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Arbuscular Mycorrhiza and Petroleum-Degrading Microorganisms Enhance Phytoremediation of Petroleum-Contaminated Soil
Alejandro Alarcón ab; Fred T. Davies Jr. a; Robin L. Autenrieth c; David A. Zuberer d a
Department of Horticultural Sciences, Faculty of Molecular and Environmental Plant Sciences, Texas A&M University, College Station, Texas, USA b c
rea de Microbiolog a. Colegio de Postgraduados, Estado de M xico, M xico Department of Civil Engineering, Texas A&M University, College Station, Texas, USA d Department of Soil and Crop Sciences, Texas A&M University, College Station, Texas, USA Online Publication Date: 01 July 2008 To cite this Article: Alarcón, Alejandro, Davies Jr., Fred T., Autenrieth, Robin L. and Zuberer, David A. (2008) 'Arbuscular Mycorrhiza and Petroleum-Degrading Microorganisms Enhance Phytoremediation of Petroleum-Contaminated Soil', International Journal of Phytoremediation, 10:4, 251 — 263 To link to this article: DOI: 10.1080/15226510802096002 URL: http://dx.doi.org/10.1080/15226510802096002
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International Journal of Phytoremediation, 10:251–263, 2008 C Taylor & Francis Group, LLC Copyright ISSN: 1522-6514 print / 1549-7879 online DOI: 10.1080/15226510802096002
ARBUSCULAR MYCORRHIZA AND PETROLEUM-DEGRADING MICROORGANISMS ENHANCE PHYTOREMEDIATION OF PETROLEUM-CONTAMINATED SOIL Alejandro Alarc´on,1 Fred T. Davies Jr.,1 Robin L. Autenrieth,2 and David A. Zuberer3 1
Department of Horticultural Sciences, Faculty of Molecular and Environmental Plant Sciences, Texas A&M University, College Station, Texas, USA 2 Department of Civil Engineering, Texas A&M University, College Station, Texas, USA 3 Department of Soil and Crop Sciences, Texas A&M University, College Station, Texas, USA While plants can phytoremediate soils that are contaminated with petroleum hydrocarbons, adding microbes to remediate contaminated sites with petroleum-degrading microorganisms and arbuscular mycorrhizal fungi (AMF) is not well understood. The phytoremediation of Arabian medium crude oil (ACO) was done with a Lolium multiflorum system inoculated with an AMF (Glomus intraradices) and a mixture of petroleum-degrading microorganisms—the bacterium, Sphingomonas paucimobilis (Sp) and the filamentous fungus, Cunninghamella echinulata (Ce, SpCe)—or with a combination of microorganisms (AMF + SpCe). Based on an earlier study on screening plants for phytoremediation of ACO, L. multiflorum (Italian ryegrass) was selected for its tolerance and rapid growth response (Alarc´on, 2006). The plants were exposed to ACO-contaminated soil (6000 mg kg−1) for 80 d under greenhouse conditions. A modified Long Ashton Nutrient Solution (LANS) was supplied to all treatments at 30 µg P mL−1, except for a second, higher P, control treatment at 44 µg P mL−1. Inoculation with AMF, SpCe, or AMF + SpCe resulted in significantly increased leaf area as well as leaf and pseudostem dry mass as compared to controls at 30 µg P mL−1. Populations of bacteria grown on a nitrogen-free medium and filamentous fungi increased with AMF + SpCe and SpCe treatments. The average total colonization and arbuscule formation of AMF-inoculated plants in ACOcontaminated soil were 25% and 8%, respectively. No adverse effects were caused by SpCe on AMFcolonization. Most importantly, ACOdegradation was significantly enhanced by the addition of petroleum-degrading microorganisms and higher fertility controls, as compared to plants at 30 µg P mL−1. The highest ACOdegradation (59%) was observed with AMF + SpCe. The phytoremediation of ACO was also enhanced by single inoculation of AMF or SpCe. The effect of AMF and petroleum-degrading microorganisms on plant growth and ACOdegradation was not attributable to differences in proline, total phenolics, nitrate reductase levels, or variation in plant–gas exchange. KEY WORDS: Glomus intraradices, Sphingomonas, Cunninghamella, Lolium multiflorum, phosphorus, total petroleum hydrocarbon degradation
