Review of Literature - Shodhganga

Review of Literature In Hanahan, South Carolina in 1975 a massive leak from a military fuel storage facility released about 80,000 gallons of kerosene-based jet fuel. Immediate and extensive recovery measures managed to contain the spill, but could not prevent some fuel from soaking into the permeable sandy soil and reaching the underlying water table. Soon, ground water was leaching such toxic chemicals as benzene from the fuel-saturated soils and carrying them toward a nearby residential area. By 1985, contamination had reached the residential area, and the facility was faced with a serious environmental problem. Studies by U.S. Geological survey had shown that microorganisms naturally present in the soils were actively consuming fuel-derived toxic compounds and transforming them into harmless carbon dioxide. By "stimulating" the natural microbial community through nutrient addition, it was theoretically possible to increase rates of biodegradation and thereby shield the residential area from further contamination. In 1992, this theory was put into practice by USGS scientists. Nutrients were delivered to contaminated soils through infiltration galleries, contaminated ground water was removed by a series of extraction wells, and the arduous task of monitoring contamination levels began. By the end of 1993, contamination in the residential area had been reduced by 75 percent. Nearer to the infiltration galleries (the source of the nutrients), the results were even better. Ground water that once had contained more than 5,000 parts per billion toluene now contained no detectable contamination. Bioremediation had worked! This was the first and best-documented example of intrinsic bioremediation in which naturally occurring microbial processes remediates contaminated ground water without human intervention (USGS, 1995). Fungi and bacteria are known to be the principal agents of hydrocarbon biodegradation. Such organisms are readily isolated from soil and it was reported that the introduction of oily wastes into soil caused appreciable increases in the numbers of both groups (Jensen, 1975; Lianos and Kjoller, 1976; Pinholt et al., 1979). Although soil counts cannot be used for analysis of biodegradability of the spilled hydrocarbons, the diversity and the number of microorganisms at a given site Mycoremediation of Crude Oil Contaminated Soil

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may help to characterize that site with respect to the toxicity of these hydrocarbons to the microbiota, age of the spill and concentration of the pollutant. In addition, microbial isolates from the soils that are historically exposed to hydrocarbon pollution exhibit a higher potential of biodegradation than others with no history of such exposure. For that matter, in a crude petroleum-polluted soil, the biodiversity and microbial prevalence of certain microbe(s) may indicate how well the soil is supporting the growth of that microbe(s) (Bossert and Compeau, 1995). Uncontrolled and catastrophic releases of petroleum pose ecological and environmental repercussions as a lot of hydrocarbon components are toxic and persistent in terrestrial and aquatic environments. Several physico-chemical methods of decontaminating the environment have been established and employed. Biological degradation, a safe, effective and an economic alternative method, is a process of decay initiated by biological agents, specifically in this case by microorganisms. Bioremediation refers to site restoration through the removal of organic contaminants by microorganisms. The factors, that influence the rates of microbial degradation of hydrocarbons, include temperature, pH, salinity, oxygen, nutrients, and physical and chemical composition of petroleum. Due to complexity of crude oil, biodegradation involves the interaction of many different microbial species. It could be attributed to the effects of synergistic interactions among members of the consortium (Salleh et al., 2003). 2.1

Isolation and screening of soil fungi Fluorescein diactetae hydrolysis was studied in soil and straw incubated for

up to 3 h. Hydrolysis was found to increase linearly with soil addition. FDA hydrolysis by pure cultures of Fusarium culmorum increased linearly with mycelium addition both in shake cultures and after inoculation into sterile soil. FDA hydrolysis by Pseudomonas denitrificans increased linearly with biomass addition. The FDA hydrolytic activities in soil samples from different layers of an agricultural soil were correlated with respiration. Acetone was found to be suitable for terminating the reaction (Schnurer and Rosswall, 1982). An

extracellular

lignin-degrading

enzyme

from

the

basidiomycete

Phanerochaete chrysosporium Burdsall was purified to homogeneity by ionexchange chromatography. The 42,000-dalton ligninase contains one protoheme IX Mycoremediation of Crude Oil Contaminated Soil

Page 42

per molecule. It catalyzed, nonstereospecifically, several oxidations in the alkyl side chains of lignin-related compounds: Ca-Cp cleavage in lignin model compounds of the type aryl-C.HOH-CpHRCyH20H (R = -aryl or -0- aryl), oxidation of benzyl alcohols to aldehydes or ketones, intradiol cleavage in phenylglycol structures, and hydroxylation of benzylic methylene groups. It also catalyzed oxidative coupling of phenols, perhaps explaining the long-recognized association between phenol oxidation and lignin degradation. All reactions required H202. The Ci-Cp cleavage and methylene hydroxylation reactions involved substrate oxygenation; the oxygen atom is from 02 and not H202. Thus the enzyme was an oxygenase, unique in its requirement for H202 (Tien and Kirk, 1984). Ligninolytic enzymes of the white rot fungi Coriolopsis polyzona, Phanerochaete chrysosporium, and Trametes versicolor growing on wheat straw under nearly natural conditions were investigated by Vyas et al. (1994a). Manganese peroxidase (MnP), secreted as early as on day 3, was dominant over other activities during the initial phase (the first 10 days). Its activity profile was similar in all the three fungi. Lignin peroxidase (LIP) was not detected in the extracellular enzyme extracts of C .polyzona and P. chrysosporium cultures. T. versicolor secreted LIP after 10 d of growth. Laccase activity appeared at basal levels without any significant changes. The white-rot fungus Phanerochaete chrysosporium produces ligninolytic enzymes during secondary metabolism in response to nutrient carbon, nitrogen or sulfur starvation (Glenn and Gold, 1985; Paszczynski et al., 1985; Tien and Kirk, 1988). P. chrysosporium mineralized poplar wood lignin to CO2 better at 28ºC compared with that at 39ºC (Hattaka and Uusi-Rauva, 1983). Asther et al. (1988) and Liebeskind et al. (1990) achieved higher productivity of lignin peroxidase by employing down-shifts of temperature to that suboptimal for growth, prior to the productive phase. But for this, there is no other information about the influence temperature of growth exerts on the production of ligninolytic enzymes (Vyas et al., 1994b). The in vitro oxidation of the two polycyclic aromatic hydrocarbons anthracene and benzo[a]pyrene, which have ionization potentials of 1,000)

P. chrysosporium, T. versicolor

Total ligninolytic activity, Lacc. Lacc., LiP, MnP

Simazine, trifluralin, dieldrin (10 each) 3-7 ring PAHs (6.3-53 each

P. ostreatus, P. chrysosporium, T. versicolor

Lacc., LiP, MnP

Sum of 3 PAHs (150)

95(P.ostr.) 78(P.chry.) 69(T.vers.)

Slight No LiP activity

Straw in tube separated with nylon web (1:1)

10Mm Phosphate buffer, Ph 6.5, 40ºC Deionized H20 + 0.1 M KPhosphatecitric acid, pH4.5 and 7.0 50mM Succinate lactate buffer, pH4.5

P. ostreatus, P. chrysosporium, T. versicolor

Lacc., MnP

ANT (50) PYR (50)

ANT-PYR 95-97 (P. ostr.) 60-82 (P. chry.) 60-53 (T. vers.)

MnP (P. ostr.) Slight (P.chry.)

Polyurethane or pinewood cubed + straw

50mM Succinate lactate buffer, pH4.5

Novotny et al., 2004

Lentinus edodes

Lacc., MnP

PCP (200)

99(sterilized soil + fungus) 42(non-sterilized soil + fungus)

Lacc. and MnP

Sawdust (1:5)

Distilled water 4ºC

Okeke et al., 1997

P. chrysosporium

LiP, MnP

7 WRF and 5 LDF

Eggen, 1999

Novotny et al., 1999

PHE (10) 73(PHE) LiP and Sawdust Distilled Wang et PYR (10) 51(PYR) MnP (1:10) water al., 2009 BaP (10) 25(BaP) WRF = white-rot fungus; LDF = litter-decomposing fungus; Lacc. = laccase; LiP = lignin peroxidase; MiP = manganese independent peroxidise; MnP = manganese peroxidise; SMC = spent mushroom compost; MC = mushroom compost; PCP = pentachlorophenol; PCA = penthachloronoanisole; ANT = anthracene; PYR = pyrene; PHE = Phenantherene; BaP = benzo [a] pyrene.

Mycoremediation of Crude Oil Contaminated Soil

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The abilities of ten bacterial and five fungal isolates indigenous to polluted mechanic soils to utilise and degrade crude oil and gasoline was investigated by Sebiomo et al. (2011). Of all the bacterial and fungal isolates Pseudomonas sp., Bacillus sp. and Aspergillus sp. were found to be more predominant in the polluted mechanic soils. Bacillus subtilis, Pseudomonas aeruginosa and Aspergillus ochraceus have shown the best abilities to utilise and degrade crude oil and gasoline in-vitro. Fraga et al., 2011 studied the isolation and selection of filamentous fungi from petroleum contaminated soil. Two hundred and thirteen filamentous fungi were isolated from contaminated and uncontaminated soil samples. In uncontaminated soil, 12 genera and 29 species were identified, whereas in contaminated soil eight genera and 12 species were identified. The presence of oil in the soil significantly decreased the number of CFU (44.39 x 103 vs. 8.83 x 102) and consequently, the diversity of soil fungi. Four fungi strains viz. Aspergillus niger, A. terreus, Rhizopus sp. and Penicillium sp. were isolated from soil and tarball samples collected from mangrove forest of Alibaug and Akshi coastal area, Maharashtra, India (Lotfinasabasl et al., 2012). These strains were assessed for their degradation capability of petroleum hydrocarbons measuring growth diameter in Potato Dextrose Agar (PDA) solid media for different concentrations of kerosene (5%- 20% (v/v)). Table-9 depicts various advantages and disadvantages of using biochemical based methods for studying microbial diversity in soil.


