Biodegradation Of Oil. Milan C. Vavrek and William J. Campbell. School of Biological Sciences. Louisiana Tech University. ABSTRACT. Plants have been shown ...
Identification Of Plant Traits That Enhance Biodegradation Of Oil Milan C. Vavrek and William J. Campbell School of Biological Sciences Louisiana Tech University
ABSTRACT Plants have been shown to accelerate biodegradation of oil. If specific plant traits that stimulate degradation can be identified, species selection for remediation can be simplified. To identify traits, species differing in growth rate, root morphology, water-use efficiency, photosynthetic pathway and nitrogen fixation were grown in a greenhouse, in oil contaminated (6 L m-’) soil. After 20 weeks, TPH indicated that plants contributed little to bioremediation. However, a bioassay indicated a reduction in soil toxicity. Panicum virgatum, Festuca arundinacea and Cajanus cajan tolerated oil and enhanced oil degradation. These species are perennial, drought tolerant and tolerant of warm temperatures. Drought tolerance may be advantageous because of the hydrophobic nature of oil-contaminated soils. Thus, water-use efficiency may be an important trait for oil tolerance. The species differed in photosynthetic pathway, nitrogen fixation, and root mass. Because these species represent an array of traits, planting a combination of species may accelerate remediation.
INTRODUCTION Several studies have demonstrated that spilled oil degrades more rapidly on vegetated soils than on soils lacking vegetation (e.g., 1-7), but the mechanisms of this acceleration are not clearly defined. Direct uptake of oil by plants does not occur (8, 9), although low concentrations of low molecular weight polynuclear aromatic hydrocarbons were detected in root peels of Daucus carota (10). Plants may polymerize contaminants via peroxidase activity on root surfaces (11). Eichhornia crassipes (water hyacinth), for example, polymerized guaiacol (11). While the direct action of plants in remediation of oil is limited, plants may enhance bioremediation of oil indirectly via their interaction with bacteria and fungi (12). Identification of plant traits important to these interactions would provide a mechanism to rapidly screen plant species for use in restoration plans. Species from the local flora could be screened using data from the literature for possession of one or more traits that enhance biodegradation. This subset of species could then be tested for tolerance to oil. This method of species selection would be more rapid and site specific than greenhouse screening of all local species. The interaction between plants and soil microbes occurs in the rhizosphere. Therefore, root characteristics may be the most important plant trait involved with remediation. Enhanced microbial activity within the rhizosphere may be the result of plant-derived compounds that contain resources limiting to microbial growth and metabolism. As much as 20% of photosynthates can leak from roots (13, 14). Root exudates include sugars, organic acids, amino acids, flavonoids, siderophores and enzymes (15-19). Additional plant contributions to the rhizosphere include cells that are shed from roots surfaces as roots enlarge and turnover, and release of mucigel (a lubricant that aides root penetration through the soil). Together these exudates and sloughed cells comprise 7 to 27% of a plant’s mass (20). The exudates and plant material may serve directly or indirectly as a source of carbon, nitrogen and phosphorus for bacteria and fungi (21, 22). Organic substances excreted by roots may also serve as cometabolites (15, 23, 24). A cometabolite provides an energy source for microbial metabolism or satisfies some other metabolic requirement unlike the xenobiotic compound (23). Lastly, roots provide surface area for colonization by microbes (25). Therefore root system size and shape may be particularly important for bioremediation because root influence on the soil environment is limited generally to a distance of 20 mm or less from the root (26). The greatest rhizosphere volume may occur with plant species possessing dense, deep roots (27), high root:shoot ratios (28), fibrous roots (1, 29) and abundant root hairs (30). The quality and quantity of root exudates including cometabolites is also species specific, e.g., enzyme production has been shown to be high in horseradish, tomato, water hyacinth and cotton (11, 14, 31, 32). Release of these compounds is limited primarily to just behind the root cap (21, 33), thus a highly branched, rapidly, and continuously growing root system may also contribute large quantities of exudates (34). Further, rapid root growth will result in more rapid root turnover increasing rhizodeposition (30). A second group of plant traits that may enhance aerobic metabolism of petroleum involves the oxygen content of soil. Oxygen concentration can be improved directly by diffusion from plant roots (35-37). Rates of oxygen release from plant roots of 0.5 mol O2 m-2 soil surface day-1 have been recorded (38). Plants tolerant to flooding, in particular, maintain an oxygen supply at the roots for aerobic respiration of belowground