´ The current address of A. Alarc´on is Area de Microbiolog´ıa. Colegio de Postgraduados. Carretera M´exicoTexcoco km. 36.5. Montecillo 56230, Estado de M´exico. M´exico. Address correspondence to F.T. Davies, Jr., Dept. of Horticultural Sciences, Faculty of Molecular & Environmental Plant Sciences, Texas A&M University, College Station, TX 77843-2133, USA. E-mail:
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INTRODUCTION The enhanced degradation of petroleum hydrocarbons via phytoremediation is dependent on abiotic and biotic conditions including: 1) soil type and the nutrient and water availability, 2) type and concentration of petroleum hydrocarbons in soil, and 3) proliferation of petroleum-degrading microorganisms that reduce available contaminants in the soil and rhizosphere (Schnoor et al., 1995; Cunningham et al., 1997; Siciliano and Germida, 1998). Plant sensitivity to contaminants, low soil fertility, slow plant growth rates, and reduced microbial populations in soils due to chronic exposure to contaminants are factors that limit the phytoremediation of petroleum-contaminated soils (Alkorta and Garbisu, 2001; Susarla, Medina, and McCutcheon, 2002; Pilon-Smits, 2005). During phytoremediation, plants may improve soil aeration via their root system, which enhances rhizosphere microbial activity and contaminant degradation. Microbial activity is stimulated by root exudates that serve as sources of carbon and energy for microorganisms that oxidize and/or degrade organic contaminants. However, in spite of the apparent benefit for plants in the phytoremediation of petroleum-contaminated soils, there is little information about the effect of combining phytoremediation that utilizes arbuscular mycorrhizal fungi (AMF) and petroleum-degrading microorganisms (Huang et al., 2004). Microorganisms that have the ability to degrade petroleum hydrocarbons are ubiquitous; however, when their populations are depleted, inoculation with specific microbes to enhance bioremediation and/or phytoremediation is usually recommended. Free-living bacteria such as Sphingomonas paucimobilis, as well as filamentous fungi such as Cunninghamella spp., are ideal microorganisms that oxidize and/or degrade petroleum hydrocarbons, including polycyclic aromatic hydrocarbons (Cerniglia, 1992; van Hamme, Singh, and Ward, 2003). Although some physiological and biochemical mechanisms by which these microorganisms degrade petroleum hydrocarbons have been reported, no information is available about their effects on plants during phytoremediation. AMF are ubiquitous rhizosphere microorganisms that form mutually beneficial symbiosis with the root system of approximately 80% of terrestrial plants (Smith and Read, 1997). This symbiosis can have important effects on the phytoremediation of soils contaminated with inorganic and organic compounds (Cabello, 2001; Meharg, 2001; Joner and Leyval, 2003a, 2003b). Direct benefits are related to enhanced plant adaptation and growth, including enhanced nutrition, and abiotic and biotic stress resistance. Indirect benefits may include modification of microbial groups in the mycorrhizosphere and the potential proliferation of petroleum-degrading microorganisms via extraradical hyphal exudation. In addition, the interaction of AMF and petroleum-degrading microorganisms has received little attention (Gaspar et al., 2002). Little is known about the role of AMF and their interaction with petroleum-degrading microorganisms on plant growth, physiological responses, and rhizosphere microbial populations during the phytoremediation of petroleum hydrocarbons in soils. Therefore, the objectives of this study were to: 1) determine the growth and selected physiological barometers of abiotic stress (gas exchange, total phenolics, nitrate reductase, and proline) of AMF-colonized plants of Lolium multiflorum, inoculated with Sphingomonas paucimobilis and Cunninghamella echinulata var. elegans in a soil contaminated with Arabian medium crude oil (ACO), and 2) determine the phytoremediation of ACO via inoculation with petroleum-degrading microorganisms and AMF.
