Grantee Research Project Results

Final Report: Anaerobic Intrinsic Bioremediation of Whole Gasoline

EPA Grant Number: R827015C004
Subproject: this is subproject number 004 , established and managed by the Center Director under grant R827015
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: HSRC (1989) - Northeast HSRC
Center Director: Sidhu, Sukh S.
Title: Anaerobic Intrinsic Bioremediation of Whole Gasoline
Investigators: Thoma, Greg , Beyrouty, Craig , Wolf, Duane
Institution: University of Oklahoma
EPA Project Officer: Aja, Hayley
Project Period: February 1, 1999 through January 31, 2000 (Extended to June 30, 2001)
Project Amount: Refer to main center abstract for funding details.
RFA: Integrated Petroleum Environmental Consortium (IPEC) (1999) RFA Text |  Recipients Lists
Research Category: Hazardous Waste/Remediation , Targeted Research

Objective:

The objectives of this research project were to: (1) conduct greenhouse studies to screen plants for their ability to germinate and grow in weathered crude oil-contaminated soil with or without amendments; (2) survey and collect plant species currently growing on contaminated sites and screen the plants and rhizosphere microorganisms for their ability to enhance biodegradation of petroleum contaminants; (3) conduct an onsite field study to evaluate likely combinations of plants and management systems to enhance phytoremediation of weathered crude oil-contaminated sites; and (4) develop a model that can be used to summarize and aid in the interpretation of experimental data collected in both the laboratory and field during the first experimental season.

Summary/Accomplishments (Outputs/Outcomes):

Field Study

The field site in El Dorado, Arkansas, is located in a bermed area that is the site of an intentional spill by vandals in 1997. The experimental plots consist of four replicates of the following treatments: (1) nonvegetated-nonfertilized control, (2) fescue-ryegrass-alfalfa plus fertilizer, and (3) fescue-bermudagrass plus fertilizer. Each field plot has 12 microplots (soil socks) that contain homogenized soil that allow monitoring of the field treatments, on a smaller scale, with less effect of field variability of the contaminant levels.

On July 10-12, 2000, 6 months after establishment of vegetation at the site, soil and plant samples were collected from the plots. Plant shoot biomass and root biomass, length, surface area, and volume for each of the treatments are summarized in Table 1. All plant species appeared to be exhibiting adequate plant growth.

Table 1. Shoot Biomass and Root Biomass, Length, Surface Area, and Volume for Samples Collected July 12, 2000—6 Months After Initiation of the Field Study in El Dorado, Arkansas

Treatment	Biomass		Root
	Shoot	Root	Length	Surface Area	Volume
	g/m2	g/m3	km/m3	m2/m3	cm3/m3
Control* No Fertilizer No Vegetation	0	18.40±14.32	1.48±1.58	0.93±0.88	4,838
Fescue/Rye* + Fertilizer	1.05±0.81	162.93±67.46	27.37±12.78	20.98±9.53	1,231±621
Bermudagrass** + Fertilizer	4.26±3.66	264.89±95.85	50.70±11.54	35.85±10.21	1,914±872
*Values are means of 4 samples ±1 standard deviation. **Values are means of 3 samples ±1 standard deviation.

The sampling that was scheduled for December 2000 was canceled because of ice storms that made travel nearly impossible. A percent plant cover evaluation of the plots was attempted February 25, 2001, but was unsuccessful as the result of flood conditions that prevented the researchers from reaching the field site.

An additional survey was conducted to identify plant species currently growing on petroleum-contaminated sites near the El Dorado, Arkansas, field site. Plants observed to be growing at the various sites included bermudagrass (Cynodon dactylon L.), yellow nutsedge (Cyperus esculentus L.), fall panicum (Panicum dichotomiflorum Michx.), brome grass (Bromus spp.), and witchgrass (Panicum capillare L.).