plant parts. Oxygen will often diffuse from these roots into the rhizosphere (e.g., 35, 3942). Diffusion of oxygen often occurs to the greatest extent at the root tip (21, 35, 43, 44). Thus similar to exudates, highly branched root systems with many root tips or systems with rapid growth of lateral roots probably increase the amount of oxygen released into the soil. Additional plant adaptations to inundation that may result in increased soil oxygen concentration include presence and/or hypertrophy of lenticels, adventitious roots, aerenchyma, air roots, pneumatophores, mycorrhizae, as well as thermoosmosis and convective flow (38, 45-48). These adaptations provide a mechanism to supply roots with oxygen from the atmosphere or enhance gas exchange in the inundated soil. Plants with high transpiration rates may also oxygenate soils, particularly in inundated soil. Dacey and Howes (49) and Howes et al. (50) showed that a decline in the water table of a Spartina alterniflora marsh was the result of root uptake and evapotranspiration. The reduction in the water table was accompanied by an influx of air into the soil and an increase in sediment redox potential. The high evapotranspiration rates of hybrid poplars have been used to prevent contaminant leaching in a landfill (51). Hybrid poplars grow rapidly and form dense root mass (51). Plant traits associated with high mean daily water uptake, e.g., high rooting density (52), may therefore promote bioremediation. Other traits associated with high water uptake may, however, slow bioremediation by reducing oxygen diffusion from roots and reducing root surface area available for colonization. A large root radius contributes to high mean daily water uptake (53), but would slow diffusion and limit root surface area. Also rapidly growing root systems would require greater quantities of oxygen for respiration reducing the amount of oxygen available for microbial metabolism (28). Biological nitrogen fixation potentially contributes nitrogen to microbes in the rhizosphere (54, 55) as well as reducing plant/microbe competition for soil nitrogen (32, 56). Biological nitrogen fixation associated with soybean, clover and alfalfa, for example, can contribute 50 to 200 kg of nitrogen per hectare to the rhizosphere each year (55). This may be why Banks et al. (7) found that Trifolium repens, a species with coarse roots, contributed to greater acceleration of oil degradation than species possessing fine, fibrous root systems. An increase in soil nitrogen from nitrogen-fixing bacteria associated with leguminous and other plant species increases plant growth, root exudation and bioremediation (30, 32, 55, 57). Biological nitrogen fixation occurs in association with a wide range of plant species and can be improved via selection. Selection for improved fixation in Lotus corniculatus resulted in larger plants possessing more fibrous roots and improved nodulation (58). Similar studies with alfalfa also revealed increased nitrogen fixation (59). Plants may contribute to remediation by catalyzing the binding of petroleum constituents to humus particles. Humification decreases bioavailability of contaminants (60). Plant roots release enzymes such as oxidoreductases and laccases that contribute to humification into the rhizosphere (11, 51, 61). The increase of soil organic matter by plants also contributes to humification (32), as well as improving and stabilizing soil structure (5, 32), reducing leachability (62) and improving diffusion of oxygen (54). Improving soil structure reduces contaminant adsorption on clay particles in high-clay content soils (32). Rapid plant growth may allow for rapid leaf turnover and nutrient cycling (63) which in turn increases nutrient availability, stabilizes soil structure and enriches soil organic matter. Further, soil porosity may be improved by the presence of plants contributing to gas flux between the atmosphere and soils via void root channels (38, 64).
