Article pubs.acs.org/est
Microbial Metabolism and Community Structure in Response to Bioelectrochemically Enhanced Remediation of Petroleum Hydrocarbon-Contaminated Soil Lu Lu,† Tyler Huggins,† Song Jin,‡ Yi Zuo,§ and Zhiyong Jason Ren*,† †
Department of Civil, Environmental, and Architectural Engineering, University of Colorado Boulder, Boulder, Colorado 80309, United States ‡ Department of Civil and Architectural Engineering, University of Wyoming, Laramie, Wyoming 82071, United States § Chevron Energy Technology Company, San Ramon, California 94583, United States S Supporting Information *
ABSTRACT: This study demonstrates that electrodes in a bioelectrochemical system (BES) can potentially serve as a nonexhaustible electron acceptor for in situ bioremediation of hydrocarbon contaminated soil. The deployment of BES not only eliminates aeration or supplement of electron acceptors as in contemporary bioremediation but also significantly shortens the remediation period and produces sustainable electricity. More interestingly, the study reveals that microbial metabolism and community structure distinctively respond to the bioelectrochemically enhanced remediation. Tubular BESs with carbon cloth anode (CCA) or biochar anode (BCA) were inserted into raw water saturated soils containing petroleum hydrocarbons for enhancing in situ remediation. Results show that total petroleum hydrocarbon (TPH) removal rate almost doubled in soils close to the anode (63.5−78.7%) than that in the open circuit positive controls (37.6−43.4%) during a period of 64 days. The maximum current density from the BESs ranged from 73 to 86 mA/m2. Comprehensive microbial and chemical characterizations and statistical analyses show that the residual TPH has a strongly positive correlation with hydrocarbon-degrading microorganisms (HDM) numbers, dehydrogenase activity, and lipase activity and a negative correlation with soil pH, conductivity, and catalase activity. Distinctive microbial communities were identified at the anode, in soil with electrodes, and soil without electrodes. Uncommon electrochemically active bacteria capable of hydrocarbon degradation such as Comamonas testosteroni, Pseudomonas putida, and Ochrobactrum anthropi were selectively enriched on the anode, while hydrocarbon oxidizing bacteria were dominant in soil samples. Results from genus or phylum level characterizations well agree with the data from cluster analysis. Data from this study suggests that a unique constitution of microbial communities may play a key role in BES enhancement of petroleum hydrocarbons biodegradation in soils.
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INTRODUCTION Petroleum hydrocarbon contamination in soil and groundwater is a widespread environmental problem, especially in areas with high industrial activities.1 Remediation of hydrocarbon contaminants using in situ bioremediation, such as biosparging, biostimulation, and bioaugmentation, is usually considered cost-effective and environmentally friendly compared to physical and chemical remedies.2,3 However, the performance of bioremediation is primarily governed by the interplay between the abundance of electron acceptor (e.g., nitrate, sulfate, Fe(III), or oxygen)4,5 and hydrocarbon contaminant as the electron donor and carbon source for microorganisms. A common challenge for bioremediation is the limitation of electron acceptor availability, especially in the contaminated subsurface where natural electron acceptors are usually depleted. The delivery of oxygen or alternative electron © 2014 American Chemical Society
acceptors such as nitrate could be limited by matrix permeability and require relatively high energy and material input. Alternative electron acceptors may also cause secondary contaminations depending on specific site conditions.6 It was recently discovered that electrodes can be used as the electron acceptor by electrochemically active bacteria, also known as exoelectrogens, electricigens, or anode-respiring bacteria5,7,8 during their anaerobic oxidation of substrates. In recently developed bioelectrochemical systems (BESs),8 such as “microbial fuel cells” (MFCs), the electrons collected on the anode are transferred to the air-cathode for oxygen reduction, Received: Revised: Accepted: Published: 4021
December 29, 2013 February 26, 2014 March 5, 2014 March 14, 2014 dx.doi.org/10.1021/es4057906 | Environ. Sci. Technol. 2014, 48, 4021−4029
Environmental Science & Technology
Article
