Grantee Research Project Results

Final Report: Biodegradation of Petroleum Hydrocarbons in Salt-Impacted Soil by Native Halophiles or Halotolerants and Strategies for Enhanced Degradation

EPA Grant Number: R827015C019
Subproject: this is subproject number 019 , 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: Center for the Study of Metals in the Environment
Center Director: Allen, Herbert E.
Title: Biodegradation of Petroleum Hydrocarbons in Salt-Impacted Soil by Native Halophiles or Halotolerants and Strategies for Enhanced Degradation
Investigators: Fathepure, Babu Z.
Institution: Oklahoma State University
EPA Project Officer: Aja, Hayley
Project Period: January 1, 2002 through June 30, 2002 (Extended to November 30, 2002)
RFA: Integrated Petroleum Environmental Consortium (IPEC) (1999) RFA Text |  Recipients Lists
Research Category: Hazardous Waste/Remediation , Targeted Research

Objective:

There are many exploration and production (EP) sites in Oklahoma and throughout the world that are contaminated with both oil and salt. This poses a problem for cleaning up these sites using bioremediation technologies since the harsh conditions caused by high salinity will not support externally added bacteria. In addition, information on the degradation of petroleum compounds in oil-brine soil is scant. Therefore, the main purpose of this project was to demonstrate the potential of indigenous halophilic or halotolerant bacteria to degrade petroleum hydrocarbons and to try to optimize conditions for enhanced rates.

Summary/Accomplishments (Outputs/Outcomes):

Oil-Brine Soil: This project was initiated with soil samples obtained from five contaminated sites in Oklahoma including Stephens and Seminole counties. They are labeled Stephens, Sem-1, Sem-2, Sem-3, and Sem-4. Table 1 shows analytical results for benzene, toluene, ethylbenzene, total petroleum hydrocarbon (TPH), and chloride for each oil-brine soil used in this study.

Table 1. Soil Analysis

 

Soil Analysis*	Method (EPA)	Stephens	Sem-1	Sem-2	Sem-3	Sem-4
Sample depth (ft)		0-1.5	4	0-3	4	4
Benzene (mg/Kg)	8021B	BDL	4.48	3.41	1.72	0.149
Toluene (mg/Kg)	8021B	0.241	9.72	10.4	7.48	0.715
Ethylbenzene (mg/Kg)	8021B	BDL	30.2	7.95	14.1	3.04
TPH (mg/Kg)	8015M	125580	72744	6071	25198	120276
Chloride (mg/Kg)	325.3	1270	42200	14800	3740	220

* Soil analyses were performed by the Beacon Environmental Assistance, Corp., Edmond, OK.

Since, Sem-2 has considerably less TPH, it was used to assess aerobic benzene degradation, while the other four soils were used to assess biodegradation under anaerobic conditions.

Microcosm Preparation: Microcosms were prepared using 70-ml capacity serum bottles containing 10g (wet weight) soil and 40-ml mineral salts medium (MSM) containing high concentrations of NaCl (2.5 M). The bottles for the aerobic studies were supplemented with 100 mg of magnesium peroxide (MgO2) as a source of oxygen. Anaerobic microcosms were prepared similarly in an anaerobic glove box filled with N2. Microcosms were also prepared as described above to evaluate the effect of osmolytes or NaCl concentrations on the rate of benzene biodegradation. The bottles were closed with Teflon-coated septa and aluminum caps and incubated static in the dark.

Mineralization of ¹⁴C-Benzene in Brine Soil: In order to determine conclusively that benzene is mineralized to CO2, microcosms were set up for each of the anaerobic soils obtained from the Seminole and Stephens counties, OK. Also, microcosms were setup using an enrichment culture from the Seminole county to test if the culture mineralized the added benzene to CO2. Each bottle was spiked with 100µL stock ¹⁴C-benzene (specific activity of ¹⁴C-benzene = 33.2 mCi/mmole). This amounts to 27500 dpm/ml. At the end of 4, 8, 12 weeks, triplicate active and duplicate control bottles were sacrificed and stored at -20°C until analyzed for radioactive CO2.

At the end of three months of incubation, roughly 5 to 10% of the radiolabeled benzene was mineralized over the controls under anaerobic conditions in samples from the Seminole county (Table 2). However, no degradation was seen under same growth conditions in microcosms prepared with soil from the Stephens County. Although, the removal of benzene under anaerobic conditions seem poor, the rate and extent of degradation could be enhanced under more optimal growth conditions. Results show that significant amount of benzene was mineralized under aerobic conditions by the Sem-2 enrichment culture.

Table 2. Mineralization of Benzene by Halophiles Under Aerobic and Anaerobic Conditions

Site	Soil Type	Time incubated	Percent1 14C-benzene Mineralized to 14C-CO2
Sem-1	Anaerobic	>12 weeks	5.93 ± 0.94
Sem-2	Aerobic	4 weeks	46.83 ± 14.09
Sem-3	Anaerobic	12 weeks	10.16 ± 5.87
Sem-4	Anaerobic	12 weeks	4.6 ± 0.12
Steph	Anaerobic	12 weeks	<1%

¹Percentage of ¹⁴C-CO2 recoveries are percents above the control

Sem-2 Enrichment Culture: Using the Sem-2 soil, we have obtained a highly enriched aerobic culture. The enrichment rapidly degraded benzene as the sole carbon and energy source in mineral salts medium containing 2.5 M NaCl. For quantification of benzene, 100-µl of headspace gases from bottles was analyzed by gas chromatography. After more than 10 months of continuous enrichment, the Sem-2 culture consistently degrades benzene completely within 2.5 weeks at room temperature.

