Microbial Activity of Soil Following Steam Enhanced Soil Vapor Extraction of Hydrocarbons

Transcription

1 Microbial Activity of Soil Following Steam Enhanced Soil Vapor Extraction of Hydrocarbons A Master s Thesis Presented to the Faculty of California Polytechnic State University San Luis Obispo In partial fulfillment of the requirements for the degree of Master of Science in Civil and Environmental Engineering By Barbara C. Orchard May 24, 2005

2 AUTHORIZATION FOR REPRODUCTION OF MASTER S THESIS I hereby grant permission for the reproduction of this thesis in its entirety or any of its parts, without further authorization, provided acknowledgement is made to the author(s) and advisor(s). Barbara C. Orchard Date ii

3 MASTER S THESIS APPROVAL TITLE: Microbial Activity of Soil Following Steam Enhanced Soil Vapor Extraction of Hydrocarbons AUTHOR: Barbara C. Orchard DATE SUBMITTED: May 24, 2005 THESIS COMMITTEE MEMBERS: Dr. Yarrow Nelson Date Dr. Christopher Kitts Date Dr. Nirupam Pal Date iii

4 ABSTRACT MICROBIAL ACTIVITY OF SOIL FOLLOWING STEAM ENHANCED SOIL VAPOR EXTRACTION OF HYDROCARBONS Barbara Orchard The effect of steam extraction of light non-aqueous phase liquid (LNAPL) hydrocarbons on subsurface aerobic and anaerobic microbial communities was investigated using multiple microbial assays. Soil samples were gathered and analyzed prior to, one month after, and eight months after a five-month field pilot test of steam injection and extraction at the former Guadalupe Oil Field site. Aerobic soil samples were analyzed by respirometry, plate counts, and direct microscopic counts. Anaerobic microbial activity was examined by monitoring methane generation in anaerobic microcosms using gas chromatography. Respirometry showed significant pre-steam CO 2 production, poststeam (one month) CO 2 production below detection, and post-steam (eight months) CO 2 production comparable to the blank, which contained no sample. However, the eight month respirometry results are questionable, since they show CO 2 production for the blank. Post-steam (one and eight month) plate counts were one to four orders of magnitude lower than the pre-steam samples. Direct microscopic counts showed poststeam (one and eight month) cell numbers higher than the pre-steam counts, but based on plate counts these cells were mostly non-viable. Significant amounts of methane and hydrogen were generated from pre-steam anaerobic microcosms, but post-steam microcosms (one and eight months) had no detectable methane, and only trace amounts of hydrogen. Terminal restriction fragment (TRF) analysis was performed to determine the diversity of the microbial community before and after steam treatment. Pre-steam TRF analysis of soil showed distinct differences in the microbial communities above and below the smear zone. Post-steam soil TRF analyses were not possible because insufficient DNA could be extracted from the soil. However, sufficient DNA was extracted from post-steam (one and eight month) groundwater. When compared to presteam groundwater TRF patterns, the post-steam (one and eight month) TRF patterns showed a decrease in the diversity of the microbial community. iv

5 ACKNOWLEDGEMENTS Special thanks to: Dr. Yarrow Nelson for his guidance, encouragement, and insight. Dr. Chris Kitts and Dr. Nirupam Pal for their support. Alice Hamrick and all the biology students at the Environmental Biotechnology Institute. Lynne Maloney for perfecting the analytical methods with pre-steam assays and for her assistance during post-steam experiments. Greg Ouellette, Inland Empire Analytical Bob Pease, LFR Funding for this project was provided by Unocal. Thanks to everyone at Unocal, especially Dr. Paul Lundegard, Marlea Harmon, and Gonzalo Garcia. Family and friends. v

6 Table of Contents List of Tables... ix List of Figures... x Chapter 1 Introduction Project Scope... 3 Chapter 2 Background Field Site Site Remediation Studies Steam Pilot Test Soil Vapor Extraction (SVE) Steam Enhanced Soil Vapor Extraction (SESVE) Effect of Steam Treatment on Microbial Activity of Soil Natural Attenuation Aerobic Natural Attenuation Anaerobic Natural Attenuation Review of Monitoring and Microbial Analysis Methods Terminal Restriction Fragment (TRF) Pattern Analysis Enhanced Bioremediation Chapter 3 Materials and Methods Soil Samples vi

7 Aerobic Soil Samples Anaerobic Soil Samples Respirometry Plate Counts Direct Microscopic Counts Anaerobic Microcosms Total Petroleum Hydrocarbon (TPH) Analysis Terminal Restriction Fragment (TRF) Analysis Chapter 4 Results Respirometry Plate Counts Direct Microscopic Counts Anaerobic Microcosms Terminal Restriction Fragment (TRF) Results Chapter 5 Discussion Aerobic Method Conclusions and Comparisons Anaerobic Microcosms Terminal Restriction Fragment (TRF) Analysis Chapter 6 Conclusions and Recommendations vii

8 6.1. Conclusions Recommendations References Appendix A Fixed Gas Analysis Protocol... A-1 Appendix B Cal Poly Environmental Biotechnology Institute TRF Protocol... B-1 viii

9 List of Tables Table 2-1: Average Properties of Guadalupe Diluent*(Lundegard and Garcia 2001)... 7 Table 3-1 TPH results for aerobic soil samples (Core 8) Table 3-2: TPH results for aerobic soil samples (Core 6) Table 3-3: TPH results for anaerobic soil samples (Core 4) Table 3-4: TPH results for anaerobic soil samples (Core 6) Table 4-1: Average Respiration Rates for Core 6 before and after steam treatment Table 4-2: Average Respiration Rates for Core 8 before and after steam treatment Table 4-3: Comparison of Pre-steam and Post-steam Plate Counts Table 4-4: Comparison of all Core 6 plate counts Table 4-5: Direct count results for Core Table 4-6: Direct count results for Core Table 4-7: Pre-steam anaerobic microcosm GC results (Maloney 2003) Table 4-8: Post-steam (one month) anaerobic microcosm GC results Table 4-9: Post-steam (eight months) anaerobic microcosm GC results Table 4-10: Comparison of peaks in groundwater TRF patterns before and after steam.57 ix

10 List of Figures Figure 2-1: Location of the former Guadalupe Oil Field... 6 Figure 2-2: Guadalupe Site Map Figure 2-3: Steam Pilot Test Site Figure 2-4: Anaerobic biodegradation pathway (Brock et al. 1994) Figure 3-1: Micro-Oxymax respirometer Figure 3-2: Nitrogen-purged anaerobic glove box Figure 3-3: Anaerobic microcosms Core 4 (51, 54.4, 63 ft) Figure 4-1: Pre-steam cumulative carbon dioxide production (Maloney 2003) Figure 4-2: Post-steam (one month) cumulative carbon dioxide production Figure 4-3: Post-steam (eight months) cumulative carbon dioxide production Figure 4-4: Core 8 plates in order of decreasing dilution from left to right (62 ft) Figure 4-5: Core 6 (post-steam one month) plate count results Figure 4-6: Core 8 (post-steam one month) plate count results Figure 4-7: Post-steam (eight months) plate counts (Core 6) Figure 4-8: Comparison of pre-steam and post-steam plate counts (Core 6) Figure 4-9: Comparison of pre-steam and post-steam plate counts (Core 8) Figure 4-10: Comparison of pre and post steam direct counts (Core 6) Figure 4-11: Comparison of pre and post steam direct counts (Core 8) Figure 4-12: Comparison of groundwater TRF patterns before and after steam x

