X-s 1.14:; m nu- {.I‘ ,. um: E g. 1 1- 4 1r ‘1 ‘3‘. "a. .3 i} .—v-4 -a- r“: :'" szu‘c- u a 3 vi- #9 on .L::-.:r- ~* u LIBRARY Michigar‘ State University This is to certify that the dissertation entitled REDOX GRADIENTS, TCE REDUCTIVE DECHLORINATION AND Cr(V|) DETOXIFICATION IN BIOAUGMENTED MODEL AQUIFER SYSTEMS presented by Timothy Joseph Mayotte has been accepted towards fulfillment of the requirements for the Ph.D. degree in Environmental Engineering M3167 Pr67essor’s Signature I / Date MSU is an Affinnative Action/Equal Opportunity Institution PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/01 cJClRC/DatoDuopes-pJS REDOX GRADIENTS, TCE REDUCTIVE DECHLORINATION, AND Cr(VI) DETOXIFICATION IN BIOAUGMENTED MODEL AQUIFER SYSTEMS By Timothy Joseph Mayotte A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Civil and Environmental Engineering 2003 ABSTRACT REDOX GRADIENTS, TCE REDUCTIVE DECHLORINATION, AND Cr(VI) DETOXIFICATION IN BIOAUGMENTED MODEL AQUIFER SYSTEMS By Timothy Joseph Mayotte A mixed microbial culture enriched for tetrachloroethene or tn'chloroethene (TCE) dehalogenation activity was introduced into and conveyed through granular aquifer materials under an imposed hydraulic gradient. Over time, colonization of the aquifer solids by the enrichment culture resulted in a permeable stationary-phase biologically active zone (BAZ) within which complete reductive dechlorination of TCE occurred when provided organic carbon and nutrients. During a period of acclimation, a unique stationary-phase community profile was expected to result from successive depletion of electron acceptors and consumption and/or production of electron donors in pore fluids passing through the BAZ. It was postulated that the near steady-state community profile within the BAZ would exhibit spatially- and/or temporally discrete intervals of sulfate-reduction, methanogenesis, and halorespiration. Batch and continuous-flow column systems containing solvent- and hexavalent chromium (Cr{VI})-impacted soil and groundwater from the Schoolcraft, Michigan site were used to transiently and spatially examine: 1) redox gradients; 2) TCE transformation Patterns; and 3) successional adaptations of mobile- and stationary-phase microbial communities following inoculation of the aquifer materials with a TCE-dechlorinating enrichment culture from the Bachman Road site in Oscoda, Michigan. Real-Time SYBR Green Polymerase Chain Reaction (RT-PCR) analyses were employed to measure the mobile-phase community structure at temporally discrete intervals over the acclimation WM and upor. an anempl l0 hi: the fate and quaz‘ treatment and a: Data fro." inoculation. TCE; rate coetlicient 0 lion column \x‘cr following each 0 levels (Em-250 l presumably by r sulfate reductio: of the expenme: Occurrence of la mechanism of r the Corresnond: and Vinyl chlon‘ appared l0 dcc lo il'lillbll dech] Optimized feed “film the B. A-Z period and upon attainment of near steady-state treatment conditions across the BAZ. In an attempt to identify the primary zone(s) of halorespiration, RT-PCR was used to track the fate and quantify the mass of Dehalococcoides ethenogenes during inoculation, treatment, and as part of the post-treatment BAZ characterization of the aquifer solids. Data from the semi-batch column experiments indicated that within one week of inoculation, TCE dechlorination to vinyl chloride was rapid with an apparent first-order rate coefficient of 0.6 day]. TCE concentrations (1-4 mg/L) entering the continuous flow column were reduced by approximately 30-40% within a seven-day interval following each of nine deliveries of lactate and nutrients to the BAZ. Influent Cr(VI) levels (ZOO-250 pg/L) consistently decreased by over 90% during these intervals, presmnably by reduction to less soluble Cr(III). Following lactate fermentation, iron and sulfate reduction were the predominant redox processes within the BAZ over the duration of the experiment. Reductive dechlorination of TCE was spatially correlated to the occurrence of lactate fermentation. Halorespiration appeared to be the dominant mechanism of reductive dechlorination, based on the presence of Dehalococcoides sp., the corresponding rates of TCE depletion and production of both cis-1,2-dichloroethene and vinyl chloride. However, the rates and completeness of TCE transformation appeared to decrease with an increase in sulfidogenic activity. Methanogenesis appeared to inhibit dechlorination. Fate and transport modeling simulations suggest that an optimized feed delivery scheme may result in greater than 90% TCE treatment efficiency within the BAZ over a one-week period of time. Cepyn'ght by TIMOTHY JOSEPH MAYOTTE 2003 To Kathleen For all her love, sacrifices, and selfless dedication to my goals. To the support and gut. My \Vilt‘ constant sources facilitate my edt Ctptrirnce um i have nex'er purg Mike D} leaning expem ability to think . to [ethnical pm addremd OVcr Commit Pl0\'ided "aludi and EXpefienCe Haekfli: and generous m Quickly to the 12 Similar}. ACKNOWLEDGMENTS To the following individuals, I express sincere gratitude and appreciation for their support and guidance during the completion of this work. My wife Kathleen, son Daniel and daughters Mary Grace and Anna have been constant sources of support and inspiration. Kathleen has given much of herself to facilitate my educational and professional goals throughout our marriage. This experience was certainly no exception. Without her support and encouragement, I would have never pursued this opportunity. Mike Dybas was responsible for making possible this valuable and enjoyable learning experience. Observing and experiencing Mike’s practical view of research and ability to think and act on his feet were significant in helping me transform my approach to technical problems related to this study as well as many consulting issues that I have addressed over the past two years. Committee members Dave Wiggert, Syed Hashsham and Jim Tiedje have provided valuable insight and patience throughout this study. Their collective knowledge and experience have been of great benefit to this study and to me in general. Haekyung Kim has been a most enjoyable and helpful lab partner. Her patience and generous assistance was invaluable. More than anyone, Kim helped me adapt quickly to the laboratory environment. Similarly, Leslie Dybas has been a tremendous source of laboratory education and guidance. I have truly appreciated Leslie’s cool and professional approach to procedures. She has provided me many insights into molecular microbiological laboratory techniques. vi Without her help, this study would not have been nearly as fulfilling as it ultimately came to be. Xianda Zhao and Yan Pan have always been generous and gracious in offering assistance with experimental nuances and analytical procedures. Both have tremendous knowledge. But I most appreciated their approachability and affability, which always made technical discussions enjoyable and rewarding experiences. To my parents, Bernie and Jean Mayotte, I express heartfelt appreciation. Throughout my life, they have been strong sources of inspiration and encouragement. Even with this experience coming so much later in my life, they continued to express such great interest and pride in all that I have been doing. Pleasing them with this accomplishment is one of the most gratifying experiences of my life. Finally, to Northern Environmental Technologies, Inc. of Mequon, Wisconsin, and, specifically, to David Rautmann and Dale Buser I offer thanks for their willingness to enter into an employment arrangement that made the pursuit of this academic goal possible. vii llSl OF TAB: i llSl OF HGL'E llSl OF SYM’: ClikPTER l - 1.1 l.'.’ (Hum r.) ya F.) f.) H I 2.4 TABLE OF CONTENTS LIST OF TABLES ............................................................................................................. xi LIST OF FIGURES .......................................................................................................... xii LIST OF SYMBOLS ....................................................................................................... xiii CHAPTER 1 - INTRODUCTION ................................................................................. 1 1.1 Introduction ............................................................................................ 1 1.2 Background ............................................................................................ 1 1.2.1 Plume G ..................................................................................... 2 1.2.2 Plume F ...................................................................................... 3 1.2.3 Plume F/G Bioremediation Remediation Project ...................... 4 1.2.4 Previous Bioremediation Research ............................................ 5 1.3 Plume F/G Bioaugrnentation Studies ................................................... 10 1.3.1 The BR Enrichment Culture .................................................... 10 1.3.2 Summary of Preliminary Results ............................................. 13 1.4 Hypotheses and Research Objectives .................................................. 18 CHAPTER 2 - EXPERMENTAL DESIGN ................................................................. 21 2.1 Introduction .......................................................................................... 21 2.2 Small-Scale Column Microcosm Experiment ..................................... 22 2.2.1 Column Design and Assembly ................................................. 23 2.2.2 Column Operation and Monitoring .......................................... 26 Task 1.1 — Characterization of F low-Through Properties ....... 26 Task 1.2 -— TCE Mass Loading and Sorption Capacity Evaluation .............................................................. 27 Task 1.3 — Treatment Strategy Evaluation ............................... 27 Task 1.4 — Post-Treatment Characterization ............................ 29 Operational and Sampling Procedures ..................................... 30 Data Reduction ......................................................................... 32 Kinetics of Biotransforrnation .................................................. 34 2.3 Large-Scale Column Experiment ......................................................... 35 2.3.1 Column Design and Assembly ................................................. 38 2.3.2 Colmnn Operation and Monitoring .......................................... 42 Task 2.1 — TCE Mass Loading and Sorption Capacity Evaluation .............................................................. 42 Task 2.2 - Inoculation/Characterization of F low-Through Properties ............................................................... 43 Task 2.3 — Treatment ............................................................... 43 Task 2.4 — Post-Treatment Characterization ............................ 44 Operational and Sampling Procedures ..................................... 45 2.4 Soil and Groundwater .......................................................................... 51 viii 2.5 2.6 2.7 CHAPTER 3 - 3.1 3.2 3.3 3.4 3.5 3.6 CHAPTER 4 - 4.1 4.2 4.3 4.4 CHAPTER 5 - 5.1 5.2 APPENDIX A - APPENDIX B - APPENDIX C - C-1 C-2 C-3 C-4 Analytical Procedures .......................................................................... 53 2.5.1 Aqueous Solutes ....................................................................... 53 2.5.2 Headspace Gasses .................................................................... 55 2.5.3 Biomass and DNA ................................................................... 56 Measurement of TCE Transformation Kinetics ................................... 60 Fate and Transport Modeling ............................................................... 62 RESULTS AND DISCUSSION .......................................................... 67 Characterization of Aquifer Materials ................................................. 67 F low-Through Properties ..................................................................... 67 Sorption Characteristics ....................................................................... 71 Inoculum Transport .............................................................................. 73 Biotransformation ................................................................................ 74 3.5.1 Small-Scale Column Experiment ............................................. 75 TCE Transformation Kinetics and Mass Removal .................. 83 3.5.2 Large-Scale Column Experiment ............................................. 87 Operational Phase A ................................................................ 88 Operational Phase B ................................................................. 95 TCE Dechlorination Kinetics and Mass Removal ................. 102 3.5.3 Redox Gradients and Halorespiration Activity ...................... 105 3.5.4 Hexavalent Chromium Reduction and Immobilization ......... 110 Summary ............................................................................................ 1 1 1 FATE AND TRANSPORT MODELING ......................................... 116 Introduction ........................................................................................ 1 16 Model Calibration .............................................................................. 116 Sensitivity Analyses ........................................................................... 120 4.3.1 Variation Coefficients ............................................................ 121 4.3.2 Bio-kinetics and Sorption ...................................................... 123 4.3.3 Summary ................................................................................ 124 Predictions .......................................................................................... 124 CONCLUSIONS AND RECOMMENDATIONS ............................ 126 Conclusions ........................................................................................ 126 Recommendations .............................................................................. 1 3 1 Large Scale Column A Operational History ...................................... 136 Retec Group, Inc. TCE Transformation Kinetics Study .................... 144 Large Column Study Analytical Results — Operational Phase A ...... 155 Fatty Acids ......................................................................................... 156 Major Anions ..................................................................................... 165 Soluble Metals ................................................................................... 174 Chlorinated Aliphatics ....................................................................... 183 ix APPENDD( D - Large Column Study Analytical Results — Operational Phase B ....... 193 D-1 Fatty Acids ......................................................................................... 194 D-2 Major Anions ..................................................................................... 201 D-3 Soluble Metals ................................................................................... 208 D-4 Chlorinated Aliphatics ....................................................................... 215 APPENDIX E - Daily Solute Concentrations within a Control Volume Conveyed through Column A, Incubation Periods 4B-6B .................................. 222 APPENDD( F - Daily Solute Concentration Profiles Over Column A, Incubation Periods 4B-6B .................................................................. 225 APPENDIX G - Reactive Transport Modeling Results ................................................ 230 G-l Calibration .......................................................................................... 231 G-2 Sensitivity Analyses ........................................................................... 233 G3 Predictions .......................................................................................... 237 BIBLIOGRAPHY ............................................................................................................ 240 Table 2.1. Table 2.2. Table 3.1. Table 3.2 Table 3.3. Table 3.4. Table 4.1. Table 4.2. LIST OF TABLES Summary of Aqueous Sample Analyses — Small-Scale Column Microcosm Experiment. Summary of Aqueous Sample Analyses — Large-Scale Column Experiment. Characteristics of Schoolcraft Aquifer Materials. RT-PCR results for small- and large-scale column samples. First order rate coefficients, k ', within control volumes of Column A between October 3 and October 22, 2003. TCE mass treatment rates and efficiency over the length of Column A over the duration of incubation periods 5A-8A and BB-6B. Numerical model input parameter values and data sources. Variation coefficients for key model parameters on day 7 of Incubation Period 6B. xi Figure 1.1. Figure 2.1. Figure 2.2. Figure 3.1. Figure 3.2. Figure 3.3. Figure 3.4. Figure 3.5. Figure 3.6. Figure 3.7. Figure 3.8. Figure 3.9. Figure 3.10. LIST OF FIGURES Central Schoolcraft, Michigan and groundwater contaminant Plumes F and G. Small-Scale Column Microcosm Design (Dolan and McCarty, 1996). Large-Scale Column Design. Composite breakthrough profiles of bromide, TCE and inoculum through small-scale columns. Bromide breakthrough profiles at sample Ports 2 and 4 of large-scale Column B. Dashed line with open boxes represent bromide data fiom each port. Solid line represents numerical simulation. Bromide and major anion breakthrough profiles for large scale Column A. Dimensionless equilibrium partitioning coefficients composited from small-scale columns D-U. Column B port-specific inoculum breakthrough based on optical density (OD660) measurements. Lactate and phosphate data for Columns P, Q, and R. Lactate is indicated by the open triangles; acetate is indicated by the closed triangles. Phosphate trends are presented by the closed circles and bold tie-lines. Nitrate and sulfate data for Columns P, Q, and R. Nitrate data indicated by open circles; sulfate data indicated by open triangles and bold tie-lines. Cumulative TCE mass removed in each small-scale column set. TCE removal and cis-DCE and VC accumulation in columns P, Q, R (a), J, K, L (b), and M, N, O (c). Apparent first-order rate coefficient trend, Columns P, Q, and R. xii aL bDC C0 C1 C2 Cliquid Ccolumn CBr CTCE CDCE CVC CETH CH2 (1 Ddis f Kat Kde Mr M dissolved M TCEm Msor'l M TCE MHz LIST OF SYMBOLS Longitudinal dispersivity, m Microbial decay rate, l/day Initial pore volume concentration of TCE within small column, ug/L Aqueous solute concentration at start of small column exchange, pg/L Aqueous solute concentration at end of small column exchange, ug/L Concentration of TCE within pore volume of small column, ug/L Initial pore volume concentration of TCE within small column, ug/L Concentration of bromide within pore volume of large column, ug/ L Concentration of TCE within pore volume of large column, ug/L Concentration of DCE within pore volume of large column, ug/L Concentration of VC within pore volume of large column, pg/L Concentration of ethene within pore volume of large column, ug/L Concentration of hydrogen within pore volume of large column, ug/L Soil particle diameter, cm Distribution coefficient, cmZ/day Fraction of exchange sites at equilibrium Microbial attachment rate, l/day Microbial detachment rate, 1/day TCE half-saturation coefficient, mg/L Hydrogen half-saturation coefficient, mg/L Distribution coefficient, L/mg TCE distribution coefficient, L/mg DCE distribution coefficient, L/mg VC distribution coefficient, L/mg Ethene distribution coefficient, L/mg Hydrogen distribution coefficient, L/mg TCE reaction rate, ng'lday'l DCE reaction rate, ng'lday‘1 VC reaction rate, ng' day’l Ethene reaction rate, ng'lday‘l TCE kinetic (de) sorption rate, l/day DCE kinetic (de) sorption rate, l/day VC kinetic (de) sorption rate, 1/day Ethene kinetic (de) sorption rate, 1/day Characteristic length of a control volume, cm Total mass of solute, g Mass of solute retained on solids, g Mass of solute dissolved in pore fluids, g Mass of TCE within column, g Mass of soil within column, g Monod-type saturation term for TCE Monod-type saturation term for hydrogen, g xiii Vcolumn Vpore V Vconram. v9},— U X Xm Xi YTCE-DCE YDCE- VC YVC-Em yTCE-X yHZX W AW x Bacterial growth coefficient, day'l Maximum specific growth rate, l/day Model parameter Change in model variable Reynolds coefficient Dimensionless equilibrium partitioning coefficient Retardation factor TCE retardation coefficient DCE retardation coefficient VC retardation coefficient Ethene retardation coefficient H2 retardation coefficient Soil bulk density, mg/L Solid-phase TCE concentration, mg/mg Solid-phase DCE concentration, mg/mg Solid-phase VC concentration, mg/mg Solid-phase ethene concentration, mg/mg Sediment porosity Time, days Variation coefficient Empty bed volume of small column, mL Pore volume within small column, mL Superficial velocity of groundwater flow, cm/min Average linear velocity of solute transport, cm/hr Average linear velocity of groundwater flow, cm/hr Kinematic viscocity, cmZ/hr Total biomass concentration, mg/L Biomass concentration in aqueous phase, mg/L Biomass concentration on the solid phase, mg/L TCE/DCE stoichiometeric constant TCE/DCE stoichiometeric constant DCE/ethene stoichiometeric constant TCE yield Hydrogen yield Model variable Change in mode] variable Distance along one-dimensional length of column, cm xiv Chapter 1 INTRODUCTION 1.1 Introduction The experimental work summarized herein is directed at understanding relationships within and between, and the extent of biodegradation associated with, mobile- and stationary-phase microbiological communities resulting from bioaugmentation of an aquifer system contaminated with trichloroethene and hexavalent chromium. Knowledge gained through these efforts could be of significant benefit to the design and maintenance of bioremediation strategies for treating chlorinated solvent- irnpacted groundwater with non-native enrichment cultures. The research has been conducted as part of a comprehensive bioremediation technology development program funded by the State of Michigan, and to partially fulfill the requirements for the degree of Doctor of Philosophy in Environmental Engineering at Michigan State University. 1.2 Background The Village of Schoolcrafi is a rural community of some 1,200 persons located approximately 10 miles south of the City of Kalamazoo in southwestern Michigan (Figure 1.1). The area is underlain by a regionally extensive, glacial outwash plain that forms a large shallow aquifer system. The aquifer represents a valuable regional resource used extensively for domestic, industrial, and agricultural purposes. Since 1979, Schoolcrafi has been the focal point of a series of State-sponsored investigations into the causes and distributions of several occurrences of groundwater contamination. Through 1995, a total of seven distinct plumes of impacted groundwater had been identified and delineated during these investigations. The plumes have been designated A through G by the State of Michigan (Figure 1.1). Of the seven plumes, five (Pltunes A, C, D, E and G) have resulted from releases of chlorinated solvents into the environment. With the exception of Plume A, all of these plumes include trichloroethene (TCE) as the primary contaminant of concern. TCE is a suspected human carcinogen, based on studies performed with laboratory animals. It has also been linked to mutagenic effects in humans (Mallinckrodt Baker, Inc., 2000). Due to its widespread occurrence as an environmental pollutant and its potential threat to public health, TCE was ranked 15th of the 275 compounds comprising the 2001 CERCLA Priority List of Hazardous Substances (ASTDR, 2001). Consequently, there is a critical need to understand the processes that control the fate of TCE in the environment and to develop effective strategies to manage or remediate TCE- impacted environmental media. 1.2.1 Plume G Plume G is distinctly the largest and most severe example of TCE-impacted groundwater in Schoolcrafi. The plume originated at a now-defunct flexible rubber parts fabricating facility positioned in the center of the Village. From its source, Plume G extends downgradient approximately 1.5 miles, is nearly a quarter mile in width, and in some locales penetrates the entire saturated thickness of the aquifer (e. g., 7 O to 90 feet). Chromium \ Fixing ”it“ Zone “xi. i \ Current Testing System Figure 1.1. Central Schoolcraft, Michigan and groundwater contaminant Plumes F and G 1.2.2 Plume F Plume F is the only known occurrence of groundwater contamination by toxic metals in Schoolcrafi. Its source is a former wood pressure treatment operation at which extensive quantities of chromated copper arsenate solutions were released to the subsurface, resulting in hexavalent chromium and arsenic contaminated groundwater. The site of the facility at which Plume F originated is located hydraulically downgradient of the source of Plume G. Consequently, Plumes F and G are commingled and are henceforth referred to as Plume F/G. Soluble ionic species of hexavalent chromium, including chromate and dichromate, are extremely toxic and exhibit mutagenic and carcinogenic effects on biological systems due to their strong oxidizing nature (McLean and Beveridge, 2001). These ions are the dominant species of chromium in most aqueous environments. However, under suitably reducing conditions, hexavalent chromium {Cr(VD} may be reduced to trivalent chromium {Cr(III)}. Cr(III) is less toxic and bioavailable than Cr(V I), as it readily forms insoluble oxides and hydroxides in aqueous systems above pH 5 (Rai, et. al, 1987). 1.2.3 Plume FIG Bioremediation Research Program The commingled nature of Plumes F and G represents a unique challenge to the regulators responsible for the management and remediation of these impacts. In fact, since 1990 the State of Michigan has struggled to develop a technically- and cost- effective strategy for the combined clean up of these plumes. Remediation of groundwater impacted with TCE by conventional means, such as through pore fluid extraction and ex-situ treatment by air stripping or activated carbon, in most cases, has proved to be excessively costly and of limited effectiveness. TCE has the tendency to sorb to soil particles owing to its low aqueous solubility and relatively high octanol-water partitioning behavior. Consequently, the ratio of TCE mass per unit volume of groundwater extracted for treatment is inefficiently low. Due to these phenomena, alternative remedial strategies for TCE-impacted groundwater are desired. Favorable alternative remedies include those that emphasize in-place (in-situ) treatment. To expand the potential for development of an innovative solution, the State has contracted with researchers at MSU to formulate and evaluate bioremediation strategies for Plume F/G. Natural bioattenuation strategies and biological treatment processes such as bioenhancement and bioaugrnentation offer the potential for transforming TCE to non- toxic byproducts in-situ, eliminating the need to remove contaminated media from the subsurface for treatment. Accordingly, MSU is engaged in conducting a series of bioenhancement and bioaugmentation experiments designed to completely reductively dehalogenate TCE and create reducing conditions favorable for the in-situ conversion of Cr(VI) to Cr(III). 