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Journal of Scientific & Industrial Research Vo1.58. October 1999. pp 764-772
Biodegradation of Soil Contaminants
Dimitre G Karamanev Department of Chemical and Biochemical Engineering. The University of Western Ontario. London.
Ontario. Canada N6A 5B9
The paper reviews the current technologies for the biological degradation of recalcitrant soil pollutants.
Introduction
Many industrial and other human activities resulted in a widespread contamination of soils with various contaminants. Most of these contaminants are toxic to humans and animals . They reach humans and animals through migration into the atmosphere, groundwater and crops I. The pollution became especially disturbing in the past few decades, when the awareness of thi s problem resulted in the enormous growth of environmental Science and Technolc.gy (S&T). While some of the contaminants can be eas i!y degraded by the soil microflora, other are much more persistent and can remain in soil for decades, continuously contaminating groundwater and/or air.
There are many different chemical, physica l and thermal methods for the elimination of recalcitrant soil pollutants such as incineration, vitrification, chemical oxidation, etc2,3 , Unfortunately, most of these methods are expensive and not quite environmentally friendly. The natural capacity of microorganisms to degrade a huge variety of organic and some inorganic compounds, even the most tox ic ones (e.g. cyanides), is the bas is for the microbial methods for degradation (biodegradation) of soil contaminants, or bioremediation4
. These methods are generally much less expensive than chemical and physical ones. Another very interesting feature of microorganisms is that they are capable of degrading organic substances, which never existed in the nature and were produced only synthetically I. Biodegradation is. in fact , a biocatalytic process of the transformation of soil contaminants into harmless products. The biocatalyst are the microbial cells. The carbon, oxygen, hydro-
gen, and chlorine atoms in the contaminant molecule are usually converted into inorganic substances such as H
20,
CO~ , CH4 ' and HC\. The microbial methods for contaminants degradation can be considered as a basis of "green technologies" 4.
Soil Contaminants that Can Be Treat,ed Biologically
There are two major types of soil pollutants : organic and inorganic ones. Among the major sources of inorganic pollutants is the mining industry, while organic pollutants are often associated with petroleum and chemical industries and agriculture. The most widely spread biodegradable soil pollutants are given subsequently.
Alcanes - The major source of both normal and cyclic alcanes are petroleum and its derivatives . In addition to petroleum, food-grade oils also contaminate some soilsv,; however both their spread and toxicity are much smaller compared to those of petroleum. One of the main reasons for the pollution by petroleum products globally is the fact that the biggest oil producers are not the biggest consumers which means that huge amounts of oil have to be transported. The largest oil spills occur during transportation7
• This type of spills usually occurs in sea, but spilled oil quickly moves towards the coastlines, where it pollutes soils, The list of the recent spills is given in Table I (ref.7), Leaking underground tanks while releasing smaller quantity of petroleum products per unit, produce a significant pollution because of their large numberH
The toxicity of petroleum hydrocarbons to any type of fauna and flora has long been established~, However, there are many types of microorganisms that can degrade
KARAMANEV: SOIL CONTAMINANTS 765
Table I - The largest oil spills
Source Place Year Oil in
tonnes
Iraq/Quwait Persian Gulf 1991 1000000
Exxon Valdez Alaska 1989 33000
IXTOC I well Campeche Bay, Mexico 1979 350000
Amoco Cadiz Brittany, France 1978 223000
Torrey Canyon Cornwall, England 1967 117000
them. The microbial metabolism of normal and cyclic alkanes has been discussed in details by Atlas 'o. Several microbial species, mainly bacteria , fungi and microalgae, that degrade petroleum hydrocarbons have been isolated and studied " . A typical brut reaction of aerobic degradation of alcanes is shown below :
. . . ( I )
While most of the microorganisms degrade alcanes aerobically, ap"''''robic degradation also occurs, where sulphate ions can be used as alternative electron acceptorl ~ . n .
A romatic Hydrocarbons - There are two major types of aromatic soil pollutants: monocyclic and polycyclic ones. The typical monocyclic aromatic hydrocarbons are benzene, toluene, ethy lbenzene and xylene. Their mixture (BTEX) is considered to be among the most toxic component of petroleum hydrocarbons . Polycyclic aromatic hydrocarbons (PAHs) such as anthracene, phen-
anthrene, naphthalene, and pyrene are found mostly in creosote.