11 CHAPTER 1 INTRODUCTION Mineral oil hydrocarbons are common environmental contaminants and include everything from gasoline to tars (Boopathy, 2004). Petroleum has contaminated soils and the subsurface extensively around the world (Kaufmann et al. 2004) leading to extensive research on methods of remediation. Microorganisms capable of degrading hydrocarbons are frequently found in environments impacted by hydrocarbons (Margesin et al. 2000). Biological remediation of hydrocarbon contamination by these in-situ microorganisms leaves the environment intact, and, thus it is usually preferable to physical and chemical methods (Margesin et al. 2000). However, in-situ bioremediation can be slow, so physical/chemical methods to remove non-aqueous phase liquid (NAPL) contaminants more rapidly have been investigated. One new method for physicalchemical removal of NAPLs is steam-enhanced soil vapor extraction (SESVE). SESVE facilitates non-aqueous phase liquids (NAPLs) extraction by the injection of steam into the subsurface, and recovery of vapor and liquid through extraction wells. When applied appropriately, SESVE may significantly reduce the time required for the remediation of sites contaminated with volatile organic compounds (VOCs) and semi-volatile organic compounds (SVOCs) (Bouchard et al. 2003). However, one potential drawback of SESVE is that the elevated subsurface temperatures may inhibit microbial activity following treatment, and thus hinder bioremediation following SESVE (Davis 1997; Krauter et al. 1996). This research evaluates the impact of an in situ steam injection and extraction pilot test for the removal of petroleum hydrocarbons on the microbial community and microbial activity of soil at a former oil field. 1

12 Even after successful SESVE, a site will probably have residual contamination, especially where SVOCs are the target. In these cases, natural attenuation is important as a polishing step. However, SESVE may be detrimental to the indigenous microbial communities due to the high temperatures employed. The effect of SESVE on the microbial activity of soil has been studied with bench-top experiments such as those of Dablow et al. (1995), Huesmann et al. 2002, Friis et al and Richardson et al. (2002). The number of active microorganisms in soil prior to cooling following benchtop steaming was below the detection limit (Richardson et al. 2002). Another laboratory study showed significant reductions in microbial populations in soil following steam treatment (Dablow et al. 1995). An enhanced bioremediation experiment was then conducted on the treated and non-treated soil to determine if bacteria could rebound under the right conditions. The treated soil required nutrient amendments to stimulate growth (Dablow et al. 1995). There have been a few field experiments to determine microbial activity directly following treatment, but in these experiments the subsurface remained at elevated temperatures (Krauter et al. 1996). The soil (still at elevated temperatures) experienced significant decreases in total microbial populations of up to 98%. The microbial community also shifted from gram negative to gram positive organisms, since some gram positive cells form spores, which are capable of surviving high temperatures. Two years after steam treatment commenced, the groundwater temperature was still elevated to temperatures ranging from C (Krauter et al. 1996). Thus, although microbial populations may rebound from SESVE after the subsurface cools, during this extended 2

13 cooling period the microbial population and activity are still decimated. Additional studies are needed to examine the recovery of microbial populations after SESVE. Further, anaerobic microbial activity was not addressed in these studies, and this may be important because anaerobes may have a different tolerance to the high temperatures Project Scope To further test the effect of SESVE on soil microbial communities, aerobic and anaerobic microbial activity were evaluated before and after a steam pilot test. The pilot test was conducted at the former Guadalupe Oil Field site to remove light non-aqueous phase liquid (LNAPL) hydrocarbons and lasted five months with subsurface temperatures reaching 115 C. Pre-steam microbial assays were conducted previously (Maloney 2003; Maloney et al. 2004). Post-steam soil samples were collected one and eight months after cessation of steam injection when the soil temperatures were up to 91 C and 58 C, respectively. Soil samples from multiple depths were analyzed to determine the aerobic and anaerobic microbial activity, population, and species diversity. This comparison was used to determine the effect of steam treatment on the microbial activity of soil. This research included several objectives: Quantify aerobic populations using plate counts, and direct microscopic counts. Quantify aerobic microbial activity by measuring carbon dioxide production. Quantify anaerobic microbial activity by monitoring methane and hydrogen generation in anaerobic microcosms. Characterize the diversity of the aerobic and anaerobic microbial populations using terminal restriction fragment (TRF) analysis 3

14 Compare the post-steam results to the pre-steam results Evaluate recovery of the microbial community during cooling 4

15 CHAPTER 2 BACKGROUND 2.1. Field Site The former Guadalupe Oil Field (GOF) is located on the Central Coast of California, approximately half-way between Los Angeles and San Francisco (Figure 2-1) (Lundegard and Garcia 2001). The GOF produced a dense and viscous crude oil (Santa Maria Valley crude) from 1953 until The thick crude required a diluent before it could be pumped. Diluent was pumped to the GOF from a nearby petroleum refinery (and several other sources) for use as a thinning agent to facilitate the flow of the heavy crude through pipelines. In 1990, an oil sheen and odor was noticed at the beach leading to investigation of the GOF. In 1994, production was ceased and all of the wells were plugged and abandoned. Unocal settled the subsequent Natural Resources Damage lawsuit for $43.8 million. During the operation of the GOF, approximately 8.5 million gallons of diluent were spilled within the 3000 acre GOF (Lundegard and Garcia 2001). The diluent leaked from pipes and tanks over the 40 years it was used. Other chemicals of concern such as metals and poly chlorinated biphenyls (PCBs) were present in minor amounts. Thus, the diluent is the focus of the ecological risk assessment and remediation activities. 5

16 Figure 2-1: Location of the former Guadalupe Oil Field The diluent used was a mid-range petroleum distillate product, straight-run gas oil. As the crude slate varied over time at the refinery, the diluent sent to Guadalupe changed as well. This along with different degrees of environmental weathering and biodegradation accounts for the high variability of the separate-phase product currently found at Guadalupe. To characterize the properties and extent of the subsurface contamination an extensive network of monitoring wells (over 800) were installed, and soil borings (over 3000) were taken. The diluent is mainly found as LNAPL in the capillary fringe zone. The properties of the diluent present at the site are summarized in Table 2-1 (Lundegard 6