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MATERIALS AND METHODS Cultural Conditions, Soil Contamination, Transplant, Microbial and Mycorrhizal Inoculation The study was conducted under greenhouse conditions at Texas A&M University, College Station, Texas, USA for 80 days. Temperature and relative humidity were monitored with a watchdog data logger (Model 150, Spectrum Technologies, Inc., Plainfield, IL, USA) and photosynthetic photon flux density (PPFD) was determined with a LI-190SA R Quantum/Radiometer/Photometer and Sensor (LI-COR Biosciences, Lincoln, NE, USA). Average minimum/maximum temperature and relative humidity were 22.3/25.7◦ C and 64.9/72.4%, respectively. The average maximum PPFD determined at solar noon was 583.5 µmoles m−1 s−2. A 14-h photoperiod was maintained by artificially lighting plants from 18:00 to 22:00 during October and November 2005. A mixture of sand and sandy loam soil (1:1 v/v) was utilized as the container substrate, with chemical properties of: (µg g−1) 0.9 NO3 -N, 2.1 NH4 -N, 1.5 P, 17 K, 9468 Ca, 72 Mg, 161 Na, and 53 S. The electrical conductivity was 0.17 dS·m−1, pH of 7.7, and a textural analysis of sand 85%, clay 10%, and silt 5%. The substrate was steam-pasteurized at 70◦ C for 8 h on 2 consecutive days. The Arabian medium crude oil (ACO) concentration utilized for contaminating the substrate was 6000 mg kg−1, based on preliminary studies (Alarc´on, 2006). Physical properties of ACO were as follows: specific gravity 0.87, API gravity 30.7 API degree, Reid vapor pressure 2.5 kPa, viscosity at 15◦ C 21.4 CST, pour point −23◦ C, and sulfur content 2.6 wt.% (Simon et al., 2004). The viscosity of the contaminant was reduced through the R application of dichloromethane solvent (Sigma , < 0.002% of residue after evaporation) to ensure its homogenization in the container substrate. One-week-old Lolium multiflorum Lam. cv. Passerel Plus seedlings were transplanted, with one seedling per container with 2 kg of contaminated soil. Thus, selected treatments were inoculated with 500 spores of Glomus intraradices Schenck & Smith (AMF) R (Mycorise ASP, PremierTech Biotechnologies, Quebec, Canada) and/or with 2 mL of liquid inoculum in sterile water (8.8 × 108 CFU mL−1) of the flouranthene-preadapted bacterium, Sphingomonas paucimobilis (Sp, EPA505; Mueller et al., 1990) and 2 mL of liquid inoculum in sterile water (5.5 × 104 CFU mL−1) of the filamentous fungus, Cunninghamella echinulata var. elegans (Ce, ATCC-36112). Microbial inocula were applied directly to the root system of the seedlings. Non-inoculated seedlings in ACOcontaminated soil were utilized as controls. Plants were fertilized weekly with Long Ashton Nutrient Solution (LANS) at 1× (Hewitt, 1966) modified to supply 30 µg P mL−1. Plants were watered as needed with deionized water. An additional control treatment with LANS modified to supply 44 µg P mL−1 was included to determine if high P was substituted for or was equivalent to AMF effects on plant development, selected physiological responses, and phytoremediation. Plant Growth Evaluation After 80 days, plants were harvested to determine the leaf area (cm2) and dry mass (DM) of leaves, pseudostems, roots, and total plants. The leaf area ratio [(LAR): leaf area/total plant DM, cm2 g−1], specific leaf area [(SLA): leaf area/leaf DM, cm2 g−1], and root- to-shoot ratio [(RSR): root DM/leaf + pseudostems DM, g g−1] were also estimated.