Aromatic hydrocarbons can be degraded aerobically by various microorganisms, including bacteria, fungi and microalgae. The monocyclic compounds can be biodegraded much easier than polycyclic ones. The recalcitrance of PAHs increases with increase in the number of aromatic rings7. This is probably related to the solubility of the compound, which also decreases with increase in the number of rings'4. The anaerobic biodegradation of aromatic hydrocarbons has been studied by many authors. The alternative electron acceptors which replace oxygen are sulphates, nitrates and ferric ions '5 .
Chlorinated Aliphatic Compounds - These are substituted hydrocarbons in which one or more hydrogen atoms have been replaced with chlorine. Most of these compounds are used as industrial solvents . This is probably the largest group of man-made environmental pollutants. Many of these compounds are not only toxic, but are also suspected human carcinogens and/or mutagens Ir,. This is the reason why chlorinated aliphatics make the largest group ,of priority pollutants in the li st of the US Environmental Protection Agency. The industrially produced chlorinated aliphatics in c lude ch loroalcanes , chloroa lkenes and chlorinated cyc loaliphatic compounds '7 . Typical examples of ch loroalcanes are I , I-trichloroethane, I , I ,2 -trichloroethane, carbon tetrachloride, etc. Chloroalkenes, and es pec ia lly trichloroethene (TCE) a nd tetrachloroethene (PC E) are the most widely spread contaminant in groundwater and soil woridwide 'K • The chlorinated cycloaliphatics such as lindane (y-l ,2,3,4,5,6-hexachlorocyclohexan) were used in the past as pesticides, but are currently being rarely used because of their toxicity. .
It has to be mentioned here that a large amount (es timated to 5x I O~ kg) of chlorinated aliphatics such as chlo-
DimitrI.' G Karamanev is an Assistant PIVJessor at the Department oJChemical Engineering at the University oj Western Olllario in London, Ontario. Canada. He received his doctoral degree in Chemical Engineering JlVm the Bulgariall Academy oj Sciences, Sofia. Dr Karamanev is a specialist, in the fields oj EnvilVnmental Bioengineering (desigll oj novel bioreactor systems Jor the treatment oj contaminated soil, gas and water; hydrodynamics, mass-transJer, kinetic studies. [md mathematicallllodelling oj these systems, scaling-up oj bioreaclors), Chemical Engineering (lwsic studies oj different multiphase systems such as in verse fluidized beds, single solid parlie/es, and gas bubbles ill liquid), Biohydromelallurgy (biological production oj uranium and gold). He is an a/./lhor oj more than 90 papers published in scholarly journals and conJerence proceedings. Dr Karamanev is lIl ember oj the Canadian Sociely Jor Chemical Engineering, American InslitUle oj Chemical Engineers, and the Inlenwlional Sociely Jor EnvilVllmental Biotechnology.
766 ] SCI IND RES VOL.58 OCTOBER 1999
romethane are being produced naturally, mainly by soil fungi 17. However, they are only intermediates in the global carbon cycle and are being degraded quickly after
being produced by soi l bacteria. Therefore, they do not have any negative effect on the flora and fauna.
In add ition to their toxic ity, chlorinated aliphat ics are also very persistent to biodegradation under natural conditions. That is why they are an object of extensive studies. Twenty years ago, most of the ch lorinated a l iph atics were considered non-biodegradable. Since then significant progress has been made. It has been shown that the aerobic biodegradati on rate of ch lorinated alcanes decreases with the increase of the number of CI atoms . At the same time, anaerobic biotransformations of these compounds show an opposite effect - the biological reactivity increases with the increase in the number of CI atoms 17 . Chloroalkenes are more chemical ly stable, and therefo re more res istant to biodegradati on. The direct microbial degradation of TCE is impossi ble . However, it has been shown that methanotrophic microorganisms can cometabolize TCE in the presence of methane I9.20 . Some other microbial-cosubstrate systems are also being studied recently. Presently, thi s is the most popular way to treat TCE biologically. The most studied mi croorganism is Methilosinus trichosporium OB3b which produces the enzyme methanomon oox igenase (MMO), responsible for the TCE biodegradation21. The
biodegradation can lead to total minerali zation, resulting in the production of CO2, HP, and CI-
Chlorinated Aromatic Compounds - Because of the overall toxicity of monocyclic ch lorinated aromatics , they have been used ex tensively as herbicides, fungicides, and insecticides22 . The most important among them are c hlorinated benzenes, such as monochlorobenzene, and chlorinated phenol s such as pentachlorophenol and 2,3,4,6-te trachlorophenol. Among the chlorinated compounds containing more than one aromatic ring, the Illost environmentally important ones are po lychlorinated biphenyls (PCBs), DDT, dioxins and furans. These are extremely toxic chemicals which are very persistent to biodegradation: they are detected in soil several years after being disposed there l
. That is why even if their production has been discontinued in most countries, there is still a significant environmental pollution.
