Pollut. 4, 89–112.Microbiol. 33 (4), 269–277.Klug, M.J., Markovetz, K., 1971. Utilization of aliphatic hydro-

Salam, A.A., 1996. Remediation and rehabilitation of oil-lakecarbons by microorganisms. Adv. Microbial Physiol. 5, 1–39.beds. In: Al-Awadhi, N., Balba, M.T., Kamizawa, C. (Eds.),

Krume, M.L., Smith, R.L., Egestorff, J. et al., 1994. Behaviour ofEnvironmental Disaster, Elsevier, Amsterdam, pp. 117–139.

pollutant-degrading microorganisms in aquifers: prediction ofSarkhoh, N.A., Ghannoum, M.A., Ibrahim, A.S., Stretton, R.J.,

genetically engineered organisms. Environ. Sci. Technol. 28,Radwan, S.S., 1990. Crude oil and hydrocarbon degrading

1134–1138.strains of Rhodococcus: Rhodococcus strains isolated from soil

Leahy, J.G., Colwell, R.R., 1990. Microbial degradation ofand marine environments in Kuwait. Environ. Pollut. 65, 1–18.

hydrocarbons in the environment. Microbiol. Rev. 54, 305–315.Shell International Ltd., 1983. The chemistry of petroleum. In:

Lenhard, G., 1956. Die dehydrogenaseaktivitat des Bodens alsThe Petroleum Handbook, 6th ed., Elsevier, New York, pp.

Mass fur die Mikroorganismentatigkeit im Boden. Z. Pflan-223–264.

zenernaehr. Dueng. Bodenkd. 73, 1–11.Singer, M.E., Finnerty, W.R., 1984. Microbial metabolism of

Lorch, H.J., Benckieser, G., Ottow, J.C.G., 1995. Basic methods straight-chain and branched alkanes. In: Atlas, R.M. (Ed.),for counting microorganisms in soil and water. In: Alef, K., Petroleum Microbiology, Macmillan Inc., pp. 1–59.Nannipieri, P. (Eds.), Methods in Applied Soil Microbiology Song, H.G., Bartha, R., 1990. Effect of jet fuel spills on theand Biochemistry, Academic Press, New York, pp. 146–161. microbial community of soil. Appl. Environ. Microbiol. 56 (3),

Maron, D.M., Ames, B.N., 1983. Revised methods for the 646–651.Salmonella mutagenicity test. Mutat. Res. 113, 173–215. Standard Methods for the Examination of Water and Wastewater,

Mathew, E., Hastings, L., 1987. Evaluation of toxicity test 1985. APHA-AWWA-WPCF, pp. 496–503.procedure for screening treatability potential of waste in soil. Suga, S.F., Lindstrom, J.E., 1997. Braddock, EnvironmentalToxic. Assessment 2, 265–281. influences on microbial degradation of Exxon Valdez oil on the

National Academy of Science, 1975. Petroleum in the Marine shorelines of Prince William Sound Alaska. Environ. Sci.Environment, National Academy Press, Washington, D.C., p. Technol. 31 (5), 1564–1572.107. Swanell, R.P.J., Head, I.M., 1994. Bioremediation come of age.

O’Donnell, A.G., Hopkins, D.W., 1993. Extraction, detection and Nature 368, 396–397.identification of genetically engineered microorganisms from Thomas, J.M., Ward, C.H., 1994. Introduced organisms forsoils. In: Edward, C. (Ed.), Monitoring Genetically Manipulated subsurface bioremediation. In: Norris, Hinchee, Brown et al.Microorganisms in the Environment, Wiley Publishers, pp. (Eds.), Handbook of Bioremediation, Lewis Publishers, Lon-111–131. don, pp. 227–339.

Page, A.L., Miller, R.H., Keeney, D.R., 1982. Methods of Soil US Environmental Protection Agency, 1988. Technology Screen-Analysis, part II, 2nd ed., ASA-SSSA, Wisconsin, pp. 937–970. ing Guide for Treatment of CERCLA soils and sludges. EPA/

Paul, E.A., Clark, F.E., 1989. Soil Microbilogy and Biochemistry, 540/288/004, Washington DC.Academic Press, New York, pp. 46–48. US Environmental Protection Agency, 1990. Bioremediation of

Perry, J.J., 1981. Microbial metabolism of cyclic alkanes. In: Hazardous Wastes, EPA/600/9-90/041, Office of Research andAtlas, R.M. (Ed.), Petroleum Microbiology, Macmillan Inc., pp. Development, Washington DC.62–128. Veal, D.A., Stokes, H.W., 1992. Genetic exchange in natural

Potter, L., 1993. Analysis of petroleum contaminated soil and microbial communities. In: Marshall, K.C. (Ed.), Advances inwater: An Overview. In: Calabrese, E., Kostecki, P. (Eds.), Microbial Ecology, vol. 12, Plenum Press, New York, pp.Principle and Practices for Petroleum Contaminated Soils, 383–430.Lewis Publishers, London, pp. 1-24. Wang, Z., Fingas, M., Li, K., 1994. Fractionation of a light crude

Prichard, P.H., Bourquin, A.W., 1984. The use of microcosms for oil and identification and quantification of aliphatic, aromatic,evaluation of interactions between pollutants and microorga- and biomarker compounds by GC–FID and GC–MS Part I. J.nisms. In: Marshall, K.C. (Ed.), Advances in Microbial Ecolo- Chromatogr. Sci. 32, 361–366.gy, vol. 7, Plenum Press, New York, pp. 133–215. Weissenfels, W.D., Beyer, M., Klein, J., 1990. Degradation of

Prichard, H.P., Costa, C.F., 1991. EPA’s Alaska oil spill phenanthrene, fluorene, fluoranthene by pure bacterial cultures.bioremediation report. Environ. Science Technol. 25, 372–379. Appl. Microbiol. Biotechnol. 32, 479–484.

Prince, R.C., Clark, J.R., Lindstrom, J.E. et al., 1994. Bioremedia- Yateem, A., Balba, M.T., Al-Awadhi, N., El-Nawawy, A.S., 1998.tion of the Exxon Valdez oil spill: Monitoring safety and White rot fungi and their role in remediating oil contaminatedefficacy. In: Hinchee, R.E., Alleman, B.C., Hoeppel, R.E., soil. Environ. Int. 24 (1), 181–187.