17 and Garcia 2001). Polar organic compounds (containing nitrogen, sulfur, and oxygen (NSOs)) make up a significant fraction of the diluent and are the primary compounds that dissolve in the groundwater. Table 2-1: Average Properties of Guadalupe Diluent*(Lundegard and Garcia 2001) Property Average Value Mono Aromatic Content Benzene 9.5 mg/kg Toluene 15 mg/kg Ethyl benzene 45 mg/kg Xylenes 124 mg/kg Equivalent Boiling Point Distribution <n-c 11 <1% n-c 11 -n-c 14 9% n-c 14 -n-c 22 65% n-c 22 -n-c 30 20% >n-c 30 5% Compound Class Composition Saturated hydrocarbons 41% Aromatic Hydrocarbons 29% Polars + Asphaltenes (NSOs) 30% Total Poly Aromatic Hydrocarbons 12,900 mg/kg Total Naphthalenes 7,760 mg/kg Specific Gravity (60 F) Viscosity 14 cp (70 F) Reid Vapor Pressure 1 psia Apparent Solubility (in water) ~20 mg/l *samples collected as free product from monitoring wells The site has both salt and freshwater habitats; the Pacific Ocean borders the western edge of the site, and the Santa Maria River lies to the south. Ground water generally flows at approximately one-foot per day west toward the Pacific and is at a depth of zero to 150 feet. The site is primarily sparsely to un-vegetated dune sand, a rare habitat in coastal California. At least 17 different wildlife habitats have been identified. Since public 7

18 access was restricted during production and the subsequent remediation efforts, the ecosystem has been protected and remains well-preserved. Of the hundreds of species present at the site, over 40 are threatened and endangered species of plants and animals, including the western snowy plover, the California red legged frog, and the La Graciosa thistle. Thus, it is important that the remediation of the diluent contamination at the former Guadalupe Oil Field does not further damage this ecosystem Site Remediation Studies Petroleum hydrocarbons can be treated or removed from the subsurface with several techniques both in-situ and ex-situ. Many in-situ, or on-site, treatment techniques utilize bioremediation, whether enhancing natural attenuation (NA) or monitoring NA. Ex-situ techniques include excavation followed by landfilling, land treatment, or incineration. Remediation techniques are generally evaluated on their effectiveness of removing contaminants, time required for cleanup, cost and sometimes the environmental impacts of the technique. The former GOF is a complex site with over 90 identified diluent source zones (Figure 2-2). Since contamination of the marine environment was an immediate concern, excavation of soil from source zones adjacent to the Pacific was completed in Until a final remediation method is selected, contaminated groundwater is being pumped and the NAPL is removed by letting the oil and water separate. Several pilot-scale remediation techniques have been tested at the former GOF including phytoremediation, land treatment units, biosparging, and steam treatment. Natural attenuation is the 8

19 emphasis of recent and on-going studies (Cunningham 2004; Dreyer 2003; Lundegard and Johnson 2003; Maloney et al. 2004). 9

20 Steam Pilot-Test Location Figure 2-2: Guadalupe Site Map 10

21 Steam Pilot Test The Regional Water Quality Control Board required a pilot test of SESVE to test the removal of diluent from the diluent tank source zone at the former GOF (Figure 2-2). The pilot test was conducted on a 71 by 71 foot area with an approximate treatment volume of 2,220 cubic yards (Figure 2-3). The soil was composed almost entirely of wind-deposited sand that is very uniform in size. Groundwater was approximately 55 to 65 feet below ground surface, and separate-phase diluent was in the smear zone from about two feet above the water table to a depth of eight to ten feet below. The test lasted five months with subsurface temperatures reaching a maximum of 115 C. Figure 2-3: Steam Pilot Test Site 11

22 Following hot water injection, steam was injected into four injection wells (one in each corner, 50 feet from the central extraction well). At the same time, liquids and vapors were extracted from the center well and eight other extraction wells adjacent to the test site. The extracted vapors were treated with activated carbon, and the liquids were treated by oil/water separation and were then injected into deep injection wells (Personal Communication: Steam Tech Environmental Services, Inc. Work Plan, 2003). To evaluate the effect of the test on the subsurface contamination levels and composition, soil samples were taken and analyzed for several constituents. The greatest pre-steam concentrations of TPH (in the 50,000 to 150,000 mg/kg range) were located in the smear zone. After the pilot test, these concentrations decreased to peak in the 4,000 to 7,000 mg/kg range. The post-steam TPH concentrations were lowest in borings located the closest to injection wells. Total petroleum hydrocarbon (TPH) composition also changed as a result of the pilot test. TPH of the pre-steam soil samples from the saturated zone was typically in the C10 to C32 equivalent range. The shorter equivalent range hydrocarbons (C14 to C20) were generally absent from post-steam soil samples from the saturated zone. The loss of the shorter hydrocarbons was greater in soil samples collected between the pre-steam and post-steam water tables, and in post-steam soil from nearby injection wells. Benzene, toluene, ethyl benzene and xylene (BTEX) and polycyclic aromatic hydrocarbon (PAH) concentrations were initially highest near the pre-steam water table. However, the post-steam samples from these elevations contained no detectable BTEX and lower concentrations of PAHs (Personal Communication: Levine Frick Post-Test Site Characterization, 2004). 12

23 2.3. Soil Vapor Extraction (SVE) SESVE is an enhanced form of soil vapor extraction (SVE). SVE is a process in which steam injected into the subsurface, while vapor and liquid are recovered through extraction wells. A vacuum is pulled through vapor extraction wells (perforated pipes) to transfer the VOCs from the soil and water to the gas stream. The extracted gas stream must then be contained and treated. The gases may be passed through a knockout drum to remove moisture and then some form of treatment to remove the VOCs from the gas stream, such as activated carbon. The effective radius for SVE wells has been seen to vary from 6-45 meters (m) with effective depths up to 7 m; these depend on soil characteristics. The contaminant, soil, and site environmental properties affect the contaminant transport between phases and thus the effectiveness of the SVE system. Some of these important properties include: soil permeability, porosity, ph, and organic content; the contaminant s concentration, solubility, Henry s law constant, adsorption coefficient, vapor pressure, and diffusion coefficient; and the site temperature, humidity, rainfall, and vegetation (La Grega et al. 2001). Many types of enhancements can be made to SVE to increase the recovery of the contaminants including: groundwater extraction wells, placing an impermeable barrier over the surface, installing air recharge wells, and air sparging (La Grega et al. 2001). Thermal enhancements are increasingly popular. There are three general methods of thermal based treatment methods, injection of hot gases (steam or air), electromagnetic 13

24 energy heating, and hot water injection. All of these methods were developed by the petroleum industry to enhance the recovery of oil from the subsurface (Davis 1997) Steam Enhanced Soil Vapor Extraction (SESVE) Steam is injected into the subsurface, while contaminants are extracted in the vapors and liquid, often as free-product. SESVE works via two main mechanisms: distillation of subsurface contaminants and the displacement of non-aqueous phase liquids (NAPLs) (Davis 1997). Heat based techniques improve recovery by several mechanisms (Davis 1997): Thermal expansion of the liquid and a decrease in viscosity allow it to flow more readily; Vaporization of liquids to gases increase the mobility; Expansion of compounds helps move fluids out of pore space; Increased diffusion of contaminants moves contaminants from areas of low permeability to areas of higher permeability SESVE is most economical and effective on large volumes of moderately contaminated soils (Environmental Protection Agency 1995). It can be used to treat at depths up to 100 ft. A confining layer is desirable below the area of contamination, to prevent the downward migration of contaminants, particularly dense non-aqueous phase liquids (DNAPLs). A confining layer above the treatment area is also necessary when treating close to the surface. SESVE is not applicable to sites with fractured geology, shallow contamination, or soils with no or very low permeability (Environmental Protection 14