Both aerobic and anaerobic biod eg radation of monocyclic chlorinaQed aromatics have been reported. For example, the anaerobic treatment of one of the most widely spread members of that group, pentach lorophenol (PCP), results in the dehalogenation and formation
of phenol and benzoates23• Aerobic biotreatment of the
same compound is much more popular4.2'~ . The total min
eralization of PCP yields CO2, HP, and CI - (ref.27).
Polycyclic compounds are much more persistent to biological treatment. However, anaerobic dehalogenation of PCBs by mixed cultures has already been reported2X . The aerobic degradation of PCBs is relat ively quick only in the case of less chlorinated PCBS29.
Nitroaromatic Compounds - The main source of soil pollution by thi s c lass of chemicals is associated with military activities, and in particular, explosives lO
. The most important from env ironmental point of view are 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenol (picri c acid). They are considered environmental hazard because of their toxic ity and recalcitrance. TNT is also a mutagen31. The mic robial treatment of nitroaromatic explosives is a promising technology which has been under development in the recent past. Both aerobic and anaerob ic biotreatment are poss ible. During the aerobic biotreatment of TNT, a significant decrease in both toxicity and mutagenicity has been observed·'o. However, no significant mineralization has been observed by measuring the amount of CO2 released. T he fate of the biotransformed TNT is not clear yet. The microorganisms used for nitroaromatics biodegradation are mostly Pseudomonas33 sp . and Phanerochaefe chrysosporium.32 •
The anaerobic treatment of nitroaromatics results in the reduction of aromatic nitro groups34.
Inorganic Pollulants - While majority of the biotreatable soi l and groundwater pollu tants are organic compounds, there are some inorganic pollutants that can be treated biologically. These include metals (Fe, Cu , Zn , Mn , AI, Co), usually in ionic form, as well as anions (NO]-, CN-, SO/ -) . The above mentioned cations are usua·lly found in acid mine drainage, which is associated with minin g activities. When these metals are present in soi l, they are usually first leached and then the ca ti ons in leachate are treated biologically. One of the most popular methods for biological treatment of these cati ons is by sulphate reduction34-37. It is based on the reduction of sulphates (which are also present in the acid mine drainage) by anaerobic microorganisms such as Desulfovibrio . The resulting sulphide ions combine with the cations, formi ng insoluble sulphides. Ferrous ions can also be removed from water by aerobic treatment using the bacterium Thiobacillus ferrooxidans, which oxidized them to ferric ions37.w. The latter can be
easily precipitated as hydroxide . Nitrates can be reduced
anaerobically to nitrogen gas. Cyanide ions are amena-
KARAMANEV: SOIL CONTAMINANTS 767
ble to biological treatment by the fungus Pseudomonas putidia resulting in ammonia and carbon dioxide40
.4I.
Methods for the Contaminant Treatment
The physical methods of treatment of soil contaminants are associated usually with the change of the phase of the contaminant. The most important among them are:
• Soil washing - The contaminant is transferred from the soil particles to a liquid phase (usually aqueous solution);
• Venting - This method is used for the transfer of volatile components such as gasoline. from soil to gas phase (usually air);
• Stripping - In this method, volatile compounds are stripped from liquid by a gas;
• Adsorption - The contaminant is adsorbed by a solid particles (sorbent), usually activated carbon ; and
• Solvent extraction. The biological methods include:
• Aerobic oxidation; • Anaerobic treatment; • Biosorption - This method is actually a combina
tion between a biological method (cell cultivation) and a physical method (adsorption). It is based on the adsorption of different, usually inorganic, compounds by either live or dead microbial cells. The pollutant is not involved in biological reaction .