1.2.4 Previous Bioremediation Research Research conducted over the past 20 years confirms that a variety of soil microorganisms possess enzyme systems capable of directly metabolizing or co- metabolically dehalogenating chlorinated solvents such as TCE (Lee, et. al, 1998). Vogel and McCarty (1985) performed one of the first studies that revealed the potential for mineralization of perchloroethene (PCE) in soil and aquifer systems under anaerobic conditions. They conducted column experiments using both glass beads and quartzite rock to demonstrate step-wise dechlorination of PCB to TCE, cis-1,2-dichloroethene (cis- DCE), and vinyl chloride (V C), respectively, under methanogenic conditions. VC conversion to C02 was also observed, suggesting that complete detoxification of PCB is possible under these conditions. Barrio-Lage, et al., (1986 and 1987), later demonstrated the conversion of TCE to cis-DCE in a variety of sediments maintained under anoxic conditions in batch microcosms. Their study offered clear evidence that soil microorganisms from a variety of environmental settings may reductively dechlorinate TCE. DeBruin, et. al. (1992) observed complete reductive dechlorination of PCB to ethene and ethane in Rhine River sediments fed anaerobic granular sludge within a flow- through column system. The implication of their work was that mixed cultures of microorganisms could be introduced into non-native sediments to effect in-situ remediation of PCB (e.g., bioaugrnentation). Since these studies, work by numerous other researchers has served to establish bioenhancement and bioaugrnentation as viable treatment strategies for many sites with solvent-impacted groundwater. Over the past ten years researchers have demonstrated the ability to use aquifer materials from solvent-contaminated sites to develop enrichment cultures capable of accelerated and/or complete transformation of PCB and TCE. In some cases the strain(s) responsible for the reductive dechlorination have been isolated and characterized. Holliger, et al. (1993) isolated a Dehalobacter restrictus species (PER K23) from anaerobic packed-bed column materials that is capable of coupling the reductive dechlorination of TCE to cis-DCE to growth. Hydrogen and forrnate were the only electron donors that could support growth with TCE as an electron acceptor. Wild, et al. (1997) identified another D. restrictus species from a mixed culture that completely reduces chlorinated ethenes to ethene. This organism, strain TEA, also coupled reductive dehalogenation of TCE to cis-DCE to growth using molecular hydrogen as the electron donor. Neumann, et al. (1994) discovered Dehalospirillum multivorans, a strictly anaerobic bacterium that is able to dechlorinate PCE to TCE and cis-DCE as part of its energy metabolism. Studies with D. multivorans by Miller, et al. (1996) identified a cytoplasm-associated dehalogenase that facilitates the transfer of electrons from hydrogen or forrnate to PCE, resulting in a reduction to TCE. Gerriste, et al. (1999) isolated Desulfitobacteriumfrappieri TCEl from a PCE-dechlorinating soil slurry. Like D. restrictus and D. multivorans, TCEl couples the dehalogenation of TCE to cis-DCE to growth. Sung, et al. (2003) describes an acetate-oxidizing, PCE to cis-DCE dechlorinating Desulfilromonas species (Desulfuromonas michiganensis strain BRS 1) that was cultivated from contaminated aquifer materials from eastern Michigan. Maymo- Gatell, et al., (1997) described the isolation of a bacteritun, referred to as strain 195, that dechlorinates PCE the ethene. To date, strain 195, a Dehalococcoides species, is the only known bacterium that is able to completely dechlorinate PCB and TCE to ethene. Dehalococcoides populations that couple hydrogen oxidation with VC respiration have also been identified (He, et al., 2003). However, in virtually all of these cases, the reductive dechlorination activity expressed by the isolate appears to depend directly or indirectly on mutualistic interactions with other members of the community from which it was isolated (Bouwer, 1995). For example, the PCB transformation activity of strain 195 (Dehalococcoides ethenogenes) is inferred to depend on a supply of vitamin B12 in culture media. D. ethenogenes does not produce vitamin B12 and, therefore, must depend on other microorganisms for a supply of this co-factor in the environment. A recent study by Drzyzga and Gottschal (2002) revealed that TCE dehalogenation by TCEI appears to depend on the activity of a sulfate-reducing bacterium. Specifically, sulfate reducers capable of fatty acid fermentation produce hydrogen and grow in syntropic association with TCE] . This study suggests that bacterial dehalogenation and/or halorespiration of TCE may be indirectly stimulated by the activity of fermentative sulfate reducers under sulfate-depleted conditions in soil environments. Recent studies reveal that dehalogenating and halorespiring microorganisms may exist within a variety of soil types and conditions. Hendrickson, et al. (2002) conducted a study on the environmental distribution of the Dehalococcoides group of organisms in association with chloroethane-contaminated sites across North America and Europe. They used PCR assays to detect Dehalococcoides 16S rRNA gene sequences and demonstrated that members of this group are widely distributed in nature and can be found within a variety of geologic formations and in different climatic zones. For these reasons, the natural biodegradation of chlorinated solvents, including TCE, is suspected at many sites. In some cases, the rates and completeness of compound transformation is deemed sufficient to allow these natural processes to clean up the site without direct human intervention. Conversely, at other sites, natural degradation is slow and/or incomplete. At such sites, natural attenuation processes are not favorable to remediate chlorinated solvents without anthropogenic enhancement or augmentation. For these sites, bioenhancement or bioaugrnentation techniques may be employed to accelerate the natural rates and extent of dehalogenation such that bioremediation may be considered an effective treatment alternative. Bioenhancement strategies often require a significant investment in time, effort, and associated cost to accelerate treatment rates or enhance the activity of certain microbes and ensure that the pathway of TCE transformation is complete. Alternatively, mixed cultures of native soil microorganisms enriched for dechlorinating activity may be introduced into aquifer systems to augment the native dechlorinating microflora and thereby promote more rapid and complete treatment. The acclimation of enrichment cultures introduced into non-native sediments during bioaugnentation may be accelerated if environmental conditions within the host medium are first adjusted to favor the growth and activity of the dehalogenators. This may be accomplished through pH- adjustment, or provision of a grth substrate preferred by the enrichment. Most experimental work to evaluate the dechlorination behavior of mixed and pure cultures in native and non-native aquifer materials has been performed in laboratory microcosms in which environmental and nutritional conditions are strictly controlled. Work by Isalou and Sleep (1998), Harkness, et. a1. (1999), and Cirpka, et. al. (1999), examined dehalogenation activities expressed by non-native mixed enrichment cultures and halorespiring isolates in flow-through systems packed with aquifer materials and maintained under natural conditions. These studies emphasized the spatial measurement of electron donors and acceptors, chlorinated solvent dehalogenation and metabolic byproducts associated with the activity of the augmented microbial community. However, with the exception of work done in fi'actured and granulated basalts by Lehman, eta]. (2001), few studies have attempted to evaluate the transport behavior of inoculates during delivery, or correlate the spatial variability of aquifer geochemistry and contaminant transformation with the structures of mobile- and stationary-phase microbial communities resulting from bioaugnentation. Such evaluations could significantly advance the current understanding of the successional adaptations of in-situ treatment regimes following inoculation of aquifer materials. Further, the knowledge gained from this experimentation could benefit strategies for optimizing biotransformation capacities within bioaugnentation treatment zones. 1.3 Plume FIG Bioaugmentation Studies The bioaugnentation research being conducted at MSU for the Plume F /G project is focused on assessing the efficacy of introducing a mixed culture of non-native microorganisms enriched for PCE dechlorination activity into site aquifer materials to effect complete dehalogenation of TCE. It is postulated that a concomitant decrease in oxidation-reduction potential will result sufficient to cause the reduction of Cr(VI) to Cr(III). The initial phase of the assessment entailed the completion of small-scale, semi- batch column microcosm studies desigred to compare the effects of bioaugnentation against bioenhancement and sorption controls. This phase was followed by experiments using large-scale, continuous-flow columns to examine transient and spatial TCE transformation and successional adaptations of mobile- and stationary-phase microbial communities following inoculation of the Plume F/G aquifer materials. 1.3.1 The BR Enrichment Culture The enrichment culture used for the Plume F/G experiments was developed using PCE-impacted aquifer materials from the Bachman Road site in Oscoda, Michigan. Like Plume F/G, the Bachman Road site yielded data indicating that natural attenuation of PCB has been occurring, but not at favorable rates. Further, there are disproportionally higher levels of dichloroethene isomers (primarily cis-DCE) than VC or ethene (ethene) concentrations in the goundwater, suggesting that the pathway of PCB transformation by native aquifer flora is incomplete over much of the site. Activity measurements from batch microcosm studies with sediment and goundwater from a variety of locations and 10 depths within the PCB plume reveled a heterogeneous distribution of dechlorinating populations in the Bachman Road aquifer (Lendvay, et al., 2003). From a series of experiments in which Bachman Road aquifer materials were exposed to an anaerobic culture medium and elevated concentrations of PCB, cis-DCE and electron donor, the activities of the native dehalogenating microorganisms were geatly enhanced. The resulting mixed culture (“the BR Enrichment”) can completely dechlorinate PCE when fed lactate as a primary carbon source. Recent work to characterize the structure of the microbial community in, and cultivate dechlorinating members from, the mixed culture revealed the presence of Desulfuromonas and Dehalococcoides species (Sung, et al., 2003; He, et al., 2003), including strain BRSl and D. ethenogenes. Further, it appears that the activity of D. ethenogenes is directly, if not exclusively, responsible for PCE dechlorination to ethene in the enrichment. The enrichment has been successfully re-introduced into a portion of the impacted Bachman Road aquifer to accelerate PCE detoxification in those sediments. The BR Enrichment and associated PCE transformation activity has been maintained for over three years within a 75 L chemostat operated by EF X Systems of Lansing, Michigan. Similar to the Plume F/G site, the Bachman Road sediments consist of glacial and post-glacial alluvium. The hydraulic characteristics of these materials are compatible with those measured in the Schoolcrafi aquifer. Sediments sampled at the two sites also exhibit similarities with respect to geocherrristry and microbiology. For these reasons, it has been inferred that the BR Enrichment may be effectively introduced into Plume F/G aquifer materials where it will readily acclimate and express similar TCE-transformation characteristics. 11 In the completely—mixed reactor system used for culture gowth, the BR Enrichment is maintained in either municipal water or Plume F/G goundwater. In both instances, water is re-circulated through the reactor so that the residence time of the fluids is extended many-fold. Periodically, PCB and electron donor (lactate) and nutrients (e.g., urea and phosphoric acid) are added to the re-circulating water and the subsequent extent and magnitude of dehalogenation is monitored. It is assumed that dehalogenation is maximized only after culture gowth and metabolically induced redox conditions within the re-circulating fluids have reached appropriate levels. As it acclimates to fresh make- up water or following feeding, the enrichment undergoes successional changes during which the activities of certain key community members respond to progessive changes in the concentrations of electron donors and acceptors. For example, when fresh goundwater is added to the reactor, the activities of nitrate reducers and fermenting organisms are expected to be at their maximum. Over time, as a decrease in the levels of nitrate occurs and the levels of dissolved hydrogen increase fiom fermentation, the activities of iron, manganese and sulfate reducers will dominate. Finally, as the concentrations of the more energetically favorable electron acceptors such as soluble Fe(III) and sulfate begin to diminish, the activity of hydrogen-utilizing methanogens will prevail. Similarly, the magnitude of dechlorination activity will be time-dependent in response to the addition of fresh water or feed to the reactor. Specifically, incomplete dechlorination activity is expected to predominate amongst sulfate-reducing populations and methanogens utilizing organic carbon for reducing power. At this stage, TCE concentrations will give way to an accumulation of cis-DCE. As the partial pressure of 12 dissolved hydrogen increases, the activities of Hz-utilizing halorespiring microorganisms, including D. ethenogenes, is expected to also increase, resulting in a geater occurrence in the conversion of cis-DCE to ethene. The metabolic activity and transformation capacities observed over time in the reactor environment are likely to be manifested over space in a flow-through system, such as downgadient of aquifer injection points. This largely is due to the lack of completely mixed conditions between microflora and goundwater. Therefore, in a model aquifer system fed TCE-impacted goundwater, we would expect to observe a spatial-dependence in community structure that generally correlates with concentrations of electron donors and acceptors along the path of goundwater flow. A monitoring system with spatial resolution downgadient of the point of inoculation would provide a unique opportunity to measure TCE transformation activity as well as geochemical conditions and microbiological diversity along the longitudinal path of contaminant transport. The information derived from such observations and measurements could improve the current understanding of how to establish, maintain, and monitor efficient biologically active treatment zones during the implementation of bioaugnentation strategies. 1.3.2 Summary of Preliminary Results As noted above, an experiment with a series of small-scale column microcosms was completed as part of the MSU bioaugmentation research progam. The details associated with this work are presented in the following chapter (Experimental Design). 13 Comprehensive treatment of the data from the small column experiment is provided in Chapter 3 (Results). General observations of the data set include the following: o A composite profile of biomass breakthrough resulting from the inoculation of triplicate columns is provided on Figure 3.1. This figure reveals that the inoculum traveled through the subject column systems at a rate very close to the average linear velocity of goundwater flow, as the breakthrough of biomass occurred nearly co- incident with that of a conservative tracer. However, the profile also indicates that a significant fraction (e. g., nearly 50%) of the biomass introduced to the columns was apparently retained in the sediments during the inoculation event. 0 As expected, nitrate levels in the Plume F/G goundwater fed to the columns were depleted during the incubation periods following the weekly exchange of pore fluids, suggesting that nitrate reduction by the inoculum was complete during these periods. 0 Conversely, sulfate reduction was minimal, suggesting that the duration of pore fluid contact with the inoculated sediments was not sufficient to permit successional development of predominantly sulfate reducing conditions by the microflora in the small-scale columns. ' Organic carbon (e. g., lactate) concentrations fed to the inoculated columns were consistently reduced by approximately 60% to 90% during the incubation periods between pore fluid exchanges. Given that lactate was the only carbon source added 14 to the feed water, it is inferred that acetate and hydrogen produced through lactate fermentation were also sources of reducing power available to the microflora within the column systems, including BRSl and D. ethenogenes. The analyses of headspace gases revealed that hydrogen levels within the atmosphere of the anaerobic glove box in which the experiments were conducted interfered with the accurate measurement of hydrogen partial pressures resulting from bioactivity within the columns. Specifically, hydrogen concentrations measured within control samples of the glove box atmosphere were consistently geater than levels detected within the samples of the head space of the small columns, rendering these results suspect due to the potential for cross-contamination of sample containers stored within the glove box. Other than hydrogen, methane was the only gas detected in head space samples. However, methane was only measurable in samples collected from bioaugnented columns. In general, the relative amounts of methane observed were geatest within the columns that received an undiluted inoculate suspension. These results suggest that methanogenic conditions developed in at least a portion of each of these columns during the incubation periods between exchanges, even though sulfate levels appeared to persist within the pore fluids. During a ten-week period immediately following inoculation, TCE transformation to cis-DCE and VC was evident. In the columns that received undiluted inoculum, TCE removal was complete during this period. In these columns, TCE appeared to be converted to cis-DCE (approximately 10%) and VC (approximately 50%), suggesting 15 that efficient dechlorination was occurring but was incomplete due to insufficient residence times between pore water exchanges within the columns. No significant TCE removal was observed in radiation-sterilized and bioenhancement control columns during the period parallel with inoculation and subsequent feeding exchanges. Cr(VI) levels in post-incubation period effluent samples were, in general, significantly lower than the concentrations in the Plume F/G goundwater fed to the inoculated controls during each exchange. Increases in nutrient and TCE loadings during the late stages of the small-scale column experiment had little to no observable effect on TCE transformation activity within inoculated columns sets. From these observations, it is inferred that a mixed microbial culture enriched for PCE or TCE dehalogenation activity, such as the BR enrichment, can be introduced into and conveyed through the Plume F/G aquifer materials under an imposed hydraulic gadient. Along the inoculum path of mi gation, a fraction of the introduced biomass will be retained within the sediment, thereby promoting colonization of aquifer solids. The biomass retained in the aquifer will irnitially exhibit spatial segegation of key community members owing to differences in physical transport characteristics including sorption, straining and dispersion. Over time, colonization of the aquifer solids by the enrichment l6 culture is expected to result in the establishment of a permeable stationary-phase biologically active zone (BAZ) across the width of which complete reductive dechlorination of TCE will occur when provided appropriate sources and dosages of organic carbon and nutrients. During a period of acclimation leading to near steady-state treatment conditions within the BAZ, a more pronounced spatial dependency of the stationary—phase community structure is expected to result from successive depletion of electron acceptors and consumption and/or evolution of electron donors in pore fluids passing through the BAZ. It is postulated that the near steady-state community profile within the BAZ will exhibit spatially- or temporally-discrete intervals of sulfate- reduction, methanogenesis, and halorespiration. Resolution of the spatial relationships between these regions and redox gadients will provide valuable insight into how the microbial community structure within the BAZ can be maintained to optimize TCE dechlorination. For example, there may be a benefit in controlling the influx of lactate conveyed through the BAZ to limit stimulation of non- dehalogenating organisms and maintain dissolved hydrogen concentrations at or near threshold levels associated with TCE halorespiration. Conversely, a higher input of orgarnic carbon could stimulate rapid TCE reduction to cis—DCE through partial dehalogenation by acetate-oxidizers, sulfate reducers and methanogens. The predominance of cis-DCE could serve to increase the rates of transformation to ethene, as downgadient halorespiring organisms, such as D. ethenogenes, can dechlorinate cis-DCE to ethene more rapidly alter TCE is depleted. It is further postulated that goundwater passing through the treatment zone will become progessively reduced due to the increase in anaerobic metabolic diversity and 17 activity. The decrease in oxidation-reduction potential is expected to become sufficient to favor the reduction of Cr(VI) to Cr(III). Due to its insoluble nature, immobilization of Cr(III) will ensue, potentially resulting in a reduction in the concentrations of total dissolved and hexavalent chromium to be below State-mandated cleanup levels. 1.4 Hypotheses and Research Objectives A goal of the Plume F/G bioaugnentation research is to explore issues such as those summarized above and thereby improve the understanding of the structure of microbial diversity within biological treatment zones resulting from inoculation of aquifer systems with non-native enrichment cultures. Accordingly, through completion of the studies summarized herein, the following hypotheses were tested: 1. The inoculation of the Plume F/G aquifer materials with the BR Enrichment will produce a spatially dependent, stationary-phase microbial community profile. 2. Within the community profile, a biologically active zone (BAZ) consisting of spatially and/or temporally discrete regions of sulfate-reduction, methanogenesis, and halorespiration will exist wherein transformation of TCE to non-toxic end products will occur. 3. Within the BAZ, the goundwater redox chemistry will favor for the reduction of Cr(V I) to and immobilization of Cr(III). 18 To prove these hypotheses, it was necessary to: 1. Define the general transport characteristics of the BR Enrichment within the Plume F/G aquifer materials. 2. Verify that the completeness of TCE reductive dechlorination associated with inoculation of the Plume F/G aquifer materials is consistent with that achieved by the BR Enrichment under controlled laboratory conditions. 3. Establish a range of substrate and nutrient loadings that are practical for enhancing the reductive dechlorination activity expressed by the BR Enrichment within the Plume F/G aquifer materials. 4. Estimate the dimensions of the BAZ for reductive dechlorination activity established by the BR Enrichment in the Plume F/G aquifer materials under enhanced delivery and maintenance conditions. 5. Characterize the profile of the augnented microbial community structure across the widtln of the BAZ at specific time intervals leading up to and including the period when near steady-state treatment conditions were established. 6. Identify the portion of the BAZ and associated redox conditions in which halorespiration predominates. l9 7. Estimate the rates of reductive dechlorination expressed within the BAZ between feeding events. 8. Identify correlations between: a) nutrient and redox gadients and the extent of reductive dechlorination, and b) the microbial community structure across the steady- state BAZ. 9. Evaluate the occurrence and extent of in-situ chromium detoxification and immobilization associated with the development of reducing conditions within the BAZ. 20 Chapter 2 EXPERIMENTAL DESIGN 2.1 Introduction Based on the observations and the objectives summarized in Chapter 1, a research progam, consisting of the following experiments and associated tasks, was undertaken: l. Small-Scale Column Microcosm Experiment Task 1.1 - Characterization of F low-Through Properties Task 1.2 - TCE Mass Loading and Sorption Capacity Evaluation Task 1.3 - Treatment Strategy Evaluation Subtask 3.1 - Baseline Maintenance Exchange Events Subtask 3.2 - Optimization Exchange Events Task 1.4 - Post-Treatment Characterization 2. Large-Scale Column Experiment Task 2.1 - TCE Mass Loading and Sorption Capacity Evaluation Task 2.2 - Inoculation/Characterization of Flow-Through Properties Task 2.3 - Treatment Task 2.4 - Post-Treatment Characterization 3. Measurement of TCE TraLsformation Kinetics 21 4. TCE Transport Modeling The small-scale colnunn experiment was conducted between July of 2001 and April of 2002. The Large-scale column experiment began in June of 2002 and concluded in late October of 2002. Measurements of TCE transformation kinetics associated with the BR Enrichment and fate and transport modeling activities were performed concurrent with and subsequent to the large-scale column experiment, respectively. Ms. Leslie Dybas and Baolin Sun performed DNA analyses associated with the small- and large- scale experiments. DNA extraction from environmental samples and all other analyses were performed by the author. Retec Group, Inc. was responsible for completion of the TCE transformation kinetics study. Dr. Mantha Phanikumar assisted with the reactive transport model development. 2.2 Small-Scale Column Microcosm Experiment Between July of 2001 and April of 2002, a four-phase experiment was performed using small-scale soil column microcosms. The objectives of the experiment were to verify that the BR Enrichment could be effectively delivered through Plume F/G sediments under an imposed hydraulic gadient, and to temporally measure the levels of dechlorination in inoculated columns as well as sterile and non-sterile controls (e.g., Objective #3 1, 2, and 3 from Chapter 1). Further, the fate of Cr(VI) was examined to verify that reduction to Cr(III) was occurring in inoculated coltunns (Objective #9). 22 A total of 21 columns were operated during this experiment. These were gouped into seven (7) distinct sets, each consisting of three (3)—columns. The seven column sets were organized into the following categories: i. Radiation-sterilized Columns (Columns A, B, and C) 2. Natural Attenuation Columns I (Columns D, E, and F) 3. Natural Attenuation Columns 11 (Columns G, H, and I) 4. Inoculated Columns I (1% inoculum concentration; Columns J, K, and L) 5. Inoculated Columns II (10% inoculum concentration; Columns M, N, and O) 6. Inoculated Columns III (100% inoculum concentration; Columns P, Q, and R) 7. Biostimulation Columns (Columns S, T, and U) 2.2.] Column Design and Assembly The design and operation of the small-scale column microcosms were consistent with those described by Dolan and McCarty (1996) and Mayotte, et al. (1996). A :chematic of an example column is provided on Figure 2.1. Columns consisted of 85 L borosilicate glass test tubes (Pyrex). Each column was filled partially with site nundwater prior to assembly to ensure that internals (e.g., soil and plumbing) were rrted under saturated conditions. The base of each column was then packed with a offinsed silica wool (Restek Corporation Cat. # 20790) over which a disk of glass paper (VVhatman Glass MicroFibre) was carefully placed. Plume F/G aquifer .al was deposited over the support medium in 1 to 3 cm thick lifts by allowing soil [es to gravity settle through the goundwater. After a lift had settled, the bottom of 23 each column was tapped on a solid surface to dynamically compact the soil. The final soil bed depth within each column measured approximately 10 cm, resulting in a packed soil volume of about 58-60 mL. Once packed, each column was fitted with internal and external plumbing. Effluent Collection Stainless Steel End Cap % F lange-F ree Fittings Influent Addition —’ Rubber Stopper ~i F Stainless Steel Needle ‘ Aquifer Material F33: :33; Stainless Steel Tubing .; .33.] 3:2? 23:3 43%? '«3‘. Glass Wool Figure 2.1. Small-Scale Column Microcosm (Dolan and McCarty, 1996). First, an effluent line (1/ 16-inch stainless steel tubing, Alltech Associates, Inc.) .shed through the soil and into the support medium at the base of each column. A of 0.027 -in. piano wire (Small Parts, Inc.) inserted inside the tubing prevented soil 24 coring and plugging of the line during placement. The wire was removed immediately after the terminal end of the tubing had been positioned within the support medium at the base of the column. A butyl-rubber stopper (No. 4) was then used to fix the tubing in- place and seal the contents of the column. To further secure the seal, electrical tape was wrapped around the outside of the stopper and column. To facilitate the delivery of groundwater into a column, a two-inch long, 18-gauge stainless steel needle (VWR Scientific Products) with a luer lock coupling was also inserted through the rubber stopper and positioned at a depth approximately 2 cm above the water level over the soil. Groundwater was delivered at a fixed volumetric flux through 100 mL capacity, gas- tight, glass syringes (SGE, Inc.) and the stainless steel tubing (1/ 16-inch inside dia.) and the influent needles. The controlled delivery of goundwater was accomplished using Harvard syringes pumps (Harvard Apparatus Model #22), which could be progarnmed to ompress the plungers in the glass syringes at a constant rate. External plumbing onnections consisted of leak-tight stainless steel compression fittings (Swagelok). .ssembled columns were placed in triplicate sets within customized wood boxes to inimize disturbance of internals during operations. All column components were autoclaved under 20 lbs/in2 (psi) of pressure at 9 °C for a minimum of 25 minutes prior to column assembly. Column assembly 'vities were performed within an anaerobic glove box (Coy Laboratory Products, Inc.) er an atmosphere of approximately 95% N2 and 5% H2 and at a temperature of 18 °C. trol columns A, B, and C were radiation sterilized using a cobalt-6O energy source ting approximately 1.2 nrillirads per hour for 1.7 hours. Radiation sterilization was rmed at the Phoenix Memorial Laboratory at the University of Michigan in Ann 25 Arbor. Assembled columns were maintained under anoxic conditions at 18 °C within the glove box throughout the duration of the experiment. 