25 Agency 1995). VOCs and semi-volatile organic compounds (SVOCs) are the best candidates for SESVE. Higher molecular weight and less volatile compounds are removed at lower efficiencies (Environmental Protection Agency 1995). SESVE not only improves the recovery compared to SVE but is also able to recover contaminants with higher boiling points and contaminants in soils with clays (Davis 1997). The Research Facility for Subsurface Remediation at the University of Stuttgart, Germany conducted research examining the environmental impact of Brownfield redevelopment, specifically focusing on different remediation approaches (Schrenk 2002). As part of this study, a life cycle assessment (LCA) compared steam treatment or thermally enhanced soil vapor extraction (TSVE) to regular soil vapor extraction. Although TSVE requires substantial resources for heat production, due to its enhancement of recovery it was found to consume the same or less total resources than SVE to remove more contamination in shorter time periods. The study also compared several SVE use scenarios to TSVE over seven environmental impact categories; cumulative energy demand, total waste, fossil resources, land use, global warming, acidification, and photo-oxidant formation. TSVE had a lower impact across all categories compared to all but one of the SVE scenarios, which was slightly lower than TSVE in two categories. One of the first full-scale demonstrations of SESVE was at the Rainbow Disposal Site in Huntington Beach, CA (Environmental Protection Agency 1995). Over 70,000 gallons of diesel fuel had been released at this site. The Rainbow Disposal Site remained in 15

26 operation during the SESVE treatment. The SESVE treated an area of approximately 2.3 acres including the soil underneath structures, around underground storage tanks, and utilities. As with any new technology, there were numerous operational problems; these resulted in the planned one year treatment actually taking two years. About 16,000 gallons of diesel were removed from the subsurface; the recovered liquid phase diesel was recycled, and the gaseous phase was treated with a thermal oxidation unit. The cleanup objective of 1000 ppm TPH was not met, and the estimated overall removal efficiency was 40 %. The total cost was $4.4 million with an estimated cost of $43/yd 3. SESVE has been implemented successfully to remediate hydrocarbon contamination, reducing project cleanup times from decades or centuries using traditional remediation methods to months and years (Bouchard 2003). The Southern California Edison Pole Yard Superfund Site in Visalia, CA, a former wood treatment facility contaminated with creosote, and oil compounds containing pentachlorophenol (PCP), was the site of two different applications of TSVE. The first method was conventional SESVE, which was effective but left residual DNAPLs. The second method, in situ hydrothermal oxidative destruction, which is similar to SESVE, except oxygen is injected along with the steam, accelerated the remediation in comparison to SESVE by oxidizing the residual DNAPLs. Using pump and treat technologies, it was estimated the site would take over 100 years to be cleaned up; however, in sharp contrast the combination of TSVE techniques remediated the site to closure levels in about two years (Leif et al. 1998). 16

27 2.5. Effect of Steam Treatment on Microbial Activity of Soil Following SESVE, natural attenuation is essential as a polishing step. However, the extreme temperatures of SESVE are detrimental to the indigenous microbial communities. The effect of SESVE on the microbial activity of soil has been studied mostly with bench-top experiments and a few experiments directly following treatment, while the subsurface remained at elevated temperatures. Dynamic Underground Stripping (DUS) is a method of SESVE combined with electrical energy heating of the subsurface. Prior to and four weeks after the DUS at Lawrence Livermore National Lab, soil samples were taken and assessed to determine the effect of the DUS on the indigenous microbial community (Krauter et al. 1996). The DUS rapidly removed about 3600 gallons of gasoline. Several methods were used to assess the microbial population of the soil: direct epifluorescent microscopic (DEM) total cell counts, heterotrophic plate counts, degradation in aerobic microcosms spiked with BTEX, and classification of microbial isolates using the Microbial Identification System (MIDI). The DEM and plate counts showed a 90-98% decrease in total microbial populations in the treatment area and a 20-85% decrease in the periphery of treatment. The microbial community shifted from gram negative to gram positive organisms. This is because some gram positive cells form spores, which can withstand extreme conditions, including high temperatures. The microcosms experienced degradation of BTEX, leading the researchers to conclude thermotolerant microorganisms survived which were capable of degrading gasoline compounds (Krauter et al. 1996). Two years 17

28 after the DUS the groundwater temperature at the site was still elevated to temperatures ranging from C. Bench-scale steaming experiments were conducted on three different soil types (Richardson et al. 2002). The soil was steamed for only about two hours. The pre-steam and post-steam soil (hot and cooled to room temperature) was analyzed using DEM total and active counts, enrichment cultures of various compounds, and fluorescent in situ hybridization (FISH). The hot soil samples had DEM active counts below detection levels, while the cooled samples had DEM total and active counts lower than pre-steam. FISH showed the surviving microbial community still included both Archea and Bacteria. Using the same methods, soil cores from a six-week field-scale SESVE treatment were analyzed (Richardson et al. 2002). The soil was sampled before and after treatment. The soil collected after treatment was tested while still hot and after a gradual cool down under laboratory conditions. The field-scale results were similar to the bench-scale with the hot samples having no detectable microbial activity and the cooled samples having activity but at decreased levels compared to pre-steam. Richardson suggests these studies show the native microbial community can rebound following steam treatment. However, bench-scale experiments are not a good approximation of field conditions, and in the field, cooling occurs much more slowly, possibly taking years (Krauter et al. 1996). Thus, although microbial populations may rebound from SESVE after the subsurface cools, during this extended cooling period the microbial population and activity are still decimated (Davis 1997). 18

29 2.6. Natural Attenuation Natural attenuation (NA) is the reduction in quantity of a chemical by natural processes without human intervention. Generally, NA is used to refer to all natural processes which reduce contaminant concentrations. These natural processes can be divided into chemical and biological processes. The main chemical processes are adsorption, volatilization, dispersion, dissolution, transformation, and evaporation. Volatilization can cause a significant reduction in the volatile hydrocarbons (specifically BTEX). Hydrocarbons are not very soluble which limits dispersion and dissolution, while most of the other chemical processes can depend on the site characteristics. The biological portion of natural attenuation is biodegradation, the microbial degradation of the contaminant to ultimately produce microbial cells, carbon dioxide, and water (Dragun 1998). For petroleum hydrocarbons, the chemical processes are responsible for a minor part of natural attenuation when compared with biodegradation. Intrinsic bioremediation of a petroleum-contaminated estuarine wetland in southeast Texas was investigated. Since biodegradation in wetlands is often limited by anoxia (the absence of oxygen) and lack of nutrients, petroleum contamination can be persistent. During the flood in 1994, a pipeline ruptured releasing gasoline, heating oil, and Arabian light crude oil into the San Jacinto River. These burned and resulted in primarily in the byproducts and crude oil contaminating the wetland (Mills et al. 2003). Sediments from the contaminated wetland were analyzed by gas-chromatography-mass spectrophotometry (GC-MS). The composition of poly-aromatic hydrocarbons (PAHs) increased relative to saturated hydrocarbons, suggesting preferential biodegradation with 19