Since most of the physical methods result just in the change of phase of the pollutant, and not in its destruction, it is beneficial to supplement them with a destructive, biological method. For example, the air, containing pollutant after the venting of gasoline-contaminated soil can be treated biologically in order to mineralize the hydrocarbon molecules. The resulting overall process is called bioventing. Or, process water used in soil washing can be treated in bioreactors in order to mineralize the organic pollutants washed from soil. This shows that biodegradation of soil pollutants can be performed not only in solid (soil) phase, but also in liquid and in gas phase.
Engineering Aspects of Soil Pollutants Biodegradation
Two major methods for the biodegradation of soil pollutants are being used in practice :
• In-situ methods. There are two major types of in-situ methods : (i) Natural attenuation and (ii) Active in-situ bioremediation
• Ex-situ Methods
Bioreactor Methods: Slurry reactors; Immobilized soil reactors; Rotating drums ; Composting; and Landfarming.
Natural Attenuation - Also called intrinsic remediation is the most passive among the above mentioned groups of methods42
. It is based on the ability of the microflora, naturally present in soil, to degrade the pollutants without any significant human intervention . The course of the natural processes can be analyzed ("monitored natural attenuation") and some corrective action can be performed if necessary. This type of soil treatment is applicable only in the cases when no significant contaminant migration is expected, when the degradation under natural conditions is reasonably fast and when there is enough time to wait until the completion of the process. In general, the natural attenuation plan is based on a risk-based approach43.
Active ill-sitll Bioremediation - In this case the soil and groundwater stay in place, but they are subjected to some kind of engineer.ing action, usually aimed at the improvement of microbial activity in soil:
• Bioaugmentation: addition of active microbial culture to soil in the cases when the natural microflora is not sufficient;
• Addition of chemicals required for the microbial growth, which are not sufficient in the native soil44
:
electron acceptors (oxygen, hydrogen peroxide, nitrates), nutrient salts (nitrogen, phosphorus sources), co-substrates (methane), moisture (water sprinkling; pumping humid air);
• Addition of agents improving bioavailability (sUIfactants);
• Adjustment of pH of soil (addition of acids or bases);
• Temperature control (radio frequence heating, covering soil with thermal insulation);
• Toxicity reduction by removal of some of the pol-lutant by44:
• In-situ soil flushing; • Soil venting; • In-situ soil vapour extraction ; • Steam extraction ;
768 J SCI IND RES VOL.58 OCTOBER 1999
• Enhancement of bioremediation by parti al chemical oxidatjon4~.
The methods related to toxicity reduction are usually coupled with biodegradation of the pollutant which has been transferred to liquid or gas phase.
The ex-s itu methods are most important from engineering point of view, and they are discussed subsequently ..
Bioengineering Methods for Ex-Situ Treatment of Soil Pollutants
The advantages of ex-situ methods include the ability to intensify and control the major process parameters of the biodegradation such as oxygen input, nutrient sa lts addition, spatial di stribution (mixing) , temperature, and pH. As a result, these methods have much higher volumetric efficiency than pass ive (in-situ) methods, but they are also more costly.
The different methods for biological so il treatme nt can be cl ass ified according to:
• The movement of so il part ic les. There are two
maj or types of processes: (i) F ixed bed (particles are fix ed in space) : (ii ) Moving soil partic les .
• The continuous phase can be: (a) L iquid (soil particles are fl ooded or dispersed in liquid) ; (b) Gas (the space between soi l particles is occupied by air or another gas).
Biotreatment of Soil in Slurry Reactors (2a) According to the above classification , thi s is a mov
ing-particle process (2) with liquid as a continuous phase (a), so it was class ified as (2a) . The slurry treatment is presently among the most popul ar methods for ex-situ soil bioreme(liati on. It has been tested to treat aromatic and aliphatic hydrocarbons, chlorinated mono- and polycyclic aromatic compounds, ha logenated a liphati cs, nitroarornatics46
-4x
• Therefore, so il s polluted with pesticides, fue ls, wood-preserving wastes, PCBs, coal tars , refinery wastes , explosives as well as many other pollutants can be treated.