2.2.2 Column Operation and Monitoring Columns were operated by periodic semi-batch exchange of soil pore fluids. Specifically, this entailed a series pore fluid exchanges performed once weekly. Each exchange involved a rapid transfer of pore fluids (Plume F/G goundwater spiked with TCE and amendments, as necessary) within each column, followed by a week-long incubation (no flow) period. After incubation, the pore fluids were re-exchanged. The small-scale column microcosm experiment consisted of four (4) tasks, as described below. In total, approximately 35 weeks of pore-fluid exchanges were aerformed during this experiment. ‘ask 1.1 - Characterization of Flow-Through Properties Task 1.1 entailed determining the flow-through characteristics of the soil within ch coltunn. Specifically, the flow-through characteristics were estimated by changing goundwater spiked with 50 mg/L of sodium bromide (NaBr) through each umn and tracking the effluent concentrations of the tracer by ion chromatogaphy. : breakthrough profile of bromide thus obtained revealed the approximate internal : volume of the packed soil in each column. Similarly, the breakthrough of TCE ally conveyed through a subset of columns was used to estimate the “apparent” nal pore volume, which is a crude measure of the magnitude of the combined effects rption and dispersion relative to the transport of the conservative tracer. 26 Measurement of both parameters was necessary to calculate the volume of TCE-spiked groundwater necessary to ensure complete contaminant breakthrough during an exchange event. In addition to TCE and bromide, the pore fluids at both the onset and conclusion of each exchange were analyzed by ion chromatogaphy to measure acetate, chloride, nitrite, nitrate, phosphate and sulfate levels and thereby establish the baseline geochemical conditions within the columns prior to inoculation with the BR Enrichment. An additional component of Task 1.3 was the tracking of biomass breakthrough during inoculation. Accordingly, a triplicate set of biomass breakthrough profiles was obtained by measuring the optical density of effluent fractions during the inoculation of a set of three columns with a non-diluted suspension of BR Enrichment. These data were used for estimating the inoculum transport characteristics and quantity of biomass retained within the columns during inoculation. Biomass concentrated from replicate samples were used to track the breakthrough of D. ethenogenes using RT-PCR. 'ask 1.2 - TCE Mass Loading and Sorption Capacity Evaluation Task 1.2 included a series of exchange events during which each soil column was ised with approximately 1,000 ug/L of TCE until solid and aqueous phase ncentrations of the solute appeared to be near equilibrium. :k 1.3 - Treatment Strategy Evaluation Task 1.3 entailed inoculation and subsequent pore fluid exchanges in BR-enriched :ontrol columns to evaluate TCE transformation as well as mobile-phase biomass ransient geochemical gadients resulting from the bioaugnentation. During this 27 phase, the weekly exchange of pore fluids, including the input of TCE (500-1 ,000 pg/L), organic carbon, and nutrients, were performed as follows: 0 All columns (A through U) received the specified dosage of TCE on a weekly basis; 0 Every two weeks, sterile (e. g., sorption) controls (Columns A, B, and C) received organic carbon and nutrients; o Inoculated columns (J, K, L, M, N, O, P, Q, R) received organic carbon and nutrients bi-weekly; o Bioenhancement controls (Columns S, T, and U) received organic carbon and nutrients bi-weekly; Back-up columns, D, E, F, G, H, and I, received the weekly dosage of TCE only. Bi-weekly feeding entailed loading the relevant columns with organic carbon (240 II DL-lactic acid), as well as nitrogen (15.4 p.M NH4C1) and phosphorus (2 uM [2P04). During the final three weeks of Task 1.3, a series of exchanges were conducted n g which the influent concentrations of TCE and feed to the inoculated and control mns were adjusted to evaluate the associated effects on inoculum performance task 3.2). Specifically, the TCE dosage to columns C, R and U was increased 10- 28 1d, and the feeding of columns B, Q, and T was increased to weekly. Conversely, no terations were made to the TCE loading or feeding protocols for columns A, P, and S uring this period. Analytes measured in samples collected during the majority of the exchange :vents conducted as part of Task 1.3 are summarized on Table 2.1. Task 1.4 - Post-Treatment Characterization Task 1.4 entailed the disassembly of the inoculated and biostimulated control sets. During these activities, soil within the drained columns was collected for measurement of biomass levels and characterization of the solid-phase microbial community structure by PicoGreen® DNA Quantification, and T-RFLP and RT-PCR analyses, respectively. Table 2.1. Summary of Aqueous Sample Analyses — Small-Scale Column Microcosm Experiment. Aqueous Solutes Headspace Gasses Biomass Gas Chromatogaphy (electron Reduced Gas Analysis Optical Density captnue detection)/Mass Hydrogen Spectrometry PicoGreenO dsDNA Quantification PCE Gas Chromatogaphy TCE (flame ion detection) Polymerase Chain Reaction cis-DCE Methane (PCR)-based Terminal Restriction trans-DCE Ethene Fragment Length Polymorphism DCE (T-RF LP) VC Real-Time (S YBR Green) PCR Ion Chromatogaphy Acetate Chloride Nitrite Bromide Nitrate Phosphate Sulfate Atomic Absorption Hexavalent chrorrnium 29 'perational and Sampling Procedures During a pore fluid exchange, 100 mL of amended goundwater was conveyed 1rough each column at a rate of 1.00 mL/min. Effluent samples were collected in 2 mL 'ractions of the irnitial 12-16 mL and final 70-100 mL of goundwater exchanged. Triplicate samples from these volumetric ranges were collected in: 1) volatile orgarnic analysis (VOA) vials (40 mL capacity), containing 3 mL of sodium bisulfate (2%) as preservative; and 2) in 15 mL, sterile plastic centrifuge vials (Corning). During exchange events that included feeding amendments, two sets of centrifuge vials were filled. Following sampling, VOA vials were stored at 4°C pending analysis by GC/MS. The contents of the centrifuge vials were filtered through 0.2 urn syringe filters (Titan Filtration Systems) and decanted into l-mL glass or polyethylene ion chromatogaphy vials (Alcott) and stored at -20°C pending analysis for the major anions listed above. During exchange events that entailed feeding or targeted biomass analyses, the 2 mL volume within the each centrifuge vial was split, with approximately 1 mL of the sample being transferred to a 1.5 mL vial (Eppendorf) for subsequent centrifugation at 1,400 rpm for 20 min (Eppendorf Centrifuge 5415 C) to concentrate planktonic biomass for DNA extraction and the molecular procedures listed above. The remaining sample 1 mL was processed for ion chromatogaphy analysis, as described above. The 2 mL of groundwater collected within the second centrifuge vial was filtered, as previously described. Approximately 1 mL of the filtrate was then transferred to a 1.5 mL glass vial (SUN Catalog #200 000) and sealed with a Teflon-lined butyl rubber septa secured with a snap cap (Alltech). These samples were stored at —20 °C pending analysis for volatile fatty acids by high performance liquid chromatogaphy (HPLC). To facilitate the 30 teasurement of soluble chromium, the residual 1 mL volume of the filtrate was delivered n a 1 .5 mL Eppendorf vial that was pre-preserved with 20 uL of concentrated nitric acid Fisher Scientific Products). The preserved samples were stored at 4 °C pending analysis 'or soluble chromium by atomic adsorption. Periodically, samples of the headspace gasses in each column were collected by withdrawing triplicate 1 mL aliquots of the atmosphere above the soil within the vessels. These samples were acquired using sterile, 1 mL or 3 mL plastic syringes (Becton Dickinson) with luer tips. By detaching the influent conveyance plumbing fi'om a goundwater delivery syringe, the sampling syringes could be affixed to facilitate the extraction of headspaces gas by suction. Immediately following sampling, the syringes were fit with sterile, 22-gauge disposable needles (Becton Dickinson), which were then inserted into oversized butyl rubber stoppers to seal the contents of each syringe prior to analysis. Headspace samples were immediately analyzed for the presence of hydrogen, methane and ethene, as prescribed above. Theoretically, the average concentration of the target analytes measured in a triplicate set of goundwater samples obtained from the initial 12-16 mL of goundwater exiting each column represented the solute concentrations in the pore fluids at the conclusion of the week-long incubation period. Conversely, the average solute concentrations measured in triplicate samples of the final 70-100 mL of goundwater exiting each column at the conclusion of an exchange represented the initial conditions of the subsequent incubation period. By comparing the initial conditions of an exchange with the post-incubation concentrations measured during the following exchange, the change in solute mass during the incubation period can be estimated. From these 31 comparisons over the duration of the small-scale column experiment, mass balances were calculated for select analytes. Data Reduction In order to evaluate the fate of TCE within the three inoculated column sets (J -L, M-0, and P-R) and the natural attenuation and stimulated controls (D-1 and S-U, respectively), mass balances were developed using effluent concentrations measured during each exchange. The protocol for examirning the conservation of the mass of each of these solutes was summarized by Mayotte, et al. (1996). Similar mass balances were established for cis—DCE and VC within Columns P, Q, and R, which were inoculated with non-diluted BR Enrichment. During the initial exchange of fluids in each column, the breakthrough of both bromide and CT were evaluated. For this first exchange, C, = 0. Using the bromide data, the porosity, 0, of the aquifer media within each column was calculated by: 9 = M ( I = V pore (C2 ' CI) Vcolumn Vcolumn (2.1) when C2 = C0 = Ccozm, and M = Ccolm Vcolmd = C2 me. Unlike bromide, TCE sorbed to the aquifer solids during exchange. In addition, TCE transport appeared slightly more dispersed. These two mecharnisms were reflected in the shape of the TCE breakthrough profile, which, upon inspection, lagged behind and sloped less than the profile for bromide (Figure 3.1). 32 Mass removal between exchange events was assumed to occur as the result of either sorption or biotransformation. Thus to determine the cumulative mass removed between exchange events it was only necessary to know C2 resulting at the end of an exchange event (H) and C; measured at the beginning of the following exchange event (i). These data enabled the estimation of cumulative mass removal using: ’1 . . Mr = Vcolumng .Zl(Cf2-l ' Cal)- (22) t: Prior to inoculation, cumulative mass removal data were used to quantify sorption. Sorption was estimated by comparing the total mass of TCE that had been removed to the liquid phase concentration of TCE present in the pore fluid once the solids were saturated. This ratio is defined as the dimensionless equilibrium partition coefficient, RP, where: (2.3) V columno _ l "( i] i) C - ’C M, _ =1 2 ’ . M diSSOIVCd Vcolumn 6 Cl] R p = With knowledge of RP, the retardation factor, R,, and distribution coefficient, K; (cm3/g), for TCE in the Plume F/G aquifer materials could be estimated from: v 2.4 Rt: efl =Rp+1 ( ) Vcontaminant and 33 (2.5) where veg = average linear velocity of goundwater flow through the aquifer solids within the column (cm/hr); vwmam, = the average linear velocity of the solute front in the aquifer solids within the column (cm/hr); p2, = soil bulk density (g/cm3). Kinetics of Biotransformation Assuming a first-order kinetic expression and equilibrium between the sorbed and solid phases, the following mass balance was used to describe TCE removal attributable to transformation by the Bachman Road Enrichment: dMTCEm _ r r MICE It Cliqurd column (Vliqui l Kd M3011) 1'4““! (MTCEm)= _( k'Vcolumng )t (2,7) M TCE Vcolumng + K d M soil where k' = first order rate coefficient (day'l); C12,,“ = concentration of TCE in liquid-filled volume of the colrunn (rag/L); M205," = mass of TCE within column at (i -1) and i (pg); Mm, = mass of soil in column (g); t = time interval between (i - 1) and i (days). 34 2.3 Large-Scale Column Experiments In contrast to small-scale columns receiving semi-batch pore fluid exchanges, experimentation with large-scale sediment columns delivered continuous flow of goundwater is believed to be more conducive for both the development and examination of a BAZ and associated redox and TCE transformation gadients resulting from bioaugnentation. Therefore, laboratory-scale evaluations of spatial and temporal redox gadients and mobile- and stationary-phase microbial diversity during and subsequent to inoculation of Plume F/G aquifer materials with the BR Enrichment were accomplished using large-scale continuous flow column systems. Large-scale continuous flow column systems were assembled using Plume F /G sediments and goundwater. The expanded scale of these columns enabled more detailed examination of the transport characteristics of the BR Enrichment and D. ethenogenes in site aquifer materials. Further, the larger columns facilitated observations and measurements of mobile and stationary phase contaminant, geochemical, and microbiological profiles both prior to inoculation and during the period leading up to and including steady-state treatment. Other researchers have performed large-scale column studies in attempts to evaluate the spatial relationships between donor and acceptor consumption and the extent of reductive dechlorination. However, to date no single study has been designed to: 1) measure or characterize the successional adaptations of microbial communities in treated sediments following inoculation; and 2) spatially and temporally correlate the occurrence of near steady-state dechlorination with electron donor and redox gadients, and microbial community structure. Accordingly, for this study an attempt was made to 35 characterize the solid-phase microbial community within the Plume F /G aquifer solids prior to inoculation of the column systems with the BR Enrichment. The migation of the mobile-phase of the enrichment was then tracked during inoculation. Immediately following inoculation, the spatial distribution of both the mobile-and solid-phase commurnity structure was examined over the length of the column. Post inoculation, the mobile-phase community structure was measured at temporally-discrete time intervals over the period leading up to and including the time at which near steady-state treatment activity was observed. To identify the primary zone(s) of dehalogenation activity, attempts were made to track the transport, fate and gowth of D. ethenogenes during inoculation, treatment, and as part of the post-treatment characterization effort. Two columns of a similar scale (designated Columns A and B) were used to complete the first two tasks associated with the experiment. Two columns were deemed necessary to provide spatial resolution and duplicate results of the transport behavior of the BR Enrichment in the Plume F/G sediment, and to facilitate examinations of solid- phase contamination, geochemistry and microbiology immediately following inoculation. This was accomplished by sacrificing Column B for soil sample extraction immediately following inoculation. With the database produced from the large-scale column experiment, it was intended that the following information would be elucidated to fulfill Objective #3 4, 5, 6, 7, 8, and 9 outlined in Chapter 1: 36 Baseline concentrations of VOCs (PCE, TCE, cis-DCE, trans-DCE, DCE, VC) and soluble Cr and Fe in soil and goundwater over the length of the column prior to bioaugnentation. Geochemical (redox) gadients in goundwater over the length of the column prior to bioaugnentation. The effective porosity (internal pore volume) of the aquifer solids within the two column systems. The TCE sorption characteristics of the aquifer solids within both large column systems. General structure of the indigenous microbial community within the soil and goundwater over the length of the column prior to bioaugnentation. The bulk transport behavior of the BR Enrichment and D. ethenogenes in the Plume F/G sediments over the length of the two large column systems. Spatial VOC and Cr(VI) mass removal in Column A following bioaugnentation. Spatial and temporal redox gadients across the Column A BAZ following bioaugnentation. 37 0 Spatial and temporal shifts in microbial community structure in Column A following bioaugnentation based on suspended-phase biomass measurements. 0 The solid-phase microbial community profile and localization of a discrete region(s) of dehalogenation activity within the BAZ after a near steady-state level of TCE dechlorination was established in Column A. 2.3.1 Column Design and Assembly The two column systems used for this experiment were constructed of glass tubes (2.54 cm inside dia.) encased within glass jackets (5 cm inside dia.) and fit with length- specific sampling ports positioned on 13.97 cm to 15.24 cm centers. The MSU Glass Shop had manufactured these columns at the request of the Environmental Engineering Laboratory for previous applications. Column A measured 1.22 m in length; Column B was 1.83 m long. Schematics of assembled column systems are presented on Figure 2.2. In total, Column A had seven sampling ports. Column B, which was assembled from two, 0.91 m long segnents compression (U-clamp) sealed together with a Teflon-lined butyl rubber O-ring, had 10 ports. Each sampling port was plugged with pre-sterilized silica wool and Teflon-faced butyl rubber septa secured in-place with aluminum crimps. The temperature of the contents within each column was controlled by circulating non- contact cooling water through the outer jackets at 15°C, using a combination refiigerated water batln/centrifugal pump (Market Forge Sterilrnatic). Cooling water was conveyed to and from the columns with Tygon tubing. 38 €01 A T Col B 05m) aom) Sample ‘— v\ / Sample Ports on 5- to 6-inch centers Continuous I groundwater flow Figure 2.2. Large-Scale Column Design. A butyl-rubber stopper (No. 4) was affixed to the base (influent) of each column. Two lines of stainless steel tubing (1/16- and l/8-inch dia.) were inserted through each stopper to facilitate goundwater delivery into the base of the column systems. The stopper/influent tubing assemblies were secured in-place with U-clamps. A wad of sterilized fused-silica wool was placed over the stopper within each column. A layer 39 (approximately 2.54 m in thickness) of pre-sterilized, 3 mm diameter solid glass beads (Baxter Scientific Products G-6000-1) was placed over the silica wool. The combination of glass beads and silica wool prevented clogging the influent lines with fines from overlaying soil. The stainless steel influent plumbing extended from each column to an anaerobic glove box within which the goundwater and pumping equipment were maintained. The atmosphere of the glove box consisted of 95% N2/5% H2 and a temperature of 18 °C. The lengths of tubing extending fi'om the glove box to each column were seamless, thereby preventing leakage of fluids out of or atmospheric oxygen into the lines. Following assembly, each column system (including influent plumbing) was filled and sanitized with a solution of 200 mg/L sodium hypochlorite for a minimum of eight hours. The sterilization fluid was then drained by suction using pre-sterilized tubing (Tygon #3603) inserted through the top of each column. The columns and associated plumbing were then rinsed by batch cycling a minimum of three empty bed volumes of autoclaved, high purity de-ionized water (16 mega ohm/cm) through the vessels. After draining the final rinse solutions, non-sterile, anoxic Plume F/G goundwater was delivered to each column from gas-tight, glass syringes attached to the influent plumbing. Groundwater delivery was facilitated by Harvard syringe pumps. Syringe pumps and contents were stored within the anaerobic glove box under an atmosphere of approximately 95% N2 and 5% H2 and at a temperature of 18 °C. Anoxic conditions were maintained while filling the columns by pumping filtered N2 through pre-sterilized Teflon tubing inserted through a No. 4 rubber stopper secured within the top of each 40 vessel. Once filled with goundwater, the columns were packed with Plume F/G aquifer materials. The placement of Plume F/G soil into the two columns was performed under anoxic conditions by securing a disposable sterile glove bag (Aldrich AtmosBag) over the top of each and providing a continuous feed of N2 to the bag headspace. Soil and pre- sterilized (autoclaved) tools and funnels were placed inside the glove bag. Soil was then placed into the columns in small lifts (approximately 5 to 8 centimeters deep) by spooning small quantities into the water coltunn and allowing soil particles to settle. While each lifi was settling, the column was lightly tapped with a plastic rod or touched with a mecharnical vibrator to dynamically compact the soil. Once filled with soil, the top (effluent) of each column system was sealed with a pre-sterilized butyl rubber stopper (No. 4). Inserted within each stopper were two stainless steel effluent lines. The entry point of one of the lines (1/8-inch inside dia.) was positioned approximately 5 cm above the top of the soil to permit the discharge of goundwater conveyed vertically upward through the column. The exit point of this line was set at an elevation approximately 0.5 m below the entry point to facilitate gavity drainage. The effluent plumbing was physically conformed into a trap to exclude entry of the external atmosphere into the columns during periods when the water level within the vessels dropped below the entry point of the line. The second line (1/16-inch outside dia.) permitted periodic withdrawal of headspace gasses. The entry point of this line was positioned immediately below the bottom of the rubber stopper at the top of each column. The exit was fit with two (2) polycarbonate, two-way valves with luer lock connections (Cole-Parmer) to either seal-off the line or facilitate attachment of 5 mL-capacity, gas— 41 tight, glass syringes (Popper & Sons, Inc.) for headspace sampling. Upon completion of assembly, Column A was wrapped with aluminum foil to prevent light penetration during the experiment. 2.3.2 Column Operation and Monitoring The large—scale column experiment consisted of four (4) tasks, as outlined below. Column A was operated and monitored during each of these tasks. As introduced previously, Column B was utilized during Task 2.2 exclusively. Task 2.1 - TCE Mass Loading and Sorption Capacity Evaluation Task 2.1 included a series of batch pore fluid exchanges within Column A during which TCE-spiked Plume F/G goundwater was conveyed through the sediments to bring the solid and aqueous phase solute concentrations to near equilibrium levels. As with the small-scale columns, the exchanges entailed the rapid replacement of several pore volumes of goundwater (e. g., 600-800 mL), followed by a period of incubation. The incubation periods were 2-3 days in length to allow time for TCE to sorb to the aquifer solids. GC/MS measurements of TCE concentrations in column effluent samples were performed to verify that equilibrium conditions were approximated. Task 2.1 also entailed effluent sampling and measurement of acetate, chloride, nitrite, nitrate, phosphorus and sulfate concentrations by ion chromatogaphy to establish the backgound geochemical conditions within Column A prior to inoculation with the BR Enrichment. 42 Task 2.2 - Inoculation/Characterization of Flow-Through Properties Task 2.2 entailed inoculation of Columns A and B with the BR Enrichment and characterizing the transport behavior of the inoculum relative to specific anions and a conservative tracer. This was accomplished by tracking the breakthrough of biomass, nitrate, phosphate, sulfate and NaBr along the length of Column B and within effluent samples of Coltunn A during inoculation. This included measuring the port-specific (Column B) and effluent (Columns A and B) biomass by optical density, PCR-based molecular analyses, and ion chromatogaphy of goundwater samples collected at specific volumetric throughput intervals. Once breakthrough was achieved, length-specific goundwater samples were obtained and processed for the biomass analyses identified above. Also during Task 2.2, Column B was used to obtain soil samples from each sampling port immediately upon the conclusion of inoculation. DNA extracted from the aqueous-phase biomass and soil samples was then analyzed by PCR-based T-RF LP and Real-Time (CYBR Green) PCR using primers specific to D. ethenogenes. Task 2.3 - Treatment Task 2.3 encompassed a four-month period during which maintenance of the inoculum and developing BAZ occurred and the extent of associated aqueous-phase geochemical and microbiological changes and dechlorination activity was monitored. Task 2.3 included two operational phases (A and B) corresponding with two separate inoculations of Column A with the BR Enrichment. A comprehensive summary of the operational history of Task 2.3 is summarized in Appendix A. 43 Throughout the duration of Task 2.3, goundwater was continuously conveyed through the Column A at a rate of 1.27 mL/hour. The resulting volumetric flux of goundwater corresponds to average linear velocity of goundwater flow at the Plume F/G site (e. g., approximately 15 cm/day). Table 2.2 summarizes the analyses conducted on samples collected daily to weekly from the length-specific ports and effluent headspace of Column A. Task 2.4 - Post-Treatment Characterization The fourth and final task of the large-scale column experiment consisted of solid- phase characterization of TCE mass and spatial microbial diversity. This was accomplished by retrieving soil samples from the length-specific sampling ports of Column A using 1 mL sterile syringes. TCE measurements were conducted by GC/MS. Microbial diversity was assayed by: PicoGreen® DNA Quantification to estimate biomass concentrations; PCR-based T-RFLP for examination of community structure; and RT- PCR for D. ethenogenes quantification. Table 2.2. Summary of Aqueous Sample Analyses — Large—Scale Column Experiment. Aqueous Solutes Headspace Gasses Biomass Gas Chromatogaphy/Mass Reduced Gas Analysis Spectrometry Hydrogen (ion capture detection) PCE Gas Chromatogaphy TCE (flame ion detection) cis-DCE Methane trans-DCE Ethene DCE VC Ion Chromatogaphy Acetate Chloride Nitrite Bromide Nitrate Phosphate Sulfate High Performance Liquid Chrorrnatography Acetate Lactate Propionate Atorrnic Absorption Hexavalent chromium Ferrous Iron Operational and Sampling Procedures Optical Density PicoGreenQ dsDNA Quantification Polymerase Chain Reaction (PCR)-based Terminal Restriction Fragment Length Polymorphism (T-RFLP) Real-Time (S YBR Green) PCR The operation of the large-scale column systems consisted of a series of batch pore-fluid “acclimation” exchanges followed by an initial inoculation event (Columns A and B). To facilitate post-inoculation soil sampling, Column B was then taken out of service at the conclusion of Task 2.2. For Column A, inoculation was followed by an “incubation” period during which TCE-spiked Plume F/G goundwater was continuously delivered through the system. Typically, the flow of goundwater through Column A was interrupted every seven days to permit the rapidly delivery of substrate and nutrients to 45 feed the BAZ. After each feeding, a new “flow-through” incubation period was initiated. Approximately mid-way through the experiment, Column A was re-inoculated during one of the scheduled “feeding” events. The complete operational history of Column A is summarized in Appendix A. Operational Phase A included all of the initial inoculation and all of the incubation periods leading up to the second inoculation event. The second inoculation represented the start of Operational Phase B. Inoculation and Feeding Inoculation and feeding events entailed the rapid delivery (5-10 mL/min) of Plume F/G goundwater amended to contain 100-250 mg/L of DL-lactate, 3.1 mg/L of nitrogen (fi‘om urea), and 0.6 mg/L of phosphorus (phosphoric acid). For inoculation, dilute concentrations of the BR Enrichment (10% during Operational Phase A and 20% during Operational Phase B) were also mixed into the goundwater feedstock. The total volume of amended goundwater delivered during individual inoculation and feeding events are summarized in Appendix A. Inoculation of Column A required an exchange of approximately 700 mL of pore fluid. Column B received approximately 1,100 mL of nutrient- and enrichment-amended goundwater during inoculation. Typically, feeding events entailed delivery of 75-100 mL of nutrient amended goundwater. During the initial inoculation, biomass sampling required extracting 2 mL of goundwater fi'om select ports on Column B and hem the effluent of botln columns. One milliliter of each sample was extracted from the ports using 3 mL-capacity sterile plastic syringes and decanted into a 1 mL cuvette for immediate analysis of the optical density of the solution. The remaining 1 mL was transferred to a pre-sterilized, 1.5 mL Eppendorf 46 vial for centrifugation at 1,400 rpm for 20 rrninutes to concentrate the planktonic biomass. The processed biomass samples were then stored at —20 °C pending DNA extraction and the prescribed molecular analyses. Coincident with the biomass sampling activities, 1 mL fractions of goundwater were collected and processed for ion chromatogaphy analysis, as previously described. Routine Groundwater Delivery and Pore Fluid Sampling During the incubation periods that followed each inoculation and feeding event, TCE-spiked Plume F/G goundwater was continuously fed to Column A at a rate of 1.27 mUhr. Before use in the columns, the Plume F/G goundwater from storage carboys was transferred to pre—sterilized lmL Wheaton bottles and stripped with 80% N2/20% CO2 to remove residual TCE and adjust pH to within the range measured in Plnune F /G goundwater (e.g., 7.0 — 7.2). The goundwater was then either amended for periodic feeding purposes or decanted directly into four (4) 100 mL-capacity, gas-tight, glass syringes. After elimination of the headspace within the syringes, each was loaded into a Harvard syringe pump. An additional gas-tight glass syringe (10 mL-capacity) containing pre-stripped site goundwater spiked with 500 mg/L of TCE was loaded onto an adjacent pump and connected to a 1/ 16-inch stainless steel feed line extending from a header system of tubing connected to goundwater feed syringes. A static in-line mixer positioned immediately downstream of this confluence received the combined discharge the spiked and non-spiked goundwater syringes. The in-line mixer consisted of a 15.24 cm long section of 1.25 cm diameter stainless steel tubing packed with 3-mm diameter glass beads. Stainless steel screen was placed at the ends of the tube to hold the glass 47 beads in place. The upstream end of the tube was fit with a stainless steel reducing union (l/4-inch to 1/ 16-inch). The downstream end of the tube was fit with a straight union (‘A- inch to ‘A-inch). As noted previously, lAt-inch tubing extended from the mixer to the base of each column. The in-line mixer, internals and fittings were heat sterilized prior to being affixed to the goundwater conveyances. Unless specified otherwise in Appendix A, the duration of each incubation period was seven days. Port-specific sampling of pore fluids occurred prior to and at the conclusion of each inoculation and feeding event. Sampling was also performed at the conclusion of each incubation period. During certain incubation periods, sampling was performed daily. Sampling of the length-specific ports on the large-scale columns was accomplished using 2.5 mL-capacity, gas-tight, glass syringes (Hamilton) fit by luer connection with 5.08 cm long, 20—gauge, stainless steel, side-port needles (Hamilton). Prior to each use, sampling syringes were cleansed by triplicate flushing with methanol followed by triple rinsing with ultra—high purity de-ionized water. The cleansed syringes were then disinfected by heat drying at 60 °C for at least 24 hours. During sampling, a sterile sampling syringe was inserted through the septa until the entire length of the needle penetrated to column. Groundwater was then retrieved in 2-2.5 mL volumes and decanted into pre-labeled sample containers. Depending on the objectives for a given sampling event, up to 6 mL of goundwater was extracted fiom a port in this manner. Samples for GC/MS analysis required 40 mL glass VOA (pre- preserved with 2% sodium sulfate) vials with Teflon-lined septa. Fatty acid samples for HPLC measurement were collected in 2 mL capacity, glass serum vials with Teflon-lined 48 butyl rubber septa and snap cap. For concentrating planktonic biomass, a 1.5 mL capacity Eppendorf centrifuge vial was filled. Similarly, sample portions used for analyses of anions and fatty acids required a separate Eppendorf vial. A third set of Eppendorf vials was needed for collecting samples for soluble metals. Typically, column sampling entailed the extraction of 4 mL of pore fluid from select sample ports. 