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Table-9: Advantages and disadvantages of biochemical-based methods to study soil microbial diversity (Kirk et al., 2004) Method

Advantages

Disadvantages

Selected references

Plate counts

Fast Inexpensive

Community level physiological profiling (CLPP)/ Sole carbon source utilization patterns

Fast Highly reproducible

Unculturable microorganisms not detected Bias towards fast growing Individuals Bias towards fungal species that produce large quantities of Spores Only represents culturable fraction of community

Relatively inexpensive Differentiate between microbial communities Generates large amount of data Option of using bacterial, fungal plates or site specific carbon sources (Biolog)

Trevors (1998b), Tabacchioni et al. (2000)

Garland and Mills (1991), Garland (1996a), Classen et al. (2003)

Favours fast growing organisms Only represents those organisms capable of utilizing available carbon sources Potential metabolic diversity, not in situ diversity Sensitive to inoculum density

Soil biological activities are vital for restoration of contaminated soil with hydrocarbons by biotransforming petroleum compounds into harmless types. Activities of dehydrogenase, urease, lipase and phosphatase enzymes, the number of heterotrophic degrading bacteria and soil respiration rate, during the bioremediation of contaminated soil by crude oil were determined. The results showed that the numbers of degrading and heterotrophic microorganisms were increased with decreasing amount of contaminant especially in those samples treated with mycorrhiza inoculation (Bahrampour and Sarvimoghanlo, 2012). Twenty one fungal isolates including four new fungal isolates capable of degrading polycyclic aromatic hydrocarbons (PAHs) in soil were recovered from Mycoremediation of Crude Oil Contaminated Soil

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Orman Garden, Wadi Degla Protectorate and benzene station soils by (Ali et al., 2012). All tested fungi exhibited lignin peroxidase and manganese peroxidase activities in solid as well as in liquid cultures. However, laccase was detected in low amounts by some of the tested fungal isolates. Aspergillus terreus was superior in ligninolytic enzyme production and was chosen for the further studies. The statistical optimum temperatures for lignin peroxidase and manganese peroxidase production by A. terreus were 33.6 and 33.1°C, respectively. Meanwhile lignin peroxidase and manganese peroxidase yields were maximal at pH 4.1 and 5.8, respectively. Highest ligninolytic enzyme secretions were established on D-glucose and sodium nitrate. A. terreus was able to degrade 98.5% of naphthalene and 91% of anthracene in soil models. Five lignin degrading white rot fungi were selected (Bjerkandera sp., Phanerochaete chrysosporium, Physisporinus rivulosus, Phlebia

radiata and

Phlebia sp. Nf b19) and studied for MnP production in small-scale. Extracellular MnP activity was followed and cultivations were harvested at proximity of the peak activity. The production of MnPs varied in different organisms but was clearly regulated by inducing liquid media components (Mn2+, veratryl alcohol and malonate). The highest specific activities were observed with MnPs from P. chrysosporium (200 U mg-1), Phlebia sp. Nf b19 (55 U mg-1) and P. rivulosus (89 U mg-1) and these MnPs are considered as the most potential candidates for further studies. The molecular weight of the purified MnPs was estimated to be between 45–50 kDa (Järvinen et al., 2012). Thenmozhi et al., 2013 studied the isolation and screening of fungi from engine oil contaminated soil samples from various automobile workshops in Pudukkottai, Tamilnadu, South India. From the collected samples indigenous fungi were isolated using malt extract medium. Totally sixteen isolates were isolated among which Aspergillus and Rhizopus sp. were found to be predominant. Morphological studies of isolated fungal isolates showed different growth pattern on different media. Preliminary hydrocarbon degradation analyses were done to evaluate the growth diameter of fungal colonies on minimal media supplement with used engine oil.


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2.2 Physico-chemical properties of soil Crude hydrocarbons were added to the surface of wet tundra soils at Barrow, Alaska at volumes of 5 and 12 l/m2 to study physical and chemical effects in the soil for three years (Everett, 1978). Soils treated with 5 l/m2 had no alteration in chemical and physical properties. Whereas treatment with 12 l/m2 recorded an increase in seasonal thaw, organic carbon and available phosphorus, shift in soil pH toward neutrality and decrease in water infiltration rate and in plant available cations (Ca, Mg, K). Crude oil exploration and production has been the largest anthropogenic factor contributing to the degradation of Momoge Wetland, China (Wang et al., 2010). All contaminated areas had significantly higher (p < 0.05) contents of TPH and TOC, but significantly lower (p < 0.05) TN contents than those of the uncontaminated areas. Contaminated sites also exhibited significantly higher (p < 0.05) pH values, C/N and C/P ratios. For TP contents and EC, no significant changes were detected. Loamy sand of agricultural land in an oil producing and processing area was polluted with crude oil and the fouled soil samples were reclaimed using chemical degreasers

and

detergents

(Essien

and

John,

2010).

The

chemical

degreaser/detergent emulsion effectively recovered soil properties and plant growth in the reclaimed soil and is recommended for short-duration restoration of crude-oil degraded soil for productive agriculture. The effect of crude oil spill on soil causes decrease in soil moisture , porosity, water holding capacity, soil pH and extractable phosphorous whereas increase in total nitrogen, organic carbon and exchangeable potassium. Spilled crude oil is certainly responsible for alterations of soil physico-chemical properties (Barua et al., 2011). The effect of crude oil pollution on physical and chemical characteristics of soil was investigated in Perisoru, Braila County by Marinescu et al. (2011). In case of physical analysis, the values obtained for granulometric fractions were not influenced by the presence of crude oil. Results obtained showed variation in chemical properties of soil. Organic carbon and C/N ratios increased from an unpolluted soil to polluted soil. Mobile phosphorous and potassium registered Mycoremediation of Crude Oil Contaminated Soil

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similar values with polluted and unpolluted soil. Crude oil at high pollution levels inhibited the growth of crops. It is generally recognized that the toxicity (human and environmental) of petroleum products increases as the molecular weights of the compounds decrease. A pilot study was conducted by Vincent et al. (2011) on soil simulated with crude oil to examine the effects of the hydrocarbon on soil properties like pH, electrical conductivity, total organic carbon and matter, total nitrogen and phosphorus, texture and heavy metals (Cd, Pb, Ni, Vand Cr), the potentials of exploring soil indigenous microbes and determining suitable conditions for effective degradation of the contaminant as well as evaluating the kinetics of the process. Crude oil pollution caused a reduction in pH, conductivity and phosphorus level with significant effect in the growth rate of soil heterotrophic microbes, but did not show any negative effect on the other properties. It was suggested that suitable pH condition and nutrient availability will enhance speedy microbial transformation of contaminant. 2.3

Collection, analysis and measurement of total petroleum hydrocarbons and polycyclic aromatic hydrocarbons A gas chromatographic (GC) method for the determination of petroleum

hydrocarbons (PHs) in the boiling range from 175°C to 525°C (Clo-C4o alkane) in soil was investigated by Delft et al. (1994). A simultaneous determination method of BTEX (benzene, toluene, ethylbenzene, o, m, p-xylene) and TPH (kerosene, diesel, jet fuel and bunker C) in soil with gas chromatography/flame ionization detection (GC-FID) was studied by Shin and Kwon, 2000. The effects of extraction method, extraction solvent, solvent volume and extraction time on the extraction performance were evaluated. The advantages of this procedure was the use of simple and common equipment, reduced volumes of organic solvents, rapid extraction periods of less than 20 min, and simultaneous analysis of volatile and semivolatile compounds. Manual

solid-phase

microextraction

(SPME)

coupled

with

gas

chromatography-mass spectrometry was investigated as a possible alternative for the determination of petroleum hydrocarbons in soils (Cam and Gagni, 2001).


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Screening by gas chromatography with flame ionization detection and gas chromatography-mass spectrometry on sludge extracts of wastewater treatment basins along with soxhlet extraction with trichlorotrifluoroethane permitted a quick assessment of petroleum pollutants in the environment (Pavlova and Ivanova, 2003). To determine TPH in soil for the evaluation of contaminated level of oil, extraction characteristics of several pretreatment methods were investigated and the practical detection limits for these methods obtained by gas chromatography/ mass spectrometry were compared by Jeon et al. (2007). Pre-treatment using mechanical shaking for extraction and the used method for extraction solvent gave the lowest detection limit and are more practical and applicable to real sample than the conventional methods. Crude oil spillage contamination of soil from the Niger Delta region of Nigeria was investigated by Okop and Ekpo (2012), three months after an extensive oil spillage. Samples were collected at depths of 0 – 15 cm, 15 – 30 cm and 30 – 60 cm. The soil samples were prepared for analysis using solvent extraction methods and were analysed by gas chromatography fitted with a flame ionisation detector. 2.3.1 Collection and preservation of environmental samples The ability to collect and preserve a sample that is representative of the site is a critically important step. Obtaining representative environmental samples is always a challenge due to the heterogeneity of different sample matrices. Additional difficulties are encountered with petroleum hydrocarbons due to the wide range in volatility, solubility, biodegradation, and adsorption potential of individual constituents. Most site investigations for assessment of petroleum hydrocarbon contamination in the environment are regulated by the states. However, sample collection and preservation recommendations follow U.S. EPA guidelines. A summary of the most commonly used guidelines is included in Table-10. It should be noted that there might be additional requirements in any given state. Before a sample is collected, the particular state requirements must be investigated. Because of holding time considerations, the laboratory must be selected and notified prior to the collection of the samples (Weisman, 1998).