On May 20-23, 2001, 17 months after establishment of vegetation at the site, soil and plant samples were collected from the plots. Plant shoot biomass and root biomass, length, surface area, volume, and plant cover for each of the treatments were determined. All plant species appeared to be exhibiting adequate plant growth. The total petroleum hydrocarbon (TPH) and biomarker (hopane) analyses of the soil samples collected 17 months after plot establishment (t=17) currently are being conducted.

The addition of fertilizer and vegetation establishment significantly increased total bacterial and polycyclic aromatic hydrocarbon (PAH) degrader numbers. Control, fescue-ryegrass, and bermudagrass-fescue treatments had PAH degrader numbers of 3.36, 3.71, and 3.78 log MPN/g dry soil, respectively, and bacterial numbers of 6.09, 6.87, and 6.80 log CFU/g dry soil, respectively. Additionally, root surface area levels were higher in the samples collected at 17 months compared to 6 months. The results suggest that agronomic practices are important considerations when developing systems to phytoremediate crude oil-contaminated sites.

Greenhouse Study

Because oil-contaminated soils have different nutrient requirements than conventional agronomic recommendations suggest, a nutrient rate study using warm-season plant species was conducted. Soil analyses for the petroleum contaminated soils amended with organic materials are summarized in Table 2.

Table 2. Soil Chemical Data From Greenhouse Study

Treatment	pH	EC	P	K	Ca	Mg	Na	NH4- N	TN	TC
		µmhos/cm	mg/kg						%
Control	5.8b	45.5bc	1.7c	32.0b	367.2c	35.0b	31.2a	2.7d	0.086c	9.976a
IF	4.9c	41.3c	60.8b	79.6a	313.6d	25.7c	15.4d	5.0cd	0.123bc	9.527b
IF+SD	5.0c	50.0b	60.0b	89.4a	337.4cd	27.7c	17.0d	11.0b	0.130b	10.799a
Sludge	6.4a	49.3b	4.4c	41.2b	623.6b	39.2b	27.3b	5.9c	0.135b	10.234a
Litter	6.3a	74.5a	114.4a	88.4a	845.4a	131.5a	23.1c	17.6a	0.191a	8.546b
Means followed by the same letter are not statistically different at the 5% level.

Total petroleum hydrocarbon (TPH) analysis was completed on soil samples following the greenhouse study where plants were grown in oil-contaminated soil amended with and without inorganic and organic amendments. The initial TPH concentration was 9 percent by weight. The amendments included broiler litter, paper mill sludge, hardwood sawdust plus inorganic fertilizer, and inorganic fertilizer. The samples were extracted in accordance with U.S. Environmental Protection Agency Method 3540C, and TPH concentration was determined gravimetrically (Table 3).

Table 3. TPH Concentrations in Soil Amended With Inorganic or Organic Amendments Following 14-Week Greenhouse Study

Treatment	Total Petroleum Hydrocarbon levels
	% (dry weight basis)
Broiler litter	6.0 a*
Sawdust + Inorganic fertilizer	6.6 b
Inorganic fertilizer	7.1 c
Papermill Sludge	7.9 d
Control (no amendment)	7.9 d
*Means (n=4) followed by the same letter are not statistically different at the 5% level.

Mathematical Model

We have consulted with a mathematician regarding the solution to the system of differential equations because we found certain input parameters cause the numerical solution to fail. We have identified the cause of this failure and currently are working to correct it. For parameter values that do not cause the failure, we have demonstrated, through grid independence testing and solutions computed with different numerical algorithms (Fourth order explicit Runge-Kutta, Gear’s method, and the modified Rosenbrock method) that the numerical solutions are valid. Specifically, this means that the solution has converged, within a prespecified error level, to the true solution of the system of equations.