2 mL of sample was transferred to pro-preserved (3 mL of 2% sodium sulfate) 40 mL VOA vials; duplicate 1 mL fractions were dispensed into 1.5 mL Eppendorf vials. Following sampling, VOA vials were stored at 4°C pending analysis by GC/MS. The contents of one of the Eppendorf vials were filtered through 0.2 pm syringe filters and decanted into a secondary centrifuge vial pre-preserved with 20 uL of concentrated nitric acid. The nitric acid preserved samples were stored at 4 °C pending analysis for soluble metals by atomic adsorption. The second Eppendorf vial was centrifuged at 1,400 rpm for 20 min to concentrate planktonic biomass for DNA extraction and the molecular procedures listed above. A 0.6 mL fraction of the supernatant of the centrifuged sample was decanted into l-mL glass or polyetlnylene ion chromatogaphy vial and stored at -20°C pending analysis for the major anions listed above. 0.1 mL of the remaining supernatant was then transferred to a 2 mL glass vial containing 0.9 mL of de-ionized water and sealed with a Teflon-lined butyl rubber septa secured with a plastic snap cap. These samples were stored at ——20 °C pending analysis for volatile fatty acids by high performance liquid chromatogaphy (HPLC). During certain sampling events, an additional 1-2 mL of pore fluid were withdrawn fiom select sample ports for analysis of soluble gasses, including hydrogen, methane, ethene. These fractions were transferred to 2 mL glass vials and sealed with 49 Teflon-lined butyl rubber septa secured with aluminum crimp caps. The samples thus obtained were allowed to sit undisturbed for approximately 1 hour prior to withdrawal of 0.5-1 mL aliquots of the headspace for gas-phase analyses, as listed on Table 2.2. Post-Treatment Soil Sampling The length-specific sampling of soil in Columns A and B was accomplished by first removing the septa and wad of silica wool on each sample port using sterile utensils. Then, a 1-3 mL capacity, sterile, pre-trimmed, plastic syringe was inserted into each port to collect soil by coring. The plastic syringes were pre-trimmed using sterile cutting tools. Trimming entailed cutting the syringe shalt just below the tip to enlarge the cross- sectional area for soil coring. Once filled, the syringe contents were ejected with the plunger into a pre-preserved (3 mL of 2% sodium sulfate) 40 mL VOA vial and/or a 20 mL serum bottle. Typically, it was necessary to re-insert and fill the syringe in this manner 3-5 times to obtain a sufficient mass of soil to complete the requisite analyses. Upon retrieval of a sufficient quantity of soil to complete a sample, the partially filled containers were sealed with Teflon-lined septa and air-tight caps. At the conclusion of Task 2.4, Column A was also opened within the anaerobic glove box to facilitate retrieval of depth-specific segnents of the bulk soil under anoxic conditions. The bulk soil samples were obtained for analysis of solid-phase chromium and iron. These segnents were transferred directly into 40-ounce jars, packed on ice and shipped to AAC Trirnity, Inc. laboratory in F armington Hills, Michigan. At AAC Trinity, the samples were analyzed for chromium and iron by USEPA Method 6020. 50 2.4 Soil and Groundwater Soil for preparation of the small-scale column microcosms was composited from samples collected at several adjacent locations within the Plume F/G field-test plot southeast of Schoolcraft, Michigan (Figure 1.1). The soils were obtained from between 65 feet and 85 feet below gade, which corresponds to the depth interval at which the highest levels of TCE and cis-DCE have been consistently measured during MDEQ- sponsored investigations (HALLIBURTON NUS Environmental Corporation, 1991). Soil samples were collected with a saturated sand (piston core) sampler that had been cleansed by high-pressure steam washing prior to each use. Once retrieved from the subsurface the soil samples were immediately transferred to a field glove box and handled aseptically under an N2—enriched atmosphere. Select portions of the samples were therein transferred to pre-sterilized aluminum cans. Sterilized can lids were fitted with two canulas, constructed of butyl rubber stoppers and 2.54 m, 20 gauge sterile needles, to facilitate headspace purging with N2. The samples were then immediately transported the MSU Engineering Research Laboratory where they were transferred to an anaerobic glove box for storage under an atmosphere of 95% N2/5% H2 and a temperature of 18 °C prior to use for column assembly. Soil used for the large-scale column studies was composited from sediments collected at the Plume F/G test site (core SB-5-15) in January of 2002 from a depth interval of 65 to 66 feet below gade. The sediment sample was obtained by roto-sonic drilling methodologies, which promoted aseptic and anoxic acquisition of soil cores. A segnent of the core from the aforementioned depth-interval was isolated and sealed witlnin the rubber sleeve from the roto sonic core. The soil sample was then placed in a 51 pre-sterilized aluminum paint can within a field glove box fed 100% N2. The lid of the can was pre-fit with a canula to allow the headspace to be purged with N2 after being firmly secured in place. The can and contents were then transported on the day of acquisition to the MSU lab and placed into the anaerobic glove box for storage pending column preparation. Before insertion into either the small- or large-scale columns, source soils were first processed by sieving to remove coarse sands and gavel particles. Soil was passed through No. 8 and No. 5 sieves (Entecotts, Ltd and Bort Longyear), which had been heat- sterilized prior to use. The well sorted, passing soil fraction was then used for column preparation. Groundwater from the Plume F/G test plot was used for both the small- and large- scale column experiments. Groundwater was routinely obtained from MSU-001, a 5.08 cm diameter well positioned at the southern edge of the test plot. This well is constructed of a 1.52 m long stainless steel well screen (0.025 cm wire-wound slot) attached to a galvanized steel riser. The screen of MSU-001 is positioned from 60 feet to 65 feet below gade (18.3 m to 19.8 m b.g.). Groundwater was retrieved from MSU-001 by extraction with a dedicated electrical submersible pump and conveyed to the gound surface through polyethylene tubing. Typically, goundwater was extracted at a rate of approximately 4 liters per minute. During sampling, goundwater was initially discharged to waste for a period of approximately one (1) hour, or until a volume of water was withdrawn approximately equivalent to the sum of three static water columns witlnin the well. The pH and oxidation-reduction potential of the extracted goundwater were measured using Cole-Panner DigiSense portable electrodes to evaluate the 52 stabilization of these general geochemical parameters during this period. Following the wasting period, the extracted goundwater was conveyed into pre-sterilized 20 L Nal gene carboys. The headspace within a sample carboy was maintained anoxic by bubbling N2 tlnrough the water accumulating within the vessel. Once filled, the carboy(s) were capped and transferred to a large, heavy-duty garbage bag. The bag was tlnen purged and filled with 100% N2 and sealed to prevent leakage. The packaged carboys were then immediately transferred to the MSU laboratory. At the lab, the carboys were either directly transferred to a storage box in which the headspace atmosphere was maintained anoxic with continuous throughout of N2, or their contents were transferred to pre- sterilized, 1 L Wheaton bottles. Groundwater contained in either carboys or Wheaton bottles was stored anoxically and at 18 °C pending use. Groundwater storage never exceeded one month in duration before use or disposal. 2.5 Analytical Procedures 2.5.1 Aqueous Solutes Identification and quantification of PCB, TCE, cis-DCE, trans-DCE, 1,1-DCE, and VC in soil and goundwater samples was accomplished using a gas chromatogaph/mass spectrometer (GC/MS) equipped with an Agilent 5973 Mass Selective Detector and combined with purge-and-trap system (Tekmar/Dohrmann Precept H and Telcmar/Dohnnann 3100 Sample Concentrator). Compound identification was subsequently confirmed by routine injection of pure material into the GC while analyzing sample degadation. GC calibration was performed by measuring the instrument response to krnown masses of PCB, TCE, cis-DCE, trans-DCE, 1,1-DCE, and VC added directly to a 53 40-mL VOA bottle containing 2 mL chilled distilled de-ionized water and 3 mL of 2% sodium bisulfate. Anions were assayed by ion chromatogaphy using suppressed conductivity detection within a Dionex model 2000i/SP ion chromatogaph fitted with a Dionex AS4A IonPac column and utilizing a 1.8 mM biocarbonate—l .7 mM carbonate mobile phase conveyed at 8 mL/min. Chromatogarns were recorded and data integated using Turbochrom R 4 software (Perkin Elmer, Incorporation). Five—point calibration curves were prepared by diluting primary arnion standards into secondary de-ionized water standards. A sample volume of 600 uL was used to accomplish the ion chromatogaphy analyses dtuing both experiments. Acetic, lactic and propionic acids were measured by high performance liquid chromatogaphy using a Supelco Discovery C8 column (No. 59354—U; 25 cm, 4.6 mm, 5 um). Eluent consisted of 3.4 g/L of KH2P04. Acetonitrile was used to regenerate the column between samples. Compound detection was accomplished through measurement of sample response to ionization with 210 nm of ultraviolet radiation at rate of 0.6 mL/min. Turbochrom R 4 software (Perkin Elmer, Incorporation) was used for chromatogaphic integation and data processing. Soluble metals samples (1 mL pre-preserved with 20 uL of concentrated nitric acid) were analyzed by atomic absorption witln a Perkirn-Ehner Analyst 800 Atomic Absoption Spectrometer operated with AA WinLab Analyst Software (version 4.1 SP1). Samples were atomized electrotlnermally through a five step temperature ramp. Radiation sources emitted wavelengths of 357.9 nm and 248.3 nm for detection of soluble chromium and iron, respectively. Quantitation of sample constituents was done through comparison with 54 standard curves developed from the analyses of standards containing pre-measured solute concentrations. For each set of analysis, a five point calibration curve was established for soluble chromium quantification; for soluble iron samples, a four point calibration curve was used. 2.5.2 Headspace Gasses Hydrogen levels within the headspace of small-scale columns or in low-volume samples taken from sample ports on Column A were measured using a RGA3 reduction gas analyzer (Trace Analytical, Menlo Park, CA) Operated with Turbochrom R4 software and data integation. Headspace samples were injected onto a 1% SP-1000 Carbopack B column through an automated switching valve timed at 2 minutes. Standards were prepared by adding measured volumes of 100 ppmv hydrogen to serum bottles purged with N2 and crimp capped with butyl rubber septa. Headspace samples for methane and ethene quantification were measured by gas chromatogaphic (GC) analysis. 0.5-mL of headspace sample was injected into a Perkin Elmer Autosystem GC equipped with a flarne-ionization detector and a 3.2-mm x 2.44-m stainless-steel column packed with 1% SP-1000 on 60/80 Carbopack-B (Supelco, Inc.) maintained isothennally at 50 °C. Compound identification and quantification was accomplished by matching the retention times of authentic material with those of peaks from the headspace samples. 55 2.5.3 Biomass and DNA Optical density measurements were performed on 1 mL samples using a Shimadzu UV-160 spectrophotometer measuring the transmittance of energy at a wavelength of 660 nm. Extraction of total community DNA fiom cell pellets of planktonic biomass samples was initiated by re-suspending and digesting pellets in a 0.5 mL solution of sucrose-NaCl-Tris (0.05 M Tris-Cl [pH 8], 0.1 M NaCl, and 25% sucrose) and lysozyme (Signa-Aldrich) (5 mg/mL). Sample digestion occurred under gentle agitation at 30°C for 10 minutes. To facilitate decomposition of extraneous cell components, approximately 0.2 g of sterile zirconium beads (Biospec #1007901Z) were added to each sample. Samples were then shaken at 5,000 rpm for 90 seconds using a Biospec Mini Beadbeater. To decompose fatty acids and other non-genomic macromolecules and improve DNA solubility, 250 uL of a solution of 0.5 M Tris, 0.05 M EDTA, 1% SDS and 6% Tris-Phenol was then mixed with each sample. Samples were then chilled at 0°C for 20 minutes. Sample contents were mixed with 750 p.L of phenol chloroform isoarnyl alcohol (25:24: 1) and centrifuged at 1,000 rpm for 10 rrninutes. After centrifugation, the supernatant was removed aseptically from each sample and replaced with 750 uL of chloroform isoarnyl alcohol (24:1). Samples were again centrifuged at 1,000 rpm for 10 minutes. Approximately 450 uL of the supernatant of each sample was transferred to a sterile 1.5 mL vial. 45 ”L of 3 M sodium acetate and approximately 1 mL of 96% ethyl alcohol was then added to each sample to precipitate DNA ovennight while stored at ~20 °C. Precipitated samples were centrifuged at 14,000 rpm for 30 nninutes. Supernatant was then removed and the pellet washed with 1 mL of chilled 70% ethyl alcohol. 56 Washed samples were centrifuged again at 14,000 rpm for 10 minutes. Supernatant was then removed and the samples were air dried in a fnune hood at room temperature for approximately 2 hours. Dried samples were then stored at -—20 °C pending analyses. Extraction of total community genomic DNA from soil samples was accomplished using Ultra-Clean Soil DNA Kits from MoBio Systems, Inc. (Solana Beach, CA) following manufacturer’s guidelines. DNA quantification was accomplished using PicoGreen® dsDNA Quantification Kits and reagents (Molecular Probes, Inc., Eugene, OR, Cat. # P-7589). PicoGreen® quantification reagent is an ultra sensitive fluorescent nucleic acid stain for quantitating double-stranded DNA (dsDNA) in solutions. Quantification assays were performed using 5-10% dilutions of sample extracts re-suspended in 50 [IL of 10 mM TRIS-HCl, [pH 7.5], following manufacturers guidelines. Sample quantitations were accomplished using an MP X Microtiter Plate F lourometer and integation software. Verification of the presence of methanogens and sulfate-reducing bacteria (SRBs) in biomass samples was accomplished by PCR amplification and electrophoretic separation of 168 rRNA genes, and comparison to positive and negative controls. Primers selected for these analyses included those specific to archea and SRBs. A non- specific eubacterial primer set was also used. For PCR, 1 [LL of each primer solution was added to each re-suspended sample (containing 10-100 ng of DNA), along with 5 uL of 10X PCR buffer (500 mM Tris [pH 8.3], 20 mM MgC12, 5-10% ficoll, 10 mM Tartrazine), 5 uL of 10X bovine serum albumin (2.5 mg/mL), 5 uL of 10X dNTP (2 mM each), 2 Units of T aq DNA polymerase, and sterile deionized water to create a 50 uL sample. 57 Archean-specifc PCR primers included 340f (5’-CCT ACG GGG CGC A(C/G)CA GG(C/G) GC-3’) and 915r (5’-GTG CTC CCC CGC CAA TTC CT-3’). Reaction conditions included a denaturing cycle of 3 minutes at 94 °C, followed by 30 cycles, each consisting of 30 seconds of denaturing at 94°C, annealing for 45 seconds at 60 °C, and extension for 2 minutes and 10 seconds at 72 °C. Final elongation occurred at 72 °C for 6 minutes. Primers for amplification of a 1.9-kb DNA fragnent encoding most of the alpha and beta subunits of the dissimilatory reductase (DSR) from all recognized lineages of sulfate-reducing bacteria (SRB) included DSRlF (5’- AC(C/G)CACTGGAAGCACG-3’) and DSR4R (5’-GTGTAGCAGTTACCGCA-3’) (Baolin, pers. comm). Reaction conditions entailed denaturing for 15 seconds at 94 °C, 30 denaturation/annealing cycles (94 °C for 15 seconds; 30 seconds at 54 °C; and 1 minute at 72 °C). Final elongation occurred for 1 minute at 72 °C. Eubacterial-specific primers included fl)1 (5’AGA GTT TGA TCC TGG CTC AG-3’) and RDl (5’-AAG GAG GTG ATC CAG CC-3’). Reaction conditions entailed denaturing at 94 °C for 3 minutes followed by 30 cycles of denaturing at 94 °C for 30 seconds, annealing at 55 °C for 30 seconds, and extension at 72 °C for 2 minutes and 10 seconds. Final elongation was at 72 °C for 7 minutes. Negative controls containing no added DNA, as well as positive controls containing pure culture genomic DNA, were included alongside reactions. Aliquots (10 uL) of PCR products were separated by electrophoresis in a 1.5% (wt/vol) agarose gel in 1X TAE buffer. The gel was stained with ethidium bromide (0.5 ag/mL) and visualized by ultraviolet excitation (Helton, 2001). T-RF LP analysis was performed as previously described by Liu, et al. (1997) and F lyrnn et al. (2000). PCR was performed with bacterial domain specific small subunit 58 (SSU) ribosomal ribonucleic acid (rRNA) primers 8-27 forward labeled with hexachloroflourescence (Hex) at the 5’-end (synthesized by Operon Technologies, Inc., Alameda, CA) and 1510-1492 reverse each having the sequences AGAGTTGATCMTGGCTCAG and RGYTACCTTGTTACGACTT, respectively. In the reverse primer the degeneration positions R and Y employed the pyrimidine and purine derivatives dK and dP, respectively (Glen Research, Sterling, VA). The SSU rRNA genes were amplified using approximately 40 ng of DNA and a Perkin-Elmer 9600 thermocycler (Perkin-Elmer, Norwalk, CT). The PCR reactions were performed under the following conditions: 3 minutes at 95 °C, 1 minute at 55 °C, and 3 minutes at 72 °C, followed by a 7 minute extension period at 72 °C. The resulting PCR products were purified using the Wizard PCR purification kit (Promega, Madison, WI) and digested overrnight at 37 °C with 20 units of HhaI, MspI, or RsaI separately (Life Technologies, Gibco BRL, Gaitlnersburg, MD). The digest fragnents were then resolved on an ABI 373A sequencer running in the gene scan mode with 6% urea-containing polyacrylarnide gels (PEApplied Biosystems, Foster City, CA). Size calibration was performed with the Tamara 2500 standard (PEApplied Biosystems). The resulting gel patterns were analyzed using the Genescan software, version 2.1 (PEApplied Biosystems). Real-time PCR procedures employed for this study followed those outlined by Griintzig, et al. (2001). The reaction mixture for real-tirne PCR consisted of IX T aqMan Universal Master Mix (contairning AmpliTaq Gold DNA polymerase, AmpErase uracil- N—glycosylase, which degades PCR carryover products from previous reactions, deoxynucleoside triphosphates with dUTP, a passive reference [6carboxy-X-rhodamine], and optimized buffer components) (PE Applied Biosystems), 300 nM forward primer, 59 900 n}. tubes u DNA po the 770i) noleIIiZt emission . threshold CIOSSC‘S gr [Cannons ) Will cross Sarnple W meet get 900 nM reverse primer, and 525 nM flourogenic probe. MicroAmp optical caps and tubes were used for the final reactions. PCR conditions were as follows: 2 minutes at 50 °C, 10 minutes at 95 °C, then 40 cycles of 15 seconds at 95 °C and 1 minute at 60 °C. Negative controls with no template DNA or no probe were run in each reaction. The increase in fluorescence emission, due to the degadation of the probe by the DNA polymerase in each elongation step, was monitored during PCR amplification using the 7700 Sequence Detector (PE Applied Biosystems). The fluorescence signal was normalized by dividing the emission of the reported dye (6-carboxyflourscene) by the emission of the passive reference dye, 6-carboxy-X-rhodamine. The parameter CT (tlnreshold cycle) is the fractional cycle number at which the fluorescence emission crosses an arbitrarily defined threshold within the logarithmic increase phase (0.1 in our reactions). The higher the amount of initial template DNA, the earlier the fluorescence will cross the threshold and the smaller will be CT. The CT values obtained for each sample were compared with a standard curve to determine the initial copy number of the target gene. PicoGreen®, PCR, T-RF LP and RT-PCR procedures were completed by Leslie Dybas. Technical assistance was provided by Baolin Sun. 2.6 Measurement of TCE Transformation Kinetics Batch microcosm experiments were performed by The Retec Group, Inc. (Retec) of Lansing, Michigan to estimate rates of PCB transformation associated with the BR Enrichment. A summary of the Retec experimental media, protocol, analytical results, and data reduction is provided in Appendix B. 60 Seven batch microcosms (4 test and 3 control) were prepared to facilitate the experiment. Microcosms were prepared using 250 mL glass serum bottles partially filled with 160 mL of either BR Enrichment (test bottles) or de—ionized water (controls). Each bottle also received: 2 mL of a mineral salt solution (50 g/L NaCl, 25 g/L MgCl2'6H2O, 10 g/L KH2PO4, 15 g/L NHaCl, 15 g/L KCl, and 0.75 g/L CaCl2‘2H2O); 0.2 mL of a trace metal solution (10 mg/L HCl, 1,500 mg/L FeCl2'4H2O, 190 mg/L CoCl2'6H2O, 100 mg/L MnCl2 '4H2O, 70 mg/L ZnCl2, 6 mg/L H3B03, 36 mg/L Na2MnOa'2H2O, 24 mg/L NiCl2'6H2O, and 2 mg/L CuCl2'2H2O); 0.2 mL of sodium sulfide (48 g/L stock) to maintain an ORP favorable for dechlorination; 0.2 mL of a 0.1% resazurin solution; 2 mL of phosphate buffer (80% H3PO4); 1 mL of 60% DL-lactic acid; and 0.4 uL of neat PCE. The total solution volume within each microcosm at the start of the experiment was 165.6 mL. Microcosms were sealed with Teflon-lined butyl rubber septa secured with aluminum crimp caps. Following preparation, each microcosm was covered in foil to prevent light penetration and stored on an orbital shaker at room temperature pending sampling. Microcosm preparation and sampling was performed aseptically and under anoxic conditions. Each microcosm was sampled once prior to the addition of PCB, and at approximately 0.5 to 1 day intervals thereafter over a period of approximately 197 hours, or eight days. In total, 13 samples were obtained from each control and test microcosm following the addition of PCE. Dechlorination was measured by following PCE, TCE, cis—, trans- and 1,1-DCE and VC concentrations over the duration of the experiment. Ethene, methane and total suspended solids concentrations were also tracked. Sampling and analyses were performed by Retec. 61 2.7 Fate and Transport Modeling A computer model was developed from a system of mass balance equations to simulate TCE movement and transformation during the operational history of Column A. Specifically, one-dimensional, numerical modeling techniques were used to simulate the fate and transport of TCE, dissolve hydrogen, and attached and planktonic D. ethenogenes. The analytical data from the spatial and temporal sampling of the column and empirical TCE transformation kinetic data were used to calibrate the model. The calibrated model was then used to predict and optimize treatment performance within the BAZ under a variety of feeding scenarios. The objectives for the numerical modeling were to verify the transport characteristics of TCE and the BR Enrichment within the Plume F/G aquifer materials, and to simulate a range of substrate and nutrient delivery strategies that could be used to optimize TCE treatment in the Column A BAZ. A series of mass balance equations describe the changes in solute concentrations associated with TCE transformation by D. ethenogenes. The equations presented below were developed from those previously described by Witt et al. (1995), Clement et al. (2000) and Phanikumar et al. (2002). Hydrogen serves as an electron donor in these equations. TCE, cis-DCE, and VC serve as electron acceptors. Modifications by the author for this study included the inclusion of rate-limited (de)sorption to describe the fate of cis-DCE, VC, and ethene. For this study, it was assumed that TCE is transformed by both attached and planktonic cells and that transformation is first order with respect to TCE concentration and first order with respect to the concentration of D. ethenogenes biomass. Further, it 62 was assumed that partial equilibrium exists between pore fluid and solid-phase TCE concentrations, with desorption limitations on the remaining mass of sorbed TCE. The following processes were mathematically described and incorporated into the numerical model for Column A: (1) advective transport of tracer, TCE (electron acceptor) and it’s transformation products and dissolved hydrogen (electron donor), as influenced by hydrodynamic dispersion and sorption; (2) sorption and desorption of TCE, cis—DCE and VC; (3) the biochemical response of D. ethenogenes as indicated by its rates of substrate utilization, TCE transformation, gowth and decay; and (4) attachment and detachment of D ethenogenes. Transport of a non-reactive tracer such as bromide is described by: 9Q: z 129191241398; 03) at 5x2 fix The fate and transport of chlorinated ethene species mediated by dechlorination reactions such as halorespiration may be represented by a set of coupled mathematical expressions. Assuming second-order biodegadation kinetics, transport and transformation of TCE, cis-DCE, VC and ethene, respectively, can be simulated by solving the following set of partial differential equations (Clement et al., 2000, Phanikumar, et al. 2002): TCE 2 p f K 6C a c 6c . (1+ b d ) TCE = D—TCE -U—TCE -kTCECTCE(Xm+Xim) 0 at 6x2 6): P K'T E ’—b_6—C;‘[(l‘f)K5CECTCE"STCEl—J—(Xm+Xim) (2'9) yTCE—X 63 beKiiCE aCDCE _ 62CDCE _ aCDCE (1 + ) —D——— U 6 at 6x2 ax . P K‘DCE - YDCE-VCkDCECDCE(Xm+Xim)'—‘Q7—l(1—f)KgCECDCE‘SDCEI (2'10) + YTCE—DCEkTCECTCE(Xm + Xim) + YDCE—VCkDCE CDCE(Xm + Xim) (1+beKZC)6CVC=D62Cyc_UaCr/C 6 0 t 6x2 5x PbKVC —YVC—ETHki/CCVC(Xm + Xim)- 9 [(1 -f)KZCCVC*SVC] (2'1 1) [,beng aCram __ 62CETH _ aClam + ) —D—-—— U— 6 at 6x2 ax _ pbKETH 6 + YVC—ETH kl/CCVC(Xm + er) (1 [(l-f)K5THCETH—SETHI (2-12) Sorbed-phase TCE is characterized by (2.13) 6 SaZCE = KTCEIO - f )K 5°15 C TCE - S TCE] The fate and transport of dissolved hydrogen is described as 6C... =Dazcfl._U6CH._ #1 at 6x2 6x szX (2.14) (Xm+Xim)' RH, 64 When microbial gowth is limited by a single substrate, mobile microbial production may be described by (Phanikumar, et al, 2002): 6X 2.15 Earl—b—Kaax. ( ) If the rate of gowth of D. ethenogenes is linnited by a single substrate (either hydrogen or TCE), it can be estimated using a Monod kinetic expression. For hydrogen, the expression is (2.16) 6 CH2:_#max( CH2 )Xz—fltnaxMHZX. at yuzX CH2+KH2 yHZX When multiple limiting solutes are present (hydrogen and TCE), the gowth rate of D. ethenogenes, pr, can be expressed by the following interactive relation (Phanikumar et al. zoozy C’” )( CTCE ). (2.17) .U=.U M2MTCE=.U ( max H mCuz’er CTCE+KTCE Mobile- and irnmobile-phase D. ethenogenes concentrations may be described by ax," _ 62 Xm 6X", _ _ X —M y at —D 6x2 _U__+[p—bDC(1 MHZ) Kat] m‘l'Kde(1 H2) im’ (2.18) and 65 aXim at =[it-bpc(1-MH2)—Kae(1—MH2)]X.