This project experimentally tested the effectiveness of particular plant traits for degradation of oil and plant growth in oil-contaminated soils. A series of species and varieties possessing one or more of the specified traits were grown in a greenhouse. Plant growth and the decline in petroleum hydrocarbons in the soil were quantified over time to assess the success of bioremediation.
METHODS The project examined three major groups of plant traits for their ability to enhance biodegradation of crude oil and oil tolerance. The traits included root morphology, photosynthetic pathway (C3 vs. C4) and presence of symbiotic nitrogen fixation. To perform these tests, 24 species or varieties were used (Table 1). When possible, varietal differences were used to test for the benefit to oil tolerance of specific plant traits among closely related individuals.
Planting system Plants were grown in a greenhouse in SC-10 Super Cells (3.8 cm diameter x 21 cm depth; 164 ml volume; Stuewe and Sons, Inc.). Pots were rinsed in a sodium hypochlorite solution and filled with sterilized commercial potting mix (Promix BX; Premier Brands, Inc.). Soil sterilization consisted of two-30 minute periods of autoclaving separated by a minimum of 24 hours. This method reduced accumulation of secondary compounds and controlled fungal spores. Soil re-inoculation was performed by adding 10 ml of a solution formed by mixing oak-hickory-pine soil (collected from the Louisiana Tech University Arboretum, Ruston, LA) with a 0.9% saline solution (to reduce osmotic shock of bacteria and fungi). Soils were flushed before seedlings were transplanted to remove any salt residue. Thus soil and growth conditions were uniform among individuals while possessing natural soil microorganisms as appropriate to the design. North Louisiana crude oil (Calumet Lubricants Co., Princeton, LA; specific gravity = 0.7) was applied at the rate of 6 L m-2 to the soil surface (equivalent to 3 g of oil per 15 g of oven-dried soil). The application rate represented a substantial spill, but was not intended to eliminate all plant growth. Applied petroleum was allowed to volatilize for ca. seven days before the experiment was begun. Seeds were sown into plug flats (TLC Polyform, Inc.; 288 square flat) also using Promix BX. Seedlings were begun in the laboratory under 24 hr fluorescent lighting (50.46 + 3.08 µmol m-2 s-1; 25 – 27 °C) and later moved to the greenhouse. Seedlings were ca. 40 days old at the time of transplanting to the Super Cells. Seedling roots were surface sterilization by dipping plug trays into a 0.26% sodium hypochlorite solution before transplanting. Once seedlings were transplanted, the Super Cells were placed in a greenhouse. Plants were generally allowed to grow for 20 weeks. Several species were harvested earlier (e.g., Avena and Sorghum) after senescence. Greenhouse temperatures ranged from a mean low of 17.0 °C to a mean high of 34.4 °C.
Plant performance
Plant size was quantified to evaluate plant performance as size is generally correlated with fate (i.e., survival and fecundity; 65, 66). Size was estimated using total shoot area and height using a CI-203 portable laser area meter (CID, Inc.). Root elongation was also measured because of the importance of the rhizosphere for microbial degradation of oil. In addition, root length is indicative of plant response to toxic chemicals (67) including polycyclic aromatic hydrocarbons (68). Soil was gently removed from the plant roots and reserved. The remaining soil particles were washed from the roots. Root length and area were then measured using the area meter. Roots and shoots were oven-dried (> 48 hr at 60 °C) and weighed. Specific root length (root length per root dry weight; e.g., 69) was also calculated and compared among treatments. Plant performance was analyzed using a series of fully factorial, fixed two-way analysis of variance (JMP, v. 3.1.6; SAS, 1995). Main effects included possession or absence of the trait and oil (0 or 6 L m-2).
Bioremediation of oil The effect of the plant trait on remediation was assessed by quantifying residual petroleum hydrocarbon in the soil. Soil removed from plants, which had been refrigerated, was pooled across replicates (to reduce cost of analysis), mixed thoroughly and sent to Environmental Conservation Laboratories, Inc. (Jacksonville, FL) to quantify TPH (total petroleum hydrocarbons; EPA method FLPRO; C8 – C40). Thus, plant trait success was determined by reduction in petroleum between treatments with species or varieties serving as replicates. Comparisons of residual petroleum were made using linear regression and fixed effect, one-way analyses of variance with possession or absence of the trait as the main effect. The mean value for each plant trait of a species or variety was used in the regression with TPH.
