Duane C. Wolf

Rhizosphere microbial populations in contaminated soils

Rhizosphere microbial populations may increase bioremediation of soil contaminated with organic chemicals. A growth chamber study was conducted to evaluate rhizosphere microbial populations in contaminated and non-contaminated soil. Alfalfa (Medicago sativa L.) and alpine bluegrass (Poa alpina L.) were grown in soil containing a mixture of organic chemicals for 14 weeks. The equal millimolar mixture of hexadecane, (2,2-dimethylpropyl)benzene, cis-decahydronaphthalene (decalin), benzoic acid, phenanthrene, and pyrene was added at levels of 0 and 2000 mg/kg. Organic chemical degrader (OCD) populations were assessed by a Most-Probable-Number technique, and bacteria and fungi were enumerated by plate count methods. Different methods for expressing OCD rhizosphere populations were investigated to determine the effect it had on interpretation of the results. At 9 weeks, the OCD numbers were significantly higher in rhizosphere and contaminated soils than in bulk and non-contaminated soils, respectively. Alfalfa rhizosphere OCD levels were 4 × 107/g for contaminated and 6 × 106/g for non-contaminated soils. Bluegrass rhizosphere OCD levels were 1 × 107/g and 1 × 106/g in contaminated and non-contaminated soils, respectively. Selective enrichment of OCD populations was observed in contaminated rhizosphere soil. Higher numbers of OCD in contaminated rhizospheres suggest potential stimulation of bioremediation around plant roots.

Sorption of heavy metals by the soil fungi Aspergillus niger and Mucor rouxii

Abstract Sorption of the nitrate salts of cadmium(II), copper(II), lanthanum(III) and silver(I) by two fungi, Aspergillus niger and Mucor rouxii, was evaluated using Freundlich adsorption isotherms and energy dispersive X-ray electron microscopy. The linearized Freundlich isotherm described the metal sorption data well for metal concentrations of 5 μm-1 Mm metal. Differences in metal binding were observed among metals, as well as between fungal species. Calculated Freundlich K values indicated that metal binding decreased in the order La3+ ⩾ Ag+ > Cu2+ > Cd2+. However, sorption of Ag+ was greater than that of La3+ from solutions of 0.1 and 1 mM metal and likely due to precipitation at the cell wall surface. At the 1 mM initial concentration, there were no significant differences between the two fungi in metal sorption, except for Ag+ binding. At the 5 μM concentration, there was no difference between the fungi in their sorption capacities for the four metals. Electron microscopy-energy dispersive X-ray analysis indicated that silver precipitated onto cells as colloidal silver. The results indicate that Freundlich isotherms may be useful for describing short-term metal sorption by fungal biomass and for comparison with other soil constituents in standardized systems.

Influence of organic and inorganic soil amendments on plant growth in crude oil-contaminated soil

Abstract Phytoremediation can be a viable alternative to traditional, more costly remediation techniques. Three greenhouse studies were conducted to evaluate plant growth with different soil amendments in crude oil‐contaminated soil. Growth of alfalfa (Medicago sativa L., cultivar: Riley), bermudagrass (Cynodon dactylon L., cultivar: Common), crabgrass (Digitaria sanguinalis cultivar: Large), fescue (Lolium arundinaceum Schreb., cultivar: Kentucky 31), and ryegrass (Lolium multiflorum Lam., cultivar: Marshall) was determined in crude oil‐contaminated soil amended with either inorganic fertilizer, hardwood sawdust, papermill sludge, broiler litter or unamended (control). In the first study, the addition of broiler litter reduced seed germination for ryegrass, fescue, and alfalfa. In the second study, bermudagrass grown in broiler litteramended soil produced the most shoot biomass, bermudagrass produced the most root biomass, and crabgrass and bermudagrass produced the most root length. In the third study, soil amended with broiler litter resulted in the greatest reduction in gravimetric total petroleum hydrocarbon (TPH) levels across the six plant treatments following the 14‐wk study. Ryegrass produced more root biomass than any other species when grown in inorganic fertilizer‐ or hardwood sawdust + inorganic fertilizer‐amended soil. The studies demonstrated that soil amendments and plant species selection were important considerations for phytoremediation of crude oil‐contaminated soil.