4 °C before use. The main characteristics of the soil samples are summarized in Supporting Information (SI) Table S1. The soil was saturated (immersed) with deionized water during regular experiments without adding buffer solution or additional hydrocarbons. Soil Tubular BES Construction and Operation. Tubular BESs were constructed by wrapping an assembly of anode, separator, and cathode layers around a perforated PVC tube (L 20 cm × D 3.5 cm), with the cathode layer facing inside and exposed to air, and the anode facing outside exposed to soil (SI Figure S1). Activated carbon cloth (Chemviron Carbon Ltd., PA) with a Pt/C catalyst layer (0.1 mg Pt/cm2) and four PTFE diffusion layers19 on carbon support was served as the air cathode, and the effective cathode projected area was 110 cm2. The anode was either made of carbon cloth (11 cm × 12 cm, Fuel Cell Earth LLC, MA) or biochar, which was manufactured in our lab as described in Huggines et al.18 and the SI. Four layers of nonconductive permeable glass fiber were sandwiched between the electrodes as the separator to prevent short circuit. The carbon cloth anode (CCA) was wrapped together with separators and the cathode, while the biochar anode (BCA) was constructed by packing biochar particles in between the separator layer and an outermost layer of nylon mesh, with an average thickness of 1 cm (Figure S1B). A graphite rod (L 6 cm × D 0.5 cm) was inserted into the biochar pack (stacking volume of 168 cm3) as a current collector. The anode and cathode were connected using a 100 Ω external resistor. The tubular BESs were inserted into 15 cm (H) × 16 cm (D) buckets containing saturated hydrocarbon-contaminated soil. The top of each bucket was covered to prevent evaporation of water and volatile hydrocarbons, and water loss was made up by deionized water to maintain the saturation condition. In one carbon cloth reactor, a nonionic surfactant Triton X-100 (500 mg/L) was mixed with soil (CCA-S) to examine if the addition of surfactant would improve desorption and transfer of hydrocarbons from soil toward the anode reaction zone.20 Table S2 (SI) summarizes the abbreviations of the different reactor setups and sample conditions. Soil samples were routinely extracted from the top, middle, and bottom of the soil layer in each sampling event in triplicates, and the composite soil samples in vertical orientation were used for chemical and biological analyses. Noncontaminated soil (NCS) obtained from pristine background and original hydrocarbon contaminated soil (OHCS) were also be sampled from the site for analyses. Anode biofilms and soil samples collected from CCA, BCA, and CCA-S reactors were used for bacterial community analysis. Indigenous microbes remained in all systems without any external inoculation. All reactors were operated at room temperature of 20−22 °C without light irradiation. Chemical, Biological, and Electrochemical Analyses. All soil samples were characterized for total petroleum hydrocarbon (TPH) by following the EPA Method 8015D21 and MA EPH.22 Briefly, 5 g of the soil sample was blended with anhydrous Na2SO4 until the soil flows freely, then the mixture was mixed with 10 mL of dichloromethane followed by extraction using vortex and sonication, each 5 min. The suspension was centrifuged at 8,000 × g for 5 min, and the supernatant was decanted. This procedure was repeated, and the combined extractants were concentrated to 1 mL and purified by using silica gel. TPH was measured using a Thermo TRACE gas chromatograph equipped with a Restek Rxi-1 ms column (20 m × 0.18 mm ID, 0.18 μm film thickness) and flame ionization detector. Aliphatic hydrocarbon calibration
and at the same time electric current can be produced.7 Such an approach is hypothesized to stimulate and enhance hydrocarbon degradation accompanied by energy production, because the electrode pair serves as a nonexhaustible electron acceptor, which eliminates the necessity of aeration in the subsurface but sustains an aerobic-like metabolic pathway on the cathode. Previous studies showed the feasibility of using an electrode approach for the removal of different hydrocarbons, such as crude oil,9 diesel,4 refinery wastewater,10 petroleum sludge,11 and aromatic compounds5,12 through direct exoelectrogenic oxidation5 or syntrophic interactions4 among microbial consortia. However, most studies were conducted using synthetic hydrocarbon substrates in sediment or groundwater, which are very different from actual soil contamination conditions due to the great variety in soil composition, permeability, and water content.13−17 For example, our early studies show that a lab scale two-chamber MFC reactor increased diesel carbon degradation by 165% (from 31% to 82%) than the open circuit control within 21 days of operation.4 Another study demonstrated that the influence radius of the anode of a U-tube MFC and soil moisture significantly affected the degradation rate, and the total petroleum hydrocarbon (TPH) removal was enhanced by 120% in saturated condition (33% water content) and close to the anode (