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Leaf area was determined using a portable area meter (LI-COR Model LI-3000, LI-COR Biosciences, Lincoln, NE, USA). Plant Gas Exchange, Chlorophyll Content, and Other Selected Physiological Barometers of Abiotic Stress To determine the detrimental effects of ACO on selected physiological barometers of abiotic stress, plant gas-exchange measurements, including photosynthesis (Pn) and stomatal conductance (gs ) were taken at 80 days. Measurements were done on individual mature leaf blades from three random plants per treatment (n = 3), with a portable photosynthesis system (LI-COR model LI-6400, LI-COR Inc., Lincoln, NE, USA) with a red/blue LED light source (LI6400–02B) at photosynthetic active radiation (PAR) levels of 500 µmol m−2 s−1 and CO2 concentration of 360 µL L−1 in the chamber. Water use efficiency (WUE) was also determined (in Pn/gs ). Total leaf chlorophyll content was determined with 80% acetone extraction (Harborne, 1998) and absorbance readings were R taken at 645 and 663 nm (Beckman CoulterTM Du Series 640 UV/Vis Spectrophotometer, Beckman Coulter, Inc., Fullerton, CA, USA). Nitrate extractable reductase activity was performed by the procedures described by Foyer et al. (1998). Briefly, leaf samples were ground with an extraction buffer solution consisting of 50 mM Mops-KOH, pH 7.8, 5 mM NaF, 1 µM Na2 MoO4 , 10 µM FAD (flavin adenine dinucleotide), 1 µM leupeptin, 1 µM microcystin, 0.2 g PVP (polyvinylpyrollidone) g−1 fresh weight, 2 mM ß-mercaptoethanol, and 5 mM ethylene diaminetetra aceitic acid (EDTA). An aliquot of 200 µL was taken and then reacted to 200 µL of a reaction mixture solution consisting of 50 mM Mops-KOH buffer, pH 7.5, supplemented with 1 mM NaF, 10 mM KNO3 , 0.17 mM NADH, and 5 mM EDTA. The reaction was terminated after 15 min by the addition of 200 µL of sulfanilamide (1% [w/v] in 3 N HCl) and 200 µL of naphthylethylene-diamine dihydrochloride (0.02% [w/v]) to the reaction mixture. R The absorbance at 540 nm was measured (Beckman CoulterTM Du Series 640 UV/Vis Spectrophotometer, Beckman Coulter, Inc., Fullerton, CA, USA). Proline was determined by following the procedures of Bates, Waldren, and Teare (1973) and Gzik (1996). Briefly, leaf fresh tissue was macerated in an iced-mortar with 3% sulfosalicylic acid. An aliquot of the extract reacted with 200 µL ninhydrin reagent and 200 µL glacial acetic acid, and was incubated with at 100◦ C for 1 h. Proline was extracted with toluene and absorbance readings were immediately taken at 520 nm (Beckman R CoulterTM Du Series 640 UV/Vis Spectrophotometer, Beckman Coulter, Inc., Fullerton, CA, USA). Total phenolics were determined by using the Folin–Ciocalteu reagent assay utilizing chlorogenic acid as a standard curve (Singleton and Rossi, 1965; Soong and Barlow, 2004). In brief, leaf fresh tissue was macerated in a chilled mortar with 80% methanol. Extracts were centrifuged for 15 min at 15,000 rpm. The reaction mixture consisted of mixing 30 µL of the extract added with 90 µL of Na2 CO3 and 150 µL of Folin–Ciocalteau reagent in a 96-well microplate. After 30 min the absorbance was measured at 725 nm (KC-4 R spectrophotometer, Biotek Instruments, Inc. Winooski, VT, USA). Total Microbial Populations and Mycorrhizal Colonization Naturally-occurring populations of bacteria and filamentous fungi were estimated by performing the dilution plate-count method (Alexander, 2005). Total recoverable bacteria
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were counted on nutrient agar plates, while filamentous fungi were counted on potato dextrose agar plates. Bacteria able to grow on a N-free medium were quantified on Rennie’s medium plates (Rennie, 1981). Colony-forming units (CFU) of each microbial group were transformed to logarithmic units for further statistical analysis. Three plants per treatment were randomly selected and assayed for AMF colonization by clearing roots with KOH and staining them with trypan blue (Phillips and Hayman, 1970). Five slides of 10 1-cm stained root segments per plant per treatment were examined under a Aphaphot YS compound microscope (Nikon, Melville, NY, USA). Three observations (top, middle, and bottom) of each 1-cm root piece at 40× were evaluated for a total of 150 (1-cm) root segments per plant per treatment (10 1-cm roots per slide, five slides per plant, and three plants per treatment). There were 450 root observations per treatment; n = 3. Percentages of root length with arbuscules, vesicles, and internal hyphae (total colonization) in root cortical cells were determined, as described by Biermann and Linderman (1981), after clearing and staining the roots with trypan blue according to Koske and Gemma (1989). Total