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Table-10: U.S. EPA-recommended sampling protocols Analytical Parameter

Trphb

Volatile Petroleum Hydrocarbons (VPH)d

Analytical Method(s) EPA 418.1 (IR); Gravimetric; GC/FID

Sample Containera Media Volume Type water 1 liter Glass jar with Teflon lined cap soil 125 mL Wide mouth glassTeflon lined cap

water Various

soil

Extractable Petroleum Hydrocarbons (EPH)f

BTEX

40 mL

40 mL

water

1 liter

soil

60 mL

water

40 mL

soil

40 mL

Glass vial with Teflon lined septum Glass vial with Teflon lined septum Glass jar with Teflon lined cap

Preservativesc

acid fix pHSonication >Supercritical fluid >Subcritical fluid. Volatile compounds (e.g., BTEX and gasoline) may be solvent-extracted from soil. EPA Method 5035, purge and trap analysis, specifies a methanol extraction, which is usually done by mechanical shaking of the soil with methanol. A portion of the methanol extract is added to a purge vessel and diluted in reagent grade water. The extract is then purged similar to a water sample. Headspace analysis also works well for analyzing volatiles in soils. The soil is placed in a headspace vial and heated. Salts can be added to more efficiently drive out the volatiles from the sample into the head-space of the sample container.


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Similar to water headspace analysis, the soil head-space technique is useful when heavy oils and high analyte concentrations are present which can severely contaminate purge and trap instrumentation. Detection limits are generally higher for headspace analysis than for purge and trap analysis. The simplest method to separate semivolatile compounds from soils is to shake or vortex (vigorous mechanical stirring) soil with a solvent. Adding a desiccant to the soil/solvent mixture can help to break up soil and increase the surface area. The extract can be analyzed directly. Simple shaking is quick and easy, making it an excellent field extraction technique. However, extraction efficiency may vary depending on soil type. Soxhlet extraction is a very efficient extraction process which is commonly used for semivolatiles. Solvent is heated and refluxed (recirculated) through the soil sample continuously for 16-24 hours or overnight. This method generates a relatively large volume of extract that needs to be concentrated. Thus, it is more appropriate for semivolatiles than for volatiles (Weisman, 1998). 2.3.3

Total petroleum hydrocarbon (TPH) measurement Petroleum compounds can be generally classified into two major component

categories: hydrocarbons and non-hydrocarbons. Hydrocarbons comprise the majority of the components in most petroleum products and are the compounds that are primarily (but not always) measured as TPH. The non-hydrocarbon components (those containing sulphur, nitrogen and oxygen heteroatoms, as well as carbon and hydrogen in the molecule) are relatively minor in most refined motor fuels as they tend to concentrate in the heavy distillation fractions. The hydrocarbon constituents can be grouped into saturated hydrocarbons, unsaturated hydrocarbons and aromatics. There are several subclasses of importance within these groups. Fig. 6 summarizes the different categories and subclasses.


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Fig. 6: TPH compounds by class (Weisman, 1998).


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Fig. 7: Summary of petroleum product types and TPH analytical methods with respect to approximate carbon number and boiling point ranges (Weisman, 1998) Fig. 7 shows the relationship between boiling range and carbon number for some common petroleum products. This figure clearly shows the overlap between carbon ranges of different products as well as the overlap in corresponding analytical methods. For example, Fig. 5 shows that an analytical method designed for gasoline range organics may report some of the hydrocarbons present in diesel fuel. The same is also true for analytical tests for diesel range organics that will identify some of the hydrocarbons present in gasoline contaminated soils. A more detailed discussion of boiling point and carbon number classification as well as a discussion of petroleum product composition, specification, product additives, and weathering is provided in Appendix II: Characterization of Petroleum Products. Total petroleum hydrocarbon (TPH) measurements are conducted to determine the total amount of hydrocarbon present in the environment. There are a Mycoremediation of Crude Oil Contaminated Soil

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wide variety of TPH methods. In practice, TPH is defined by the method used to analyze it. Different methods often give different results because they are designed to extract and measure slightly different subsets of petroleum hydrocarbons. No single method gives a precise and accurate measurement of TPH for all types of contamination. The four most commonly used TPH testing methods include gas chromatography (GC), infrared spectrometry (IR), gravimetric analysis, and immunoassay (Weisman, 1998). A TPH method generates a single number quantifying the amount of petroleum that is measured by the specified technique. GC-based methods are currently the preferred laboratory methods for TPH measurement because they detect a broad range of hydrocarbons, they provide both sensitivity and selectivity, and they can be used for TPH identification as well as quantification. IR-based methods have been widely used in the past for TPH measurement because they are simple, quick and inexpensive. However, their use is currently decreasing due to the worldwide ban on Freon production (needed for sample extraction and measurement), the nonspecificity of these methods, and their inability to provide any information on TPH identification and potential risk. Gravimetric-based methods are also simple, quick, and inexpensive, but they suffer from the same limitations as IRbased methods. Gravimetric-based methods may be useful for very oily sludge and wastewaters, which will present analytical difficulties for other more sensitive methods. Immunoassay TPH methods are gaining popularity for field testing because they offer a simple, quick technique for in-situ TPH quantification (Weisman, 1998). Table-12 briefly summarizes the information for each TPH analytical method for quick reference. This table provide more detailed information about published GC-based and non-GC TPH methods. These tables provide methodspecific information for EPA and state methods, including recommendations for method use, common interferences, procedural notes, advantages, and disadvantages (Weisman, 1998).


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Table-12: General summary of analytical methods for TPH measurement Method Name GC-Based TPH methods

Method Type/ Environmental media primarily laboratory but also field applicationscan be adapted for all media

Typical products detected

Typical carbon range detected

Primarily gasolines, diesel fuel, and fuel oil #2 - can be modified for heavier hydro-carbon mixtures (e.g., lubricating oils, heavy fuel oils)

normally between C6 and C25 or C36 (can be modified for higher carbon numbers) most hydrocarbons with very high hydrocarbons

EPA Method 8015B, State modified 8015 methods EPA Method 418.1 EPA Method 9071; EPA proposed Method 1664 EPA Method 4030

IR-Based TPH methods

laboratory and field screening most appropriate for water and soil

primarily diesel and fuel oils

Gravimetric TPH methods

laboratory - most appropriate for wastewaters, sludges, and sediment

most appropriate for heavier petroleum products (e.g., crude oils, lubricating oils, etc.)

anything that is extractable (with exception of volatiles which are lost)

Immunoassay TPH methods

Field screening – most appropriate for soil and water

Various products (but yields only screening numbers)

Aromatic hydrocarbons (e.g., BTEX, PAHs)

Detector type GC/FID

Approximate detection limits can be as low as 0.5 mg/L in water, 10 mg/kg in soil

IR spectrometer

1 mg/L in water; 10 mg/kg in soil

Gravimetric balance

5 to 10 mg/L in water; 50 mg/kg in soil

technique is simple, quick, and inexpensive

Portable test kit

200 to 500 µg/L in water; 10 to 500 mg/kg in soil

Technique is simple, quick, inexpensive, and can be done in the field

Published methods

Key advantages

Interferences/ limitations

can detect broad range of hydrocarbon compounds; simple and sensitive; can provide information (e.g., a chromatogram) for product identification technique is simple, quick, and inexpensive

normally cannot detect compounds below C6; may not detect polar hydrocarbons (e.g., alcohols, ethers, etc.); chlorinated solvents may be quantified as TPH Freon is now banned; lack of specificity; low sensitivity; high loss of volatiles; poor extraction of high molecular weight hydrocarbons; prone to interferences; provides quantitation only Freon is banned, although other solvents are available; lack of specificity; low sensitivity; high loss of volatiles; prone to interferences; provides quantitation only Low sensitivity; can detect interferences; primarily only measure aromatics; low accuracy and precision; should only be used as screening measurement; provides quantitation only