In this method, smaller particle fractioll of contaminated soil is mixed with water containing nutrient mineraI salts andlor other supplements . In order to keep the particles in suspension" the system is mechanica lly mixed using different types of impe llers and is aerated (in the
Soil in
Ai r Soil out
Figure I - Schemat ic view o f a two- impeller slurry biorcactor
cases of aerobi c treatment). The process is usually carried out in self-contained vessels, or bioreactors . In most cases the microorgani sms, naturally assoc iated with soil , are sufficient to treat the pollutant. If the soil contains no appropriate mi croorganisms, bioreactor is inoculated arti ficiall t~.
Variou s types of s lurry bi oreactors have been proposed. There are different design solutions for the mechanical mixing dev ice (type and number of impellers), aeration (surface aerators, submerged aerators, aeration through the impel ler) and the fl ow structure (shape of the vesse l, draft tube, baffles) ~. A typical two-impeller s lurry bioreac tor with baffles is shown in Figure I .
The EIMCO Biolift reactor~O can operate at very high s lurry concentrations (25-50 per cent wt) with re lati ve ly low energy inpllt. The reactor uses the airlift principle for both mixing and aeration . It a lso contains a mechanical mi xing device. The reactor was used to treat soils contaminated with pesticides, PCp, oil and other pollutants50
The Dutch Dual Inj ec ted Turbul e nt Separation (DITS)-reactor" is an another type of s lurry bioreactors. This is an air-agitated suspension reactor with a tapered bottom (Figure 2) withou t mechanical mix ing. Its geometry allows to combine the processes of soil size separation and biotreatment. The geometrical parameters of this reactor were optimized52 and treatment studies were performed" .
Slurry bioprocesses can al so be carried out in open aerated lagoons instead ofbioreactors47.4x. While thi s is
the s implest type of slurry process, it has significant di sadvantages - low volumetric efficiency and release of volatile compounds into the atmosphere. They are also
KARAMANEY: SOIL CONTAMINANTS 769
Slurry recycle Air
Fine fraction (slurry)
Gas-liquid injector
Figure 2 - The D1TS bi oreactor
affected by the variation in atmospheric conditions .
Slurry bioreactors are so popular in soil treatment, that their popUlarity was compared to that of activated
sludge process for wastewater treatment50, which was first introduced at the beginning of twentieth century54.
Both types of bioprocesses are suspension processes : suspended microorganisms in the activated sludge and
suspended soil particles in the case of soil slurry. How
ever, there are two disadvantages of the activated sludge
process: (i) wash-out of microorganisms from the reac
tor and (ii)high shear stress around the impeller. These
problems were solved by immobilization of micro organisms to the surface of inert solid surfaces~5. Similar prob
lems are also observed in soil slurry reactors. Especially
important is the shear stress . Since pollutant-degrading microorganisms usually grow on the surface of soil par
ticles forming so called biofilm ' , the particle-particle
friction in the slurry affects very negatively the growth
ofbiofilm, and therefore, the entire biodegradation process . Continuing the analogy to the wastewater treatment,
the problems in slurry reactors can also be solved by
immobilization. The immobilized soil bioreactor was first proposed a few years ago~6 ,which is discu ssed, in de
tails, SUbsequently.
Soil Immobilization ( J a) In order to eliminate both interparticle friction and
washing-out of soil particles, it was proposed to fix these particles in space by immobilizing them onto an inert
solid support27• The best support for immobilization was
found to be non-woven polyethylene or polypropylene geotextile. The process of soil immobilization was carried out in a new type of bioreactor, named immobilized soil bioreactor. It is based on an airlift princ iple: a verti
cal vessel is divided vertically into two separate secti ons by means of a geotexti Ie (Figure 3). One of the sec ti ons
Semipenneable wall
Immobilized soil
Geotextile fibres
Soil partie es in suspension
Air
Figure3 - Scheme of the immobilized soil bioreactor
is aerated. The geotextile is a highly porous material
(porosity>98 per cent) with a wide pore size di stribution, between ·several microns and 2 mm. In the
bioreactor, the geotextile wall is semipermeable: its pore
size is such that liquid can flow through it while gas
bubbles are too large to pass through the pores. Because of the semipermeability of geotextile, the flow s(Jucture
of liquid in the reactor is very specific: it flows upwards
in the aerated section, downwards in the non-aerated one and horizontally through the geotextile from the non
aerated towards the aerated section (Figure 3) . When
contaminated soil is introduced to liquid in the reactor,
the resulting slurry starts circulating as described above.