Petroleum Hydrocarbon Degradation Analysis of total petroleum hydrocarbons (TPH) from ACO-contaminated soil was performed by a modified EPA SW-846 Method 8270B (Louchouarn et al., 2000; USEPA, 1986), utilizing an automated accelerated solvent extractor (Dionex ASE-200, Dionex Corp., Sunnyvale, CA, USA) with 100% dichloromethane. The extracted TPH was collected and immediately concentrated to a volume of 1 mL. Final extracts were used in the quantitative determination of TPH by GC-MS (HP 5890 Series II Gas Chromatograph Hewlett-Packard Co., Wilmington, DE, USA). Experimental Design The experiment was a completely randomized design including five treatments: Control − 44 P µg mL−1, Control, AMF, SpCe, and AMF + SpCe at 30 µg P mL−1 of modified LANS, exposed to an ACO-concentration of 6000 mg kg−1. Data were analyzed by using analysis of variance (ANOVA) and LSD test for means comparisons (p ≤ 0.05) or standard error (± SE) (SAS Institute Inc., 2002). Each pot, containing one plant, was one replicate. The number of replications were as follows: plant DM, n = 7; gas exchange, chlorophyll, proline, total phenolics, antioxidant, and nitrate reductase activities, n = 3; microbial populations, n = 5; mycorrhizal colonization and TPHdegradation, n = 3. RESULTS Plant Growth Responses A selected plant growth response to ACO was significantly (p ≤ 0.01) enhanced by inoculation of microbes and higher P-fertilization (44 µg P mL−1) (Table 1). Plants inoculated with AMF, SpCe, or AMF + SpCe had increased leaf area, as well as leaf and pseudostem DM (p ≤ 0.01) as compared to controls at 30 µg P mL−1 (Table 1). In addition, total plant DM was significantly enhanced (p ≤ 0.05) due to SpCe-inoculation in comparison to controls at 30 µg P mL−1 (Table 1). Controls at higher P-fertility (44 µg P mL−1) had greater leaf area, but all other growth parameters were the same as 30 µg P mL−1
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Table 1 Effect of microbial inoculation (bioaugmentation) and phosphorus (biostimulation) on plant growth of Lolium multiflorum cv. Passerel Plus in the phytoremediation of soil contaminated with ACO (6000 mg kg−1), after 80 days
Treatment Control-44 P Control x AMFx SpCex AMF + SpCex Significance
Leaf Area (cm2) 121.9aw 66.6b 148.9a 152.6a 163.8a 0.01
Leaf DM (g)
Pseudostem DM (g)
Root DM (g)
Total plant DM (g)
SLA (cm2 g−1)
LAR (cm2 g−1)
RSR (g g−1)
0.3ab 0.2b 0.4a 0.5a 0.4a 0.01
0.1b 0.1b 0.2a 0.2a 0.2a 0.01
0.6 0.3 0.5 0.6 0.6 NS
1.0ab 0.6b 1.1ab 1.3a 1.2ab 0.05
392.7 321.3 363.0 327.1 398.4 NS
119.8 114.3 130.8 118.8 151.2 NS
1.2 0.9 0.9 0.9 0.8 NS
in the same column followed by the same letter are not significantly different (LSD, p ≤ .05). DM = dry mass; NS = nonsignificant, n = 7. xTreatments fertilized with long ashton nutrient solution (LANS), modified to supply 30 µg P mL−1; AMF = Glomus intraradices; SpCe = Sphingomonas paucimobilis and Cunninghamella echinulata var. elegans. wMeans
control plants. No significant differences among treatments were observed for root DM, SLA, LAR, or RSR (Table 1). Plant Gas Exchange, Chlorophyll Content and Selected Physiological Responses Total chlorophyll and gas exchange (Pn and gs ) were not significantly enhanced by either increased P-fertility or inoculation with microbes or AMF (data not presented). Leaf nitrate reductase, proline, and total phenolics were significantly (p ≤ 0.05) affected by treatments (Table 2). Nitrate reductase was lowest with controls at 44 µg P mL−1, while similar among other treatments (Table 2). The 44-µg P mL−1 controls and AMF plants had 76% lower proline, whereas controls with 30 µg P mL−1 and SpCe had the highest content of this amino acid (Table 3). Total phenolics were lowest for the controls at 44 µg P mL−1 and were comparable among other treatments (Table 2). Table 2 Effect of microbial inoculation (bioaugmentation) and phosphorus (biostimulation) on leaf nitrate reductase activity, proline, antioxidant activity, and total phenolics of Lolium multiflorum cv. Passerel Plus, established in the phytoremediation of soil contaminated with Arabian medium crude oil (6000 mg kg−1), after 80 days
Treatment Control 44 P Controlx AMFx SpCex AMF + SpCex Significance
NO3 reductase (µM NO2 g−1)
Proline (µg g−1)
Total phenolics (µg chlorogenic acid g−1)
152.7bw 233.3a 214.6a 251.1a 229.1a 0.05
0.9c 3.8ab 0.9c 5.0a 3.1b 0.01
1920.2b 3406.8a 2962.4a 2629.6ab 3171.6a 0.05
wMeans in the same column followed by the same letter are not significantly different (LSD, p = ≤ .05); NS = nonsignificant, n = 3. xTreatments fertilized with long ashton nutrient solution (LANS), modified to supply 30 µg P mL−1; AMF = Glomus intraradices; SpCe = Sphingomonas paucimobilis and Cunninghamella echinulata var. elegans.