Tracking the fate and recycling of 13C-labeled glucose in soil

A short-term incubation of soil amended with 13C-glucose was conducted to determine the extent of labeled C recycling that might occur within the microbial community. Changes in the production and isotopic composition of CO2 and biomass suggest that two phases of microbial activity occurred after the glucose addition. The initial phase due directly to the metabolism of the added glucose was characterized by an increase in biomass and a high growth efficiency. A second phase appeared to be driven by less available substrates (e.g., cell wall structures, soil organic matter) and characterized by insignificant changes in biomass but significant generation of CO2 suggestive of low growth efficiency. Glucose-C supported 12 to 73% of the CO2-C evolved and 17 to 21% of biomass-C, suggesting glucose was the principle energy rather than a C source during the 15- to 48-hour phase of the incubation. Variation in &dgr;13C composition of individual phospholipid fatty acids (PLFA) during the incubation indicated that different components of the microbial community played different roles in the cycling of the added glucose. The most enriched &dgr;13C values were initially those PLFA associated with Gram-positive bacteria, suggesting they were responsible for much of the initial incorporation. By contrast, at the end of the 48-hour incubation, 4 of 24 PLFA biomarkers were not labeled with 13C. Actinomycetes, however, probably played a larger role in the use of recycled glucose-derived C, as suggested by the enrichment in 13C of 10-methyl 18:0 PLFA after the exhaustion of glucose. Results from this study show that the element of time needs to be considered carefully in the interpretation of any stable isotope labeling and biomarker study.

Plant enhancement of indigenous soil micro-organisms: a low-cost treatment of contaminated soils

The United States has more than 1000 individual areas of petroleum-contaminated soil at formerly used defense (FUD) sites located in cold regions. This paper investigates biotreatment systems based on exploiting naturally occurring phenomena in the rhizosphere — the soil adjacent to and influenced by plant roots. Rhizosphere-based remediation systems would be inexpensive to implement and maintain and would be applicable to remote or permafrost sites. Herein, this paper provides the rationale for using rhizosphere-based biotreatment systems and some initial results. In both laboratory and field studies, successful plant germination, plant growth, and root intrusion into and through contaminated soil are demonstrated. Using a Captina silt loam in a 10-week laboratory study, the effects of vegetation and contamination on microbial numbers were compared. The vegetation treatments included an unvegetated control and a vegetated treatment seeded with bahiagrass ( Paspalum notatum ). The contamination treatments included an uncontaminated control and a treatment with 2000 mg pyrene kg -1 soil added. Microbial numbers at 10 weeks were not significantly influenced by the contaminant level of 2000 mg pyrene kg -1 soil compared to the control. However, microbial numbers were greater in the rhizosphere of the bahiagrass-vegetated soil compared to the unvegetated soil. In a 34-week field study, total petroleum hydrocarbon (TPH) concentrations of a diesel-contaminated soil decreased significantly more in the rhizosphere+nutrient treatment compared to the control that was not vegetated or fertilized. Bacterial numbers in the field study were 287 times greater in the rhizosphere+nutrient treated soils than in the control treatments. Measurable TPH compounds in the plant tissue were insignificant. The data demonstrated that rhizosphereenhanced treatment of organic-contaminated soils can be effective in reducing soil petroleum concentrations and may be a cost-effective strategy particularly suited for treating cold-region sites where remediation options are limited by cost, remoteness of the site, and/or brevity of the treatment season.

Phytoremediation of Pyrene in a Cecil Soil under Field Conditions

We evaluated the effects of annual ryegrass (Lolium multiflorum Lam.) and phosphorus (P) availability on the dissipation of pyrene added at a concentration of approximately 600 mg kg−1 dry soil in the top 7.5 cm of a Cecil loamy sand (fine, kaolinitic, thermic Typic Kanhapludults) in a 10-month experiment under field conditions in Clemson, South Carolina. Plastic canopies were installed to prevent flooding of plots and raindrop dispersion of pyrene. Treatment factors were pyrene, vegetation, and available P levels. Each of the eight treatments had four replicates. The soil was adjusted to low and high P concentrations (an average of 41 and 66 kg extractable P ha−1, respectively). After a 175-d lag period for all treatments, the rate of pyrene removal followed first-order kinetics. The first-order rate constant was significantly higher in nonvegetated (0.098 d−1) than vegetated treatments (0.034 d−1). These data suggest that the presence of easily biodegradable organic matter from plant roots slowed the removal rate of pyrene. The levels of available P did not affect the rate of pyrene dissipation. Pyrene decreased below the detection limit of 6.25 mg kg−1 dry soil in all treatments after 301 d.