Simulations to evaluate the significance of the bulk and rhizosphere kinetic rate constants and the rhizosphere volume were conducted. Several comparisons can be drawn regarding the phytoremediation reaction system. A comparison indicates that the bulk soil degradation rate constant, here taken to be 10 percent of the maximum rhizosphere rate constant, still significantly contributes to the overall hydrocarbon dissipation. Thus, even when phytoremediation is used, some management strategy focusing on increasing the microbial activity in the bulk soil should be considered. For the conditions simulated, an annual species would be recommended. Comparison of curves 3 and 5 shows the effect of increasing the rhizosphere zone to 1.5 mm from the root surface. This increase is simulated for the same total root biomass. It would represent the effect of a management strategy that enhanced the microbial biomass extent around the roots (perhaps through additional aeration, irrigation, fertilization, or plant species selection). Finally, comparison shows the impact of a management strategy that increased the degradation rate constant, k, through manipulations focused on increasing the microbial activity by, for example, increasing the number of degrader organisms in the rhizosphere through selective enhancement such as adjusting soil pH, moisture, or nutrient availability. It is clear from the model predictions that this model can be very useful in guiding the phytoremediation research effort.

We have identified the source of the instability in the model solution as resulting from simulations in which the soil becomes “root-bound” and the calculated volume of soil remaining as “bulk” drops to zero (or in extreme cases, negative values). We are correcting the acceptable ranges of certain parameters (particularly the ratio of rhizosphere to root volume) that influence this behavior. We have preliminary models of the root growth and senescence functions for annual and perennial species that more closely mimic the expected growth patterns. In particular, we have functions that can be used to simulate the root growth patterns expected when cool- season -> warm-season -> cool-season species are planted successively in a single year. We are testing the L-system analysis code to be sure that segment orientation (for single segments) does not affect the computed results and that the overlap of the rhizosphere is calculated correctly for adjacent, parallel segments.

We are continuing to improve the estimate of rhizosphere volume through the analysis of fractal root structures. The code has been validated with respect to multiple, separated roots as a function of the spatial orientation of the root segments. We have a limited ability to test analytically the overlapping of adjacent rhizosphere zones (limited to spheres and parallel cylinders only), and the accuracy testing has been acceptable. We have also proposed a submodel for the idealized behavior of roots through out the growing season. The parameters in this submodel can be determined from easily observed field parameters that characterize root growth. These are maximum rooting depth, maximum standing biomass, fractional turnover on an annual basis, and average root lifespan.

Journal Articles:

No journal articles submitted with this report: View all 11 publications for this subproject

Supplemental Keywords:

Arkansas (AR), petroleum, phytoremediation, EPA Region 6, rhizosphere,, RFA, Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, Geographic Area, Waste, Hazardous, Remediation, Engineering, State, Civil/Environmental Engineering, Chemistry, Biology, Groundwater remediation, Microbiology, Hazardous Waste, Environmental Microbiology, Northwest, Biochemistry, Bioavailability, Bioremediation, Oil Spills, Environmental Engineering, hydrocarbons, gasoline, biological markers, Ft. Lupton, CO, anaerobic biotransformation, anaerobic treatment, risk assessment, anaerobic bioremediation, biodegradation, groundwater, anaerobic biodegradation, Colorado (CO), anaerobic bioconversion, risk assessments

Relevant Websites:

http://ipec.utulsa.edu/ Exit

Progress and Final Reports:

Original Abstract

1999

2000 Progress Report

Main Center Abstract and Reports:

R827015    HSRC (1989) - Northeast HSRC

Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R827015C001 Evaluation of Road Base Material Derived from Tank Bottom Sludges
R827015C002 Passive Sampling Devices (PSDs) for Bioavailability Screening of Soils Containing Petrochemicals
R827015C003 Demonstration of a Subsurface Drainage System for the Remediation of Brine-Impacted Soil
R827015C004 Anaerobic Intrinsic Bioremediation of Whole Gasoline
R827015C005 Microflora Involved in Phytoremediation of Polyaromatic Hydrocarbons
R827015C006 Microbial Treatment of Naturally Occurring Radioactive Material (NORM)
R827015C007 Using Plants to Remediate Petroleum-Contaminated Soil
R827015C008 The Use of Nitrate for the Control of Sulfide Formation in Oklahoma Oil Fields
R827015C009 Surfactant-Enhanced Treatment of Oil-Contaminated Soils and Oil-Based Drill Cuttings
R827015C010 Novel Materials for Facile Separation of Petroleum Products from Aqueous Mixtures Via Magnetic Filtration
R827015C011 Development of Relevant Ecological Screening Criteria (RESC) for Petroleum Hydrocarbon-Contaminated Exploration and Production Sites
R827015C012 Humate-Induced Remediation of Petroleum Contaminated Surface Soils
R827015C013 New Process for Plugging Abandoned Wells
R827015C014 Enhancement of Microbial Sulfate Reduction for the Remediation of Hydrocarbon Contaminated Aquifers - A Laboratory and Field Scale Demonstration
R827015C015 Locating Oil-Water Interfaces in Process Vessels
R827015C016 Remediation of Brine Spills with Hay
R827015C017 Continuation of an Investigation into the Anaerobic Intrinsic Bioremediation of Whole Gasoline
R827015C018 Using Plants to Remediate Petroleum-Contaminated Soil
R827015C019 Biodegradation of Petroleum Hydrocarbons in Salt-Impacted Soil by Native Halophiles or Halotolerants and Strategies for Enhanced Degradation
R827015C020 Anaerobic Intrinsic Bioremediation of MTBE
R827015C021 Evaluation of Commercial, Microbial-Based Products to Treat Paraffin Deposition in Tank Bottoms and Oil Production Equipment
R827015C022 A Continuation: Humate-Induced Remediation of Petroleum Contaminated Surface Soils
R827015C023 Data for Design of Vapor Recovery Units for Crude Oil Stock Tank Emissions
R827015C024 Development of an Environmentally Friendly and Economical Process for Plugging Abandoned Wells
R827015C025 A Continuation of Remediation of Brine Spills with Hay
R827015C026 Identifying the Signature of the Natural Attenuation of MTBE in Goundwater Using Molecular Methods and "Bug Traps"
R827015C027 Identifying the Signature of Natural Attenuation in the Microbial Ecology of Hydrocarbon Contaminated Groundwater Using Molecular Methods and "Bug Traps"
R827015C028 Using Plants to Remediate Petroleum-Contaminated Soil: Project Continuation
R827015C030 Effective Stormwater and Sediment Control During Pipeline Construction Using a New Filter Fence Concept
R827015C031 Evaluation of Sub-micellar Synthetic Surfactants versus Biosurfactants for Enhanced LNAPL Recovery
R827015C032 Utilization of the Carbon and Hydrogen Isotopic Composition of Individual Compounds in Refined Hydrocarbon Products To Monitor Their Fate in the Environment
R830633 Integrated Petroleum Environmental Consortium (IPEC)
R830633C001 Development of an Environmentally Friendly and Economical Process for Plugging Abandoned Wells (Phase II)
R830633C002 A Continuation of Remediation of Brine Spills with Hay
R830633C003 Effective Stormwater and Sediment Control During Pipeline Construction Using a New Filter Fence Concept
R830633C004 Evaluation of Sub-micellar Synthetic Surfactants versus Biosurfactants for Enhanced LNAPL Recovery
R830633C005 Utilization of the Carbon and Hydrogen Isotopic Composition of Individual Compounds in Refined Hydrocarbon Products To Monitor Their Fate in the Environment
R830633C006 Evaluation of Commercial, Microbial-Based Products to Treat Paraffin Deposition in Tank Bottoms and Oil Production Equipment
R830633C007 Identifying the Signature of the Natural Attenuation in the Microbial Ecology of Hydrocarbon Contaminated Groundwater Using Molecular Methods and “Bug Traps”
R830633C008 Using Plants to Remediate Petroleum-Contaminated Soil: Project Continuation
R830633C009 Use of Earthworms to Accelerate the Restoration of Oil and Brine Impacted Sites