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Figure 1 Microbial populations in the rhizosphere of Lolium multiflorum cv. Passerel Plus established in soil contaminated with Arabian medium crude oil (6000 mg kg−1) with higher P-fertilization [44 µg P mL−1 (Control 44P)] or microbes [AMF = Glomus intraradices, SpCe = Sphingomonas paucimobilis (EPA-505), and Cunninghamella echinulata var. elegans (ATCC-36112)] and non-inoculated control plants at 30 µg P mL−1, after 80 days. BGNFM = bacteria grown with nitrogen-free medium. ± SE, n = 5. Main treatment effects were not significant for total bacteria, but significant (P ≤ 0.001) for bacteria grown on nitrogen-free media (BGNFM) and filamentous fungi.
Microbial Populations in the Rhizosphere and Mycorrhizal Colonization No significant differences were observed among treatments for the numbers of recoverable bacteria (Figure 1). In contrast, populations of naturally-occurring bacteria that were able to grow on N-free medium (BGNFM) and filamentous fungi were significantly (p ≤ 0.001) affected by treatments (Figure 1). The number of bacteria on BGNFM were greater with AMF, SpCe, AMF + SpCe than 30 µg P mL−1 control plants (Figure 1). Filamentous fungi were significantly greater for SpCe and AMF + SpCe than control plants at 30 or 44 µg P mL−1 (Figure 1). AMF colonization in root cortical cells of L. multiflorum in ACO-contaminated soil were 24%, 8%, and 9%, respectively, for total colonization, arbuscules, and vesicle formation (Figure 2). Mycorrhizal colonization was not significantly reduced by the co-inoculation with SpCe (Figure 2). AMF-structures were not found in plants without AMF-inoculation. Total Petroleum Hydrocarbon Degradation TPH-degradation in the rhizosphere of L. multiflorum was significantly affected (p ≤ 0.001) by treatment (Figure 3). The highest TPH-degradation occurred with AMF + SpCe (59%), followed by 44 µg P mL−1 controls (49%), third highest by SpCe (46%) and AMF (45%), and the 30 µg P mL−1 control plants had the lowest TPH-degradation (36%). DISCUSSION This research is one of the first studies to demonstrate the importance of utilizing a mixture of petroleum-degrading microorganisms in combination with AMF during the
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Figure 2 Effect of petroleum-degrading microorganisms on the colonization by Glomus intraradices (AMF) in roots of Lolium multiflorum cv. Passerel Plus established in soil contaminated with Arabian medium crude oil [(ACO) 6000 mg kg−1, after 80 days]. SpCe = Sphingomonas paucimobilis and Cunninghamella echinulata var. Elegans; ± SE, n = 3. Main effects of treatment were not significantly different for total colonization, arbuscules, nor vesicles.