Selecting Plants and Nitrogen Rates to Vegetate Crude-Oil–Contaminated Soil

Phytoremediation can be effective for remediating contaminated soils in situ and generally requires the addition of nitrogen (N) to increase plant growth. Our research objectives were to evaluate seedling emergence and survival of plant species and to determine the effects of N additions on plant growth in crude-oil–contaminated soil. From a preliminary survival study, three warm-season grasses—pearlmillet (Pennisetum glaucum [L.] R. Br.), sudangrass (Sorghum sudanense [Piper] Stapf [Piper]), and browntop millet (Brachiaria ramosa L.)—and one warm-season legume—jointvetch (Aeschynomene americana L.)—were chosen to determine the influence of the N application rate on plant growth in soil contaminated with weathered crude oil. Nitrogen was added based on total petroleum hydrocarbon-C:added N ratios (TPH-C:TN) ranging from 44:1 to 11:1. Plant species were grown for 7 wk. Root and shoot biomass were determined and root length and surface area were analyzed. Pearlmillet and sudangrass had higher shoot and root biomass when grown at a TPH-C:TN (inorganic) ratio of 11:1 and pearlmillet had higher root length and surface area when grown at 11:1 compared with the other species. By selecting appropriate plant species and determining optimum N application rates, increased plant root growth and an extended rhizosphere influence should lead to enhanced phytoremediation of crude-oil–contaminated soil.

Mycorrhizal colonization and microbial community structure in the rhizosphere of annual ryegrass grown in pyrene-amended soils.

Abstract In an 182‐d lightroom experiment, annual ryegrass (Lolium multiflorum Lam.) was grown in two soils under conditions of high and low fertility to examine the effect of pyrene (500 mg kg‐1) on plant shoot biomass, mycorrhizal colonization, and soil microbial community structure. Treatments were destructively sampled every 14 d. Plant shoot biomass remained relatively unaffected by pyrene in either soil. Mycorrhizal colonization was only briefly affected by pyrene in one soil, but was unaffected in the other. Changes in soil microbial community structure were measured with whole soil fatty acid methyl ester (FAME) profiles. Differences in soil microbial community structure were observed between planted and nonplanted treatments at both fertility levels, but these differences were unrelated to the presence of pyrene. The bulk soil was associated primarily with fatty acid biomarkers for Gram‐positive bacteria, while the rhizosphere was associated primarily with the fatty acid biomarkers associated with protozoa. Differences in microbial community structure were observed between the two soils. Methylene chloride‐extractable pyrene decreased in nonplanted and planted treatments of one soil, but decreased only in nonplanted treatments of the other soil. These results suggest the potential for phytoremediation to differ among soils.

A mathematical model of phytoremediation for petroleum contaminated soil: sensitivity analysis.

Phytoremediation is an attractive treatment technology for many contaminated sites due to its cost effectiveness and public acceptance. We present a sensitivity analysis of important parameters from a screening level model for phytoremediation by grass species of weathered petroleum-contaminated sites. The conceptual framework is that root movement through contaminated soil will enhance contaminant biodegradation by providing a local environment more favorable for petroleum degrading microorganisms—the so-called rhizosphere effect. Common questions in phytoremediation are, “What species should be planted?” and “What management practices should be followed?” These choices may affect degradation kinetics, root biomass (and therefore rhizosphere volume), and the root turnover. Important model parameters are the rate constants, rhizosphere volume, and the rate of root turnover. We present a sensitivity analysis with the aim of identifying the most important factors for improving phytoremediation effectiveness. For simulations of the phytoremediation of weathered diesel range organics, our results indicate that annual species, with higher root turnover, are preferred over perennial species with the caveat of equal degradation rate constants, that is, no species-dependent effects. In addition, the results suggest that the management of nonrhizosphere soil could play an important role in the overall effectiveness of phytoremediation. Finally, the effect of increasing root biomass or increasing the rhizosphere thickness is approximately equivalent with respect to the ultimate removal of the contaminants.

Influence of methanol and hexane on soil adsorption of atrazine

The increasing frequency of chemically contaminated groundwater, occurring as a result of improperly managed waste disposal or accidental spills, presents a need for research on the fate of chemical mixtures in the soil. The batch equilibration technique was used to measure adsorption of 14C ring-labeled atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) for a Palouse silt loam (Pachic Ultic Haploxeroll) and a Pembroke silty clay loam (Typic Paleudalf). The solution phase consisted of mixtures of methanol-water and hexane-water containing up to 33.3 % organic solvent by volume. Aqueous solubility limited atrazine concentrations to 100 μmol L−1 except for an additional isotherm determined in 33.3 methanol-water at up to 1542 μmol L−1 The Freundlich adsorption coefficient indicated that the Palouse adsorbed more atrazine than the Pembroke with K values of 4.95 and 0.54, respectively. Both soils showed a significant decrease in K as the percentage methanol increased. Adsorption isotherms from a 33. 3 methanol-water system were of the Freundlich type for atrazine concentrations of 0.25 to 1542 μmol L−1. In the hexane-water systems, K decreased as the fraction of hexane increased and the Pembroke soil adsorbed less atrazine than the Palouse soil. These results suggest that the introduction of nonaqueous solvents such as methanol and hexane decreased adsorption and increased the potential for atrazine mobility.