Microbial Metabolism and Community Structure in Response to Bioelectrochemically Enhanced Remediation of Petroleum Hydrocarbon-Contaminated Soil Lu Lu,† Tyler Huggins,† Song Jin,‡ Yi Zuo,§ and Zhiyong Jason Ren*,† †
Department of Civil, Environmental, and Architectural Engineering, University of Colorado Boulder, Boulder, Colorado 80309, United States ‡ Department of Civil and Architectural Engineering, University of Wyoming, Laramie, Wyoming 82071, United States § Chevron Energy Technology Company, San Ramon, California 94583, United States S Supporting Information *
ABSTRACT: This study demonstrates that electrodes in a bioelectrochemical system (BES) can potentially serve as a nonexhaustible electron acceptor for in situ bioremediation of hydrocarbon contaminated soil. The deployment of BES not only eliminates aeration or supplement of electron acceptors as in contemporary bioremediation but also significantly shortens the remediation period and produces sustainable electricity. More interestingly, the study reveals that microbial metabolism and community structure distinctively respond to the bioelectrochemically enhanced remediation. Tubular BESs with carbon cloth anode (CCA) or biochar anode (BCA) were inserted into raw water saturated soils containing petroleum hydrocarbons for enhancing in situ remediation. Results show that total petroleum hydrocarbon (TPH) removal rate almost doubled in soils close to the anode (63.5−78.7%) than that in the open circuit positive controls (37.6−43.4%) during a period of 64 days. The maximum current density from the BESs ranged from 73 to 86 mA/m2. Comprehensive microbial and chemical characterizations and statistical analyses show that the residual TPH has a strongly positive correlation with hydrocarbon-degrading microorganisms (HDM) numbers, dehydrogenase activity, and lipase activity and a negative correlation with soil pH, conductivity, and catalase activity. Distinctive microbial communities were identified at the anode, in soil with electrodes, and soil without electrodes. Uncommon electrochemically active bacteria capable of hydrocarbon degradation such as Comamonas testosteroni, Pseudomonas putida, and Ochrobactrum anthropi were selectively enriched on the anode, while hydrocarbon oxidizing bacteria were dominant in soil samples. Results from genus or phylum level characterizations well agree with the data from cluster analysis. Data from this study suggests that a unique constitution of microbial communities may play a key role in BES enhancement of petroleum hydrocarbons biodegradation in soils.
■
INTRODUCTION Petroleum hydrocarbon contamination in soil and groundwater is a widespread environmental problem, especially in areas with high industrial activities.1 Remediation of hydrocarbon contaminants using in situ bioremediation, such as biosparging, biostimulation, and bioaugmentation, is usually considered cost-effective and environmentally friendly compared to physical and chemical remedies.2,3 However, the performance of bioremediation is primarily governed by the interplay between the abundance of electron acceptor (e.g., nitrate, sulfate, Fe(III), or oxygen)4,5 and hydrocarbon contaminant as the electron donor and carbon source for microorganisms. A common challenge for bioremediation is the limitation of electron acceptor availability, especially in the contaminated subsurface where natural electron acceptors are usually depleted. The delivery of oxygen or alternative electron © 2014 American Chemical Society
acceptors such as nitrate could be limited by matrix permeability and require relatively high energy and material input. Alternative electron acceptors may also cause secondary contaminations depending on specific site conditions.6 It was recently discovered that electrodes can be used as the electron acceptor by electrochemically active bacteria, also known as exoelectrogens, electricigens, or anode-respiring bacteria5,7,8 during their anaerobic oxidation of substrates. In recently developed bioelectrochemical systems (BESs),8 such as “microbial fuel cells” (MFCs), the electrons collected on the anode are transferred to the air-cathode for oxygen reduction, Received: Revised: Accepted: Published: 4021
December 29, 2013 February 26, 2014 March 5, 2014 March 14, 2014 dx.doi.org/10.1021/es4057906 | Environ. Sci. Technol. 2014, 48, 4021−4029
Environmental Science & Technology
Article