-m+KaiXm, (2-19) respectively. In the above equations, C3,, C mg, C DCE, C Vc, C Em, and C H2 are aqueous concentrations of bromide, TCE, cis-DCE, VC, ethene and hydrogen, respectively; Xi", and X", are the immobile and planktonic concentrations of D. ethenogenes, respectively; and Sm; is the concentration of sorbed TCE. The coefficients xi and f define the kinetic (de)sorption rate (day!) and fraction of equilibrium sorption sites, respectively. 1’,- and y, are stoichiometeric and anaerobic halorespiration reaction yield coefficients, respectively. Second-order biotransformation reaction rates are defined by k ’,-. The coefficient of hydrodynamic dispersion is defined here as D = aU, where U is the average linear velocity (cm/day) of goundwater flow through Column A and or is the dispersivity (m) of the aquifer solids within the column. M,- is the Monod-type saturation kinetics term. The MODF LOW (McDonald and Harbaugh, 1988) and RT3D (Clement et al. 1998) computer codes were used to establish the hydraulic head distribution within Column A and solve the equations describing bioreactive transport, respectively. 66 Chapter 3 RESULTS AND DISCUSSION 3.] Characterization of Aquifer Materials Table 3.1 summarizes select physical and geochemical characteristics of the Plume F/G-impacted soil and goundwater, as determined from analyses performed during this study and previous investigations sponsored by the MDEQ (Mayotte, et al., 1996). These data reveal that the site aquifer materials support a significant microflora (measured in numbers of cell forming units per gam of dry soil or mL of goundwater). In fact, it is probable that the cis-DCE and VC detected in goundwater samples from the Plume F/G test gid have originated from biotransformation of TCE by indigenous microorganisms, since alternative sources of these compounds are unknown and unlikely. TCE concentrations in Plume F/G goundwater collected from site monitoring well, MSU-1, for both the small- and large-scale column experiments were consistently within the range of 300 rig/L to 500 rig/L. 3.2 Flow-Through Properties The flow-through properties of the small-scale columns are presented in Table 4.1. A composite profile for bromide transport through Columns A tlnrough F is presented on Figure 3.1. As expected, the flow-tlnrough characteristics for each of the small-scale columns are similar. In general, the effective porosities of the re-packed aquifer solids within the columns were estimated to range from 25% to 39%, indicating internal pore volumes of 14-22 mL. The average linear velocities, V317, of goundwater flow during each exchange were determined from the porosity data and the superficial 67 velocity, v (0.4 cur/min), to range from 1.29-1.74 cm/min. The Reynolds number (Re = vd/U) was 0.067, assuming an average soil gain diameter, d, of 0.1 cm, and kinematic viscosity, v, of 36 cmZ/hr at 20°C (Siegist and McCarty, 1987). A value of Re less than unity indicates that inertial forces dominate. Therefore, flow was essentially laminar during the exchanges (Freeze and Cherry, 1979). Table 3.1. Characteristics of Schoolcraft Aquifer Materials. Media Parameter Soil Groundwater pH 8.81 7.0445 Alkalinity - 331 mg/L1 Hardness - 410 mg/L1 Fraction of organic carbon 0.1%1 - Iron 37 mg/Kgl 11 mg/L Copper - 0.03 mg/L1 Nitrate <1 mg/Kg‘ 39 mg/L Phosphate - <1 mg/L Sulfate - 25 mg/L TCE 300 — 500 rig/L Microbial population (anaerobic) 2.3 x 107 CFU/gl 1 x 10° CFU/mLl 1Brown & Root Environmental CFU —- colony forming unit. The movement of goundwater through each column deviated slightly from ideal plug flow. The composite breakthrough profile (Figure 3.1) was used to evaluate the dispersion characteristics of the aquifer solids within the columns. The first 12 mL of pore fluids was essentially free of bromide, and a total throughput of 30-32 mL of bromide-spiked goundwater was necessary to achieve complete breakthrough, or saturation. The approximate spreading of the breakthrough front (as defined by the 68 dimensionless quantity Ddb/ Vefl'L, where L is the column length) was 0.01 , giving a dispersion coefficient, Ddisa of approximately 10 cmZ/hr. 1.00 l 3:“ - ' V 2: ”A 0.75 0’ -- . . a 0.50 T i 11 i V I i i Ideal Plug Flow 0'25 " 1 i 1 i —o—B..ma —— i if i l I l X' Inoculum l i l i z - A- TCE 0.00 i 1 ‘1 p % ! ! 0 10 20 30 40 50 60 70 80 90 100 Volume Exchanged (mL) Figure 3.1. Composite breakthrough profiles of bromide, TCE and inoculum through small-scale columns. Figures 3.2 and 3.3 present bromide breakthrough profiles for large-scale Column A and select length-specific sampling ports of Column B, respectively. 111 general, these profiles indicate internal pore volumes of 220 mL (Column A) and 280 mL (Column B). The effective porosity, 0, for both columns was calculated to be approximately 39%. The results fi'om numerical modeling simulations performed to simulate the transport of a non-sorptive, non-reactive solute (e. g., bromide) are presented on Figures 3.2 and 3.3. These data reveal that the re-packed Plume F/G aquifer solids within both columns are characterized by a longitudinal dispersivity, 012, of 0.01 m (1 cm). 69 _r \ ~t. O \ .g. .. .0“ W B B . , 0.8 ' 0.6 C/Co 0.4 02 f ' 0 l: Port 2 ; ! Port 4 0 200 400 600 0 200 400 600 Volume Throughput (mL) Figure 3.2. Bromide breakthrough profiles at sample Ports 2 and 4 of large-scale Column B. Dashed line with open boxes represent bromide data from each port. Solid line represents numerical simulation. C/Co - - o - - Bromide —-D—Nitrate A Phosphate ”‘ ‘—Simulation j I l l 0 100 200 300 400 500 600 700 Volume Throughput (mL) Figure 3.3. Bromide and major anion breakthrough profiles for large scale Column A. 70 3.3 Sorption Characteristics Composite breakthrough profiles were developed for TCE and bromide transport through small columns A, B, and C (Figure 3.1). As illustrated on this figure, TCE transport was retarded in comparison to bromide. TCE concentrations were first observed in these columns after approximately 14 mL of pore fluids had been exchanged. TCE breakthrough was complete during these exchange events after throughput of 60—65 mL of spiked goundwater. Therefore, for small column mass balances, TCE concentrations measured in up to three 2 mL samples obtained from the initial 14 mL and the final 30 mL of fluid emerging from each column (e.g., 70-100 mL throughput) during the pore fluid exchanges were averaged to determine C1 and C2, respectively. The profile of TCE breakthrough composited from the Column A, B, and C data is typical of flow . through a closed vessel (Levenspiel, 1979) with a dispersion coefficient, Dd), of less that 0.1 cmz/hr (Table 4.1). The ratio of solute mass sorbed to aquifer solids to mass in pore fluids defines the equilibrium partition coefficient, RP. A summary of the change in magnitude of RP for columns A, B, and C is presented in Figure 3.4. After five exchanges within these three columns, Rp values stabilized, indicating that sorption was essentially complete. The resulting “equilibrium” RP values ranged from a low of 4.56 (Column P) to a high of about 5.44 (Column Q) (Table 3.2). These results would suggest that retardation factors, R,, of 5.56 to 6.44 characterize the movement of TCE relative to the rate of goundwater flow within the repacked aquifer solids. However, from examination of the breakthrough profile for TCE within the small columns (Figure 3.1), it is evident that the values of RP and R, are unrealistically high, and most likely reflect excess solute sorption onto the base 71 of the rubber stoppers that sealed each column. Therefore, transport modeling simulations were performed to simulate TCE breakthrough in these columns and calibrate R). The calibrated modeling results indicate that an R, value of 1.3 more closely characterizes TCE transport in the repacked Plume F/G aquifer materials. 0 “k1 m 1 1 T r 0 10 20 30 40 50 60 Days Figure 3.4. Dimensionless equilibrium partitioning coefficients composited from small-scale columns D-U. From the value Rp reported on Table 3.2 and assuming linear (de)sorption kinetics, a distribution coefficient, K, of 0.07 cmz/g was calculated. This indicates that approximately 28% of the total TCE mass within the three columns was retained on the solid matrix following the completion of sorption. Given that the source of the soil and processing protocols were consistent between the two experiments, it is assumed that the 72 distribution coefficient estimated from the small-column data is valid for characterizing sorption within large-scale Column A. 3.4 Inoculum Transport As illustrated on Figure 3.1, the BR Enrichment was readily transported through the re-packed Plume F/G aquifer solids in small-scale columns A, B, and C. In fact, breakthrough was slightly more rapid that the average linear velocity of the exchange fluids (as defined by bromide breakthrough), probably owing to charge and/or pore size exclusion and the motility of some community members within the inoculum. Only about 60% of the mass of cells introduced exited the column during the breakthrough. The remaining 40% of the inoculum was retained within the column. The mechanism of retention is unknown. In general, the tracking of optical density in port-specific and effluent samples from the large-scale columns was inconclusive. Plots of these data are provided on Figure 3.5. As seen on the figure, no distinct trend in optical density increase is evident. Further, DNA concentrations from PicoGreen® analysis did not indicate trends in increasing biomass levels at Column B sample ports or within Column A effluent during inoculation. These observations were not unexpected, considering that dilute concentrations (e.g., 38 pig/L or 4 x 10° CFU/mL) of the BR Enrichment were used for inoculation of Columns A and B. Consequently, it is likely that much of the biomass introduced during inoculation was retained within the sediment upgadient of the sampling points. Dehalococcoides sp. were measured at levels within the 102 CFUs/mL range within the inoculum (Table 3.2), but were not detected in any of the aqueous 73 biomass samples collected from Columns A or B during these innitial breakthrough studies. Portl "um-X Port 2 -—1¥-—Port3 —O—Port 4 - -x- Port 5 cm0 0 200 400 600 800 1000 1200 Volume Throughput (mL) Figure 3.5. Column B port-specific inoculum breakthrough based on optical density (ODéw) measurements. 3.5 Biotransformation Biotransformation activity within both the small- and large-scale column systems was evaluated through an integated examination of several elements of data. For the small-scale columns, the primary data elements included temporal carbon source (fatty acid), major anion, and TCE transformation data. Limited sets of biomass, headspace gas, and soluble chromium data were also examined. The assessment of biotransformation within large-scale Column A was based on spatial and temporal 74 biomass, fatty acid, headspace gas (e.g., methane), major anion, soluble chromium and iron, and TCE dechlorination data. Table 3.3 RT-PCR results for small- and large-scale column samples. Sample Designation pg/mL CFU/mL Source Soil Not detected Not detected EFXReactor Sample 1.60 x 10'5 1.70 x 102 Small Column Post— Treatment Soils: P (top 1/3) 3.65 x 10'7 41.30 Q (top 1/3) 4.60 x 10'8 5.20 U (top 1/3) 1.96 x 10‘8 2.22 Large-Scale Column A: Op. Phase B pre-inoc. effluent 1 7.99 x 10'7 9.04 Op. Phase B inoculation breakthrough 1.19 x 10'3 1.35 x 104 10/1/02 Port 4 pore water 9.76 x 10'9 1.10 10/15/02 Port 5 pore water 1.50 x 10’8 1.71 10/15/02 Port 7 pore water 1.00 x 10'8 1.13 Post-Treatment Soil — Port 2 1.24 x 10'8 1.41 Post-Treatment Soil — Port 4 5.74 x 10'8 6.49 Post-Treatment Soil — Port 6 2.28 x 10'8 2.57 Post-Treatment Soil — Port 7 1.07 x 10'8 1.21 Biorrnass concentrations extrapolated from DNA quantification results assuming 8.84 x 10’” g of DNA per cell, 2.13 molecules of DNA per cell, and a typical cell mass of 9.5 x 10‘'3 g. 3.5.1 Small-Scale Column Experiment Biomass Biomass measurements during the small column experiment were restricted to the analyses of post-treatment soils fiom inoculated columns P, Q, and R, and stimulated columns S, T, and U. Curiously, these data indicate that the average solid-phase biomass accumulation within the stimulated columns (7 x 107 CFU/ g) was geater than the quantity detected in the bioaugnented columns (4 x 10° CFU/ g). Further, 75 Dehalococcoides sp. were detected in all but one soil sample fiom both column sets at approximately 10‘1 CFU/ g, which may indicate that these microorganisms are native to the Plume F/G sediment. However, a sample collected from inoculated Column P yielded a concentration of Dehalococcoides sp. within a range of 1-100 CFU/ g (Table 3.2), possibly indicating a gowth dependency on environmental conditions imposed by the BR Enrichment. Fatty Acids Although no specific fatty acid analyses were performed on samples collected from the small-scale column, the results from the IC analysis of samples collected immediately following feeding events, when lactate was the only fatty acid within the column system at detectable concentrations, exhibited organic acid concentrations consistent with those associated with the levels of lactate fed to the columns. Therefore, IC results were used to track fatty acid levels within the small-scale columns. However, interpretation of the IC- generated results required an understanding of the feeding history of each column, as these data indicated the presence of lactate in samples collected immediately following feeding, and total fatty acids in samples obtained at all other times, assuming a certain degee of lactate decomposition post-feeding. In this context, the anion (1C) data for the inoculated and stimulated small-scale columns indicate that fatty acid levels persisted during the post-inoculation incubation periods (data for column P, Q, and R shown on Figure 3.6). This trend was not evident within the sterile control columns (data not shown). However, the mass of fatty acids remaining with the bioaugnented and biostimulated columns was significantly less than 76 anticipated, assuming lactate fermentation occurred to completion during each incubation period. The depletion in mass coupled with the presence of methane (see below) may have indicated that acetate was produced through lactate fermentation and that acetate- metabolism, likely by acetotrophic methanogens, had occurred during the incubation periods. The general mechanisms of lactate decomposition resulting in production of propionate, acetate and molecular hydrogen are stoichiometrically described by: 3C3H603 ———’ 2CH3CH2C00H + CH3CO0H + C02 + H20 (Lactate) (Propionate) (Acetate) CH3CH2CO0H + 3H20 _" CH3CO0H + C02 + 3H2 C3H603 + 2H20 —‘> CH3CO0H + C02 + 2H2 Molecular hydrogen produced through lactate and propionate fermentation was, therefore, a source of reducing power for hydrogenotrophic microorgarnisms (including halorespiring bacteria) present in the re—packed Plume F/G sediments. Note that, based on stoichiometery and backgound concentrations for nitrate (22 mg/L) and sulfate (48 mg/L), an estimated 200 mg/L of 60% DL-lactate needed to be fed to the bioaugnented column systems on a weekly basis in order to completely reduce these solutes and dechlorinate a TCE concentration of 2.5 mg/L. 77 120.00 Inoculation \ l l 60.00 mg/L 40.00 20.00 0.00 Figure 3.6. Organic acid and phosphate data for Columns P, Q, and R. Organic acid levels are indicated by the open diamonds. Phosphate trends are presented by the closed circles and bold tie-lines. Headspace Gasses The analyses of headspace gas data (not shown) revealed that hydrogen levels within the atmosphere (3-5% or approximately 42 11M) of the anaerobic glove box in which the experiments were conducted likely interfered with the measurement of hydrogen partial pressures associated with bioactivity within the columns. Other than hydrogen, methane was the only gas detected in headspace samples. Methane was measurable in samples collected from both the bioaugnented columns (Columns J, K, L, M, N, O, P, Q, and R) and the biostimulation controls (Columns S, T, and U). In general, the occurrence of methane was most significant within Columns P, Q, and R, which were 78 inoculated with non-dilute BR Enrichment. These qualitative results indicate that methanogenic conditions developed in all of the columns that received bi-weekly dosages of lactate. The depletion of fatty acid levels during the associated incubation periods may indicate that acetate-utilizing methanogens were active. The occurrence of acetotrophic methanogenesis in both bioaugnented and biostimulated columns indicates that these microorganisms were likely associated with the Plume F/G aquifer materials. Phosphate During post-inoculation incubation periods, phosphorus levels measured in columns P, Q, and R consistently increased to 5-10 mg/L (Figure 3.6). This trend was not evident in other inoculated or control column sets and indicates that there was a mechanism causing phosphate solubilization that was unique to the columns receiving non-diluted BR Enrichment. The exact mechanism is unknown, but may have included chelation with ferrous iron, the concentration of which was likely increasing due to ferric iron reduction associated with the elevated bioactivity and associated decrease in ORP within these columns. Nitrate and Sulfate Influent nitrate levels (15-25 mg/L) within columns P, Q, and R were consistently depleted during the post-inoculation incubation periods. Curiously, this trend was evident within all columns (including controls) throughout the duration of the experiment. Therefore, the mecharnism of nitrate removal is unclear. Influent sulfate levels (SO-75 mg/L) consistently decreased approximately 50% during the post- 79 inoculation incubation periods associated with Columns P, Q, and R (Figure 3.7). In general, the magnitude of sulfate reduction was less pronounced in the other two inoculated column sets. No discemable reductions in sulfate levels were evident within the biostimulated or control columns (data not shown). The persistence of sulfate in the inoculated columns suggests that the bulk of the reducing equivalents resulting from lactate fermentation were consumed by other terminal electron accepting processes such as nitrate and iron reduction and halorespiration. The duration of contact of amended pore fluids with the inoculated Plume F/G sediments was apparently not sufficient to permit successional development of predominantly sulfate reducing conditions by the rrnicroflora in the columns. It is likely that sulfate-reducing conditions would have developed if a more concentrated biweekly dosage of lactate was fed to the columns to drive the bulk ORP of the system to the level of sulfidogenesis or methanogenesis, or the duration of the incubation periods were increased to produce a similar effect. 80 100.00 90.00 "\\ \ Inoculation (nitrate at 300 mg/L) 80.00 70.00 60.00 50.00 mg/L 40.00 I\ 1 1. 20.00. l ?\ ’~ \‘k . ‘ v. ‘ 10.00 0.00 A",‘, , , v ‘ XL \\ 10 20 50 0 30 40 6O 70 Days Figure 3.7. Nitrate and sulfate data for Columns P, Q, and R. Nitrate data indicated by open diamonds; sulfate data indicated by closed circles and bold tie- lines. Soluble Metals No measurements of soluble iron were made during the small-scale column experiment. However, evidence of sulfate reduction in Column P, Q, and R, albeit limited, suggests that iron reduction had occurred in the sediments that were inoculated with non-diluted BR Enrichment. Cr(VI) levels in post-incubation period effluent samples (<10 mg/L) were, in general, significantly lower than the concentrations in the Plume F/G goundwater fed to the inoculated controls (>200 mg/L) during the exchange events evaluated. These data indicate that the ORP within the inoculated columns was consistently below the level necessary to favor the reduction of chromium. 81 T CE Transformation Examination of the small-scale column time-series plots (Figure 3.8) reveals that prior to inoculation, sorption was essentially complete within column sets 2 through 7, as evident by the lack of significant TCE removal between the exchange events during weeks 4 through 6. After inoculation, TCE removal was distinct within the three bioaugnented column sets (Columns J, K, L, M, N, O, P, Q, and R), indicating biotransformation. This trend was more pronounced within Columns P, Q, and R, which were inoculated with undiluted BR Enrichment. Cumulative mass balances for these columns indicate that following inoculation between 50-100% of the TCE mass input into each vessel during an exchange was transformed during the subsequent incubation periods. Greater than 90% of the dechlorination resulted in the production of VC in Columns P, Q, and R (Figure 3.9). The accumulation of VC was significantly less in column set #’s 3 and 4 (Figure 3.9). Less than 10% of TCE dechlorination represented an accumulation of cis-DCE. The preponderance of VC accumulation relative to cis-DCE suggests that reductive dechlorination processes were trending toward completion during the incubation periods. No significant TCE removal was observed in radiation-sterilized, natural attenuation, and bioenhancement control columns during the period parallel with inoculation and subsequent feeding exchanges. Further, increases in nutrient and TCE loadings during the late stages of the small-scale column studies had little to no observable beneficial effect on TCE transformation activity in inoculated columns. In summary, the direct comparison of bioaugnentation versus natural attenuation and biostimulation within the small-scale column systems produced strong evidence of a 82 causal relationship between the dechlorination activity of the in-situ microbial community supplemented with the BR Enrichment and removal of TCE from the Plume F/G aquifer materials. 200 l l l l . . a —0— Bioaugmented (100%) ""Dm Natural Attenuation —-fi—- Natural Attenuation __4,___ ..__ -_.__._ 150 *— i 1 i go *Biostimulated k i X? :5 + Bioaugmented (1%) i «>2 —-0- BioaugrmntedUO‘VOI } fi% 0 . g 100 ~ 1P 1 ff? ‘ °‘ ‘ i ii a . v I .-' 0‘ g ' e ' 1"?“ 50 - ..... i l l 1 i I i : i 'i 0 - 1L l l J 1 fit l l L 0 50 100 150 Days Figure 3.8. Cumulative TCE mass removed in each small-scale column set. TCE Transformation Kinetics and Mass Removal Figure 3.8 illustrates the cumulative removal of TCE witlnin each of the six column sets over the duration of the small-scale column experiment. TCE removal increased sharply in column set #6 (columns P, Q, and R) following inoculation with the Bachman Road Enrichment. The increase is progessively less pronounced within column set #5 (e.g., column M, N, O, which received a 10% inoculate strength) and column set #5 (e.g., columns J, K, and L, which received a 1% inoculate strength). 83 1.2 —o—TCE I 1 —c1— C-DCE T “C - 1 DH - 1 W umoles 62 82 102 122 142 62 82 102 122 142 Day Day Figure 3.9. TCE removal and cis-DCE and VC accumulation in columns P, Q, R (a), J, K, L (b), and M, N, O (c). No substantial TCE degedation was observed in the control column sets. For the inoculated column sets, the increase in TCE removal was expected. However, there was also some expectation that feeding of the non-sterilized controls (columns S, T, and U) would result in stimulation of indigenous microorganisms capable or transforming TCE. 84 Although there was some evidence of limited TCE removal within this column set, the trend was similar to tlnose observed in the data sets for the TCE-fed control (natural attenuation) columns (Columns D, E, F, G, H, and I). This indicates that no significant biostimulation occurred over the duration of the experiment. Figure 3.8 reveals a trend that was common to columns P, Q, and R. After inoculation, TCE levels steadily decreased at a relatively consistent rate. Further, TCE transformation during this period was equal to or exceeded the mass of solute loaded into the columns during the associated exchange events, indicating removal of sorbed contaminant. Figure 3.10 illustrates changes in the apparent first-order rate coefficient, k ’, of TCE mass removal in columns P, Q, and R during the treatment period (e.g., days 60 through 130). From these data, an average k ’ of 0.62 day’1 was obtained. Laboratory studies have shown that the rate of TCE transformation in batch experiments is a firnction of culture age; decay of transformation activity occurs as cells enter a stationary phase. The k ’ value reported herein reflects this loss of activity. Apparent first-order rate coefficients estimated from data from column sets 4 (J, K, L) and 5 (M, N, 0) were 0.24 day"1 and 0.15 day”, respectively. 85 1.8 1.6 1:: .. a \\ 0.6 (am " 0.4 Rvka 0.2 u k' (l/day) / Incubation Period Figure 3.10. Apparent first-order rate coefficient trend, Columns P, Q, and R. The results from the analyses of the batch microcosm samples are summarized in Appendix B. From these data, a zero-order rate of PCB dechlorination of 29.6 uM/g (biomass)/day was measured. First-order TCE dechlorination rates of 0.58 day”I to 3.54 day’1 were also estimated from the PCB transformation data. Therefore, the range of k ’ estimated from the small column data is consistent with the range associated with the culture maintained under laboratory conditions. The cumulative mass removal data from the column experiments indicate that bioaugnentation of the Plume F/G aquifer materials with the BR Enrichment will result in TCE transformation. The small-scale column data revealed that VC was the predominant end product resulting after seven days of treatment. The range of apparent 86 first-order rate coefficients reported here suggests that favorable rates of TCE transformation may be attainable from engineered bioaugnentation of Plume F/G. 3.5.2 Large-Scale Column Experiment Appendix A summarizes the sampling dates associated with the operational history of Column A, as well as the influent concentrations of TCE, fatty acids, hydrogen, major anions, and soluble chromium and iron in the fluids delivered to the column over the duration of the experiment. As shown on Table 3.5, Column A was inoculated with the Bachman Road enrichment culture on two separate occasions. The periods of Operation that followed each inoculation event are referenced as Operational Phases A and B. The duration of Operational Phase A was 9 1/2 weeks and included a total of eight distinct incubation periods that followed the initial inoculation and seven subsequent feeding events. From a preliminary examination of the Column A database at the conclusion of Operational Phase A it was originally inferred that TCE dechlorination was not occurring to the extent anticipated, based on the treatment performance of the BR Enrichment within both the EFX reactor and the small-scale columns. On this basis, re- inoculation of Column A was considered necessary. This second inoculation event and five subsequent incubation periods extending over a duration of six weeks comprised Operational Phase B. Time series plots of sample port-specific solute concentrations (e. g. TCE/cis-DCEN C, fatty acids, anions, and soluble chromium and iron) measured during each incubation period of Operational Phase A and Operational Phase B are provided in Appendix C and Appendix D, respectively. 