 

Figure 1. Biodegradation of Benzene by the Sem-2 Enrichment

The Sem-2 enrichment degraded benzene after repeated spikes (results from only one of the two enrichment bottles are shown; results from the other bottle were similar).

Effect of Osmolytes: Microcosms were also established with the Sem-2 enrichment to evaluate the effects of different osmolytes on the rate of benzene degradation. Our data revealed that none of the tested osmolytes showed enhanced degradation activity. On the contrary, the rates are lower than when no osmolyte was present (Table 3). The exact reason for the lack of stimulation in the presence of tested osmolytes is not known. However, it is possible that perhaps bacteria were able to synthesize their own osmolytes. Alternatively, the tested osmolytes were not preferred by halophiles present in the Sem-2.

Table 3. Effect of Osmolytes on Benzene Biodegradation.

	Additive
	Concentration %	Degradation	# of days
Autoclaved Control	NA*	43.50 ± 12.95	61
No osmolyte	NA	97.77 ± 0.87	12
Glycine	1M	62.69 ± 0.22	61
Proline	1M	42.30 ± 4.20	61
Betain	1M	67.25 ± 2.38	61
KCl	1M	56.41 ± 2.11	61
Aspartic Acid	0.1M	51.41 ± 3.37	61
Glutamic Acid	0.1M	61.32 ±0.46	61

*No osmolytes were added to the controls

We also evaluated the ability of the Sem-2 culture to degrade other petroleum compounds including toluene, ethlybenzene, and o-, m-, and p- xylene by the benzene enrichment culture. As seen in Figure 2, the degradation rates of the latter two compounds were comparable to that of benzene, while toluene degraded markedly faster.

 

Figure 2. Biodegradation of (a) Ethylbenzene, (b) o-m-p-Xylenes, and (c) Toluene by the Sem-2 Enrichment.

 

(a)

 

 

(b)

 

 

(c)

 

Biodegradation of Benzene at Varied Salt Concentrations: Further tests with the highly enriched culture included determining the degradation of benzene in the presence of varied NaCl concentrations ranging from 0 to 4 M. As seen in Figure 3, benzene degradation occurred in the presence of NaCl ranging from 0.5 to 2.5 M salt and addition of higher concentrations (>3M) inhibited the degradation. Also, no degradation of benzene occurred in the absence of added salt suggesting that the culture is a true halophile and required salt for growth.

 

Figure 3. Effect of Salt on Benzene Degradation by the Sem-2 Enrichment.

Community Analysis of Sem-2 Enrichment: The purpose of this work was to identify differences in major microbial populations present in the Sem-2 enrichment when grown in the presence and absence of salt. Bacterial DNA from Sem-2 culture grown on 0 M and 2.5 M NaCl was extracted and amplified by polymerase chain reaction (PCR).

 

The differing banding patterns between the 0 M and 2.5 M NaCl samples indicate different microbial communities at the two salt concentrations.

The PCR products were then separated using the denaturing gradient gel electrophoresis (DGGE), which separates the differing DNAs by their nucleotide sequence. The DGGE profile of the enrichment’s 16S rDNA showed the presence of four major bands when cultured in the presence of 2.5 M NaCl and 30 mg/l benzene as the sole carbon source. Evidently these bands are missing when the culture was grown on benzene but no salt. Figure 4 shows bands labeled A, B, C, and D. Each of these bands was cut and sequenced. The sequences were BLASTed in GenBank and the results show > 99% sequence homology with the genus Marinobacter. The bands labeled F did not yield enough information for sequence identity.

Conclusions:

Benzene can be completely mineralized under both aerobic and anaerobic halophilic conditions. We have obtained a highly enriched microbial consortium from a brine-contaminated soil that has the ability to completely degrade benzene, as well as conclusively showed that benzene is mineralized to CO2. This work also looked at the potential of various osmolytes to stimulate benzene degradation by the enrichment culture. Our results showed that none of the tested osmolytes enhanced the rate of benzene degradation. In addition, we tested the culture’s ability to degrade benzene under varying concentrations of NaCl. Degradation occurred in all bottles with NaCl ranging from 0.5 to 2.5 M and no degradation occurred at higher concentrations (> 3M). The culture did not degrade benzene in the absence of added salt indicating that the enrichment is a true halophile and requires salt for growth. Community analysis using DGGE suggested a Marinobacter species was the dominant organism in the enrichment. Our future goals are to isolate pure cultures of halophiles and assess their ability to degrade petroleum compounds under high salinity for the development of bioaugmentation technologies. This would provide a cost-effective approach for cleaning up oil at EP sites that have been contaminated with high concentrations of salt.

Supplemental Keywords:

bioremediation, halophilic, halotolerant, BTEX, enrichment culture, osmolytes, denaturing gradient gel electrophoresis., RFA, Scientific Discipline, Toxics, INTERNATIONAL COOPERATION, Waste, Water, TREATMENT/CONTROL, POLLUTANTS/TOXICS, National Recommended Water Quality, Contaminated Sediments, Treatment Technologies, Remediation, Chemistry, Chemicals, Contaminant Candidate List, Environmental Microbiology, Microbiology, Hazardous Waste, Bioremediation, Biology, Environmental Engineering, Hazardous, degradation, waste treatment, hazardous waste treatment, contaminated sites, microbial degradation, decontamination of soil and water, napthalene, biodegradation, decontamination of soil, field studies, Naphthalene, anaerobic biodegradation, microbes, microflora, PAH, contaminated soil, soils, benzene, contaminants in soil, bioremediation of soils, soil, groundwater remediation, in-situ bioremediation, hydrocarbons, contaminated groundwater, water quality, soil microbes

Main Center Abstract and Reports:

R827015    Center for the Study of Metals in the Environment

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