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2.3.3.1 Gas chromatography (GC) total petroleum hydrocarbon methods For GC-based methods, TPH is defined as anything extractable by a solvent or purge gas and detectable by gas chromatography/flame ionization detection (GC/FID) within a specified carbon range. The primary advantage of GC-based methods is that they provide information about the type of petroleum in the sample in addition to measuring the amount. Identification of product type(s) is not always straightforward, however, and requires an experienced analyst of petroleum products. Detection limits are method- and matrix-dependent and can be as low as 0.5 mg/L in water or 10 mg/kg in soil (Weisman, 1998). Overview of the Technique Gas chromatography is a technique that separates mixtures. “A mixture of chemicals is separated into its individual components as the sample travels through a column in the gas chromatograph. Separation is achieved by a combination of factors including boiling point, polarity, and affinity differences among the different components in the sample. The time a compound spends on a specific column is called the retention time and it is reproducible. The retention time is characteristic of a compound under given experimental parameters and specified column. As the separated components elute from the column, they are detected (Swallow et al., 1988). The detector signal is proportional to the amount of compound present. Chromatographic columns are commonly used to determine TPH compounds approximately in the order of their boiling points. Compounds are detected with a flame ionization detector, which responds to virtually all compounds that can burn. The sum of all responses within a specified range is equated to a hydrocarbon concentration by reference to standards of known concentration. Two techniques are most commonly used to get the samples into the column. (a) Purge and trap systems purge components out of water or water/methanol by bubbling gas through the liquid. The components are concentrated on a very short intermediate column or “trap,” which is heated to drive them onto the analytical column where they are separated. Hydrocarbons from C5 through about C12 can be analyzed using this technique. Purge and trap sample introduction is used for light products such as gasoline and condensate. Mycoremediation of Crude Oil Contaminated Soil

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(b) Direct injection involves taking the hydrocarbon, diluted hydrocarbon, or an extract of hydrocarbon into a syringe and injecting it into the gas chromatograph. This technique can be used for any type of hydrocarbon, but it is most frequently used for distillates, lube oils and crude oils. Headspace sample introduction can be used for the determination of light hydrocarbons, and it is most often used for field screening (Weisman, 1998). 2.3.3.2 What Do GC Methods Measure? A GC/FID will detect any hydrocarbons that elute from the column and burn. The analog signal from the detector is called a chromatogram. GC/FID methods specify a certain portion of the chromatogram (a “window” or carbon number range) for quantification. The carbon number range will approximate that of the fuel of interest gasoline, diesel, or heavier hydrocarbon. The carbon number range specified for each fuel may differ from state to state. Volatile compounds that elute before the solvent peak (usually those < C6) are typically not measured (Weisman, 1998). GC-based methods can be broadly used for different kinds of petroleum releases but are most appropriate for detecting non polar hydrocarbons with carbon numbers between C6 and C25 or C36. Many lube oils contain molecules with more than 40 carbon atoms. Crude oils may contain molecules with 100 carbons or more. These heavy hydrocarbons are outside the detection range of the more common GC-based TPH methods, but specialized gas chromatographs are capable of analyzing such heavy molecules. Accurate quantification depends on adjusting the chromatograph to reach as high a carbon number as possible, then running a calibration standard with the same carbon range as the sample. The lab must also check for mass discrimination, a tendency for heavy molecular weight hydrocarbons to be retained in the injection port. Labs should be notified if a sample is suspected to be heavy oil, or to contain a mixture of light and heavy oils, so that they can use the appropriate GC method. Gravimetric or IR methods are often preferred for very heavy samples. They can even be used as a check on GC/FID results if it is suspected that heavy molecular weight hydrocarbons are present but are not being detected. Laboratories should flag data if heavy material is observed in the chromatogram, even if this material cannot be quantified. Calibration standards vary. Most methods specify a gasoline calibration standard for Mycoremediation of Crude Oil Contaminated Soil

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volatile range TPH and a diesel fuel #2 standard for extractable range TPH. Some methods use synthetic mixtures for calibration. Because most methods are written for gasoline or diesel fuel, TPH methods may have to be adjusted to measure contamination by heavier hydrocarbons - lubricating oils, heavy fuel oils, or crude. Such adjustments may entail use of a more aggressive solvent, a wider GC “window” - up to C36 or more - and a different calibration standard that more closely resembles the “heavier” contamination (Weisman, 1998). GC-based methods can be modified and fine-tuned so that they are suitable for measurement of specific petroleum products or group types. Examples of modified GC-based methods include GRO, DRO, and TPHV methods. These modified methods can be particularly useful when there is information on the source of contamination, but method results should be interpreted with the clear understanding that a modified method was used for detection of a specific carbon range. It is essential that the user understand what hydrocarbons a GC-based method can and cannot detect and how results are quantified. For example, BTEX is a subset of TPHV. If benzene, toluene, ethylbenzene, and the three xylene isomers are present in a sample, they will be quantified along with the other TPHV components. The TPHV measurement typically is greater than the sum of the BTEX measurements. Gasoline should not be quantified by adding the TPHV and BTEX quantities together (Weisman, 1998). Interpretation of GC-based TPH data can be complicated and the analytical method should always be considered when interpreting concentration data. A volatile range TPH analysis may be very useful for quantifying TPH at a gasoline release site, but a volatile range TPH analysis will not detect the presence of lube oil. In addition, a modified GC-based method which has been specifically selected for detection of gasoline-range organics at a gasoline-contaminated site may also detect hydrocarbons from other petroleum releases because fuel carbon ranges frequently overlap. Gasoline is found primarily in the volatile range. Diesel fuel falls primarily in an extractable range. Jet fuel overlaps both the volatile and semivolatile ranges. However, the detection of different kinds of petroleum does not necessarily indicate that there have been multiple releases at a site. Analyses of spilled waste oil will frequently detect the presence of gasoline, and sometimes diesel. This does not necessarily indicate multiple spills. All waste oils contain some fuel. As much as Mycoremediation of Crude Oil Contaminated Soil

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10% of used motor oil can consist of gasoline (Owen and Coley, 1990). The fuel gets into the oil as combustion chamber gases blowing past the piston rings - a more pronounced problem in high mileage engines with worn rings. Liquid fuel will also seep past rings under cold start and warm-up conditions (Rhodes et al., 1994). If the type of contaminant is unknown, a “fingerprint” analysis can help identify it. A “fingerprint”, or pattern recognition, analysis is a direct injection GC/FID analysis where the chromatogram is compared to chromatograms of reference materials. Certain fuels can be identified by characteristic, reproducible chromatographic patterns. For example, chromatograms of gasoline and diesel differ considerably. There are complicating factors as many hydrocarbon streams may have similar fingerprints for example; Diesel #2 and #2 fuel oil both has the same boiling point range and chromatographic fingerprint. A fingerprint can be used to conclusively identify a mixture when a known sample of that mixture or samples of the mixture’s source materials are available as references. These chromatograms must be interpreted by experienced analysts. While GC-based TPH and pattern recognition methods are very similar, TPH methods stress calibration and quality control, while pattern recognition methods stress detail and comparability (Weisman, 1998). 2.3.4 Polycyclic aromatic hydrocarbon pollution Polycyclic aromatic hydrocarbons (PAHs) are constituted of two or more fused benzene rings sharing a pair of carbon atoms between two adjacent rings in linear, cluster or angular arrangements. Natural inputs of PAHs occur during volcanic eruptions and forest fires. However, most PAHs originate from anthropogenic sources, such as the incomplete combustion of fossil fuels, wood and waste, automobile exhaust, and unintentional petroleum derivatives spills. PAHs contain only carbon and hydrogen atoms. Petroleum-derived heterocyclic compounds may also contain sulphur (e.g., dibenzothiophene), nitrogen or oxygen atoms. (Dabestani and Ivanov, 1999). Accidental crude oil spills in the sea are important localized sources of PAH contamination. Due the low solubility of the aromatic fraction of crude oil, which accounts for 50% of all the fractions, PAHs are mostly deposited into the sediments or transported to shorelines and other marine ecosystems, such as coastal marshes or estuaries (Dabestani and Ivanov, 1999; AECIPE, 2002). Mycoremediation of Crude Oil Contaminated Soil

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PAHs are commonly found in soil, even in remote areas without human settlements. Diffuse contamination occurs mainly via atmospheric deposition of PAHs adsorbed to particles. Wind transports these particles to further localizations and the PAHs adsorbed to particles are deposited directly onto the soil or indirectly through the vegetation. It is estimated that soil receives 0.7 - 1 mg/m2 of PAHs annually by air emissions (Johnsen and Karlson, 2007). Except for PAH compounds containing fjord regions (Fig. 8), PAHs have a planar geometry. The alternating single and double bonds give PAHs an unusual stability and consequently, resistance to microbial degradation. The water solubility and bioavailability of PAHs is low, which decrease as the molecular mass increases (Baird and Cann, 2008; Table-13). Furthermore, PAHs with a bay (e.g., chrysene or benzo[a]pyrene) or fjord regions (e.g. benzo[c]phenanthrene or dibenzo[a,l]pyrene) in their molecular structure are the most potential carcinogens. For instance, when entering the organism, benzo[a]pyrene is activated by a series of metabolic reactions that lead to the ultimate carcinogenic metabolite, a reactive diol epoxide, which may bind covalently to DNA, leading to mutations and, consequently, resulting in tumours (Mattsson, 2008).

Bay region

Fjord region

Fig. 8: Bay and fjord regions in PAH molecular structure.

Estimations predict that 1.7 - 8.8 x 106 tons of crude oil enter into coastal environments annually. As an example, the oil rig Deepwater Horizon exploded and sank in the Gulf of Mexico on the 22nd of April 2010 spreading up to 800 m3 of light crude oil per day (CEDRE, 2010). Once in the soil, PAHs may be degraded or transformed, which will determine their transport, distribution and levels of concentration.