87 Operational Phase A Biomass DNA quantification of soil and goundwater samples revealed no distinct tends in spatial or temporal biomass accumulation. Biomass concentrations were extrapolated from the DNA quantification results assuming 8.84 x 10'15 g of DNA per cell, 2.13 molecules of DNA per cell, and a typical cell mass of 9.5 x 10'13 g. In general, post- inoculation aqueous samples exhibited biomass concentrations of 4 x 104 CFU/mL to 5 x 108 CFU/mL, with an average of 3 x 107 CFU/mL. Soils samples from Column B yielded 1 x 104 CFU/g to 3 x 10° CFU/ g. Dehalococcoides sp. were measured at levels within the 102 CFU/mL range (Table 3.2) in inoculum samples, but were not detected in soil or goundwater during Operational Phase A. Headspace Gasses Throughout the duration of the large-scale column experiment, quantification of hydrogen partial pressures in aqueous samples was not possible apparently due to the limited sample volumes (0.5-1.0 mL) collected. No samples collected during Operational Phase A exhibited detectable levels of hydrogen. Conversely, methane was consistently detected over the entire length of the column. However, due to limited available sample volumes (0.5 — 1.0 mL) methane measurements were marginally above the limits of quantification. Qualitative assessment of peak heights did reveal a slight increase in methane levels over then length of the column, suggesting a progessive increase in methanogenic activity. Ethene peaks were not clearly evident in any of the Column A samples obtained during Operational Phase A, although its presence was suspected. As 88 with hydrogen, the lack of detectable concentrations of ethene is most likely due to the limited sample volumes collected during the experiment. Fatty Acids Examination of the fatty acid data collected during Operational Phase A (Appendix C-l) reveals that lactate rapidly decomposed after inoculation and each subsequent feeding event. During the two-week long incubation period immediately following inoculation (e. g. Incubation Period 1A), lactate fermentation was incomplete with propionate as the exclusive bi-product. During subsequent incubation periods, lactate in feed solutions introduced into the column was completely decomposed. Through Incubation Period 6A, propionate appeared to have been the principal byproduct of lactate fermentation. However, the maximum levels of propionate measured over this time interval were consistently less than 50% of the influent lactate concentrations delivered to the column. Subsequently, significant concentrations (e.g., > 50 mg/L [830 mM]) of acetate were measured during Incubation Periods 7A and 8A, predominantly in samples from the downgadient ports 5 and 7. This suggests that either the rate and completeness of lactate fermentation improved, or that the activity of hydrogenotrophic acetogens increased. In general, this progessive increase in lactate and propionate decomposition indicated temporal acclimation of fermenting microorgarnisms to the Column A aquifer materials. By Incubation Periods 7A and 8A, fermentation rates appeared to be essentially instantaneous. With lactate levels delivered to Column A being 100-300 mg/L (1.1-3.3 mM), Stoichiometeric dissolved hydrogen concentrations in excess of 2.5-7.5 mM were rapidly 89 and consistently produced as the result of fermentation following inoculation and each feeding event during Operational Phase A. Phosphate As shown in Appendix 02, phosphate was detected only sporadically within any port-specific goundwater samples collected during Operational Phase A. Further, these few detections revealed no distinct spatial or temporal patterns. Nitrate and Sulfate Major arnion data are presented in Appendix C-2. During the second week of Incubation Period 1A, immediately following inoculation of Column A, the concentrations of major arniorns such as chloride and sulfate steadily decreased. Nitrate levels were not measurable during this period, with the exception of a single detection (approximately 20 mg/L) in a sample collected from Port 7 at the conclusion of Incubation Period 1A. During the period following the first feeding event (e.g., Incubation Period 2A) nitrate concentrations at Ports 1 and 2 exceeded 10 mg/L. However, over the length of the column, nitrate levels decreased to below detectable levels. Incubation Period 3A revealed that the influent concentrations of nitrate (20 mg/L) were reduced to below detection limits upgadient of Port 1. Port-specific sulfate concentrations appeared to persist at levels between 10-20 mg/L. The time-series and spatial profiles of major arnion concentrations during Incubation Period 4A are similar to those associated with Incubation Period 3A. However, the sharp decrease in sulfate observed at Port 7 on August 9, 2002 suggests that sulfate reduction may have 90 temporarily been the dominant terminal electron-accepting process early during Incubation Period 4A. Similar profiles for nitrate and sulfate are associated with Incubation Periods 5A, 6A and 7A. Incubation Period 8A revealed a much stronger trend toward sulfate reduction, with sulfate concentrations below detectable levels at Ports 5 and 7 at the conclusion of the period. These data suggest that sulfate-reduction occurred during the first three days of the incubation period when acetate concentrations, produced from the rapid fermentation of lactate and propionate characteristic of this stage of Operational Phase A, were maximized. Soluble Metals Throughout the duration of Operational Phase A, ferric iron and hexavalent chromium reduction were evident (Appendix C-3). Soluble iron concentrations generally increased over the length of the column during each incubation period comprising Operational Phase A. In some instances, soluble iron levels increased from below 100 [Lg/L within influent pore fluids to 1,000-2,000 ,ug/L between Ports 2 and 4. Influent concentrations of Cr(VI) in excess of 200 jug/L were consistently reduced to less than 20 ug/L between Ports 1 and 2 by the time sampling was completed following each feeding. These data suggest that the ORP resulting from inoculation and lactate feeding was sufficiently low for promoting consistent and rapid reduction of hexavalent chromium. It is not know if the Cr(VI) reduction in Column A was biologically mediated or the result of purely physicochemical processes. However, Cr(VI)-reducing microorganisms are ubiquitous in soil and sediments. Many aerobic and anaerobic microorganisms can reduce Cr(VI) while utilizing a range of substrates at near neutral 91 pH, and several have been isolated that can use Cr(VI) as a terminal electron acceptor for growth (Criddle and Middleton, 1999). The Plume F/G aquifer materials have been exposed to Cr(VI) for decades and it is likely that the native microflora includes microbial populations capable of dichromate reduction. Alternatively, it is well known that Cr(VI) is readily reduced by ferrous iron. Ferrous iron concentrations in feed groundwater increased as much as an order of magnitude within the column system during Operational Phase A. Therefore, either mechanism for Cr(V I) reduction in Column A is plausible. T CE Transformation As described in Chapter 2, the large-scale column experiment was designed to consistently deliver Plume F/G groundwater spiked with approximately 1 mg/L of TCE to Column A. However, technical difficulties associated with stock preparation during the initial inoculation and the subsequent two weeks of groundwater delivery to Column A inadvertently resulted in the throughput of substantially lower TCE concentrations. Specifically, during inoculation, the concentration of TCE in groundwater conveyed to the column was slightly less than 50 ug/L. Groundwater fed to Column A during the first two weeks of Incubation Period A contained between 240-435 jig/L of TCE. The stock preparation difficulties were corrected prior to the initial feeding of Column A, two weeks following inoculation. Consequently, Incubation Period 2A began with the replacement of pore fluids with groundwater containing TCE in excess 2,000 jig/L. Subsequent stock preparations during Operational Phase A resulted in TCE delivery concentrations consistently greater than 1,000 ug/L. 92 A potential consequence of the early stock preparation difficulties was that the effects of TCE sorption onto the Plume F /G solids could have obscured evidence of biotransformation during the post-inoculation incubation periods. From inspection of the data presented in Appendix C-4, it appears that the effects of sorption following Incubation Period 3A are of minor consequence. TCE concentrations measured at downgradient ports (Ports 4-7) during Incubation Period 4A closely match the concentrations within the groundwater delivered to the column, suggesting that sorption was essentially complete. However, there was evidence of a localized reduction in TCE concentration that appears anomalous. Specifically, TCE concentrations at Port 3 appeared to have decreased from background levels by approximately 30%. Given the progressive increase in cis-DCE levels over the length of the column, this anomaly is believed to represent the first clear evidence of reductive dechlorination during Operational Phase A. A reduction in TCE levels consistent with that observed during Incubation Period 4A was evident within the length of column extending from Port 1 to Port 4 during the four subsequent incubation periods comprising Operational Phase A. Port-specific reductions in TCE concentrations as much as 1,000 pg/L (7.6 M) were accompanied by a coincident increase of less than 200 [Lg/L (2 pM) of cis-DCE and little to no accumulation of VC. More specifically, at the conclusion of each incubation period, cis-DCE concentrations were at maximum levels in samples from Ports 6 and 7, while TCE transformation appeared to persist between the column influent and Port 3. This indicated that cis-DCE accumulated early in the incubation period immediately following feeding, when TCE transformation rates were likely maximized. Thereafter, cis—DCE production gradually diminished, but TCE transformation appeared to remain 93 fairly constant. These observations suggest that the dominant mechanism of reductive dechlorination during these incubation periods was halorespiration by Dehalococcoides populations. Summary — Operational Phase A When viewed collectively, the anion and soluble chromium and iron monitoring data indicate that anoxic conditions prevailed within Column A during Operational Phase A. Following inoculation and through Incubation Period 2A, nitrate and iron reduction appeared to be the dominant terminal electron-accepting processes. Between Incubation Periods 2A and 4A, iron reduction prevailed. Following Incubation Period 4A, there is evidence that the oxidation-reduction potential (ORP) decreased to the level of sulfidogenesis. Specifically, these data reveal that sulfate was reduced to below detectable levels during the early stages of Incubation Periods 7A and 8A. With nitrate and iron reduction being essentially complete and sulfate reduction only occurring temporarily, it is inferred that the reducing power that persisted within the column over the duration of each incubation-period was sufficient to maintain the system ORP at or just above the level favorable for sulfidogenesis. Fatty acid data corresponding to these periods suggest that this source of reducing power was dissolved hydrogen produced through lactate and propionate fermentation. According the Loffler, et al. (1999), the hydrogen threshold concentrations associated with nitrate and iron reduction, and halorespiration are lower than those associated with sulfate reduction and methanogenesis. Therefore, it is plausible that nitrate- and iron-reduction and 94 halorespiration could occur through the duration of each incubation period Operational Phase A, without co-incident occurrence of sulfidogenesis. Dissolved hydrogen is the preferred source of electrons for halorespiration. As previously discussed, direct measurement of dissolved hydrogen concentrations within Column A was not possible due to sample volume constraints. However, if the inferences associated with the anion and fatty acid data are accurate, then the redox conditions favorable for TCE respiration would approach optimal levels after lactate and propionate fermentation was complete. However, acetate produced by these fennentations could have stimulated less efficient halorespiring populations, resulting in an accumulation of cis-DCE. It is probable that efficient halorespiration by D. ethenogenes would not predominate until acetate was metabolized within or transported out of the BAZ. Operational Period B Biomass As noted previously, DNA quantification of soil and groundwater samples revealed no distinct tends in spatial or temporal biomass concentrations. Similar to Operational Phase A, aqueous samples yielded average biomass concentrations of 3 x 107 CFU/mL. Soils samples from both Columns A at the conclusion of Operational Phase B exhibited 1x 106 CFUs/g to 7 x 106 CFU/ g. The inoculum used for Operational Phase B contained Dehalococcoides sp. within the 102 CFU/mL range (Table 3.2). During inoculum breakthrough, Dehalococcoides sp. were detected at levels of 9 CFU/mL to 1 x 104 CFU/mL. Only three aqueous samples collected during Operational Phase B, 95 collected during Incubation Periods 4B and 5B yielded detectable (e.g., 1-2 CFU/mL) quantities of Dehalococcoides sp. Conversely, soil samples collected from Ports 2, 4, 6 and 7 of Column A at the conclusion of the experiment exhibited Dehalococcoides sp. at levels ranging between 1-7 CFU/ g. Peak levels were observed at Port 4 (Table 3.2). Headspace Gasses No samples collected during Operational Phase B exhibited detectable levels of hydrogen. However, as shown on the time-series plot provided on Figure 3.11, as much as 6 mM of hydrogen should have been produced as a result of lactate and propionate fermentation. In environments saturated with hydrogen, nitrate and ferric iron are expected to be rapidly reduced. Therefore, it is plausible that hydrogen concentrations were rapidly depleted by nitrate- and ferric iron-reducing microorganisms to below levels quantifiable in the 0.5 —— 1.0 mL sample volumes obtained from Column A. Methane was consistently detected over the entire length of the column. However, the due to the limited sample volumes obtained, methane levels were only slightly above quantifiable limits. Qualitative assessment of peak heights did reveal a slight increase in methane levels over then length of the column, suggesting a progressive increase in methanogenic activity with distance traveled by pore fluids. Ethene was not detected in any of the Column A samples obtained during Operational Phase B, though its presence was suspected. As with hydrogen, the lack of detectable concentrations of ethene is most likely due to the limited sample volumes collected during the experiment. 96 Fatty Acids Column A was re-inoculated on September 3, 2002. Fatty acid data (Appendix D- 1) indicate that lactate concentrations were generally equally distributed over the length of the column immediately following re-inoculation. At the conclusion of Incubation Period 1B, lactate levels remained equally distributed but decreased over 50% at each sampling port. However, unlike during the period immediately following the initial inoculation event and subsequent incubation periods, lactate concentrations persisted above 25 mg/L. Propionate and acetate were not measured at significant levels during Incubation Period 1B. During Incubation Period 2B, lactate feed levels decreased nearly 10-fold at Port 1 to below detectable levels at Ports 5 and 7. Correspondingly, acetate concentrations increased inversely over the length of the column to approximately 400 mg/L (7 mM) at Port 7. Minor levels (< 50 mg/L [0.7 mM]) of propionate were observed at Port 5 during Incubation Period 2B. During Incubation Periods 43 through 6B, time-series and spatial gradients of lactate decomposition and byproduct formation were similar. However, with the temporal resolution of sampling increased to bi-weekly during Incubation Period 4B and daily during Incubation Periods 5B and 68, it became apparent that lactate and propionate were very rapidly decomposed following feeding, resulting in an immediate accumulation of acetate and, undoubtedly, hydrogen. Generally, the mass of acetate traveling through the column was preserved or slightly reduced, indicating that hydrogenotrophic acetogenesis did not occur to a measurable extent. With a doubling of the lactate input concentration during Incubation Period 6B, greater masses of propionate and acetate were produced. 97 Phosphate Phosphate concentrations associated with the second inoculation (200-250 mg/L) decreased to less than 10 mg/L during Incubation Period 1B (Appendix D-2). Subsequently, phosphate levels remained at or below detectable levels at Port 1. However, in contrast to the observations made during Operational Phase A, at the conclusion of each Phase B incubation period, a gradual increase in phosphate levels was evident over the length of Column A. This behavior was consistent with the phenomena observed in small-scale columns P, Q, and R, and suggests that a solubilization mechanism was associated with the elevated bioactivity in the column. Specifically, solids-associated phosphates may have chelated with the soluble ferrous iron produced from ferric iron reduction. Nitrate and Sulfate The results from the analyses of anions in Column A samples following the second inoculation (Appendix D-2) and at the conclusion of Incubation Period 1B indicate that nitrate reduction was consistent during these periods. Conversely, sulfate levels persisted at or close to background concentrations. However, as during Incubation Periods 7A and 8A, there was consistent evidence that sulfate reduction occurred temporarily during Incubation Periods 2B through 6B. The refined temporal resolution of Incubation Periods 4B-6B reveal that sulfate levels were reduced to below detectable levels during a finite interval and within a specific segment of Column A. During these periods, sulfate reduction was evident within two days of feeding and occurred most significantly within the column length between Ports 2 and 4 where and when acetate and 98 hydrogen levels were, presumably, at their maximum. Ultimately, sulfate in this interval was reduced to below detectable levels. Under these conditions, methanogenic activity was likely enhanced. However, after three days, the ORP within this portion of the column apparently increased with the infiltration of non-amended groundwater that followed each feeding. Specifically, sulfate concentrations began to gradually increase at these ports to near background levels (e. g., 50 mg/L) during days 4—7 of each incubation period. The decrease in sulfidogenesis corresponds with a progressive decrease in and competition for reducing power associated with acetate and hydrogen. Soluble Metals Soluble chromium and iron data associated with Operational Phase B (Appendix D-3) indicate that redox conditions within Column A were consistently sufficiently low and favorable for Cr(VI) reduction to concentrations below 20 mg/L over the entire length of the column, immediately following feeding and throughout the duration of each incubation period. As during Operational Phase A, iron reduction was also evident. However, iron reduction did not become significant or consistent over the length of Column A until Incubation Period ZB, presumably after nitrate-reduction was complete. The progressive increase in soluble iron concentrations over the column length following Incubation Period 2B suggests that iron reduction was gradual. The observed soluble iron gradients could, in part, have been influenced by precipitation within the upstream portion of the column. Initially, during Incubation Period 1B, this may have been the result of precipitation with phosphate remaining within the column following inoculation. During subsequent incubation periods, the lower levels of soluble iron are likely to have 99 been partly attributable to ferrous iron precipitation with sulfide resulting from the reduction of sulfate between Ports 2 and 4. T CE Transformation TCE monitoring data from Operational Phase B is summarized in Appendix D-4. For a two-week period following the second inoculation of Column A, there was no clear evidence of TCE transformation. By the first week of the two-week-long Incubation Period 3B, evidence of TCE dechlorination re-appeared. By Incubation Period 4B, the zonation and magnitude of TCE transformation was consistent with those observed during the final five weeks of Operational Phase A. Sampling was performed every 2-3 days during Incubation Period 43. The increase in temporal resolution revealed that TCE transformation was maximal during the first four days of the period. Further, co-incident cis-DCE production was observed. However, port-specific concentrations of cis-DCE did not account for the mass of TCE transformed. Specifically, a cumulative loss of TCE of approximately 9 [1M was observed at Port 7 at the conclusion of Incubation Period 4B. During this same period, a cis-DCE concentration of approximately 2 M was measured after 4 days (Port 4) of pore fluid travel time. From day 4 onward, the cis-DCE concentrations progressively decreased with no co-incident accumulation of VC. The loss in aqueous cis-DCE mass while TCE transformation continued may indicate a transition to more complete halorespiration downgradient of Port 4. The behavior revealed during Incubation Period 4B suggested that even tighter temporal and spatial sampling resolution would more accurately reveal the zonation, 100 magnitude and gradients associated with TCE transformation. Therefore, sampling was conducted daily, and, when possible, at each sampling port during Incubation Periods 5B and 6B. Time series plots of TCE, cis-DCE and VC over Incubation Periods 4B, 5B and 6B are presented in Appendix B. As seen on these figures, the data produced during Incubation Period 5B confirmed that TCE transformation occurred predominantly within the column segment bordered by the influent and Port 4 and that, although transformation appeared to occur during the entire period, k’ values were generally at maximum levels through day 3 (Port 3). Approximately 6 ptM of TCE transformation within this zone was accompanied by co-incident production of 5 uM of cis-DCE and 1 uM of VC. During the subsequent and final inoculation period (Incubation Period 6A), the concentration of lactate in the feed solution was doubled to approximately 500 mg/L (5-6 mM). This was done to evaluate if the additional lactate mass would extend the duration or increase the magnitude of TCE transformation. However, the only clearly observable result was a proportionally greater production of cis-DCE and VC through day 2 of this incubation period compared to Incubation Period 5B. By the second day of the period, 8 uM of aqueous TCE was transformed, while 10 uM of cis—DCE and 3 uM of VC were produced. VC levels continued to increase through Day 3. Thereafter, cis-DCE and VC transformation appeared to occur with consistency. Conversely, TCE levels in groundwater actually increased by approximately 2 uM through Day 5, suggesting that TCE desorption rates exceeded the transformation rates between Ports 3 and 5. Sorbed and aqueous TCE levels apparently equilibrated afier this time and, between days 5-7, TCE dechlorination rates progressively increased, resulting in a net loss of TCE. . 101 Summary — Operational Phase B In total, the fatty acid and inorganic data reveal that the ORP within Column A was highest immediately following re-inoculation, with nitrate reduction as the dominant terminal electron accepting process. Within one week of re-inoculation and throughout the remainder of Operational Phase B, iron reduction became consistent, and short-term occurrences of sulfate reduction became evident. Sulfate reduction appeared to occur for 1-2 days within the length of the column bordered by Ports 2 and 4, after which the ORP of the system slowly recovered to background levels with the influx of non-amended groundwater. As during Operational Phase A, Cr(VI) was consistently reduced, resulting in soluble chromium levels dropping from the influent concentration range of 200-250 [Lg/L to below 20 ug/L. Typically, treatment to these levels was complete at a point upgradient of Port 2. TCE Dechlorination Kinetics and Mass Removal Assuming first-order kinetics, an examination of TCE, cis-DCE and VC transformation rates was facilitated through performing mass balances on a series of discrete control volumes juxtaposed over the length of Column A. The column length associated with each control volume was defined by the distance traveled by pore fluids over a 1-day period, as determined from the average linear velocity of groundwater flow. Accordingly, a total of seven column segments approximately 15 cm in length were established. Due to the temporal resolution of the associated data sets, TCE results from Incubation Periods 4B-6B were used to complete the analysis of transformation rates. 102 Port-specific TCE results for each day of sampling during these incubation periods are presented in Appendix F. The results from these analyses are summarized on Table 3.3. Table 3.3. First order rate coefficients, k’, within control volumes of Column A between October 3 and October 22, 2002. Date Influent to Port 1 Port 1 to Port 2 Port 2 to Port 3 October 3, 2002 0.39 day'1 0.36 day7 0.25 day'r October 5, 2002 0.45 day"1 0.04 day"1 0.05 day'1 October 8, 2002 0.28 day’1 0.06 day" 0.06 day" October 9, 2002 0.20 day" 0.25 day" 0.08 day'1 October 10, 2002 0.27 day" 0.07 day'1 0.00 day" October 11, 2002 0.47 day" 0.11 day'1 0.04 day'1 October 12, 2002 0.47 day'1 0.09 day" 0.12 day" October 14, 2002 0.23 day'1 0.07 day‘1 0.01 day" October 15, 2002 0.67 day" 0.14 day" 0.00 day" October 16, 2002 0.06 day" 0.07 day'1 0.26 day" October 17, 2002 0.53 day" 0.37 day" 0.36 day" October 18, 2002 0.32 day" 0.10 day" 0.00 day" October 19, 2002 0.40 day" 0.31 day" 0.00 day" October 20, 2002 0.37 day" 0.15 day'1 0.00 day" October 21, 2002 0.36 day'1 0.40 day" 0.00 day" October 22, 2002 0.44 day" 0.27 day" 0.00 day" The rate analyses revealed that TCE transformation was most rapid within the column segment between the influent and Port 1. Here, k' values of 0.2-0.7 days'1 were measured. These rates are well within the range estimated from both the batch microcosm study and the small-scale column experiment. Beyond Port 1, the rates of transformation decrease. Specifically, between Ports 1 and 2, k’ values of 0.06 to 0.4 days’1 were observed. As exhibited in Appendix F, cis-DCE and VC accumulated upgradient of Port 4 during Incubation Periods 4B-6B. Beyond Port 4, apparent first- order rate transformation rates for cis-DCE and VC averaged 0.51 day'1 and 0.45 day", 103 respectively. Maximum k' values estimated from the batch microcosm data were 3.04 day'1 (cis-DCE) and 0.49 day" (VC). Whole-column mass balance analyses were conducted to estimate the approximate mass removal efficiency during each 1-2 week incubation period of the large—scale column experiment. The results from these calculations are presented on Table 3.4. These data reveal that, on average, 37% of the TCE mass entering Column A was removed during the eight incubation periods (e. g. Incubation Periods 5A-8A and 3B- 6B) during which transformation was most evident. Maximum TCE removal (45%) appeared to occur during Incubation Period 4B. TCE removal was lowest (26%) during Incubation Period 6A. Based on the transformation rate examination, it is inferred that TCE removal is nearly exclusively attributable to microbial dechlorination. From the analyses of transformation rates summarized above, it is evident that the bulk of TCE dechlorination occurred between the column influent and Port 3, where k" values were most significant and consistent. Downgradient of Port 2, transformation appears to be most significant between days 1 and 4 of each incubation periods. Therefore, the bulk of TCE transformation occurred within 4 days of each feeding event. 