Because of the large pore size distribution of geotextile
and the repeated circulation of soil particles through it, soil particles get entrapped ("immobilized") into the
pores of geotextile: large particles occupy larger pores
while smaller particles get entrapped into smaller pores . It has been shown that the process of soil immobilization is fast , of the order of minutes57. As soon as so il particles are immobilized, the microbial culture, exist
ing naturally in soil, begins to grow quickly because of the low shear stress, good aeration and the presence of
balanced nutrie~t salts composition in liquid . This type of bioreactor can be used for the treatment of both soil
and groundwater. When used for the mineralization of PCP in aqueous phase, the volumetric biodegradation rate was by I to 4 orders of magnitude higher than any result of PCP biodegradation reported so far~6 . The mineralization of groundwater containing II ppm PCP below detectable leve l was achieved in 90 s , while it nor-
770 J SCI IND RES VOL.58 OCTOBER 1999
E'10' = " .~ 10'" ~
.~ 10' a:;
lO-4 ~~~~
l(f ~~~~ lO-I
10'
organisms
Figure 4 - Size of the particles in different immobilized systems and the complexity of the system
mally takes hours and days to treat the same concentration by any other method27 .
It has been shown that soil immobilization can be considered as a fundamentally new level compared to the other types of immobilization from the point of view of both size, scale and complexity of the system (Figure4). Therefore, immobilized molecules, used in chemical catalysis, have the size of tens to hundreds of angstroms and represent Ithe non-living nature. They can be considered as the first level of immobilization . The next level is occupied by microbial cells with a size of several micrometers. They are single living organisms. A new level of complexity is observed in immobilized soil with a particle size between micron s and several millimeters (Figure 4). This is the most complex immobilization system, containing an entire microcosm of the soil particle.
Rotating Drum Bioreactors (2b) The rotating drum is a horizontal vessel, which oper
ates much like a concrete mixer. It is used to treat contaminated soil with low oxygen demand or for anaerobic processes'!. The soil content is high, between 65 and 75 per cent wt. Recently, drum bioreactors have been used to treat aerobically fuel-contaminated soils5K5
,!.
Rotating drums are used also for composting. This type of bioreactors still have relatively limited application.
Gas-solid Fluidized Bed Bioreactors (2b) While fluidization technology becomes more and
more popular in the bioreactor engineering for the treatment of liquid-phase pollutants(xl, its application in the
field of soil biotreatrnent is still limited. A resent study61 has shown that a gas-solid fluidized bed bioreactor can be used for the bioremediation of soil contaminated with mineral oil and hexachlorocyclohexane. The rate ofbiodegradation in the soil fluidized bed was much higher than that these in a fixed bed of soil and in a remediation heap61.
Fixed Beds of Soil ( I b) This method can either be used in contained vessels
(bioreactors)62.6J or in the field . Landfanning, biopiles and composting are the typical members of this group.
Landfarming can be applied only when the upper 30 cm of soil are contaminated'!. This method has been used successfully for the treatment of soils contaminated with petroleum hydrocarbons, wood preservation waste and many other pollutants . The method is based on the aerating the soil by agricultural tilling techniques'! . Nitrogen, phosphorus and potassium sources are added as inorganic salts in order to activate the soil microflora. pH of soil can also be controlled . The facility can either be open or covered by a roof. The efficiency of this process, as well as its cost, is intermediate between these of in-situ and bioreactor methods.
The soil composting is based on the mixing of soil with a solid organic material that is easily biodegradable, such as straw, wood chips, leafs, etc I. Additionally, inorganic nutrients can be added if necessary. The material could be placed as a heap or in a contained vessel. During the intensive biodegradation of the organic material, the temperature rises between 40-60"C. The so il pollutants are co-metabolized along with the organic material. Different contaminants such as oil hydrocarbons, chlorophenols, and TNT have been treated by compostingl.
If one analyses the present status of biotreatment of soil contaminants, it can be found that most of the efforts have been applied towards the microbiological aspect of the problem. The engineering, and in particular, bioreactor engineering aspects require more study.
Acknowledgement
This work was supported in part by the National Science and Engineering Council of Canada (NSERC).
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KARAMANEV: SOIL CONTAMINANTS 771
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