30 saturated hydrocarbons being degraded over PAHs. First-order biodegradation rates decreased with increasing molecular weight. The contaminants were biodegraded at a rate higher than expected, on average over 95 % in about 150 days. The concentrations of petroleum in the contaminated sediment after the 150 day study were similar to those of an adjacent wetland not contaminated during the 1994 flood. This rapid biodegradation was explained by increased nutrients in the wetland due to recent flooding and the adaptation of the native microbial community as a result of previous hydrocarbon exposures (both anthropogenic and non-anthropogenic). In wetlands, as in most environments, the limiting factors for biodegradation are the availability of nutrients and electron-acceptors (Mills et al. 2003) Aerobic Natural Attenuation Aerobic degradation of organic chemicals requires molecular oxygen as the terminal electron acceptor and is the most thermodynamically favorable pathway for hydrocarbon degradation. For anaerobic degradation, the electron acceptors (in order of decreasing redox potential) include NO - 3, Fe 3+, SO - 4, and CO - 2. Electron acceptors are generally depleted in order of decreasing redox potential. Aerobic NA usually degrades contaminants at rates much greater than anaerobic NA (Dragun 1998). Theoretically, approximately three to four mg/l is of oxygen is necessary to degrade one mg/l of a medium-length hydrocarbon compound (Dragun 1998). The physical, chemical, and biological processes responsible for the natural attenuation of a gasoline plume at the site were studied extensively in the field (Borden et al. 1994). 20

31 Contamination of a shallow aquifer with gasoline from a leaking underground storage tank (LUST) was discovered after complaints about the taste and odor of the water downgradient. Residual NAPLs from the LUST steadily released dissolved BTEX, creating a plume. Soil cores revealed some weathering of the NAPL with large amounts of BTEX in the source zone. Groundwater samples were taken over a year and a half. Statistical analysis indicated that the dissolved BTEX plume was stable with no significant increase or decrease in concentration (Borden et al. 1994). The down gradient decrease in BTEX concentration was attributed to biodegradation by indigenous microbes. Since the plume became narrower with distance, dispersion was not able to explain the loss of BTEX. Preferential biodegradation of the BTEX occurred, ruling out volatilization as the primary mechanism. The data fit first-order kinetics (exponential decay). The estimated degradation rates were significantly lower than previous studies reported. Possible reasons for the lower rate were the overlying clay confining layer, which limits the oxygen in the unsaturated zone and the low ph, which may inhibit biodegradation (Borden et al. 1994). The plume had reduced dissolved oxygen (DO) and redox potential compared to the non-contaminated portion of the aquifer. Dissolved carbon dioxide was found at the highest concentrations in the portion of the plume furthest from the source. This geochemical data provided further evidence of biodegradation and significant mineralization of the BTEX. The electron acceptors were primarily ferrous iron and sulfate, then nitrate and DO. Only the far edge of the plume, where the carbonate levels increased and the ph rose, had some methanogenesis, as indicated by methane (Borden et al. 1994). 21

32 Anaerobic Natural Attenuation Research on natural attenuation and enhanced bioremediation of hydrocarbons has focused on the aerobic biodegradation of contaminants with very little study of anaerobic processes. However, field conditions are often anaerobic due to previous microbial activity or natural subsurface conditions. Therefore, although anaerobic biodegradation of hydrocarbons is slow, it could be an important component of natural attenuation. High levels of CH 4 are often found in the soil near petroleum spills (Lundegard et al. 2000). Since CH 4 is a product of methanogenesis, this can be an indication of anaerobic natural attenuation. Methanogenesis uses CO 2 as the terminal electron acceptor and may be the main degradation pathway at sites that lack other electron acceptors (e.g. sites that have been contaminated for long periods of time). The potential sources of shallow CH 4 in soil may be natural or anthropogenic. Anthropogenic sources include gas pipelines, oil and gas wells, sewer and septic systems, landfills, buried compost, landfills, and spilled petroleum. The two main pathways for the biogenic formation of CH 4 are acetic acid fermentation and carbon dioxide (CO 2 ) reduction (Figure 2-4). CH 4 can be formed from petroleum under strict anaerobic conditions with both molecular hydrogen (H 2 ) and CO 2 or with certain oxygen containing compounds (organic acids). Forensic data including historical land use data, isotopic analyses, and soil gas composition analyses can be used to determine the source of CH 4 (Lundegard et al. 2000). 22

33 Figure 2-4: Anaerobic biodegradation pathway (Brock et al. 1994) A soil column study was used to evaluate the rate of anaerobic biodegradation of numbertwo diesel fuel under various anaerobic conditions to identify the best conditions for removal of diesel (Boopathy 2004). The columns conditions were natural (no nutrient supplied deionized water), sulfate-reducing (sodium sulfate and ammonium chloride), nitrate reducing (sodium salt and ammonium chloride), methanogenic (sodium carbonate and nitrogen), mixed electron acceptor (sodium sulfate, ammonium chloride, sodium carbonate) and an abiotic control (0.5 % sodium azide). Columns were kept anaerobic by 23

34 purging with 100 % helium gas and were periodically flooded with nutrients and then allowed to drain and dry. TPH concentrations of the soil, headspace, and column effluent were measured with a gas chromatograph (GC) and flame ionization detector. Anaerobic bacterial plate counts were conducted to test for the presence of anaerobic microbes. Biodegradation rates were determined by a mass balance, subtracting the TPH in the column effluent and TPH adsorbed by activated carbon from the total percent of TPH removal. After 310 days, the abiotic control had a 9 % TPH removal attributed to volatilization (1.6 %) and dissolution (6.2 %). The mixed electron acceptor column had the highest biodegradation rate (81 %) followed by sulfate-reducing with 54.5 %, nitratereducing with 47 %, methanogenic with 37 %, and the natural conditions with 19 %. Analysis of the headspace gases showed evidence of anaerobic respiration (carbon dioxide and methane production). The plate counts for the abiotic control had no microbial activity. Plate counts for the nutrient-supplemented columns were at least four orders of magnitude higher than for the natural column. The results of these column experiments indicate that significant biodegradation of petroleum hydrocarbons can occur under anaerobic conditions. If a field site lacks electron acceptors, then sulfate, nitrate, or other electron acceptors could be added through injection wells. This supplementation could increase the degradation rates of diesel fuel (Boopathy 2004) Review of Monitoring and Microbial Analysis Methods Biodegradation rates are dependent on various factors: indigenous microbial populations, nutrient and electron acceptor availability, ph, temperature, water content, contaminant properties, and soil properties (Dragun 1998). Monitoring hydrocarbon levels in the 24