4 °C before use. The main characteristics of the soil samples are summarized in Supporting Information (SI) Table S1. The soil was saturated (immersed) with deionized water during regular experiments without adding buffer solution or additional hydrocarbons. Soil Tubular BES Construction and Operation. Tubular BESs were constructed by wrapping an assembly of anode, separator, and cathode layers around a perforated PVC tube (L 20 cm × D 3.5 cm), with the cathode layer facing inside and exposed to air, and the anode facing outside exposed to soil (SI Figure S1). Activated carbon cloth (Chemviron Carbon Ltd., PA) with a Pt/C catalyst layer (0.1 mg Pt/cm2) and four PTFE diffusion layers19 on carbon support was served as the air cathode, and the effective cathode projected area was 110 cm2. The anode was either made of carbon cloth (11 cm × 12 cm, Fuel Cell Earth LLC, MA) or biochar, which was manufactured in our lab as described in Huggines et al.18 and the SI. Four layers of nonconductive permeable glass fiber were sandwiched between the electrodes as the separator to prevent short circuit. The carbon cloth anode (CCA) was wrapped together with separators and the cathode, while the biochar anode (BCA) was constructed by packing biochar particles in between the separator layer and an outermost layer of nylon mesh, with an average thickness of 1 cm (Figure S1B). A graphite rod (L 6 cm × D 0.5 cm) was inserted into the biochar pack (stacking volume of 168 cm3) as a current collector. The anode and cathode were connected using a 100 Ω external resistor. The tubular BESs were inserted into 15 cm (H) × 16 cm (D) buckets containing saturated hydrocarbon-contaminated soil. The top of each bucket was covered to prevent evaporation of water and volatile hydrocarbons, and water loss was made up by deionized water to maintain the saturation condition. In one carbon cloth reactor, a nonionic surfactant Triton X-100 (500 mg/L) was mixed with soil (CCA-S) to examine if the addition of surfactant would improve desorption and transfer of hydrocarbons from soil toward the anode reaction zone.20 Table S2 (SI) summarizes the abbreviations of the different reactor setups and sample conditions. Soil samples were routinely extracted from the top, middle, and bottom of the soil layer in each sampling event in triplicates, and the composite soil samples in vertical orientation were used for chemical and biological analyses. Noncontaminated soil (NCS) obtained from pristine background and original hydrocarbon contaminated soil (OHCS) were also be sampled from the site for analyses. Anode biofilms and soil samples collected from CCA, BCA, and CCA-S reactors were used for bacterial community analysis. Indigenous microbes remained in all systems without any external inoculation. All reactors were operated at room temperature of 20−22 °C without light irradiation. Chemical, Biological, and Electrochemical Analyses. All soil samples were characterized for total petroleum hydrocarbon (TPH) by following the EPA Method 8015D21 and MA EPH.22 Briefly, 5 g of the soil sample was blended with anhydrous Na2SO4 until the soil flows freely, then the mixture was mixed with 10 mL of dichloromethane followed by extraction using vortex and sonication, each 5 min. The suspension was centrifuged at 8,000 × g for 5 min, and the supernatant was decanted. This procedure was repeated, and the combined extractants were concentrated to 1 mL and purified by using silica gel. TPH was measured using a Thermo TRACE gas chromatograph equipped with a Restek Rxi-1 ms column (20 m × 0.18 mm ID, 0.18 μm film thickness) and flame ionization detector. Aliphatic hydrocarbon calibration
and at the same time electric current can be produced.7 Such an approach is hypothesized to stimulate and enhance hydrocarbon degradation accompanied by energy production, because the electrode pair serves as a nonexhaustible electron acceptor, which eliminates the necessity of aeration in the subsurface but sustains an aerobic-like metabolic pathway on the cathode. Previous studies showed the feasibility of using an electrode approach for the removal of different hydrocarbons, such as crude oil,9 diesel,4 refinery wastewater,10 petroleum sludge,11 and aromatic compounds5,12 through direct exoelectrogenic oxidation5 or syntrophic interactions4 among microbial consortia. However, most studies were conducted using synthetic hydrocarbon substrates in sediment or groundwater, which are very different from actual soil contamination conditions due to the great variety in soil composition, permeability, and water content.13−17 For example, our early studies show that a lab scale two-chamber MFC reactor increased diesel carbon degradation by 165% (from 31% to 82%) than the open circuit control within 21 days of operation.4 Another study demonstrated that the influence radius of the anode of a U-tube MFC and soil moisture significantly affected the degradation rate, and the total petroleum hydrocarbon (TPH) removal was enhanced by 120% in saturated condition (33% water content) and close to the anode (