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Table-13: Properties of the 16 EPA PAHs, listed according to their water solubility

IARC

Concentration in soile Forest Urban (µg/kg) (µg/kg)

3.37

2B

33

39

4.00 3.24 3.92 4.18 5.22 5.18

NA 3 3 3 3 3

3.4 60 2 6.9 118 72

16 190 57 23 805 593

F

f

2B

82

387

7.44±0.06

4.54

3

8.6

58

7.53±0.30

5.91

2B

43

437

Compound

Water solubility (mg/l)a

Molecular Mass (g/mol)a

Vapor pressure (Pa at 25°C)a,b

Naphthalene

31.0

128.19

10.40

Acenaphthylene Phenanthrene Acenaphthene Fluorene Fluoranthene Pyrene Indeno [1,2,3c,d] pyrene Anthracene Benzo [a]anthracene Benzo [a]pyrene

16.1 4.57 3.80 1.90 0.26 0.132

152.20 178.23 152.21 166.22 202.26 202.26

0.90 0.020 0.30 0.090 0.00123 0.0006

8.12± 0.02 8.22±0.04 7.90 7.68±0.05 7.88±0.05 7.9±0.1 7.43±0.01

0.062

276.33

f

0.045

178.23

0.0010

Benzo [b]fluoranthene Benzo [k]fluoranthene Chrysene Dibenzo [a,h]anthracene Benzo [g,h,i]perylene

-5

Ionization Potential (eV)a

logKow

a,c

d

0.011

228.29

2.8x10

0.0038

252.31

7.0x10-7

7.10

6.04

1

39

350

0.0015

252.31

7.70

5.80

2B

158

456

0.0008

252.31

6.7x10-5 (at 20°C) 5.2X10-

F

6.00

2B

186

236

7.60±0.03

1.65

2B

117

278

10

7.38±0.02

f

2A

15

55

1.3x10-8

7.31

6.50

3

62

370

0.0006

228.29

0.006

278.35

0.00026

268.35

8 -7

5.7x10 3.7x10-

a

Dabestani and Ivanov, 1999; Steffen, 2003; U.S. National Library of Medicine, 2010. Library of Medicine, 2010. logarithm for the octanol-water partition coefficient of a specific compound. IARC is the International Agency for Research on Cancer that classified compounds with carcinogenic risk as: 1-carcinogenic to humans; 2A-probably carcinogenic to humans; 2B-possibly carcinogenic to humans; 3-not classifiable as to its carcinogenicity to humans; 4-probably not carcinogenic to humans; NA-not classified (IARC, 2010). e Values show PAH concentration in forest or temperate urban soil (Wilcke, 2000). f data not available b U.S. National c logKow is the d


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Assessment of soil pollution on the basis of their PAH concentration is presented in Table-14. Table-14: Standard limiting PAH content (µg/kg) in the soil surface layer (Malawska and Wilkoirski, 2001) Total PAHs content < 200 200-600 600-1000 1000-5000 5000-10000 >10000 2.4

Pollution class 0 I II III IV V

Soil assessment Unpolluted (natural content) Unpolluted (Increased content) Slightly polluted Polluted Heavily polluted Very heavily polluted

Comparison of bioremediation versus physico-chemical methods for decontamination of crude oil contaminated soil. Several laboratory and field tests have demonstrated that bioremediation

could be a cost-effective clean-up technology to treat oily soils and sediments containing biodegradable hydrocarbons and indigenous specialised microorganisms (Huesemann and Moore, 1993; Bossert and Compeau, 1995; Wang et al., 1995; Jackson et al., 1996; Venosa et al., 1996; Salanitro et al., 1997). Crude oil as well as other commercial hydrocarbon mixtures containing alkanes, alkenes, aromatics and polar compounds could be considered extensively biodegradable in soils. Differences in the extent of biodegradation were reported (Morgan and Watkinson, 1989; Leahy and Colwell, 1990; Bossert and Compeau, 1995; Huesmann, 1997) depending upon soil and hydrocarbon source type, concentration of total hydrocarbons, oxygen and nutrient availability. Microorganisms are capable of catalyzing a variety of reactions (Table-15): dechlorination, hydrolysis, cleavage, oxidation, reduction, dehydrogenation, dehydrohalogenation, and substitution (Suthersan, 1999). •

Dechlorination—the chlorinated compound becomes an electron acceptor; in this process, a chlorine atom is removed and is replaced with a hydrogen atom.


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•

Hydrolysis—frequently conducted outside the microbial cell by exoenzymes. Hydrolysis is simply a cleavage of an organic molecule with the addition of water.

•

Cleavage—cleaving a carbon–carbon bond is another important reaction. An organic compound is split or a terminal carbon is cleaved off an organic chain.

•

Oxidation—breakdown of organic compounds using an electrophilic form of oxygen.

•

Reduction—breakdown of organic compounds by a nucleophilic form of hydrogen or by direct electron delivery.

•

Dehydrogenation—an oxidation–reduction reaction that results in the loss of two electrons and two protons, resulting in the loss of two hydrogen atoms.

•

Dehydrohalogenation—similar to dechlorination, results in the loss of a hydrogen and chlorine atom from the organic compound.

•

Substitution—these reactions involve replacing one atom with another.

Table-15: Microbially catalyzed reactions (McCarty and Semprini, 1994) Reaction Dehalogenation Hydrolysis Cleavage Oxidation Reduction Dehydrohalogenation Substitution

Example Cl2C = CHCl + H+→ ClHC = CHCl + Cl– RCO – OR + H2O→ RCOOH + RʹOH RCOOH→RH + CO2 CH3CHCl2 + H2O→CH3 CCl2 OH + 2H+ +2e– CCl4 + H+ + 2e–→ CHCl3 + Cl– CCl3CH3→CCl2CH2 + HCl CH3CH2Br + HS→ CH3CH2SH + Br–

Bioremediation is a process in which microorganisms metabolize contaminants either through oxidative or reductive processes. Under favourable conditions, microorganisms can oxidatively degrade organic contaminants completely into non toxic by product such as carbon dioxide and water or organic acids and methane. Highly electrophilic compounds such as halogenated aliphatic and explosives typically are bioremediated through reductive processes that remove the electrophilic halogens or nitro groups. Bioremediation processes may be directed Mycoremediation of Crude Oil Contaminated Soil

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towards accomplishing: (1) complete oxidation of organic contaminants (termed mineralization), (2) biotransformation of organic chemicals into smaller less toxic metabolites, or (3) reduction of highly electrophilic halo- and nitro- groups by transferring electrons from an electron donor (typically a sugar or fatty acid) to the contaminant, resulting in less toxic compound. With increasing numbers of successfully demonstrated cleanups, biological remediation alone or in combination of other methods, has gained an established place as a soil restoration technology (Rockne and Reddy, 2003). Extensive petroleum hydrocarbon exploration activities often result in the pollution of the environment, which could lead to disastrous consequences for the biotic and abiotic components of the ecosystem if not restored. Attention to the exploration of the biological alternatives is needed. Okoh (2006) reviewed the menace of petroleum hydrocarbon pollution and its biodegradation in the environment with the view of understanding the biodegradation processes for better exploitation in bioremediation challenges. List of physicochemical methods for hydrocarbon decontamination in soil and its limitations is summerized in Table-16.


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Table-16: Physico-chemical methods for decontamination of hydrocarbon contaminated soil (Morgan and Watkinson, 1989) Variations of Method Principles Comments technique Evaporation Rotating tube Excavation needed; top Thermal and /or furnace; fluidized soils only; off gases must destruction of bed furnace; be treated; expensive hydrocarbons incinerators Removal of Percolating towers; Excavation needed; top Extraction hydrocarbons scrubbing towers; soils only; extract must into solutions fluidized beds be disposed of; efficiency unknown; hydrocarbons may be bound to soils; very expensive; little data. Removal of Volatiles only; potential Steam Rotating drum; in volatiles for subsoils; steam must stripping situ be treated; little data Removal of Volatiles only; potential Hot-air Rotating drum; in volatiles for subsoils; little data stripping situ Chemical oxidation Ground water control

Alteration of pollutant to ease removal Pumping of aquifer to prevent flow

Immobilization Binding hydrocarbons in situ Raising Flooding hydrocarbons to surface on top of water table Groundwater Adsorption pumped through activated carbon Excavated soil Detergent or in situ soil is extraction flushed with surfactant

In situ; reaction chambers

No information for hydrocarbons

Pumping with/ without physical containment; direct removal of compounds on water

Prevents migration of hydrocarbons; no removal of compounds in unsaturated zone; efficient, useful to prevent pollution spread during biotreatment; widely used Expensive; not widely tested; doesnot remove pollutants Risk of spreading pollution; inefficient

Chemical bonding; soil solidification -

-

Expensive; waste requires disposal; efficiency unknown

-

Efficiency unknown; expensive


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Alternative biological way of soil decontamination and its advantages is presented in Table-17. Table-17: In situ treatments in bioremediation (Korda et al., 1997) Treatment

Description

Applicable to

Advantages

The addition of oxygen, water, and mineral nutrients (usually combinations of nitrogen, phosphorus and trace metals). Combines Bioventing conventional soil venting with biodegradation. The less volatile compounds are biodegraded and the volatile components are vented off in conventional venting. Bioaugmentation Direct application of microorganisms originating from (a) the remediation site (b) an off-site vendor (c) genetic engineering.

Ground water, soils

Acceleration by as much as 100-fold of the reproduction of indigenous organisms.

Soils

Addresses full range of petroleum hydrocarbons. Effective method of supplying indigenous microorganisms with oxygen to support degradation.