35 environment is complicated by heterogeneity of the subsurface and contamination. Determining the loss of hydrocarbons attributable to biological processes is further complicated by other attenuating processes (volatilization and dissolution). Thus, ratios of hydrocarbons and the levels of biologically conserved compounds such as pristine and phytane have been used to determine biodegradation rates in the field (Atlas 1995). For example, a decrease in the concentration of hydrocarbons relative to pristane can be attributed to biological processes. However, if both the pristane and hydrocarbon concentrations decrease and the ratio is unchanged, then abiotic processes are responsible for the decreased concentrations. Thus, measuring bioremediation requires more than monitoring of the contaminant concentration. Monitoring the microbial processes of the soil provides information on the microbial community, the effect of the contaminant on the microbial community and activity, and the rate of bioremediation. Soil respiration, dehydrogenase activity, and microbial counts are the typical methods used to evaluate microbial activity and communities (Margesin et al. 2000). The composition of the indigenous microbial community is critical to natural attenuation. Both the type of bacteria present and the relative composition of the microbial population provide insight into the potential for natural attenuation and bioremediation. However, characterizing the microbial populations in complex environmental samples can be difficult. Many previous techniques for counting and identifying microorganisms are ineffective and time intensive. Since only a few different morphologies exist, visual observations can not provide enough information. Biochemical methods may provide more information than visual methods, but they are 25

36 only useful for cultured species. Genetic techniques are more practical than visual or biochemical methods for measuring the diversity of an environmental sample (Clement et al. 1998). Two common methods of microbial counts are direct microscopic counts and plate counts. Direct counts result in a microbial concentration in cells per volume or mass. Plate counts results are expressed as the number of colony forming units (CFU) per volume or mass. Direct counts are expected to be several orders of magnitude higher than plate counts. This is due to cells which are not culturable in plate conditions or are in a nonculturable state, as well as unknown species for which methods for culturing have not been developed (Amann et al. 1995). Also, plate counts enumerate colonies, which may have formed from several cells clumped together. This would further contribute to lower plate counts than direct counts. To evaluate the usefulness of several biological methods for assessing biodegradation, seven pans of soil were artificially contaminated with diesel fuel. One pan was a sodium azide poisoned control, one pan was not supplemented, and the other five were supplemented with varying N- and P- sources. The pans were covered and incubated in the dark at 20 C and 70 % relative humidity for 88 days (Margesin et al. 2000). The hydrocarbon removal rate of the abiotic control was 35 %, while the other pans had rates of %. No significant difference occurred in the decontamination in soils with an N- and P- source. Soil microbial counts were conducted on R2A agar and on oil-agar plates and were counted after 7 days of incubation at 20 C. The number of counted 26

37 heterotrophic microorganisms remained nearly constant, while the counts of oil-utilizing microorganisms increased with time. Hydrocarbon content correlated strongly positively with soil respiration. The biological analyses revealed the soil microorganisms appeared to adapt quickly and to use the new carbon source. In evaluating and assessing bioremediation, soil biological methods should be used to complement the chemical and geological methods (Margesin et al. 2000) Terminal Restriction Fragment (TRF) Pattern Analysis Terminal restriction fragment (TRF) pattern analysis is a polymerase chain reaction (PCR)-based method for rapidly comparing bacterial communities without culturing or cloning (Kitts 2001; Liu et al. 1997; Marsh 1999). TRF requires several steps: isolation of DNA, PCR, primer removal and amplicon concentration, amplicon digestion, electrophoresis and fragment size determination, and data analysis. The 16S rrna genes are amplified and fluorescently labeled using PCR. The resulting labeled products are digested with a restriction enzyme, leaving the labeled terminal fragments to be separated with gel electrophoresis and detected by laser-induced fluorescence using an automated gene sequencer. TRF analysis can be used to provide insight into the potential for natural attenuation and the organisms responsible, and the changes in the bacterial community related to remediation efforts. Distinctive bacterial community patterns can be rapidly generated using the TRF method. The analysis of TRFs is useful for investigating complex communities and their dynamics over time (Clement et al. 1998). 27

38 Enhanced Bioremediation Bioremediation is the active use of biological processes to remediate contaminants and can enhance degradation rates relative to natural attenuation. Bioremediation increases the natural rate of degradation and results in minimal impacts on the environment (Atlas 1995). Petroleum compounds are often treated using bioremediation (Atlas 1995). Two methods are used to enhance the natural processes: biostimulation and bioaugmentation. Biostimulation is the addition of fertilizers (nutrients) to increase the metabolic rates of the indigenous microbial community. Microorganisms require nitrogen, phosphorous, and several other mineral nutrients for growth (Dragun 1998). Bioaugmentation (seeding) is the addition of exogenous microbial populations known to degrade the pollutant (Atlas 1995). Bioremediation studies were conducted following the Exxon Valdez spill in Prince William Sound and the Mega Borg spill off the Texan coast. Commercial seed cultures were applied to test plots in the Sound with no improvement in the oil biodegradation; however, the oil spill was already degraded by the time the test was conducted. A seed culture was applied to the Mega Borg spill; however, chemical analyses did not provide evidence of enhanced biodegradation (Atlas 1995). Three types of nutrient treatments were applied to the spill in The Sound: water-soluble, slow-release, and oleophillic. The fertilizer increased the degradation and resulted in oil free spots within 10 days of treatment. Chemical analyses indicated removal of all resolvable chemical components of the oil including four-ring PAHs and substituted 28

39 chrysenes. Biodegradation of oil was nutrient limited and was enhanced by addition of fertilizer. Bioremediation monitoring following the Exxon Valdez spill revealed the degradation was significant and differences in biodegradation rates were mainly due to different concentrations of nutrients in sediment pore waters. This suggests increasing the nutrient levels, both in dosage and frequency of application (within safe limits for organisms), would increase the degradation rates (Atlas 1995). Throughout the world, petroleum hydrocarbons are among the most common environmental contaminants. It is desirable to remediate this subsurface contamination with a minimal environmental impact (i.e. consumption of resources, waste generation, emissions). Since natural attenuation of hydrocarbon contamination in situ should have the smallest ecological footprint, it is preferable to physical and chemical methods. Much research has been devoted to assessing the effect of field conditions on natural attenuation, rates of degradation, and enhancements to natural attenuation. Natural attenuation of petroleum hydrocarbons is a viable option at many sites, while others may require enhanced bioremediation (Atlas 1995; Borden et al. 1994; Kaufmann et al. 2004; Margesin et al. 2000; Mills et al. 2003). SESVE may prohibit the use of natural attenuation and enhanced bioremediation as a polishing step. This is because the extreme temperatures of SESVE decrease microbial populations, and even after SESVE, subsurface temperatures remain elevated for years, possibly inhibiting microbial activity (Davis 1997). Thus, it is important to know the effect of SESVE on the microbial community. 29

40 CHAPTER 3 MATERIALS AND METHODS 3.1. Soil Samples Pre-steam and post-steam (one month) soil samples were gathered from multiple depths at two boring locations (Core 6 and 8) for aerobic assays. An additional boring was drilled and sampled under nitrogen to maintain anaerobic conditions for the methanogenic assays and was stored in a nitrogen atmosphere. Each sleeve was sixinches long, two-inches in diameter and was treated as one sample (depth). The sample sleeves were received the same day as they were drilled. Pre-steam samples were collected and analyzed as part of a previous experiment (Maloney 2003). Since post-steam (one month) soil samples exhibited reduced microbial populations and activity, further studies were necessary to determine the recovery of the microbial community following cooling of the subsurface. Eight months after the pilot test ended, four aerobic sleeves and six anaerobically handled sleeves were gathered from the same location as Core 6. Sleeves from this core were used for both aerobic and anaerobic experiments to save money. The same (pre and post-steam) experiments were repeated, using the same methods and materials. However, to prevent leakage, a different crimper was used to seal the post-steam (eight months) anaerobic microcosms than the post-steam microcosms. Prior to any experiments, soil from each sleeve was homogenized by mixing all of the soil from the sleeve. For anaerobic samples, this homogenization was conducted in an 30