Germination assay To confirm the relationship between the chemical analysis of oil reduction and biological activity, a seed germination test (bioassay) was performed on selected soils (soils that had grown Avena, Cajanus, Cynodon, Cyperus, Festuca, Glycine, Panicum and Triticum). Germination was also quantified on oil-contaminated (before remediation) and control soils to test the sensitivity of the assay. The tests consisted of 20 lettuce (Lactuca sativa iceberg) and 20 oat seeds (Avena sativa ‘Bob’) sown on contaminated soils in petri dishes lined with filter paper (5 gm moist soil per dish; n = 10 per assay). Seeds were exposed to 24 hr fluorescent lighting (50.46 + 3.08 µmol m-2 s-1; 25 – 27 °C). Germination is reported for four days after plating. To relate germination with bioremediation of soils, linear regression and analyses of variance were performed. Data included the mean number of seeds for each species or variety, TPH values and mean values of the plant trait per species or variety.
RESULTS Bioremediation of oil Quantification of residual total petroleum hydrocarbon in soils provided a wide range of values. Therefore, detecting differences in residual oil as a function of individual plant traits with a limited sample size was difficult. However several effects, primarily relating to plant size, were documented. Linear regression indicated that
residual petroleum was positively related to plant size. Slope of regression lines were significant relating TPH with root area (p = 0.0012; R2 = 0.30), length (p = 0.0036; R2 = 0.25), and weight (p = 0.0035; R2 = 0.25), shoot area (p = 0.0439; R2 = 0.24), length (p = 0.0298; R2 = 0.21), and weight (p = 0.0027; R2 = 0.26), and total plant weight (p = 0.0022; R2 = 0.27) (Figs. 1 – 3). A one-way analysis of variance also found greater TPH in soils associated with C4 species relative to C3 species (Fig. 4). The effect of photosynthetic pathway is confounded, however, with plant size. Plants with the C3 pathway had less root area (p = 0.0008), root weight (p = 0.0006), shoot area (p = 0.0157), shoot weight (p = 0.0038) and total plant weight (p = 0.0033) than the C4 plants. C3 and C4 plants did not differ in proportion of total weight that was root (p = 0.84) or specific root length (p = 0.36). Analyses of variance did not reveal an effect of nitrogen fixation (p = 0.59) or water-use efficiency (p = 0.91).
Plant performance Oil-contaminated soil generally had a negative effect on plant size resulting in a 54% reduction in biomass (dry weight) on average across species (p < 0.0001). Additionally, several species did not survive after exposure to oil (Eremochloa ophiuroides, Lespedeza sericea and Medicago sativa) and were not included in the analysis. Crude oil did not affect the size (total dry weight) of Festuca, Sorghum or Glycine (p > 0.05). Allocation to belowground weight (root dry weight/total dry weight) differed only between species (p < 0.0001). Exposure to oil did not affect allocation (p = 0.80).
Germination assay The germination assay (or bioassay) of oil-contaminated soils before remediation occurred indicated that Avena (‘Bob’) was not sensitive to oil. Percent germination did not differ among seeds grown in oil contaminated or uncontaminated soils (p = 0.51). Lettuce (Iceberg) germination was reduced 32.5% by oil-contaminated soils (p = 0.0003). Linear regression analyses of germination in oil-contaminated soils indicated that soils that possessed species or varieties with greater mean shoot weight (R2 = 0.55; p = 0.0036), mean total weight (R2 = 0.57; p = 0.0027) and mean shoot area (R2 = 0.61; p = 0.0044) improved germination of lettuce (n = 13). Greater mean root area also tended to improve lettuce germination (R2 = 0.26; p = 0.0760). No relationships between germination of lettuce with root length, root weight or shoot length were detected. Analysis of variance also did not detect an effect of whether the plants grown in the soil fixed nitrogen or used C3 or C4 photosynthetic pathways. Germination of lettuce seeds grown on soils containing plants with high water-use efficiency tended to be greater than on soils that had grown plants with low water-use efficiency (p = 0.096). In this study, plants with high water-use efficiency tended to have greater stem area, stem weight and total weight.