Ground water, soils

Synthetic or biogenic substances are used to increase the aqueous solubility of solid hydrocarbons and emulsify liquid hydrocarbons. To stimulate microbial metabolism by supplying indigenous oil-degraders with nutrients (N, P, K etc).

Solid and liquid aliphatic and aromatic hydrocarbons.

One of the most effective bioremediation techniques. The microorganisms have been cultured and adapted and their degrading capacity can be enhanced for specific contaminants and site conditions. Enhancement of contaminant accessibility to microorganisms, nutrients and possibly oxygen.

Biostimulation

Surfactants

Fertilizer application

Soil, groundwater, sediments.


Acceleration of natural biodegradation process, especially in nutrient deficient areas.

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For effective bioremediation of crude oil contaminated soil following parameters should be present in its optimum level Table-18.

Table-18: Optimum environmental condition for microbial activity during bioremediation (Vidali, 2001) Parameter Moisture content pH Oxygen content Nutrient content Temperature (ºC) Contaminants-hydrocarbons Heavy metals Soil type

Optimum value 40-60% 6.5-8 10-18% C:N:P = 100:10:1 20-30 5-10% of dry weight of soil 700 ppm Low clay or silt content

2.5 Case studies with fungi for bioremediation of oil contamination: The colonization of sandy loam soil following inoculation with spore suspensions of the white-rot fungi Phanerochaete chrysosporium ATCC 24725 and Chrysosporium lignorum CL1 by an epifluorescence microscopy-image analysis method was investigated. These fungi and Trametes versicolor PV1 mineralized 3,4dichloroaniline and benzo(a)pyrene in soil at concentrations up to 250 μg g−1(Morgan et al., 1993). Under

static,

non-nitrogen-limiting

conditions,

Phanerochaete

chrysosporium (INA-12) mineralized both phenanthrene and benzo[a]pyrene. Lignin peroxidase is not necessarily involved in the biodegradation of all PAH and that a significant factor in PAH biodegradation and/or disappearance in cultures with the intact fungus may be attributed to sorption phenomena (Barclay et al., 1995). The mineralization of polycyclic aromatic hydrocarbons by white rot fungus Pleurotus ostreatus to

14

CO2 7.0% of [14C] catechol, 3.0% of [14C] phenanthrene,

0.4% of [14C] pyrene, and 0.19% of [14C] benzo [a] pyrene by day 11 of incubation was studied. Although activity of both enzymes laccase and manganese inhibited peroxidase was observed, no distinct correlation to polycyclic aromatic hydrocarbon degradation was found (Bezalel et al., 1996a).


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The metabolism of phenanthrene by white rot fungus Pleurotus ostreatus, grown for 11 days in basidiomycetes rich medium containing [14C] phenanthrene was studied. The fungus metabolized 94% of the phenanthrene. Of the total radioactivity, 3% was oxidized to CO2. Approximately 52% of phenanthrene was metabolized

to

trans-9,10-dihydroxy-9,10-dihydrophenanthrene,

trans-9,10-dihydrodiol (28%),

phenanthrene

2,2*-diphenic acid (17%), and unidentified

metabolites (7%). Non extractable metabolites accounted for 35% of the total radioactivity (Bezalel et al., 1996b). The degradation of eight unlabeled highly condensed polycyclic aromatic hydrocarbons (PAH) and the mineralization of three 14C-labeled PAH by the whiterot fungus Pleurotus florida was investigated. Pleurotus florida mineralized 53% [14C]pyrene, 25% [14C]benzo[a]anthracene and 39% [14C]benzo[a]pyrene to 14CO2 in the presence of eight unlabeled PAH (50 µg applied) within 15 weeks (Wolter et al., 1997). A selection of 30 strains of micromycetes known as good degraders of polychlorinated aromatic compounds mostly isolated from soil and belonging to various taxonomic groups, has been investigated to degrade fluorene. Among the 30 strains tested, 12 could be considered as best degraders because of a rate of degradation at 60% or over and 3 strains of Cunninghamella genus were very efficient (mean of degradation: 96%) but, different strains from Ascomycetes, Basidiomycetes and Deuteromycetes were also found efficient. (Garon et al., 2000). Fertilizing an oily desert soil sample with a mixture of glucose and peptone resulted in enhancing hydrocarbon disappearance in that soil. In the oily desert soil, glucose/peptone addition increased microbial numbers, but after utilization of the glucose/peptone, microbial numbers remained high and enhanced attenuation of hydrocarbons was found (Radwan et al., 2000). The feasibility of bioremediation as a treatment option for a chronically diesel-oil-polluted soil in an alpine glacier area at an altitude of 2,875 m above mean sea level was investigated under natural attenuation and biostimulation (Margesin and Schinner, 2001). At the end of the third summer season (after 780 days), the initial level of contamination (2,612 ± 70 mg of hydrocarbons g [dry weight] of soil 21) was reduced by (50 ± 4)% and (70 ± 2)% in the unfertilized and fertilized soil, Mycoremediation of Crude Oil Contaminated Soil

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respectively. In the fertilized soil, all biological parameters (microbial numbers, soil respiration, catalase and lipase activities) were significantly enhanced and correlated significantly with each other, as well as with the residual hydrocarbon concentration, pointing to the importance of biodegradation. The feasibility of fungi bioaugmentation in composting of a flare pit soil was studied in lab-scale composters. The preliminary screening tests, used a range of bulking agents and white rot fungi strains, conducted to determine the best strain and bulking agent for the main experiments. Analysis of the TPH content of the soil after 98 days (using gravimetric method) showed an average of 29% reduction in most jars. Furthermore, gas chromatograph (GC) analysis of the oil extract from the samples showed 70–99% reduction in the peak area of the selected hydrocarbons. The results showed that the change in the bulking agent content was marginally significant for the hydrocarbon loss (Baheri and Meysami, 2002). The long term effects on soil microorganisms of diesel oil added to two contrasting soil systems revealed that after 22 weeks incubation at 22ºC, soil microbes in a silt loam mineralized 26% of added diesel, but only 3% was mineralized in sand. It was suggested that the use of bioremediation as a treatment strategy is more suited to those diesel contaminated soils which were initially more inherently fertile than those lacking in nutrients such as contained in organic matter (Kroening and Greenfield, 2002). The ability of Lentinus subnudus a white rot fungus to mineralize soil contaminated with various concentrations of crude oil (1, 2.5, 5, 10, 20, 40%) was tested. Organic matter and carbon were higher than the control at all concentrations of crude oil contamination in soils inoculated with L. subnudus for 3 months. As for TPH in crude oil contaminated soils, the highest rate of biodegradation was at 20% concentration after 3 months and 40% after 6 months of incubation (Adenipekun and Fasidi, 2005). Two methods of biostimulation in a laboratory incubation study with monitored natural attenuation (MNA) for total petroleum hydrocarbon (TPH) degradation in diesel-contaminated Tarpley clay soil with low carbon content were compared by Sarkar et al. (2005). One method utilized rapid-release inorganic fertilizers rich in N and P, and the other used sterilized, slow-release biosolids, Mycoremediation of Crude Oil Contaminated Soil

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which added C in addition to N and P. After 8 weeks of incubation, both biostimulation methods degraded approximately 96% of TPH compared to MNA, which degraded 93.8%. Bioremediation of diesel oil in soil can occur by natural attenuation, or treated by biostimulation or bioaugmentation was investigated by Bento et al. (2005). Bioaugmentation of the Long Beach soil showed the greatest degradation in the light (72.7%) and heavy (75.2%) fractions of TPH. Natural attenuation was more effective than biostimulation in the Hong Kong soil. The greatest microbial activity (dehydrogenase activity) was observed with bioaugmentation of the Long Beach soil (3.3-fold) and upon natural attenuation of the Hong Kong sample (4.0-fold). The yeast strain Candida maltosa EH 15 used as a biological agent in the hydrocarbon and emulsified hydrocarbon biodegradation indicated the strong ability of this fungus for hydrocarbon biodegradation. The addition of the tested surfactants influenced hydrocarbon biodegradation; however, biodegradation effectiveness varies with the type and concentration of surfactant (Chrzanowski et al., 2006). Biodegradation experiments using 0.4mM (100 mg/l) benzo(a)pyrene demonstrated that Fusarium sp. E033 was able to degrade 65-70% of the initial benzo(a)pyrene provided, and two transformation products, dihydroxy dihydrobenzo(a)pyrene and a benzo(a)pyrene-quinone, were detected within 30 days of incubation at 32ºC (Chulalaksananukul et al., 2006). Thirty-seven hydrocarbon-degrading strains of bacteria and fungi were isolated from two highly contaminated soils with total petroleum hydrocarbon (TPH) concentrations of 60,600 and 500,000 mg kg–1. Six strains showed a high ability to degrade PAHs, AHs and TPH which were identified as Pseudomonas pseudoalcaligenes, Bacillus firmus, Bacillus alvei, Penicillium funiculosum, Aspergillus sydowii and Rhizopus sp. (Mancera-López et al., 2007). Two fungal isolates named Pleurotus ostretus and Trichoderma harzianum were chosen carefully for the purpose of biotreatment of oily drilled cuttings resulted from drilling oil wells using oil based muds (OBMs). The best hydrocarbon degradation was observed in case of using both fungi together with 5% by weight microorganisms concentration ratio (MCR) and with the same ratio of nutrients expressed as C/N/P equal to 100/50/10 nutrient components ratio which gave Mycoremediation of Crude Oil Contaminated Soil