phytoremediation of soil contaminated with ACO in an L. multiflorum system. While physiological responses were not easily explained by treatments, soil microbe counts paralleled growth responses, which were negatively affected by ACO. Inoculation with microorganisms and fertilization at higher P-levels resulted in a significant two-fold increase in leaf area, pseudostem, and total plant DM in ACOcontaminated soil as compared to controls receiving 30 µg P mL−1. Thus, inoculation 6000 TPH-Degradation (mg kg-1 soil)
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5000 4000 3000 2000 1000 0 Control 44P Control 44P
Control
Control
AMF
AMF
Treatments
SpCe SpCe
AMF+SpCe AMF+SpCe
Figure 3 Effect of petroleum-degrading microorganisms [Sphingomonas paucimobilis (Sp) and Cunninghamella echinulata var. elegans (Ce), (SpCe], Glomus intraradices (AMF) at 30 ug mL−1 and higher P-fertiliztion [44 µg P mL−1 (Control 44P)] on total petroleum hydrocarbon (TPH) degradation in the rhizosphere of Lolium multiflorum contaminated with ACO at 6000 mg kg−1, after 80 days; ± SE, n = 3. Main treatment effects were significant at P ≤ 0.001.
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and increased fertilization enhanced plant growth and reduce the adverse effects of petroleum hydrocarbons on plants, as reported with non-mycorrhizal phytoremediation systems (Adam and Duncan, 2003; Malallah et al., 1996; Qui˜nones-Aguilar et al., 2003). While neither inoculation with petroleum-degrading microorganisms nor enhanced P-fertilization had significant effects on the chlorophyll content and gas exchange of plants, there was a non-significant trend of increased photosynthesis (Pn) of 19–32%. In another study, ACO decreased Pn and gs (P ≤ 0.001) (Alarc´on, 2006) of L. multiflorum, as was reported for terrestrial and marine plants (Baker, 1970; Daly, Hoddinott, and Dale, 1988; Durako et al., 1993; Macinnis-Ng and Ralph, 2003). There have been limited reports on the enzymatic and biochemical responses of plants during phytoremediation of petroleum hydrocarbons in soils (Malallah et al., 1996). The nitrate reductase enzymatic activity in plants is an important barometer in plants exposed to environmental stresses (Foyer et al., 1998; Sinha and Nicholas, 1981). In our study, no significant differences were observed between control plants at 30 µg P mL−1 and plants with petroleum-degrading microorganisms; however, controls at 44 µg P mL−1 had the lowest nitrate reductase, which was due, in part, to higher fertility. Proline can accumulate in a number of species subjected to drought and salt stress (Hare, Cress, and van Staden, 1999). The proline content was significantly lower for controls at 44 µg P mL−1 and AMF plants than controls at 30 µg P mL−1, which may indicate that these plants were less affected by the ACO-induced stress. This finding suggests that either AMF-inoculation or improved P-nutrition may contribute toward ACO-stress alleviation, as reported for other abiotic stresses (Ramakrishnan, Johri, and Gupta, 1988; Ruiz-Lozano, Azcon, and Gomez, 1996; Wu and Xia, 2005). However, there were no significant differences in proline between AMF + SpCe and controls at 30 µg P mL−1. Thus, proline was not a helpful indicator in explaining ACO-induced plant stress. Although a reduction in phenolic compounds has been observed with plants exposed to petroleum-contaminated soil (Ilangovan and Vivekanandan, 1992; Malallah et al., 1996), higher fertility (44 µg P mL−1) significantly reduced total phenolics. This finding is in agreement with reports of higher phenolics at low fertility, which provide greater host-plant resistance to insect herbivory and pathogen interaction (Dudt and Shure, 1994; Witzell and Shevtsova, 2004). The effects of AMF and petroleum-degrading microorganisms on total phenolics were not significantly different than controls at 30 µg P mL−1. Nevertheless, we found no previous reports on total phenolics of plants inoculated with either AMF or petroleum-degrading microorganisms during the phytoremediation of ACO in soils. Rhizosphere microbial populations may enhance a plant’s adaptation to petroleum hydrocarbons by detoxifying contaminated soils through direct mineralization of these organic contaminants (Barea et al., 2005; Jeffries et al., 2003; Robson et al., 2004; Siciliano and Germida, 1998). However, with the phytoremediation of ACO, we observed that populations of naturally-occurring bacteria grown on a nitrogen-free media (BGNFM) and filamentous fungi were stimulated by P-application or adding petroleumdegrading microorganisms. The addition of petroleum-degrading microorganisms and higher P-fertilization significantly increased the numbers of BGFNM population, whereas numbers of filamentous fungi population were highest in plants inoculated with SpCe or AMF + SpCe. The effect of AMF on microbial populations in the rhizosphere has been characterized as stimulatory, particularly for those beneficial rhizobacteria, including BGFNM, that may potentially contribute to N2 -fixation, plant-growth promotion, and degradation of organic contaminants in the soil (Barea et al., 2005; Perez-Vargas et al., 2000). Since our study was an open system in a greenhouse, the proliferation of