DISCUSSION Particular plant species appear to enhance remediation of oil-contaminated soils to a greater extent than other species (e.g., 70-72). Similarly, particular species are more tolerant to exposure to oil (e.g., 7). The plant traits that facilitate the greater remediation and tolerance are not clearly defined, although many traits important to the interaction
between plants and soil microbes are delineated. This research was designed to evaluate the effect of several plant traits on remediation and plant tolerance. Nitrogen fixation and water-use efficiency did not affect the rate of residual petroleum degradation. Biological nitrogen fixation was expected to reduce TPH values as the addition of fertilizer has been shown to increase rates of biodegradation of oil (4). High water-use efficiency was predicted to improve plant tolerance of the hydrophobic soils caused by oil, in turn enhancing degradation. Competition for limiting resources between the plants and microbes may account, in part, for the unexpected results. Both plants and soil microbes require the same resources, e.g., nitrogen, phosphorus, potassium and water. The small volume of the Super Cells may have resulted in enhanced competition for these resources by the plants at the cost of reduced microbial metabolism. Further, root restriction was no doubt imposed by the small pot size. Root restriction has been shown to reduce growth (73) and leaf water potentials (74). Thus, natural interactions between plants and soil microbes most likely were altered, reducing degradation of oil. In addition to larger pot size, an improved test of the contribution of symbiotic nitrogen fixation to phytoremediation would be to use cultivars of alfalfa (75), Glycine max (76, 77), Lotus japonicus (78) and Cajanus cajan (79) which effectively and ineffectively nodulate. Lastly, limitations in sample size, available varieties, and variance in TPH values most likely constrained detection of plant traits important to remediation. Variances in TPH have been problematic in many bioremediation studies (e.g., 80) and may relate to difficulties associated with sampling a heterogeneous matrix containing a substance that does not disperse uniformly. Remediation was enhanced by one plant trait. TPH was reduced in soils that grew C3 species as opposed to soils possessing C4 species. In this experiment, the C3 species tended to complete their lifecycle within the 20-week period. Allocation to above- and belowground vegetative portions of the C3 plants ceased early as energy was allocated to reproduction. Thus, the effect of photosynthetic pathway cannot be separated from plant size in this study. The relationship between reduced plant size and greater reduction in TPH was seen in the regression analysis. Greater root area, length and weight, and shoot area, length and weight slowed biodegradation of oil. These results were unexpected as generally large, fibrous root systems are thought to contribute more to remediation than small root systems because of the expanded opportunity for interaction between plants and soil microbes in the rhizosphere (e.g., 1, 27-30). Again, competition for limiting resources may explain these results. Large plants, relative to small plants, may be at a competitive advantage in obtaining resources. In addition, bacteria may preferentially metabolize plant-derived carbon compounds rather than hydrocarbons. Larger plants would provide greater quantities of carbon compounds via exudates and sloughed cells, slowing degradation of oil to a greater extent than in soils with small plants. Large plants may also promote cometabolism of petroleum constituents via exuded enzymes forming intermediate compounds. The chemical analysis of TPH may be unable to detect the partial degradation of oil. Biological assessment of the extent of remediation appeared to yield an opposite result. Soils that had grown plants possessing greater root area, shoot area and weight, total weight, and water-use efficiency resulted in greater germination of lettuce seeds than soils that had supported smaller plants. However, the effect of plant growth on degradation of oil and on germination is confounded. Germination in oil-contaminated soils that had previously supported plant growth was greater than germination on control