Page 77

average total petroleum hydrocarbon degradation of about 205 ppm per day (Akram et al., 2007). Degradation of four different PAHs by the white-rot fungus Bjerkandera adusta in a spiked marsh soil was evaluated in a slurry system. It was demonstrated that the fungus was able to grow and degrade the pollutants in a slurry phase stirred tank reactor. The system attained high PAH degradations, around 30 mg PAH/kg soil, after 30 days of operation under optimal conditions (Valentin et al., 2007). The bioremediation processes were applied to a sandy soil with a high level of contamination originated from the leakage of a diesel oil underground storage tank at a petrol station. Enhancement of biodegradation was carried out through biostimulation (addition of nitrogen and phosphorus solutions or Tween 80 surfactant) and bioaugmentation (bacterial consortium isolated from a landfarming system). Results showed that all bioremediation strategies enhanced the natural bioremediation of the contaminated soil and the best results were obtained when treatments had nutritional amendment. At the end of the experiments, two predominant bacteria species were isolated and identified (Staphylococcus hominis and Kocuria palustris) (Mariano et al., 2007). The contaminated soils inoculated with Lentinus squarrosulus had increased organic matter, carbon and available phosphorus, while the nitrogen and available potassium was reduced. A relatively high percentage degradation of Total Petroleum Hydrocarbon (TPH) was observed at 1% engine oil concentration (94.46%) (Adenipekun and Isikhuemhen, 2008). Use of chemical fertilizer and animal manure on crude oil reduction during biodegradation with Pleurotus tuber-regium was investigated by Ogbo and Okhuoya (2008). The co-substrates and inocula types used for the investigation were shredded banana leaf blades and sawdust of Albizia and sclerotium and spawn of the fungus, respectively. The reduction of total petroleum hydrocarbons was higher in treatments with a combination of fertilizers and co-substrates. Degradation of total petroleum content was higher in poultry litter treatment than the NPK treatments. The biodegradation of five polycyclic aromatic hydrocarbons (PAHs: acenaphthene, anthracene, phenanthrene, fluoranthene and pyrene) by white rot fungus Phanerochaete chrysosporium, isolated from soil sample of petroleum Mycoremediation of Crude Oil Contaminated Soil

Page 78

refinery was investigated. For maximum biodegradation, after 42 days of incubation, optimum conditions were pH 7.0, 30ºC and 5 µg/g PAHs concentration (Bishnoi et al., 2008). The ligninolytic fungus Irpex lacteus as an efficient degrader of oligocyclic aromatic hydrocarbons (PAHs) possessing 3-6 aromatic rings in complex liquid media was studied. The strain produced mainly Mn-dependent peroxidase in media without pollutants (Cajthaml et al., 2008). Two naturally occurring bacterial cultures, Exiguobacterium aurantiacum and Burkholderia cepacia, were capable of utilizing diesel oil as the sole source of carbon and energy by induction of hydrophobic cell surfaces with water contact angle greater than 70.1. The cultures demonstrated good degradation characteristics for diesel range n-alkanes (C9-C26) and were also capable of degrading pristane. A significant correlation was observed between maximum decay rate (MTR) of individual n-alkane peak area and initial abundance of n-alkanes in diesel (r2 ¼ 0.79 and 0.97 for E. aurantiacum and B. cepacia, respectively). C9, C17–C19, and C26 were completely degraded by both the cultures (Mohanty and Mukherji, 2008). Twelve fungal isolates recovered from oil-contaminated soils were screened for crude oil biodegradation activity in a shake-flask culture (Okafor et al., 2009). Aspergillus versicolor and Aspergillus niger exhibited the fastest onset and highest extent of biodegradation and selected for further study on specific polycyclic aromatic hydrocarbon (PAH) biodegradation. Both isolates exhibited above 98% degradation efficiency for polycyclic aromatic hydrocarbon moieties when grown in a culture medium incorporated with 1% crude oil (hydrocarbon) and 0.1 %Tween 80 for 7 days. A new bioremediation method for petroleum contaminated soil (PCS) using both fungi and bacteria was investigated using Glomus caledonium NW03 and Bacillus subtilis NW08, both of which were isolated from PCS of Petro China of Changqing (PCOC), Shaanxi, China (Chen et al., 2009). It was demonstrated that the growth behaviour of the inocula and the degradation of TPH were enhanced by the mix culture of both fungi and bacteria. The remediation via inoculating the fungal-bacterial consortium removed 92.6% of TPH in 60 days.


Page 79

Degradation benzo(a)anthracene,

of

the

polycyclic

benzo(a)fluoranthene,

aromatic

hydrocarbons

benzo(a)pyrene,

(PAH)

chrysene

and

phenanthrene in a soil that was sterilized and inoculated with the nonligninolytic fungi, Fusarium flocciferum and Trichoderma spp. and the ligninolytic fungi, Trametes versicolor and Pleurotus ostreatus in the presence of cadmium (Cd) and nickel (Ni) during a ten week incubation period was studied. The fungi degraded the tested PAHs between 21 and 93% by the end of the tenth week. The fungi degraded the less-soluble PAHs containing five or six aromatic rings more slowly than those containing fewer aromatic rings (Atagana, 2009). The feasibility of bioremediation of a soil, containing heavy metals and spiked with diesel oil (DO), through a bioaugmentation strategy based on the use of a microbial formula tailored with selected native strains was assessed by Alisi et al. (2009). The soil originated from the metallurgic area of Bagnoli (Naples, Italy). The application of this microbial formula allowed to obtain, in the presence of heavy metals, the complete degradation of n-C12-C20, the total disappearance of phenantrene, a 60% reduction of isoprenoids and an overall reduction of about 75% of the total diesel hydrocarbons in 42 days. Three organic wastes (banana skin (BS), brewery spent grain (BSG), and spent mushroom compost (SMC)) were used for bioremediation of soil spiked with used engine oil to determine the potential of these organic wastes in enhancing biodegradation of used oil in soil. Oil-contaminated soil amended with BSG showed the highest reduction in total petroleum hydrocarbon with net loss of 26.76% in 84 days compared to other treatments (Abioye et al., 2010). Effect of agricultural fertilizers (N, P, K) to enhance the microbial degradation of petroleum hydrocarbons in artificially polluted soil with %1 density of crude oil was investigated by Chorom et al. (2010). Results showed that the hydrocarbon-degrading and heterotrophic bacteria count in all the treatments increased with time and heterotrophic bacteria population increased from 6×103 cfu/g soil to 1.4×108 cfu/g soil. Also, soil C/N ratio decreased from 6 to 3. The results indicated that the applied fertilizer increased the degradation of the hydrocarbons compared with the control.


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Crude oil polluted soils of different concentrations (0%, 1%, 5%, 10%, 20%, 30% and 40%) were inoculated and incubated with Pleurotus pulmonarius and P.ostreatus for 0, 1 and 2 months to study and compare their effect in the bioremediation of crude oil and bioaccumulation of heavy metals from polluted soils. Results showed that both white rot fungi were able to biodegrade and ameliorate the soils by increasing the nutrient contents as the concentration of the crude oil in the soils increases with increase in incubation period. The result obtained showed the ability of P. pulmonarius and P. ostreatus to bioremediate a hydrocarbon and heavy metal polluted soil (Adenipekun et al., 2011). Lee et al. (2011) applied natural attenuation (NA), biostimulation (BS) and bioaugmentation (BA) to remediate diesel contaminated soil, with their remediation efficiencies and soil microbial activities compared both with and without surfactant (Tween 80). BA treatment employing Rhodococcus sp. EH831 was the most effective for the remediation of diesel-contaminated soil at initial remediation stage. A mixed consortium was prepared with 15 bacteria isolated by enrichment technique from the sample collected from an oil contaminated site and was incubated with crude oil to investigate the metabolic capability of bacteria. Total removal of aliphatic and aromatics was 94.64% and 93.75% respectively. Among the various components of the crude oil degradation by the bacterial consortium, the biotic removal of alkanes was maximum, 90.96% for tridecane (C13) followed by pentadecane (C15) at 77.95%, octadecane (C18) at 74.1%, while other alkanes showed 56 to 69% after 24 days of incubation (Malik and Ahmed, 2012). The effectiveness of monitored natural attenuation, bioenrichment, and bioaugmentation was evaluated using a consortium of three actinomycetes strains in remediating two distinct typical Brazilian soils from the Atlantic Forest and Cerrado biomes that were contaminated with crude oil, with or without the addition of NaCl (Alvarez et al., 2011). Degradation rate of n-alkanes was higher than TPH in both soils, independent of the treatment used. An indigenous microbial consortium was developed by assemble of four species of bacteria, isolated from various oil contaminated sites of India, which could biodegrade different fractions of total petroleum hydrocarbon (TPH) of the oily waste to environment friendly end products was studied by Mandal et al., 2012. Mycoremediation of Crude Oil Contaminated Soil

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The said consortium was applied on field scale and successfully bioremediated >200,000 tonnes of different types of oily waste in India. The bioremediated soil was non-toxic and natural vegetation was grown on the same. A review of the studies showing degradation of petroleum components by white rot fungi made by Young (2012) and is summarized in Table-19. Table-19: Studies showing degradation of petroleum components by white-rot fungi (Young, 2012) Pollutant