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naturally-occurring microorganisms was expected. However, we did not label our microbial strains in order to identify them among the microorganisms counted on the agar plates. Thus, it would be interesting for future experiments to track introduced microorganisms and to study any microbial succession that might occur in a petroleum-contaminated rhizosphere in combination with AMF. In another study with L. multiflorum, ACO significantly decreased AMF-colonization (Alarc´on, 2006). However, sufficient AMF-colonization (>30%) was observed in the roots of ACO-contaminated soil in this current study, which suggests tolerance of G. intraradices to ACO in the rhizosphere. In non-phytoremediation studies, AMF enhancement of plant growth or nutrient uptake has been reported with colonization levels as low as 5% (Smith and Read, 1997). Previous studies have reported AMF-colonization in hydrocarboncontaminated soils, which typically have negative effects on AMF-symbiosis (Cabello, 1997; Gaspar et al., 2002; Leyval and Binet, 1998). Most importantly, inoculation with petroleum-degrading microorganisms (SpCe) did not adversely affect AMF-colonization. Furthermore, Sp did not show antibiosis toward Ce, which was previously tested in vitro, in a preliminary study of both microorganisms (Alarc´on, 2006). The TPH-degradation in the rhizosphere of L. multiflorum was significantly enhanced by P-fertilization or inoculation with AMF + SpCe. The effect of fertilization indicates that, under our experimental conditions, P was a critical nutrient during the bioremediation of petroleum hydrocarbons, as suggested by Chang., Weaver, and Rhykerd (1996). Furthermore, P not only enhanced plant adaptation and growth, but also improved the phytoremediation of ACO, which may be due in part to rhizosphere microbial activity and plant growth. However, while P was an important component in our cultural conditions, the highest TPH-degradation occurred with AMF + SpCe plants at 30 µg P mL−1, followed by controls at 44 µg P mL−1. The inoculation of plants during the phytoremediation of petroleum hydrocarbons has received little attention. Huang et al. (2004) reported on the enhanced phytoremediation of organic contaminants using grass species inoculated with a mixture of beneficial bacteria such as Pseudomonas putida, Azospirillum brasilense, and Enterobacter cloacae. In addition, AMF are important rhizosphere components that contribute not only to alleviating toxic effects induced by petroleum hydrocarbons, but also to enhanced plant growth, and to tolerance and dissipation of contaminants in the rhizosphere (Cabello, 2001; Joner and Leyval, 2003a, 2003b; Leyval and Binet, 1998; Volante et al., 2005). Our study is among the first reports to describe the interaction of petroleum-degrading microorganisms (bacteria and filamentous fungi) with AMF and their effects on the growth and physiological responses of plants during phytoremediation of ACO. Furthermore, this is one of the first reports suggesting a microbial effect among AMF and two petroleum-degrading microorganisms in stimulating TPH-degradation in the rhizosphere of L. multiflorum. The results suggest that AMF can indirectly enhance phytoremediation of petroleum hydrocarbons in soils by enhancing the activity of petroleum-degrading bacteria and filamentous fungi. However, any phytoremediation or growth benefits were not attributable to differences in proline, total phenolics, nitrate reductase, or variation in plant gas exchange. Although the mechanisms are not well understood, this study supports the premise that AMF can enhance plant growth and development—and create favorable microenvironments (mycorrhizosphere/hyphosphere effect) that stimulate the activity of petroleum-degrading microorganisms during phytoremediation of petroleum hydrocarbons (Criquet et al., 2000; Cabello, 2001; Joner and Leyval, 2003a). In conclusion, the effects of AMF and petroleum-degrading microorganisms can be a significant benefit to plants used
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