104 Table 3.4. TCE mass treatment rates and efficiency over the length of Column A over the duration of incubation periods 5A-8A and 3B-6B. Incubation TCE Removal Rate Mass Removed Treatment Efficiency Period (pg/L) tug) 1%) 5A 42 292 36 6A 24 169 26 7A 36 253 30 8A 30 207 30 3B 47 656 43 4B 35 248 42 SB 20 141 29 6B 24 171 36 A verage 35 2 76 3 7 3.5.3 Redox Gradients and Halorespiration Activity Within the Column A environment, spatially distinct but not separate zones of fermentation activity, nitrate and ferric iron reduction and sulfidogenesis were evident. Under near steady-state conditions lactate fermentation occurred over less than a 24-hour period upstream of Port 1. Nitrate and ferric iron levels were maintained at low concentrations between the column influent and Port 1 due to biologically-mediated reduction reactions accelerated by hydrogen and acetate produced from lactate and propionate fennentations. However, in contrast to the sources of nitrate and sulfate, aqueous ferric iron concentrations within Column A were probably sustained by the dissolution of oxyhydroxides from the aquifer solids. Consequently, iron reduction was evident over the length of the column. Coincident with the sharp increase in fermentation byproducts, sulfate reduction was the dominant terminal electron accepting process, as sulfidogenesis in Column A was enhanced in the presence of acetate. Under such conditions, a certain level of methanogenesis was also expected, as methanogens are 105 more efficient utilizers of acetate. For the quantity of lactate typically fed to the system, nitrate- and iron-reduction were sufficient to limit the availability of reducing equivalents for sustained sulfidogenesis and methanogenesis throughout the duration of each incubation period. During Incubation Period 6B, perturbing the system with a pulse of lactate mass in excess of that to which the microflora in the column had become acclimated following previous feeding events appeared to temporarily increase the magnitude and duration of sulfate reduction, as sulfidogenesis was enhanced by the excess acetate produced from fermentation reactions. However, evidence of significant levels of sulfate reduction downgradient of Port 3 was scarce. Conversely, the qualitative assessment of methane data collected during the latter stages of the experiment suggested that methanogenesis gradually increased over the length of the column. Halorespiration activity appeared to coincide to some extent with nearly all of these redox processes. However, it was most strongly associated with the fermentation activity and sulfidogenesis. During the large column study, TCE dechlorination was not clearly evident until efficient lactate- and propionate-fermenting conditions prevailed. Such conditions were initially evident during Incubation Periods 4A and 3B, which were also the first instances of distinct TCE transformation during Operational Phases A and B, respectively. However, the completeness of TCE dechlorination increased as acetate concentrations became limited. Specifically, less complete TCE dechlorination was consistently evident immediately behind the pulse of acetate passing through Column A dllring each incubation period. For example, during Incubation Period 6B, when the lactate loading to the column was increased from approximately 200 mg/L to over 500 Itlg/L, acetate and propionate concentrations more than doubled over levels measured 106 during previous incubation periods. The reducing power associated with the excess acetate, and residual propionate, served to locally and temporarily decrease the ORP to the level of the sulfate reduction and methanogenesis as the pulse of these fermentation byproducts traveled through the column. Once sulfate levels became limited, the activity of acetate-oxidizing, halorespiring populations like Desulfirromonas probably increased. In general, wherever sulfate levels were depleted in the presence of acetate, incomplete reductive dechlorination prevailed. The result was an accumulation of cis-DCE. However, downstream of Port 4, evidence of sulfate reduction was scarce and may have been inhibited by an increase in methanogenic activity. Consequently, evidence of TCE transformation from Port 4 to the column effluent is limited. As acetate and propionate levels decreased due to bulk transport through the column segments in which incomplete reductive dechlorination activity was occuning, respiration of TCE by more efficient hydro genotrophic dechlorinators such as Dehalococcoides became evident and cis—DCE levels began to decline. This was expected, based on the presumption that halorespiring microorganisms such as D. ethenogenes prefer dissolved hydrogen as an electron donor for their energy-deriving TCE dechlorination reactions. An environment possessing limited levels of dissolved hydrogen as the predominant source of reducing power is likely to be selective for Dehalococcoides sp. populations over microorganisms capable of partial TCE halorespiration such as Desulfitromonas, which prefer acetate as an electron donor. Concurrently, VC accumulated in response to cis-DCE dechlorination. With continued Constunption and transport, hydrogen levels within the column segment between the influent and Port 3 eventually dropped to sub-micromolar concentrations. Under these 107 conditions, halorespiration activity was apparently exclusively attributable to Dehalococcoides, as TCE dechlorination occurred without coincident production of cis- DCE. With time, TCE dechlorination gradationally decreased over the column length in correspondence with the spatial and temporal decline in hydrogen partial pressures. As previously discussed, a symbiotic relationship appeared to exist between TCE to cis-DCE dechlorinating populations and Dehalococcoides during this study. Specifically, the dechlorination of TCE to cis-DCE was accomplished rapidly by acetate- oxidizing microorganisms such as Desulfuromonas sp. Under acetate-limited conditions, the corresponding reduction in TCE levels facilitated cis-DCE and VC dechlorination by Dehalococcoides sp. at accelerated rates. However, because of differences in scale and hydrodynamics, the balance of this relationship was markedly different within the small- scale columns than within large-scale Column A. The incubation periods during the small-scale column experiment were hydraulically stagnant. During these “no-flow” periods, acetate and hydrogen produced from lactate and propionate fermentation were continuously in direct contact with the Plume F/G aquifer materials. Due to this prolonged exposure to the acetate and hydrogen pulses, the efficiency of the symbiotic relationship between the two sets of dechlorinating populations was probably less than within the continuous flow system of Column A. Consequently, evidence of reductive dechlorination exhibited a more pronounced transition from 1)TCE respiration and cis-DCE accumulation to 2) cis-DCE respiration and 3) VC dechlorination. This may, in part, explain the disproportionately greater accumulation of VC at the conclusion of each incubation period of the small-scale 108 column experiment compared to the minor accumulations of VC observed at any point within Column A over the same interval of time. It is also plausible that VC dechlorination rates in the small columns were significantly less than those observed in Column A because of the comparatively lower soil mass and, therefore, mass and availability of iron in those systems. The increased availability of ferric iron associated with the scale and flow-through operation of Column A may have served to promote complete TCE dechlorination by creating a hydrogen sink, thereby maintaining H2 partial pressures at levels that permitted D. ethenogenes to out- compete other dechlorinators in the system. Acetate and hydrogen produced through fermentation may have been sufficient to drive fenic iron reduction to near completion near the column influent. However, time-series data reveal that iron reduction continued over the length of the column. Acetate and hydrogen pulses migrating through the column decreased the ORP within the system and promoted solubilization of iron hydroxides off the aquifer solids. This provided a surplus of electron acceptors for hydrogenotrophic iron-reducing bacteria, thereby contributing to consumption of hydrogen reducing equivalents. Consideration must also be given to the possibility that the elevated hydrogen partial pressures within the spatially constrained small columns resulting from continuous exposure to the anaerobic glove-box environment served to upset the relationship between complete and incomplete dechlorinating populations. Under hydrogen-rich conditions the rates of TCE and cis-DCE respiration and subsequent VC dehalogenation Would likely be suppressed as a result of competitive inhibition by other hydrogenotrophic populations, including sulfidogens and methanogens. 109 From an engineering standpoint, these observations emphasize the need to collect a temporally relevant set of soil and groundwater samples for characterization of major anions and dissolved gasses, and on which to perform biomolecular analyses such as T- RF LP and RT-PCR to assess microbial community structures and identify the presence of dechlorinating populations in solvent—impacted aquifer materials. This information will provide the insight necessary to strategize the magnitude and frequency of hydrogen loadings to promote complete TCE dechlorination. If an organic acid such as lactate is delivered to aquifer materials to release hydrogen for stimulation of dechlorination activity, establishment and acclimation of a BAZ will be most dependent on the colonization of the aquifer materials by fermenting microorganisms. 3.5.4 Hexavalent Chromium Reduction and Immobilization During both the small- and large-scale column experiments, the decrease in ORP resulting fiom bioaugmentation and lactate feeding appeared sufficient to consistently provide conditions favorable for Cr(VI) reduction. At the pH range typical of the Plume F/G groundwater (e.g., 6.8 to 7.4), Cr(VI) reduction to Cr(III) is favored when the redox potential of a system is less than 60 mV (Palmer and Wittbrodt, 1991). Eh measurements periodically obtained during the large-scale column experiment revealed that the redox potential of the Column A environment was consistently less than —50 mV, and frequently as low as —200 mV. Therefore, conditions favorable for Cr(V I) reduction prevailed within the bioaugrnented Plume F/G aquifer materials throughout the duration 0f the experiment. Further, soluble chromitun concentrations entering the column were 110 consistently reduced by more than an order of magnitude to less than 20 rig/L, indicating fixation of Cr(III) to the aquifer solids. 3.6 Summary From the interpretations summarized above, the following observations may be made regarding Cr (V I) detoxification, TCE transformation, redox gradients, and microbial community structure within the continuous flow model aquifer system, Column A: l. The decrease in ORP resulting from inoculation and periodic feeding of Column A resulted in rapid and consistent decreases in soluble chromium levels within pore fluids conveyed through the system. Therefore, bioaugmentation of the Plume F/G aquifer materials with the BR Enrichment appears beneficial for reduction Cr(VI) and immobilization of Cr(III). 2. Substrate and nutrient stock fed to Column A each week was restricted to a volume sufficient to displace the pore fluids in the segment of column between the influent and sampling Port #3. Evidence of lactate fermentation was restricted to this column segment (e.g., “the fermentation zone”). 3 - Reductive dechlorination activity observed during the experiment was highly associated with the fermentation zone. Outside this zone, including within column 111 segments through which residual propionate and acetate were transported, evidence of dechlorination was sparse. The association of reductive dechlorination and fermentation activity may indicate a nutritional dependency of halorespiring populations on other members of the consortia. This distinct spatial relationship also suggests that the bulk of TCE transformation resulted from solids-associated dechlorinators. Stoichiometrically excessive quantities of lactate will result in accumulations of acetate and propionate favorable for the stimulation of sulfidogenesis and methanogenesis. A redox shift to methanogenic conditions appears to inhibit dechlorination activity. It is plausible that excess levels of acetate may create conditions that are selective for acetate-oxidizing halorespiring microorganisms that dechlorinate TCE to cis-DCE, such as Desulfuromonas sp. When sulfate reduction was the dominant redox process within the fermentation zone, halorespiration rates were accelerated, but with a greater incidence of cis-DCE accumulation. More specifically, halorespiration appeared to be distinctly bi-phasic, with interspecies transfer of cis-DCE occurring between dechlorinators such as Desulfuromonas sp. and population(s) like Dehalococcoides sp. This was most evident at the downgradient edge of the fermentation zone and over other portions of 112 10. the Column A system temporarily affected by residual acetate levels, elevated hydrogen partial pressures, and increased rates of sulfate reduction. The rates and completeness of TCE dechlorination appeared to increase as residual acetate levels within the fermentation zone diminished due to metabolism by sulfate reducing bacteria (SRB) and flushing. This was likely due to a corresponding decrease in methano genesis and the low hydrogen thresholds associated with halorespiring microorganisms. Within the upgradient portion of the fermentation zone, following the passage of acetate and propionate pulses, hydrogen partial pressures were at trace levels. Under these conditions, sulfate reduction was limited. Conversely, TCE dechlorination was rapid and complete. Under acetate- and hydrogen-limited conditions, TCE transformation was apparently less dependent on the transfer of cis-DCE between distinct populations of dechlorinating microorganisms, and more a result of halorespiration by microorganisms such as Dehalococcoides sp. In summary, TCE dechlorination was spatially associated with the fermentation zone. Temporally within the fermentation zone, three phases of dechlorination activity were evident: I. Fennentafion: At near steady-state conditions, lactate fermentation and nitrate reduction were complete within 24 hours of feeding. During this period, the 113 apparent rates of reductive dechlorination were comparatively low, and TCE transformation resulted in the accumulation of cis-DCE. . Ironfiand Sulfate Reduction: Following the decomposition of lactate, the activities of iron- and sulfate-reducing bacteria appeared to increase within the fermentation zone. The magnitude and completeness of reductive dechlorination associated with this period appeared to be a function of the extent of sulfate reduction. Specifically, when sulfate reduction was apparently complete, methanogenesis occurred and the rates and completeness of reductive dechlorination were very low, suggesting inhibition. Such conditions occurred when excess lactate concentrations were fed to the column resulting in an increase in acetate levels and apparent stimulation of acetate-oxidizing microorganisms including SRB and methanogens. Conversely, when the ORP of the system was insufficient to drive sulfate reduction to completion, but the activity of SRB appeared consistent, the rates of TCE dechlorination were high, and resulted in a rapid accumulation of cis- DCE. This suggested that TCE dechlorination was attributable to acetate- oxidizing bacteria like Desulfuromonas sp. The cis-DCE produced was further dechlorinated, but at comparatively lower rates, resulting in minor and temporary accumulations of VC. cis-DCE dechlorination was apparently associated with the activities of halorespiring microorganisms like Dehalococcoides sp. 114 III. Substrate-Limited Conditions: After a 3 to 4 day period, the time required for the acetate and propionate pulses produced during fermentation to travel through the BAZ, high rates of TCE dechlorination were evident, with little to no evidence of cis-DCE and VC accumulations. During the 3 to 4 day period leading up to the next feeding period, hydrogen partial pressures within the BAZ progressively decreased. This likely created environmental conditions that were selective for TCE halorespiring microorganisms like Dehalococcoides sp. 115 Chapter 4 FATE AND TRANSPORT MODELING 4.1 Introduction Numerical modeling was performed to quantitatively examine biotransformation processes, and evaluate substrate/nutrient delivery schemes to optimize TCE treatment efficiency within the Column A system. The mathematical bases of the governing hydrodynamic and solute transport and fate processes and the computer codes used to solve the equations describing these processes, are summarized in Chapter 2. The physical, hydraulic and bio-kinetic parameters and associated values provided as input into these codes are presented in Chapter 3 and on Table 4.1. As identified on this table, these data were determined through direct measurement, scientific literature, or model calibration. The hydraulic model established for this study was calibrated through simulations of tracer and TCE breakthrough, as described in Chapter 3. The numerical transport and fate model was calibrated using solute and biomass monitoring data obtained during Incubation Period 6B, due to the representativeness and abundance of both the spatial and temporal measurements associated with that period. 4.2 Model Calibration To simulate the transport and transformation of TCE and growth and movement of biomass over the seven day duration of Incubation Period 6B, the eight mass balance equations summarized in Chapter 2 were solved numerically by finite difference 116 approximations using the RT-3D computer code (Clement, et.al., 2000). The spatial domain of Column A, as defined by the soil mass confined laterally within the 2.54 cm diameter glass tube and bordered longitudinally by the column influent and effluent, was discretized into a grid of 300 rectangular blocks, or cells. Each cell measured 2.25 cm in both height and width, and 0.37 cm in length. The total length of the column domain was 110.49 cm. The simulation period of seven days was discretized into 7,500 time steps, each 4 x 10'4 days in duration. The Euler F orward-Time Center-Space (FTCS) forward- stepping numerical integration method was used to solve the finite difference equations. Initial and boundary conditions were imposed on each of the eight finite difference equations. The initial conditions specified that the concentrations of each parameter over the length of the column corresponded to direct laboratory measurements in samples collected from Column A on day 0 of Incubation Period 6B. Boundary conditions were specified at the influent and effluent ends of the column. These included constant flow conditions at both ends, and constant TCE flux at the influent. The initial and boundary conditions imposed on the model are summarized in Appendix A. Assumptions implicit with these conditions include: 0 Solid— and aqueous-phase TCE concentrations are in equilibrium. 0 Dehalococcoides sp. are the exclusive agents of TCE, cis-DCE and VC dechlorination. 0 Hydrogen is produced exclusively through lactate fermentation. o Lactate fermentation is instantaneous. 117 The computer model simulated biomass growth and mobility, and the transport and utilization/transformation of TCE and hydrogen within Column A under both natural and imposed hydraulic gradients. For calibration and sensitivity analyses, model output, including TCE (both aqueous and sorbed concentrations), cis-DCE, VC and biomass (both attached and unattached) were compared to experimental data collected daily from sampling ports 1-7 during Incubation Period 6B. The model also produced hydrogen and ethene results. To calibrate model-predicted with measured concentrations and gradients, second-order rate coefficients for TCE, cis-DCE and VC and spatial biomass concentrations (both planktonic and solid) were adjusted within ranges considered plausible from literature-derived values and measured quantities, respectively. Calibrated rates coefficients are presented on Table 4.1. The calibrated biomass distributions are graphically presented in Appendix G. In summary, the second-order rate coefficients calibrated from the modeling efforts were 4 ng'lday'l for both TCE and cis-DCE transformation and 2 ng'lday'l for VC dechlorination. As expected, the calibrated biomass distributions reveal significant accumulations of both solid phase and planktonic microbes within and immediately downgradient of the BAZ. Experimental and predicted concentrations for all eight-model parameters (e. g., TCE, cis-DCE, VC, ethene, sorbed TCE, hydrogen, and attached and detached microbes) are presented in Appendix G-l. Both the discrete concentration data and chemical gradients associated with these constituents compare within a favorable level of tolerance to direct measurements, suggesting that the transport and fate model was well posed. In general, the model accurately predicted the transformation of TCE within the BAZ. 118 Downgradient of the BAZ, the model appears to underestimate TCE removal. The model results for cis-DCE and VC are also slightly under-predicted compared to measured concentrations. However, the spatial and temporal gradients of cis-DCE and VC production are consistent with measured results. The model results indicate that as much as 2.5 mg/L of dissolved hydrogen were consumed during the incubation period by the halorespiring microorganisms resident in the column. Further, approximately 16 pig/L of ethene were produced by the dechlorination reactions. Sorbed TCE levels decreased 3-fold within the BAZ during the seven—day simulation. Predicted immobile biomass concentrations reached a maximum of 1.1 x 10'1 mg/L (approximately 106 CFU/mL) three days following feeding. Mobil biomass levels increased to a peak of 1.6 x 10'3 mg/L (approximately 104 CFU/mL) on day four of the simulated incubation period. These values appear excessive when compared to measured concentrations of Dehalococcoides sp. in Column A (refer to Section 3.5.2, Large-Scale Column Experiment, Operational Phase B, Biomass). However, it is likely that these assays were successful in quantifying only a small percentage of the population of halorespiring microorganisms within the column. Future studies should include RT-PCR assays with a broader array of primers to identify and quantify not only Dehalococcoides sp., but also partial TCE dechlorinators such as, but not limited to, Desulfuromonas sp. 119 Table 4.1. Numerical model input parameter values and data sources. Input Definition Value Procedure Source Parameter 0 Sediment porosity 0.39 Measured This study at Longitudinal dispersivity, m 0.01 Calibration This study Kd Distribution coefficient, L/mg 7 x 10‘8 Measured This study pr, Soil bulk density, mg/L 1.4 x 10° Measured Mayotte, et al., 1996 f Fraction of exchange sites at equilibrium 0.437 Literature Phanikumar, et al., 2002 RDCE DCE retardation coefficient 1 Measured This study ch VC retardation coefficient 1 Estimated This study Rm, Ethene retardation coefficient 1 Estimated This study RH; H2 retardation coefficient 1 Estimated This study It,- Kinetic (de) sorption rate, l/day 0.3 Literature Phanikumar, et al., 2002 k 'mg TCE reaction rate, ng'lday'l 4 Calibration This study k '0“ DCE reaction rate, Lm "day" 4 Calibration This study It ’yc VC reaction rate, ng' day'I 2 Calibration This study k '5”, Ethene reaction rate, ng"day'I 0 Calibration This study um, Maximum specific grth rate, l/day 0.5 Literature Ballapragada, et al., 1997 K mg TCE half-saturation coefficient, mg/L 6.6 x 10'2 Literature Haston, McCarty, 1999 K”; H; half-saturation coefficient, mg/L 2 x 10-5 Literature Ballapragada, et al., 1997 YTCE-DCE TCE/DCE stoichiometeric constant 0.74 Calculation This study Y DCE- VC TCE/DCE stoichiometeric constant 0.64 Calculation This study Y my” TCE/DCE stoichiometeric constant 0.45 Calculation This study er£-x TCE yield 0.5 Estimated This study yHJX Hydrogen yield 0.5 Estimated This study bDC Microbial decay rate, l/day 0.1 Literature Phanikumar, et al., 2002 K0, Microbial attachment rate, l/day 0.9 Literature Phanikumar, et al., 2002 K4,, Microbial detachment rate, l/day 0.018 Literature Phanikumar, et al., 2002 4.3 Sensitivity Analyses The sensitivities of variables to key bio-kinetic parameters and conditions were analyzed following model calibration. Specifically, these analyses included evaluating numerical changes in model variables, such as TCE, cis-DCE, VC, ethene, sorbed TCE, hydrogen and solid-phase and planktonic biomass concentrations, to a range of values for parameters such as: TCE, cis-DCE, and VC second-order rate coefficients; TCE and 120 hydrogen half-saturation and yield coefficients; the maximum specific microbial growth rate; and the endogenous decay rate. Additional sensitivity analyses included performing simulations by excluding or modifying certain terms of the mass balance equations describing the fate of the aforementioned model variables. 4.3.1 Variation Coefficients The sensitivities of model variables to changes in the input values of the bio- kinetic parameters were evaluated through a series of independent model simulations. For each simulation, the value of a single parameter was increased by 50%. The resulting values for each variable were then systematically compared to calibrated values. The comparison included calculation of variation coefficient, 4), using the following relationship (Witt, 1998): AWW (0 = , AP //P (4.1) where P is a model parameter (k 768, k 'DCE, k ’yc, um, K TCE, K H2, yrcgvy, ymx and b) and Wis a variable (CTCE, CDCE, C VC, Cgm, CH2, Srcg, Xi". and X," ). Variation coefficients associated with each parameter were calculated by first determining the largest change in W over the length of the column resulting from the change in P. This value was then divided by the original variable quantity. As indicated by equation 4.1, the result was further divided by the value of the magnitude of the change in P divided by the unchanged value of P. Since each parameter was increased by 50%, the value of the 121 denominator in equation 4.1 was fixed at 0.5. Therefore, a variation coefficient of less than unity indicated that the value of a variable changed less than two-fold (e. g., 50%) in response to the 50% increase of the value of a parameter. In general, a value (b of less than 0.80 indicated that changes in the magnitudes and distributions of the model variables were insignificant. Variation coefficients determined through these sensitivity analyses are summarized on Table 4.2. These data indicate that CTCE, C DCE, Cyc, Cgm, CH2, Srcg, Xi”, and X", are all insensitive to changes in half-saturation and yield coefficients (K TCE, K H2 and, yrcwr, szx respectively). Only CH2 is sensitive to the changes in the decay coefficient, ch. C VC and Cgm are only slightly sensitive to changes in the second-order rate coefficients for TCE (k ’TCE) and DCE (k ’DCE); but both variables are significantly sensitive to changes in the maximum specific growth rate, ,umm. Similarly, both solid- phase and planktonic biomass concentrations (X,-,,, and Xm, respectively) are highly sensitive to a change in pm“. Table 4.2. Variation coefficients for key model parameters on day 7 of Incubation Period 6B. k ’TCE k 'DCE k 'VC ,umax KTCE KHz yTCE-X yHZX bDC C TCE 0.56 0.00 0.00 0.58 0.02 0.00 0.03 0.08 0.20 CDCE 0.01 0.13 0.03 0.19 0.01 0.03 0.05 0.22 0.00 C yc 0.79 0.24 0.01 0.50 0.02 0.01 0.02 0.09 0.33 C5771 0.84 0.92 0.01 1.70 0.05 0.01 0.01 0.22 0.68 CH2 0.84 0.91 1.00 4.05 0.09 0.02 0.02 0.38 0.93 STCE 0.33 0.01 0.03 0.33 0.02 0.01 0.02 0.04 0.06 X,- 0.07 0.01 0.02 12.15 0.18 0.01 0.02 0.34 0.44 X", 0.07 0.00 0.02 10.70 0.14 0.01 0.02 0.25 0.41 122 4.3.2 Bio-kinetics and Sorption The responses of model variables to the exclusion or modification of certain terms in the eight mass balance equations were determined through additional sensitivity simulations. Specifically, these equations were numerically solved using calibrated model input values and initial and boundary conditions, after certain terms were manipulated to impose: 1) hydrogen starvation; 2) the absence of biochemical reactions; and 3) instantaneous TCE sorption/desorption. Time series plots of the results from these simulations are presented in Appendix G-2. As seen on these plots, both hydrogen starvation conditions and the absence of biochemical reactions, as described by the energy terms associated with equations 2.9 through 2.19, have profound effects on C mg, C DCE, C yc, Cgm, CH2, STCE, Xi", and Xm. These results were expected. Specifically, the absence of hydrogen imposes a no-growth condition on biomass within the column, which in turn reduces the utilization of electron acceptor, TCE. Therefore, TCE transformation is severely limited. Similarly, the lack of biochemical reactions in the mass balance equations essentially eliminates all mechanisms of TCE transformation. Therefore, as expected, both the presence of hydrogen and inclusion of energy terms in the balance equations are necessary for providing accurate predictions of the TCE transformation observed in Column A. Conversely, the absence of rate-limited TCE sorption/desorption had a minimal affect on the predicted values of Crag, CDCE, C Vc, CETH, CH2, STCEr Xim and Xm- 123 4.3.3 Summary The results from the sensitivity analyses support the assertion that biochemical reactions are the predominant mechanisms for TCE removal in Column A. Further, the products of these reactions are dependent on the selection of, and highly sensitive to changes in, the value of the maximum specific growth rate, a...“ for halorespiring biomass. Further, the reactions affecting ethene and hydrogen concentrations are sensitive to changes in second-order rate coefficients for TCE and cis-DCE and the microbial decay rate. Rate-limited sorption, although important for accurately simulating the rate of TCE transport through the column, appears to have minimal affect on the predictions of TCE transformation or microbial growth. 4.4 Predictions The calibrated flow and reactive transport models were used to evaluate two (2) distinct substrate and nutrient delivery schemes. These schemes and the rationale associated with each included: 1. Increasing the dimensions of the BAZ through doubling the weekly feed volume conveyed through the coltunn. The resulting expansion of the BAZ is expected to increase contaminant residence time and thereby improve TCE removal efficiency. 