soils that had not grown plants. Thus while the role of specific plant traits is unclear in this study, the presence of plants may enhance natural recovery of spill sites by promoting germination of seeds. Seeds are generally readily available in the seed bank for recolonization after disturbance (e.g., 81). Further the addition of fertilizer to limit competition may further enhance revegetation and biodegradation of spill sites. Plants will not be able to contribute to degradation of oil unless they tolerate the spill environment. Plant performance in this study was generally reduced by exposure to oil. Responses however were species-specific and variety-specific. Festuca arundinacea, Triticum aestivanum, Glycine max, Panicum virgatum, Guatemala sorghum and Potoroo oat demonstrated some tolerance to oil. The plant traits that provide oiltolerance may also enhance microbial degradation. Spilled oil often causes hydrophobic and anaerobic soils, reducing availability of moisture and oxygen for plants. Thus, traits that promote shoot and root gas exchange and absorption of water and minerals will benefit the plant. Greater flux of gases and moisture may also benefit microbes if resources are not limiting. Plant traits imparting oil tolerance may also reduce biodegradation. Water-use efficiency is one example. Particular traits associated with high water-use efficiency, e.g., small leaf area, narrow leaves, and reduced vertical leaf extension, lower rates of evapotranspiration (82) may limit oxygenation of soils. Reestablishment of vegetation and plant-enhanced degradation therefore consist of a series of beneficial and competing plant traits and processes. A combination of the remediation and tolerance data indicates that Panicum, Festuca and Cajanus were at least partially tolerant to oil and enhanced bioremediation of oil. These species represent a range of characteristics including C3 and C4 photosynthetic pathways, biological nitrogen fixation and non-fixation, and small and large root systems. The three species have in common: a perennial life-history, and tolerance to drought and moderate to high temperatures. Drought and temperature tolerance is necessary for Louisiana’s climate and because of the hydrophobic nature of oil-contaminated soils. Experimental methods and oil analyses will have to be refined to further delineate common traits of phytoremediators. However, because these plant species represent an array of traits, effective remediation may occur by employing a mix of species. Panicum and Festuca will be examined in the future. Both species are common in Louisiana and may be valuable for restoration plans. Cajanus is not native to Louisiana (originating in India) and would not be appropriate for restoration. A legume species, however, possessing the same characteristics (e.g., drought and high temperature tolerance) may be advantageous, e.g., Vicia villosa. Refinements to the experimental system and assessment of the effectiveness of a combination of species are currently being researched. While the roles of soil microbes, plants and particular plant traits were difficult to discern in this study, sufficient differences were detected to suggest that underlying mechanisms of plant/bacterial/fungal interactions in bioremediation must be delineated in future studies. The delineation of these traits will allow restoration plans to be developed which enhance bioremediation of oil spill sites. In addition, identification of plant traits contributing to biodegradation will allow for rapid screening of regional flora for species that will effectively contribute to re-vegetation and remediation. Thus, the need to individually screen large numbers of species will be eliminated. Lastly, delineation of plant traits associated with remediation and their genetic basis is the first step in developing plant breeding and genetic engineering of species specifically for phytoremediation.