41 anaerobic glovebox. Soil for TRF analysis and total petroleum hydrocarbon (TPH) analysis was frozen until analysis Aerobic Soil Samples Aerobic samples from Core 6 and Core 8 were collected for plate counts, respirometry, and direct epifluorescent microscopy. The TPH concentrations of pre-steam aerobic soil samples were as high as 73,000 mg/kg. The TPH concentrations of post-steam soil samples were lower than pre-steam soil samples (Tables 3-1 and 3-2). Post-steam aerobic cores had decreased TPH levels from 31 to 7100 mg/kg. Table 3-1 TPH results for aerobic soil samples (Core 8) Unocal Label Depth TPH concentration (ppm) (ft bgs) Pre-steam Post-steam (one month) POS X POS X PRE-8D 58X PRE-8D 58X POS-8 61X POS-8 61X Table 3-2: TPH results for aerobic soil samples (Core 6) Unocal Label TPH concentration (ppm) Depth Post-steam Post-steam (ft bgs) Pre-steam (one month) (eight months) POS X POS X POS-6 55X POS-6 55X POS X

42 Anaerobic Soil Samples Soil samples for anaerobic experiments were gathered under anaerobic conditions. During boring, the boreholes were flooded with nitrogen. Once obtained, the sleeves were kept in a nitrogen atmosphere. The original pre-steam microcosms were setup using Core 4 soil samples (Anaerobic Microcosm Experiment I, Maloney, 2003); however, these microcosms suffered problems with leakage of oxygen and poor mixing of soil. Thus, twenty anaerobic sample sleeves were gathered from SBH2-16 (the nearby diluent tanks site) to perform additional anaerobic experiments (Anaerobic Microcosm Experiment II, Maloney, 2003). Post-steam anaerobic experiments were conducted using the methods of the second pre-steam microcosm experiment. Post-steam (one month) anaerobic soil samples were gathered from Core 4, and post-steam (eight months) anaerobic soil samples were gathered from Core 6. Pre-steam anaerobic soil sample TPH concentrations ranged from non-detect (ND) to 150,000 mg/kg, while post-steam TPH concentrations ranged from ND to 23,000 mg/kg (Tables 3-3 and 3-4) 32

43 Table 3-3: TPH results for anaerobic soil samples (Core 4) Depth (ft bgs) TPH concentration (ppm) Unocal Label Pre-steam Post-steam (one month) POS x ND ND POS x ND ND POS 4-52x ND POS 4-52x ND POS x POS x POS 4-55x POS 4-55x POS 4-61x POS 4-61x POS x POS Table 3-4: TPH results for anaerobic soil samples (Core 6) Unocal Label Depth (ft bgs) TPH concentration (ppm) Pre-steam Post-steam (eight months) POS X POS X POS 6-58X POS 6-58X POS X POS X Respirometry A Columbus Instruments (Columbus, OH) Micro-Oxymax respirometer with a CO 2 /CH 4 detector, sample pump, and expansion interface was used to measure carbon dioxide production in the aerobic samples (Figure 3-1). The respirometer draws and analyzes gas samples from each chamber every two and a half to three hours. The respirometer analyzes the air from each of the chambers with a calibrated CO 2 sensor. After every two 33

44 samples, fresh CO 2 -free air is provided to the samples by purging ambient air of CO 2 using a soda lime column. The air is desiccated using a Drierite column. Duplicate 50-gram samples of each depth from aerobic Cores 6 and 8 and a blank containing no sample were tested in the respirometer for about 48 hours. The respirometer measured temperature, CO 2 production rates, and cumulative CO 2 production. An external water bath was used to maintain a constant temperature of 20 C. Figure 3-1: Micro-Oxymax respirometer 3.3. Plate Counts To transfer the bacteria from the soil to solution, 10 g homogenized soil sample, 100 ml (autoclaved) phosphate buffer solution (PBS, ph 7.2) and 0.1 g sodium pyrophosphate 34

45 were combined in a 125 ml Erlenmeyer flask. The flask was covered and mixed for 15 minutes with a magnetic stirrer. This solution serves as the stock soil solution for each soil sample with 0.1 g of soil in each milliliter. Each soil stock solution was serially diluted with autoclave-sterilized PBS. As required, lower dilutions were prepared (e.g. 10g soil in 10 ml PBS). For each soil sample, 100 µl of each dilution was plated in triplicate. Control plates were plated with 100 µl of sterilized PBS. The plates were incubated at 20 C for four days before counting colonies on each plate Direct Microscopic Counts Direct microscopic counts were performed to enumerate the total number of cells per gram of soil for comparison with the number of CFU per gram from plate counts. Direct microscopic counts of bacteria were performed using a method modified from (Bhupathiraju et al. 1999). Using epifluorescent microscopy with 5-(4,6- dichlorotriazinyl) aminofluorescein (DTAF) as a stain, total cell counts were performed. DTAF works by staining the cell walls of bacteria, which then appear bright green when viewed using epifluorescent microscopy. DTAF stains the cell walls of both respiring and non-respiring cells and thus provides a total cell count. Pre-steam direct microscopic count experiments included counterstaining with 5-cyano- 2,3-ditolyl tetrazolium chloride (CTC), which reacts to form fluorescent red-orange formazan deposits within actively respiring cells. This counterstaining gives an active cell count. However, the results were unreliable, so active counts were not repeated for post-steam samples. 35

46 To transfer bacteria from the soil to solution, 10 grams of homogenized soil from each sleeve, 25 ml sterile PBS (ph 7.2), 75 ml DI water, and 0.1 sodium pyrophosphate were combined in a 125 ml Erlenmeyer flask and stirred soil for four hours with a magnetic stirrer. Dilutions of this soil solution were prepared as necessary to result in countable fields in the microscope. In a 2.0 ml microcentrifuge tube, 1.0 ml of soil solution was combined with 0.5 ml sterile phosphate buffered saline solution (100 ml DI water, 120 mg NaH 2 PO 4, 807 mg NaCl, and 20 mg KCl), and 0.5 ml DTAF dye solution (10 ml of DI water, 10 mg DTAF, and 47.3 mg Na 2 HPO 4 ). Each sample was centrifuged at 1000 rpm for 20 minutes, while protecting the samples from light. After centrifuging, each sample was passed through a 0.2 µm black polycarbonate filter (25mm diameter, 2.9 cm 2 effective filtration area, Millipore, Bedford, MA ) sealed in a 50-mL autoclavable polysulfone filter apparatus (Pall Corporation, East Hills, NY). Each filter was rinsed with at least 200 ml of sterilized wash solution (1.0 L DI, 7.1 g Na 2 HPO 4, and 8.5 g NaCl). The filters were air dried and mounted on a microscope slide using about 50 µl of Tris-buffered glycerol with 2% 1,4-diazabicyclo [2,2,2] octane (0.2 g 2% 1,4-diazabicyclo [2,2,2] octane, 5.0 ml glycerol, 5.0 ml 2M Tris-buffer). Each filter was covered with a cover slip and observed using the 100X objective lens with immersion oil and the UV light in a dark room. The ocular lens was 10X, providing a total magnification of 1000X. Stained bacterial cells on five randomly selected fields were counted for each slide. An Olympus BX50 Microscope, BX-FLA Reflected Light 36