Type

Alkanes

Aromatics

2-3 rings

Fungi

References

Pleurotus ostreatus, Phanerochaete chrysosporium, Pleurotus tuber-regium, Polyporus circinata Agrocybe aegerita, Bjerkandera adusta,

Pozdnyakova et al., 2008; Pozdnyakova et al., 2011a. Kanaly and Hur, 2006. Ogbo and Okhuoya, 2008. Markovetz et al., 1968. Sack et al., 1997. Field et al., 1992; Gramss et al., 1999a; Pickard et al., 1999; Novotny et al., 2000; Field et al., 1992; Kotterman et al., 1994. Morgan et al., 1991. Pickard et al., 1999. Field et al., 1992. Pickard et al., 1999. Gramss et al., 1999a. Gramss et al., 1999a. Gramss et al., 1999a. Gramss et al., 1999a. Novotny et al., 2000; Bhatt et al., 2002; Lamar et al., 2002; Cajthaml et al., 2008. Sack et al., 1997; Gramss et al., 1999a. Pozdnyakova et al., 2011a. Gramss et al., 1999a. Bumpus, 1989; Morgan et al., 1991; Sutherland et al., 1991; Dhawale et al., 1992; Field et al., 1992; Hammel et al., 1992; Moen and Hammel, 1994; Bogan and Lamar, 1995; 1996; Andersson and Henrysson, 1996; Bogan et al., 1996a; Bogan et al., 1996b; 1996c; Novotny et al., 1999;

Bjerkandera sp., Sporotrichum pulverulentum, Coriolopsis gallica, Daedaleopsis confragosa, Ganoderma applanatum, Gymnopilus sapineus, Hypholoma fasciculare, Hypholoma frowardii, Hypholoma sublaterium, Irpex lacteus, Kuehneromyces mutabilis, Lentinula edodes, Lenzites betulina, Phanerochaete chrysosporium,


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Phanerochaete laevis, Phanerochaete sordida, Pleurotus ostreatus,

Pleurotus sajor-caju, Pleurotus sp. Florida, Pleurotus tuber-regium, Polyporus pinsitus, Stereum sp., Trametes sp., Trametes trogii, Trametes versicolor

4+ rings

Agaricales sp., Agrocybe aegerita, Bjerkandera adusta,

Coriolopsis gallica , Dichomitus squalens, Irpex lacteus,

Ganoderma applanatum, Gymnopilus sapineus, Hypholoma fasciculare, Hypholoma frowardii, Hypholoma sublaterium, Kuehneromyces mutabilis, Lentinula edodes, Lenzites betulina, Phanerochaete chrysosporium, Mycoremediation of Crude Oil Contaminated Soil

Pickard et al., 1999; Novotny et al., 2000; Chigu et al., 2010; Lee et al., 2010; Bogan and Lamar, 1996. Lee et al., 2010. Andersson and Henrysson, 1996; Bezalel et al., 1996a; Bezalel et al., 1996b; Bezalel et al., 1997; Gramss et al., 1999a; Novotny et al., 1999; Pickard et al., 1999; Novotny et al., 2000; Bhatt et al., 2002; Lamar et al., 2002; Pozdnyakova et al., 2008; Pozdnyakova et al., 2011a; Pozdnyakova et al., 2011b. Andersson and Henrysson, 1996. Wolter et al., 1997. Ogbo and Okhuoya, 2008. Field et al., 1992. Field et al., 1992. Field et al., 1992. Levin et al., 2003. Morgan et al., 1991; Field et al., 1992; Collins and Dobson, 1996; Sack et al., 1997; Novotny et al., 1999; Pickard et al., 1999; Novotny et al., 2000. Field et al., 1992. Sack et al., 1997. Field et al., 1992; Gramss et al., 1999a; Pickard et al., 1999; Novotny et al., 2000. Pickard et al., 1999. in der Wiesche et al., 1996. Novotny et al., 2000; Bhatt et al., 2002; Lamar et al., 2002; Cajthaml et al., 2008. Pickard et al. 1999. Gramss et al., 1999a. Gramss et al., 1999a. Gramss et al., 1999a. Gramss et al., 1999a. Sack et al., 1997;Gramss et al., 1999a. Pozdnyakova et al., 2011a. Gramss et al., 1999a. Bumpus, 1989; Field et al., 1992; Bogan and Lamar, Page 83

Phanerochaete laevis, Phanerochaete sordida, Pleurotus ostreatus,

Pleurotus sajor-caju, Pleurotus sp., Pleurotus tuber-regium, Ramaria sp., Trametes versicolor.

BTEX

Total hydrocarbons

Diesel fuel Creosote Crude oil

Phanerochaete chrysosporium, Pleurotus ostreatus Phanerochaete chrysosporium Irpex lacteus, Pleurotus ostreatus Coriolus sp., Lentinula edodes.

1995; Andersson and Henrysson, 1996; Novotny et al., 1999; Pickard et al., 1999; Novotny et al., 2000; Lee et al., 2010. Bogan and Lamar, 1996. Lee et al., 2010. Field et al., 1992; Andersson and Henrysson, 1996; Novotny et al., 1999; Pickard et al., 1999; Novotny et al., 2000; Pozdnyakova et al., 2011a; Pozdnyakova et al., 2011b; Pozdnyakova et al., 2008. Andersson and Henrysson, 1996. Lang et al., 1996. Ogbo and Okhuoya, 2008. Field et al., 1992; Andersson and Henrysson, 1996; Sack et al., 1997; Novotny et al., 1999; Pickard et al., 1999; Novotny et al., 2000; Yadav and Reddy, 1993; Teramoto, 2004; Pozdnyakova et al., 2008. Kanaly and Hur, 2006

Lamar et al., 2002. Lamar et al., 2002. Pozdnyakova et al., 2011a. Pozdnyakova et al., 2011a

Results of experimental work elucidated that the fungi like Phanerocheate chrysosporium and Aspergillus niger were capable of producing enzymes at a faster rate to decompose the substrate hydrocarbons in soils contaminated with petrol and diesel and released more CO2 and hence these potential fungi can be utilized effectively as agents of biodegradation in waste recycling process and bioremediation of oil contaminated sites (Maruthi et al., 2013). 2.6

Phytotoxicity assessment The white rot fungus, Pleurotus tuber-regium was examined for its ability to

ameliorate crude oil polluted soil (Isikhuemhen et al., 2003). This was inferred from Mycoremediation of Crude Oil Contaminated Soil

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the ability of the polluted soil to support seed germination and seedling growth in Vigna unguiculata at 0, 7 and 14 days post treatment. Results showed that bioremediation of soil contaminated with crude oil was possible, especially when the fungus had been allowed to establish and fully colonize the substrate mixed with the soil. There were significant improvements in % germination, plant height, biomass accumulation and root elongation values of test plants and decrease in total hydrocarbon content, when seeds were planted 14 days post soil treatment. The phytotoxic effects of crude oil and oil components on the growth of red beans (Phaseolus nipponesis OWH1) and corn (Zea mays) was investigated by Baek et al. (2004). In addition, the beneficial effects of bioremediation with the oildegrading microorganism, Nocardia sp. H17-1, on corn and red bean growth in oilcontaminated soil was also determined. It was found that crude oil-contaminated soil (10,000 mg/kg) was phytotoxic to corn and red beans. In contrast, obvious phytotoxicity was not observed in soils contaminated with 0–1000 mg/kg of aliphatic hydrocarbons such as decane (C10) and eicosane (C20). Phytotoxicity was observed in soils contaminated with 10-1000 mg/kg of the poly aromatic hydrocarbons (PAHs) naphthalene, phenanthrene, and pyrene. It was observed that phytotoxicity increased with the number of aromatic rings, and that corn was more sensitive than red beans to PAH contaminated soil. Bioremediation with Nocardia sp. H17-1 reduced phytotoxicity more in corn than in red bean, suggesting that this microbial species might degrade PAHs to some degree. The bioremediation process efficiency was evaluated directly by an innovative, simple phytotoxicity test system and the diesel extracts by Daphnia magna and nematode assays. Contaminated soil samples revealed to have toxic effects on seed germination, seedling growth, and Daphnia survival. After biostimulation, the diesel concentration was reduced by 50.6%, and the soil samples showed a significant reduction in phytotoxicity (9%–15%) and Daphnia assays (3fold), confirming the effectiveness of the bioremediation process (Molina et al., 2005). An effort to enhance crop production in crude oil contaminated soils, the effect of the addition of cow dung on the growth and performance of Glycine max grown in soil contaminated with various concentrations of crude oil was investigated


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by Njoku et al., 2008. There was a general improvement on the growth, dry weight, chlorophyll content, leaf area and pod production of the crop by the addition of cow dung to crude oil polluted soil. The performance of the crop also improved as the period of study increased suggesting that the toxicity of crude oil to the crop reduced as the period of study increased. Ekpo et al., 2012 investigated the effect of crude oil on germination and growth of Glycine max in Calabar, Nigeria. Three seeds of soybean (Glycine max) were planted into each of the soil sample treated with varying concentrations of crude oil. The result indicated that crude oil pollution significantly reduces (p< 0.05) the growth of the soybean plant at higher pollution rate than at lower pollution rate. This thus implies that the higher the quantity or concentration of the crude oil in the soil the more effect it would have on the growth and germination of soybean plant.


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