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Table 1. Plant species/varieties and traits used in assessing the role of particular plant traits on remediation of crude oil-contaminated soils. Family Species Variety or cultivar Trait examined Cyperaceae Cyperus esculentus Non-nitrogen fixing, C3 Poaceae Festuca arundinacea Kentucky 31 Non-nitrogen fixing, C3 Poaceae Festuca arundinacea Asheville Non-nitrogen fixing, C3, low water-use efficiency Poaceae Festuca arundinacea AVS Non-nitrogen fixing, C3, low water-use efficiency Poaceae Festuca arundinacea NY 1146 Non-nitrogen fixing, C3, low water-use efficiency Poaceae Festuca arundinacea Turkey Non-nitrogen fixing, C3, low water-use efficiency Poaceae Cynodon dactylon Non-nitrogen fixing, C4, high water-use efficiency Poaceae Panicum virgatum Non-nitrogen fixing, C4 Poaceae Avena sativa Thousand dollar Non-nitrogen fixing, C3 Poaceae Avena sativa Red Rustproof Non-nitrogen fixing, C3 Poaceae Avena sativa Texas Non-nitrogen fixing, C3 Poaceae Avena sativa Bob Non-nitrogen fixing, C3 Poaceae Avena sativa Potoroo Non-nitrogen fixing, C3 Poaceae Triticum aestivaum ssp 5 Non-nitrogen fixing, C3, high water-use efficiency vulgare Poaceae Triticum aestivaum 8262-AR3-G Non-nitrogen fixing, C3, high water-use ssp. durum efficiency Poaceae Triticum aestivaum DF 4/72 Non-nitrogen fixing, C3, high water-use ssp. durum efficiency Poaceae X Triticosecale sp. M2A Non-nitrogen fixing, C3, high water-use efficiency Poaceae Sorghum bicolor Pakistan 14151 Non-nitrogen fixing, C4 Poaceae Sorghum bicolor Guatemala 6913 Non-nitrogen fixing, C4 Poaceae Sorghum bicolor S. Korea 1257 Non-nitrogen fixing, C4 Poaceae Sorghum bicolor Sudan IS 19041 Non-nitrogen fixing, C4 Fabaceae Cajanus cajan Norman Nitrogen fixing Fabaceae Cajanus cajan ICPL 87 Nitrogen fixing Fabaceae Glycine max Hardee Nitrogen fixing, C3 Fabaceae Glycine max Jackson Nitrogen fixing, C3, high water-use efficiency Malvaceae Gossypium herbaceum Pima S-5 Non-nitrogen fixing, C3, high water-use efficiency Malvaceae Gossypium herbaceum ACALA Non-nitrogen fixing, C3 Malvaceae Gossypium herbaceum All-Tex Express Non-nitrogen fixing, C3 Malvaceae Gossypium herbaceum D & PL 50 Non-nitrogen fixing, C3, low water-use efficiency Malvaceae Hibiscus cannabinus Non-nitrogen fixing
(a) 140000
TPH (mg/kg)
120000 100000 80000 60000 40000 20000 0 0
10
20
30
40
50
Mean root area (cm2)
(b) 140000
TPH (mg/kg)
120000 100000 80000 60000 40000 20000 0 5
10
15
20
25
30
Mean root length (cm)
(c) 140000
TPH (mg/kg)
120000 100000 80000 60000 40000 20000 0 0
0.1
0.2
0.3
0.4
0.5
0.6
Mean root weight (g)
Figure 1. Linear regression of (a) mean root area, (b) mean root length, and (c) mean root dry weight per species or variety with residual petroleum in a greenhouse study.
(a) 140000
TPH (mg/kg)
120000 100000 80000 60000 40000 20000 0 0
20
40
60
80
Mean shoot area (cm2)
(b) 140000
TPH (mg/kg)
120000 100000 80000 60000 40000 20000 0 -5
5
15
25
35
45
55
65
Mean shoot length (cm)
(c) 140000
TPH (mg/kg)
120000 100000 80000 60000 40000 20000 0 0.0
0.2
0.4
0.6
0.8
1.0
1.2
Mean shoot weight (g)
Figure 2. Linear regression of (a) mean shoot area, (b) mean shoot length, and (c) mean shoot dry weight with residual petroleum in a greenhouse study.
140000
TPH (mg/kg)
120000 100000 80000 60000 40000 20000 0 0.0
0.5
1.0
1.5
Mean plant weight (g)
Figure 3. Linear regression relating mean plant weight per species or variety with residual petroleum in a greenhouse study.
TPH (mg/kg)
120000 100000 80000 60000 40000 20000 0 C3
C4
Photosynthetic pathway
Figure 4. Residual petroleum in soils that had grown plants with C3 or C4 photosynthetic pathways in a greenhouse study. Bars represent + SE.