47 Fluorescence Attachment, and BH2-RFL-T3 Power Supply Unit were used with Omega Optical filter set XF Anaerobic Microcosms The anaerobic soil samples were collected and handled under a nitrogen-purged atmosphere to prevent exposure to oxygen. Microcosms were setup in an anaerobic (nitrogen purged) glovebox (Figure 3-2). Four to eight microcosms were setup for each depth depending on the expected TPH concentrations (four for lower concentrations and eight for higher concentrations). The soil from each core was homogenized. Forty grams of soil was placed into each 150-mL serum bottle (Figure 3-3). The lip of each bottle was carefully wiped and sealed with a Teflon -lined septa using a crimper. Excess soil was returned to the sleeve and frozen for TPH and TRF analyses. Figure 3-2: Nitrogen-purged anaerobic glove box 37

48 The anaerobic microcosms were analyzed after incubating at 20 C for 30 to 211 days. The headspace of duplicate microcosm bottles was analyzed by Greg Ouellette (Inland Empire Analytical, Norco, CA) using gas chromatography with a thermal conductivity detector (TCD) for CH 4, O 2, H 2, CO 2, N 2 and N 2 O (details in Appendix A). Figure 3-3: Anaerobic microcosms Core 4 (51, 54.4, 63 ft) 3.6. Total Petroleum Hydrocarbon (TPH) Analysis Zymax Envirotechnology (San Luis Obispo, CA) performed the TPH analysis on the soil samples using EPA Method 3510 extraction with methylene chloride, followed by gas chromatography with mass spectrometry detection (GC/MS) (State of California method similar to EPA Method 8015). The analytical range was C8 to C40. TPH was quantified against standards prepared from Guadalupe diluent. The practical quantification limit (PQL) was 10 mg TPH per kg soil. Results below the PQL are reported as Non-Detect (ND). 38

49 3.7. Terminal Restriction Fragment (TRF) Analysis Terminal Restriction Fragment (TRF) analysis was performed by Alice Hamrick and Dr. Chris Kitts (Environmental Biotechnology Institute, Cal Poly) using the method described in Appendix B. TRF analysis is a method based on the size of DNA fragments produced using restriction. TRF requires several steps: isolation of DNA, Polymerase Chain Reaction (PCR), primer removal and amplicon concentration, amplicon digestion, gel electrophoresis, and fragment size determination and data analysis (Kitts 2001). DNA extracted from the soil was amplified and fluorescently labeled using PCR. The resulting fluorescently labeled products (amplicons) were digested with a restriction enzyme. The digested amplicons were then separated with gel electrophoresis and detected by laser-induced fluorescence using an automated gene sequencer. TRF patterns were generated using eubacteria and archaebacteria primers. TRFs show as peaks eluting according to their base pair length. The height of a peak is relative to the abundance of that individual TRF length in a sample. The organisms responsible for individual TRF peaks can be identified by comparing the base pair length to a clone library or to a database of sequences. The microbial diversity of a sample is indicated by the number of TRF peaks. TRF patterns from different samples are compared using multivariate statistics methods (Kitts 2001). 39

50 CHAPTER 4 RESULTS Pre-steam and post-steam microbial assays are compared in this Chapter. Additional details of pre-steam experiments can be found in Maloney, Nelson et al. (2004). Aerobic microbial activity is defined by respirometry and aerobic microbial populations are quantified by plate counts and direct microscopic counts. Anaerobic microbial activity is quantified by methane and hydrogen production Respirometry Duplicate 50-gram soil samples of each depth from Core 6 and 8 were tested in the respirometer for about 48 hours. For both cores, one blank which contained no sample was run. Pre-steam average respiration rates ranged from 0.06 to 0.23 µl/g-hr, while the blank for this experiment exhibited little or no CO 2 production (Tables 4-1 and 4-2, Figure 4-1) (Maloney 2003). In contrast, the average carbon dioxide production rates of all the post-steam (one month) samples were zero (Figure 4-2). The average carbon dioxide respiration rates of the post-steam (eight months) samples ranged from 0.14 to 0.20 µl/g-hr, but the blank for this respirometry run also exhibited CO 2 production at an average rate of 0.08 µl/g-hr. When the blank is subtracted, post-steam (eight months) respiration rates ranged from 0.06 to 0.12 µl/ghr. The cumulative CO 2 production in µl/g for post-steam (eight months) samples also showed evidence of recovered aerobic microbial activity (Figure 4-3). 40

51 Table 4-1: Average Respiration Rates for Core 6 before and after steam treatment CO 2 Production Rate (µl/g-hr) Post-steam (one month) Depth Pre-steam (ft bgs) Sample Sample B Average A * Blank *Note: The duplicate for 54 was thrown out due to error Table 4-2: Average Respiration Rates for Core 8 before and after steam treatment CO 2 Production Rate (µl/g-hr) Depth Pre-steam Post-steam (one month) (ft bgs) Average Sample A Sample B Average Blank

52 CO 2 Accumulation, µl/g ' 54.5' ' ' 6 Blank Time, hrs Figure 4-1: Pre-steam cumulative carbon dioxide production (Maloney 2003) CO 2 Accumulation, µl/g ' 54.5' 55.5' 56.0' Blank Time, hrs Figure 4-2: Post-steam (one month) cumulative carbon dioxide production CO 2 Accumulation, µl/g ' 55.5' 56.0' 57.0' Blank Time, hrs Figure 4-3: Post-steam (eight months) cumulative carbon dioxide production 42

53 4.2. Plate Counts Serial dilutions of soil suspensions were plated and incubated at 20 C for four days. For some post-steam sample depths, the initial dilutions did not have enough colony forming units (CFU) to be countable, so lower dilutions were plated. Colonies were mostly round in shape (Figure 4-4) and varied in color from pink to blue to yellow. All of the control plates were free of any growth, bacterial or otherwise. Pre-steam plate counts varied from 1.6 x 10 6 to 2.2 x 10 7 CFU per gram of soil (Table 4-3) (Maloney 2003). The post-steam (one month) counts ranged from 3.4 x 10 3 to 1.98 x 10 5 CFU/g and were lower by one to four orders of magnitude than the pre-steam counts (Figures 4-5, 4-6, 4-8 and 4-9). The percent reduction ranged from 97.87% to 99.96% (Table 4-3). The post-steam plate counts at eight months were even lower, ranging from 5.7 x 10 2 to 5.4 x 10 3 CFU/g (Table 4-4, Figure 7). Figure 4-4: Core 8 plates in order of decreasing dilution from left to right (62 ft) 43