2. Using pore water from downgradient of the non-expanded BAZ as make-up water for feed solution rrrixing and delivery. Doing so may reduce the initial TCE load, and therefore the overall mass removal burden, within the BAZ immediately following 124 feeding. In effect, this should increase the rate and efficiency of solute treatment under the natural gradient conditions occurring between feeding events. The model-generated results from simulations of each scenario are presented in Appendix G-3. These results suggest that both substrate/nutrient delivery strategies will enhance TCE treatment. However, although treatment rates are achieved rapidly when the BAZ is fed solutions mixed with BAZ-treated groundwater, the overall efficiency of TCE removal is not greatly improved. Conversely, TCE removal is clearly increased after the BAZ is widened through consistently delivery of an increased quantity of feedstock. BAZ dimensions required for achieving requisite treatment objectives at field sites can be established and preliminarily evaluated by numerical analyses such as described herein. From the model-generated results presented in Appendix E-3, it appears that a 3-foot wide BAZ will reduce influent TCE levels of 2,500 rig/L in Column A by approximately 90% within a four to seven day period following each feeding event. 125 Chapter 5 CONCLUSIONS AND RECOMENDATIONS 5.1 Conclusions From the data presented in Chapter 3 and the results of the reactive transport modeling discussed in Chapter 4, the following conclusions may be made regarding the stated hypotheses (presented in bold text) of this study: 1. The inoculation of the Plume F/G aquifer materials with the BR Enrichment will produce a spatially dependent, stationary-phase microbial community profile. Due to unexpected analytical difficulties associated with quantifying biomass within the limited discrete quantities of soil obtained during the treatment phase of the study, it was not possible to directly characterize the solid-phase community profile within large-scale Columns A and B. However, dsDNA and RT-PCR analyses revealed that biomass, including Dehalococcoides sp., was approximately equally distributed on both solids and in the liquid phase over the length of Column A. Further, evidence associated with substrate utilization, redox, and TCE transformation gradients indirectly revealed spatial and temporal relationships between key biologically mediated processes, without discretion to phase associations. Specifically, upon development of near steady- state TCE transformation conditions, fermentation was most evident between the column influent and Port 3. Nitrate reduction occurred to completion within the column segment between the influent and Port 1. Iron reduction was consistently evident over the length 126 of the column and appeared to be the dominant terminal electron accepting process during this period. Sulfate reduction occurred on a temporary basis between Port 1 and Port 4. The duration and magnitude of sulfate reduction was dependent on the mass of lactate delivered to the column. Similarly, methanogenesis was evident on a temporary basis between Port 2 and the column effluent. 2. Within the community profile, a biologically active zone (BAZ) consisting of spatially and/or temporally discrete regions of sulfate-reduction, methanogenesis, and halorespiration will exist wherein transformation of TCE to non-toxic end products will occur. Substrate and nutrient stock fed to Column A each week was restricted to a volume sufficient to displace the pore fluids in the segment of column between the influent and sampling Port 3. As discussed above, evidence of lactate fermentation was confined to this column segment (e.g., “the fermentation zone”). Reductive dechlorination activity observed during the experiment was highly associated with the fermentation zone. Outside this zone, including within column segments through which residual propionate and acetate were transported, evidence of dechlorination was sparse. Delivery of a quantity of lactate in excess of that used to establish near steady- state TCE dechlorination activity resulted in accumulations of acetate and propionate favorable for the stimulation of sulfidogenesis and methanogenesis. A redox shift to methanogenic conditions appeared to inhibit dechlorination activity. When sulfate reduction was the dominant redox process within the fermentation zone, halorespiration 127 rates were accelerated, but with a greater incidence of cis-DCE accumulation. The rates and completeness of TCE dechlorination appeared to increase as residual acetate levels within the fermentation zone diminished due to metabolism by sulfate reducing bacteria (SRB) and flushing. Within the upgradient portion of the fermentation zone, following the passage of acetate and propionate pulses, hydrogen partial pressures were at trace levels, based on modeling results. Under these conditions, sulfate reduction was limited. Conversely, TCE dechlorination was rapid and complete. In summary, three temporally distinct phases of dechlorination activity were evident within the fermentation zone/BAZ: I. Fermentation: At near steady-state conditions, lactate fermentation and nitrate reduction were complete within 24 hours of feeding. During this period, the apparent rates of reductive dechlorination were comparatively low, and TCE transformation resulted in the accumulation of cis-DCE. H. Irowd Sulfatg Reduction: Following the decomposition of lactate, the activities of iron- and sulfate-reducing bacteria appeared to increase within the fermentation zone. The magnitude and completeness of reductive dechlorination associated with this period appeared to be a function of the extent of sulfate reduction. Specifically, when sulfate reduction was complete, methanogenesis occurred and the rates and completeness of reductive dechlorination were low, suggesting inhibition. Such conditions occurred when excess lactate concentrations were fed to the column resulting in an increase in acetate levels and stimulation of acetate-oxidizing 128 microorganisms including SRB and methanogens. Conversely, when the ORP of the system was insufficient to drive sulfate reduction to completion, but the activity of SRB appeared consistent, the rates of TCE dechlorination were high, and resulted in a rapid accumulation of cis-DCE. This suggested that TCE dechlorination was attributable to acetate-oxidizing bacteria. The cis- DCE produced was further dechlorinated, but at comparatively lower rates, resulting in minor and temporary accumulations of VC. . Substrate-Limited Conditiorg After a 3 to 4 day period, the time required for the acetate and propionate pulses produced during fermentation to travel through the BAZ, high rates of TCE dechlorination were evident, with little to no evidence of cis-DCE and VC accumulations. 3. Within the BAZ, the groundwater redox chemistry will favor for the reduction of Cr(VI) to and immobilization of Cr(III). The decrease in ORP resulting from inoculation and periodic feeding of Column A consistently resulted in rapid decreases in soluble chromium levels within pore fluids conveyed through the system. Specifically, soluble chromium levels in excess of 200 rig/L entering the column were consistently reduced to below 20 pg/ L before pore fluids traveled to Port 1 of Column A. Therefore, bioaugmentation of the Plume F/G aquifer materials with the BR Enrichment was beneficial for reduction Cr(VI) and immobilization of Cr(III). 129 To date, most experimental work to evaluate the dechlorination behavior of mixed and pure cultures in native and non-native aquifer materials has been performed in laboratory microcosms in which environmental and nutritional conditions are strictly controlled. Departures from this trend include work by Isalou and Sleep (1998), Harkness, et. al. (1999), and Cirpka, et. al. (1999), which examined dehalogenation activities expressed by non-native mixed enrichment cultures and halorespiring isolates in flow-through systems packed with aquifer materials and maintained under natural conditions. These studies emphasized the spatial measurement of electron donors and acceptors, chlorinated solvent dehalogenation and metabolic byproducts associated with the activity of the augmented microbial community. However, with the exception of work done in fractured and granulated basalts by Lehman, et.al. (2001), few studies have attempted to evaluate the transport behavior of inoculates during delivery, or correlate the spatial and temporal variability of aquifer geochemistry and contaminant transformation with the structures of mobile- and stationary-phase microbial communities resulting from bioaugmentation. The work summarized herein was an attempt to examine such relationships and advance the current understanding of the successional adaptations of in- situ treatment regimes following inoculation of aquifer materials. Specifically, the results produced from this experimentation reveal that TCE transformation by halorespiring microbial populations is closely associated with fermentation activity, and that dechlorination rates and completeness will depend on redox conditions. Evidence associated with substrate utilization, redox, and TCE transformation gradients indirectly revealed spatial and temporal relationships between key biologically mediated processes, without discretion to phase associations. Reductive dechlorination activity observed 130 during the experiment was highly associated with the fermentation zone. Outside this zone, including within column segments through which residual propionate and acetate were transported, evidence of dechlorination was sparse. A redox shift to methanogenic conditions appeared to inhibit dechlorination activity. When sulfate reduction was the dominant redox process within the fermentation zone, halorespiration rates were accelerated, but with a greater incidence of cis-DCE accumulation. The rates and completeness of TCE dechlorination appeared to increase as residual acetate levels within the fermentation zone diminished due to metabolism by sulfate reducing bacteria (SRB) and flushing. Within the upgradient portion of the fermentation zone, following the passage of acetate and propionate pulses, hydrogen partial pressures were at trace levels, based on modeling results. Under these conditions, sulfate reduction was limited. Conversely, TCE dechlorination was rapid and complete. This knowledge could benefit strategies for optimizing biotransformation capacities within bioaugmentation treatment zones. General recommendations developed from these observations are presented below. 5.2 Recommendations Engineering considerations associated with the use of the Bachman Road Enrichment for firll-scale bioaugmentation of non-native aquifer materials include: 1. Design of full-scale bioaugmentation strategies will depend on the TCE concentrations within target treatment zones, regulatory cleanup criteria, and the viability and rates of TCE dechlorination expressed by the enrichment in site aquifer 131 materials. Apparent dechlorination rates estimated from small-scale column studies may be used to establish the width of the BAZ (and, therefore, zone of fermentation) necessary to accomplish regulatory cleanup objectives. . Any bioaugmentation strategy that requires lactate as a carbon and energy source should include substrate delivery schemes that maximize the production of hydrogen and minimize accumulation of acetate and propionate. Ideally, the feeding scheme should minimize the activity of SRB and promote a swift transition from fermentation to acetate-starvation within the BAZ. In general, this may be accomplished by pulsed injection of a lactate mass that is stoichiometrically less than the mass required to drive sulfidogenesis to completion, but sufficient to provide residual hydrogen at levels above threshold concentrations for halorespiring microorganisms like Dehalococcoides sp. . Design of full-scale bioaugmentation strategies using the Bachman Road Enrichment should include the following general steps: 3. Establish range of TCE dechlorination rates from column microcosm studies using the enrichment and aquifer materials from the treatment site. b. Estimate the width of the fermentation zone (e. g., BAZ) required to accomplish the desired level of treatment, including consideration of an appropriate safety factor. 132 c. Develop strategies to deliver inoculum and feed rapidly and uniformly over the desired width of the BAZ. Use of a three well injection/extraction/re- injection strategy is recommended to maximize the uniformity of inoculum and feed delivery. (1. If practical, assess performance of the BAZ with a multi-well, multi-level monitoring system installed across the width of the BAZ to facilitate sample acquisition at spatial and temporal resolution of the redox and dechlorination dynamics within the zone of fermentation. Based on the experiences during the experiments summarized herein, the following recommendations should be considered in the design of column studies for research on the relationships between redox gradients, microbial community structure and dechlorination activity: 1. Improve gas-monitoring capabilities to facilitate more accurate measurement of headspace gasses and thereby improve the assessment of hydrogen consumption and completion of TCE transformation mass balances. 2. Improve resolution of longitudinal sampling to improve the tracking of inoculum transport and breakthrough and the understanding of redox zonation within fermentation zones. 133 . Improve temporal monitoring under near steady-state conditions by sampling daily and at every port. Aqueous analyses of these samples should include soluble metals. . Extend the duration of similar experiments to more precisely resolve trends in dechlorination activity, redox gradients, and the occurrence and completeness of halorespiration. . Improve liquid and solid microbiological monitoring of communities structure and halorespiring microorganisms to refine interpretations of correlations between redox conditions and halorespiration activity. . Utilize molecular techniques such as RT-PCR to identify and quantify the presence of both Desulfuromonas sp. and Dehalococcoides sp. . Comprehensively evaluate the individual sorption characteristics of TCE, cis-DCE and VC within the subject aquifer materials to improve the validation and accuracy of numerical models used to simulate and optimize the performance of in-situ bioaugmentation treatment zones. . Measure the yield of subject enrichment cultures when grown in the presence of TCE, cis—DCE and VC to provide plausible input to and accuracy of numerical modeling efforts. 134 9. Future modeling efforts should endeavor to include consideration of acetate utilization and cis-DCE production by Desulfuromonas sp. and hydrogen utilization and cis-DCE respiration by Dehalococcoides sp. 135 APPENDIX A Large Scale Column A Operational History 136 5... .2. 8.... 8... 8.... 8... 8.8 8... 8... 8... 8... 8...... 8... 8... a... «83.38.. 2 88.... 8 8... 8 88... a. 8... 8... 8.... 8... 8.... 8... 8..... 8... 8... 8... 8.8 8... 8... S... 8.. .8. .8 .5. 8.. 88.. 8 S... .w... 8..... 8... 8..... 8... 8..... 8... 8... 8... 8... 8... 8... 8... o... .8. .c. .5. a... 8.... c. 8... 8 88... .. 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NOONNRF 98 ac... .502»: .332; assign» 148 Schoolcrafl Kinetic Protocol Manager Jing Date 12/2/2002 PCE initial concentration 4 mg/L Kinetic aqueous volume 165.6 mi PCE density 1.69 g/ml PCE injection amount in aqueous 0.4 uL Control-1 Control-2 Control-3 water 160 160 160 from reactor mineral salt trace element Nazs solution .1 % buffer lactate PCE otal Sampling Time Days Iample Name PCE TCE DCE VC ettheth. before pce 12/03/02 9:0 0 2-1 8.0 3.5 0.0 0.0 0.0 after pce 12/03/02 11:00 0.1 2-2 2030.0 5.7 0.0 4.5 0.0 12/03/02 16:(&pm 0.3 23 2048.0 10.0 0.0 0.0 0.0 12/04/02 8:00am 1.0 2-4 2510.0 35.0 23.5 13.0 0.0 12/04/02 3:00pm 1.3 2-5 2815.0 40.0 34.0 34.5 0.0 12/05/02 9:00am 2.0 2-6 2356.0 31.0 26.5 33.0 0.0 12/05/024200 pm 2.3 2-7 2180.0 30.5 0.0 47.0 0.0 12/06/02 9:003m 3.0 28 2260.0 33.0 26.0 42.5 0.0 12/6/02 4:00PM 3.3 2-9 2234.0 49.0 33.5 69.0 21.5 12/07/02 5:00pm 4.3 2-10 2265.0 55.5 41.5 67.5 0.8 12/08l02 6:00pm 5.4 2-11 2190.0 70.5 57.0 81.5 4.5 12109/02 4:00pm 6.3 2-12 1936.5 73.0 53.5 100.5 5.0 12/10/02 3:00pm 7.3 2-13 1684.0 75.0 56.0 135.0 6.0 12/11/02 2:00pm 8.2 2-14 1005.0 50.5 37.5 138.5 4.5 do not count on the final data summary 149 . 9o8« . 988 9m 9.... 9o 9o 90 v..." «.c 5&3 «3%. 9m 98. 9o 9» 9o 8..» 9. .886 «9a a. 9m 98« 9o 9« 9o «...o. 9» .589 «9%. ..m 9«.n 98 9«« 9.. ...n .8 5819.. «33.1 9o 98. 9«o« 98« 98« 2... 9e 6988 «9887. 90 98 98. 9o«« 98o. a.» 9» 589.. «99%| 9o 93 9o 9«... 9.8. 3.. 9m 5:89.. «98E 9o «.8 9o 98. 989 «A 9« Eu 85.8892 9o 98 98 98. 988. o... 9« 2489.. «98981.1 9o 98 98 9««. 988 3 9. 589m «93$. 9o 99.. 9«.. 9 E 988 1.. 9. 5988 «9..9«. 9o 98 9o 9.« 988 .3 3 .888. «98m. 9o 9.. 9o 9.. o.«o«« «.n ..o 88”.. «9.834% 9o 90 9o «.n no .... c 88 «98m. 88 cal}! .5330 o> moo 8. won. gala...“ nan S... a 98. 98. 98. 98. 98. 98. 98. 95 .88. . . . . . . . 3: won. . . . . . . . ...5 938. « « « « « « « as .83 «.o «.o «.o «.o «.o «.o «.c 3. ..9 8582 «.o «.c «.o «.o «.o «.o «.o .35 88.8 32 «.o «.o «.o «.o «.o «.o «.o 5.5 8.8.8 88. « « « « « « « as =8 .288 8. 8. 8. 8. as? 52. 8.99.8. 8. 8. 8. 2.5 as; 3% v _n _« _. 22.80 «-688 22.80 .5 ..Ho 3033 s :50...» 580.5 won. :56 3.. 3.88 won. .8 o 8. 85.9 88:8 882 do... v 5.5.88 .3: won a 89$. 38 29 .8.... 150 988 Y 0.98. .. 0.8m. .. 988 + odomm 9m 98 .98 9o 9o 8.... «.8 =3] . 98 98 9o 9« 9.. 2+ 9. 88 «9o .8. 9m 9 .« 9o 98 9. «.... 98 88.. «98m. 98 98.« 9.8 98 98 ...v 9... .888 «988.4 98. 98. 98. 98. 988 2+ 9.. .888 «9.88. 9.. 98. 98. 9.8. 9.8. 8+ 98 .2888 «989M. 9. 9 .8 98. 9... 98.8. 8... 98 .888 «981881. 9.. 9... 9o «.8. 988. .... 88 .8 888.88% 98 98 98 98 9 .8« o... 9« .888 888.1. .98 98 9 .8 98 988 84 9. .888 «938. 9o 98. 9.8 98 988 I. 9. a8... 8% 9o 98 9. 9.. 98M 8... 9a .888. 889M. 9o 9.. 9o 98 9.8.. «... .8 Ema..88981.88fl 9o 90 9o 9o 98 ..e a =9 88 8898 8a 228 .8598 o> moo mo. won. 9% goo ...... mammal.» 98. 98. 98. 98. 98. 98. 98. ...... .8. . . . . . . . Symon . . . . . . . 2.5 28.8. « « « « « « « 8.5 88.. «.o «.o «.o «.o «.o «.o «.o 3.. ..9 5:882 «.o «.o «.o «.o «.o «.o «.o ...... 88.8 882— «.o «.o «.o «.o «.o «8 88 « « « « « « « 8. 8. 8. 8. 8. 8. 8. 28% v 7. _~ _. 9.228 «.880 .880 a . .... “We 38...... s 23...... 5.8... 8.. .8 e. ...... 8.. 2.22, 38.8 889 8... e scumbag 3:. won «8«88. son 3.5.. 151 9. 9o 9o 08 9... 9o 9.. 9o 3 «8 9. 9o 9.. 9o ...8 9o 9. 9« n. 9. 68.50 .. o> : moo a wok a mom . 8.. .8 .98 9o ...8. ..o. 9. 9' «. 98 88 9o .8 9o ...8 98 93 98.. .. 8.... 9o .8 ...o .8 ..8 8.8 98 .8.... o. 9.. 8.60 9° «8 9° ..« 98. ..m« 98 .88 8 98 0.9 o.m .8 9o .8 9« 9m 98 9.. 9.8 8 9n «8 9o «8 9« 88 9o 9.« 9mm.” . 9« . . ..o ..o ..o .... 98 98 .8. ..«In« 8 9« ..o «.o «.o «.« 98 9.... 98 9.8 m 9. 9o 98 ..o 9. 9. 9c 9.. 9n.« .. 9. 9o 9.. 9o .8 9. 9. 9. 9:. 8 9° 9° 9° 9o 98 9« 9« 9n ...8 « .8 9o 90 98 9.. 9° 9° 9. 9.. . 8 name o> woo mo. mom o> moo mo. won 28 9.... :4. 98 8.8 8.8. 8.8. o :2.- 8.5. mg §.§§§8§§2§§|£82§3 T 0.9 u U 10. _ a no. 0 U . Y 98. \n, m . . .8 r odm gflir 9mm 152 Schoolcraft Project Dechlorination Kinetic Date 12/16/2002 Zero order of PCE dechlorination rate uMIg biomass)lday 29.60 1 with linear range data TSS 0.15 L g/L average stdev 1 1.2457 1 .2616 0.106 0.1 13 0.009 3 1.2325 1.2511 0.124 4 1.2436 1 .2601 0.1 10 Linear fitting , , , , PCE dechlorination rate (With linear range of data) Time PCE days uM 20.0 0'1 1 1'2 i C = -3.3544 x Days + 18.741 0.3 14.6 a 15.0 « R2 = 03., 1.0 16.1 § 1.3 14.9 g 2.0 11.0 g 1” 2.3 9.6 8 3.0 9.4 8 5-0 1 3.3 8.6 ‘L 4.3 4.7 00 I r , , 5.4 0.2 0.0 1.0 2.0 3.0 4.0 5.0 6.3 0.0 time (days) 7.3 0.0 8.2 0.0 153 mm new.” 88.3 88?? 88.5 838.? $8.?“ 58:5,? 333. o A062 050 oE_i._.l o> o o 23” . _ S o macaw 888.» 880.2 3826 $88; 583...“. awkward 2: e833 .. w. .-.Emmzmg $8.85 0 985 E63 ... >03...” 260 oEFI mood o 2m.» N 885.2». mm 888.. 582.8 5825.? mam o 333 250 DEF 154 APPENDD( C Large Column Study Analytical Results — Operational Phase A 155 APPENDIX C-l Fatty Acids 156 FATTY ACIDS COLUMN A Incubation Period 1A June 28 - July 11, 2002 June 28, 2002 A 2500.00 .5, 2000.00 \ c “ 1500.00 5.; \. 25) 1000.00 ”'0" Propionic Acid _ 9.3 -a- Acetic Acid § 500.00 'O-Lactic Acid _2 '8 - - -C00627 (I) 0.00 A - . I- A - L 15.24 56.52 100.97 Port Position (cm) July 5, 2002 A2500.00 g) ""0"“ Propionic Acid 52000.00 - _ - - - _ -f- w - - - +Acctic Acid __ ,8 -o- Lactic Acid 2' 1500.00 R - - °C00627 c. 5 / \ 0 5 1000.00 - ° / \ 8 <2. (6 500.00 3, 1 a. E / \ = / m .... ...... .. 0.00 "7““ 0 . ~ 15.24 29.21 42.55 56.52 71.76 86.36 100.97 Port Position (cm) July 11, 2002 “0" Propionic Acid -0- Acetic Acid ‘0' Lactic Acid " ' 'C00627 15.24 56.52 100.97 Port Position (cm) 157 FATTY ACIDS COLUMN A Incubation Period 2A July 12 - 26, 2002 July 12, 2002 A 400.00 E!) 350 00 ‘g 300.00 °\; """"" § 250.00 fl" § 200.00 5 150.00 *0“ Propionic Acid _2 g 100 00 Hit-Acetic Acid g ' '0-I.actic Acid 3 50-00 - - -C00711 ”“ 0.00 0 - i - c i c l5.24 29.21 42.55 Port Position (cm) July 19, 2002 A 400.00 ..1 3 350.00 E 300.00 """"""""""" § 250.00 200.00 g ""0"- Propionic Acid Lo) 150'00 +Acetic Acid H 2 100.00 -0-Lactic Acid H '3 50,00 ' ' 'C007ll .— m 0.00 <> . A..- . ;-- A 15.24 56.5 100.97 Port Position (cm) July 26, 2002 A 400.00 $1 350.00 E, ............................ 8 300.00 E 250.00 § 20000 ”'0‘” Propionic Add “-1 8 150 00 -¢— Acetic Acid 93 ' -0-Lactic Acid 2 100.00 - - °C007ll H 3 50.00 m .6 8 W 15.24 29.21 42.55 56.52 71.76 86.36 100.97 Port Position (cm) 158 FATTY ACIDS COLUMN A Incubation Period 3A Julv 26 - August 2. 2002 July 26, 2002 400.00 350.00 0-\ % 3m.m - - - - - - - - - - - - - - - - - - - - - - - - 5 m 5 250.00 5 E \0\ 3 200.00 5 W0“ Propionic Acid \ 3 150.00 *Acetic Acid § -0—Lactic Acid \ .0 V3, 10000 " ' 'C00726 \ 50.00 b 0 00 0 1 0 1 ' , <> ' . T - (\f _ 0 0 ....¥:a-izm*mo 15.24 29.21 42.55 56.52 71.76 86.36 100.97 Port Position (cm) August 2, 2002 400.00 350.00 ”’0'” Propionic Acid H —A- Acetic Acid 300.00 '0-Lactic Acid H ' ' 'C00726 N u. .° 8 Substrate Concentration (mg/L) 5.- N p 8 8 8 .§ 8 8 86.36 100.97 15.24 29.21 42.55 56.52 71 .76 Port Position (cm) 159 Substrate Concentration (mg/L) Substrate Concentration (mg/ L) FATTY ACIDS COLUMN A Incubation Period 4A August 2 - 9, 2002 August 2, 2002 400.00 ““0“" Propionic Acid 35000 —A— Acetic Acid -0-I.actic Acid 30000 - - C00802 250.00 200.00 150.00 100.00 50.00 0.00 15.24 42.55 56.52 100.97 Port Position (cm) August 9, 2002 400.00 350.00 ”“0““ Propionic Acid H —-A- Acetic Acid —0- ' ' 300.00 lactic Ac1d H " ' C00802 250.00 200.00 150.00 100.00 15.24 29.21 42.55 56.52 71 .76 86.36 100.97 Port Position (cm) 160 FATTY ACIDS COLUMN A Incubation Period 5A August 9 - 16, 2002 August 9, 2002 400.00 WPr-opionic Acid 35000 Acetic Acid Lactic Acid § 300.00 - coogog E s: 250.00 0 '5 E § 200.00 0 L) D "’ 150.00 g - - - - - - - - - - - - - - - - - - - - - - - - ..D 53 100.00 50.00 0.00 15.24 29.21 42.55 56.52 71 .76 86.36 100.97 Port Position (cm) August 16, 2002 400.00 350.00 ““0““ Propionic Ac1d —0— Acetic Ac1d A 300 00 .0-Iactic Acid u—i ' I I B C00809 5 5 250.00 '3: E 0 20000 8 o L) 5. ..D :3 100.00 50.00 ’1 000 " 1 I 1 V II r V 1 I 15.24 29.21 42.55 56.52 71.76 86.36 100.97 Port Position (cm) 161 Substrate Concentration (mg/L) Substrate Concentration (mg/L) 400.00 350.00 é a 250.00 § 8 150.00 100.00 50.00 0.00 400.00 350.00 6’ a 250.00 200.00 150.00 100.00 50.00 0.00 FATTY ACIDS COLUMN A Incubation Period 6A August 16 - 24, 2002 August 16,2002 aria-now Propionic ACid —A-— Acetic Acid -'0-Lactic Acid " ' C00816 K""""""""""" cl':“’-'filg. "mm-a ' my“ "‘"fii- 9:9,“. _ winner!!! “shoe; A 15' "gird" '!-‘ 15.24 29.21 42.55 71.76 86.36 100.97 Port Position (cm) August 24, 2002 “‘W'Pmpionic Acid —0— Acetic Acid "0-Iactic Acid ' ' C008l6 gunman I “0"“ T ‘0 r M‘d) 15.24 42.55 71.76 100.97 Port Position (cm) 162 Substrate Concentration (mg/L) Substrate Concentration (mg/L) 400.00 350.00 400.00 350.00 250.00 200.00 150.00 100.00 50.00 — 0.00 FATTY ACIDS COLUMN A Incubation Period 7A August 24 - 30, 2002 August 24, 2002 mason-n Propionic Acid —A- Acetic Acid -0-Lactic Acid " ' “C00824 , , «a Iivl‘r'flw'"hmuom’m-VNumb...» _ r ["""""Fg:;im.<>flw 1‘“"'\ 1 Wmuntwmgo—1 15.24 29.21 42.55 56.52 71.76 86.36 100.97 Port Position (cm) August 30, 2002 “two-m Propionic Acid —A—- Acetic Acid -0—Lactic Acid - " 'C00824 “—0: I; /A W e“ A 45 ——-f T r V'— f V 15.24 42.545 71.755 218 Port Position (cm) 163 Substrate Concentration (mg/L) Substrate Concentration (mg/L) FATTY ACIDS COLUMN A Incubation Period 8A August 30 - September 3, 2002 August 30, 2002 400.00 350.00 mo"""I’rop10n1c Ac1d #— -A-— Acetic Ac1d 300 00 "0.1.3ch Acid - ' ' C00830 250.00 200.00 150.00 100.00 50.00 / \ \ k “-../x. , 0.00 4 I - .. - \W 15.24 42.55 71.76 100.97 Port Position (cm) September 3, 2002 400.00 350.00 WPropionic Acid F“ + Acetic Acid 300.00 -0"'Lactic Acid H ' ' C00903 250.00 150.00 / 100.00 C I; / A 0 50.00 “ I ” 0.00 - 15.24 42.55 71 .76 100.97 Port Position (cm) 164 APPENDIX C-2 Major Anions 165 33 season tom A88 SE8.” tom 3.2: amen 3.2 3.2: omém on. _ 5 «new 3.? 3.3 ema— . 85 _ 85 1 86m 86m -1-I1111 \ Ii . . \ Sam—am i... 82: 88— mEonnmosm It! 862 . m \ 2521.1 /1 8 E W 883< IDI 8.03 D 1 . .1 1 i F 111i >1 0 8.2K \ onto—.6101 a 283m .+.. . 35: SE lol 38:2 1.1 . a ocwwwwm tIMI... 8 on 288a. 10.. 028:6 101 8 SN . 35. Lal o .58 1! 8.8m . .Z v. @191 8.8m 88 ._ _ :3 88 .m b2 6 6 8.2: N3“ an: 8.2: NEW 3.2 1 cod w a 86 . - 8.8. _111. - 11c 111- 8.8 , 8.8_ i 111 8.8_ 7 w 1 1 1 .11.:-. -.l 1 1 11:.- 1.1 1 .-1 1 1 1-1+ 862 T 11 1 -.1 1.1.1:.- .1 1111-1l1-111 862 W 11111.0 A i110 fl - . A11 .1111 -1 1 if 868 n :01 .. Bum—am .+.. 8.8m 383m ..+.. maosamoam 11 0552+ maowwmumifllr ovum—”flail 852 r 888 50.00 0.00 15.24 42.55 71.76 100.97 Port Position (cm) 172 AN IONS COLUMN A Incubation Period 8A August 30 - September 3, 2002 August 30, 2002 400.00 “”0"” Bromide 350-00 +Nitn'te * -0'-Chlon'de 300.00 —D— Acetate i C\ + Nitrate 250.00 —0— Phosphorus “ \ ' - + ' - Sulfate 150.00 \\ 100.00 c 3 \ v o 50 00 ' 4, ------------------ + -------- \: ---------------- + 0.00 ‘3 r A I 1 E Port Position (cm) Anion Concentration (mg/L) § 8 September 3, 2002 400.00 ““’*<>"‘*Bromide 350-00 —A-—Nitrite r“ -0-Chloride A 30000 +Acetate P“ E0 +Nitrate . E 250-00 +Phosphorus ”“ 'a --+--Sulfate § 200.00 23’ 2.: 150.00 3 100.00 ‘8 50.00 f 0.00 - Q 100.97 Port Position (cm) 173 APPENDIX C-3 Soluble Metals 174 GE 828; :8 3.9: Nmém www— cod 1 I II v 8.8.0. 1 I lillll 1' iullli|lll I. aliilr 8.8V Iii! ‘lll l. I. IIIIIIIIII ll l...||ili|-.|ill|i.r 8.80 om 8580 3.: . 5 888. . . 8 8m 5358.5 via—om IOI uuuuuuuuuuuuu 8.82 i :2. 9328+ 8.8m— ~8~ .: :3 A58 .8ng tom 3.8— den cud— ri L 86 f I I- tilt 11 ll It»- ltlfioodom u I I 1..“ ..... UHF!i::l.i|.|i:...i:H..uu.i.lu:H Jul. -- - 1 code.V - 868 II. 1.l\ 9i! . .I t l r if 1 It || .- I 8.8” um 288 -..- O 888. . . . ............ . T 55.2.25 0.3—owl? co coo. 8.. 338:? oodofi NOON .mm 0:3. E3 .528.— tom 3.8. 3.3 2a: 3.3 3.? 3.3 3.2 W a \ \q/ommmooo ii I, ..U 8580. .. u 5.825 2.38 not I, :2. 0.3—om Lo! 82 .m :3 A63 eofimom com 8.8. 3.3 Eu: ~03 n23 3.3 $5 m -. E .525 228 ton 8.. 23.8 Icl L 83 KN 25.. «can .3 2.... . rm 2:... .. <— tor—om 3.52.3:— < ZEDJOU mafi—ME mam—Dacm oedew 8.8V 8.95 8.8m 868— 8.82 86 8.2K 8.8V 8.80 - 8.8» 8.25 _ 8.82 '1/fiu1 175 Metals Concentration (mg/L) Metals Concentration (mg/L) Metals Concentration (mg/L) SOLUBLE METALS COLUMN A Incubation Period 2A July 11 - July 26, 2002 July 12, 2002 1200.00 —A—— Soluble lron 100000 -0— SOlUble Chromium »— " ' 'C00712 Cr 800.00 “""C007l2 Fe 600.00 400.00 «A 200.00 / \ 0'00 _ .......... r ..................... I .......... _ 15.24 29.21 42.55 Port Position (cm) July 19, 2002 1200.00 -—-——~—-—- —— —A— Soluble Iron 1000.00 '0- Soluble Chromium -—1 " ' 'C00712 Cr 800.00 . _ _ _________ --‘--C00712Fe H 600.00 ”\ 400.00 200.00 0.00 ..-..-..-..-..-..-..-..-.._.._.T O 15.24 56.52 100.97 Port Position (cm) July 26, 2002 ”00-00 + Soluble Iron -0- Soluble Chromium 1000.00 ' ' 'C00723 Cr 8m’00 I I I I II I I I II I I I II I :';--(2(X-)'72-3fc' 600.00 400.00 200.00 15.24 29.21 56.52 71 .76 86.36 100.97 Port Position (cm) 176 Metals Concentration (mg/L) Metals Concentration (mg/L) 1200.00 1000.00 600.00 400.00 200.00 0.00 SOLUBLE METALS COLUMN A Incubation Period 3A July 26 - August 2, 2002 July 26, 2002 -A- Soluble Iron -0-Soluble Clmomium ' " 'COO726 Cr --°-'C00726 Fe 15.24 29.21 42.55 56.52 71.76 86.36 100.97 Port Position (cm) August 2, 2002 800.00 700.00 —-A— Soluble lron 1L *SOIUblCChIOn‘u'um- I I I — I I I I I I : I—‘F- .- ' ' 'C00729Cr 600.00 -----C00729Fe / \. 500.00 400.00 A / /\ / \ 15.24 29.21 42.55 56.52 71 .76 86.36 100.97 Port Position (cm) 177 Metals Concentration (mg/ L) Metals Concentration (mg/L) SOLUBLE METALS COLUMN A Incubation Period 4A August 2 - 9, 2002 August 2, 2002 800.00 700.00 600.00 -A- Soluble lron -0-Soluble Chromium 500.00 "' " 'C00802 Cr -"-'C00802 Fe 400.00 300.00 200.00 15.24 42.55 56.52 l00.97 Port Position (cm) August 9, 2002 800.00 —-A— Soluble Iron 700.00 -'0-'Soluble Chromium " " 'COO806 Cr 600.00 - ° ° - - C00806 Fe 500.00 A\ 400.00 \ f/E———u’ Me, 300.00 \ / 200.00 _ ...... X_/ .............................................. 100.00 K 000 “W 15.24 29.21 42.55 56.52 71.76 86.36 100.97 Port Position (cm) 178 Metals Concentration (mg/L) Metals Concentration (mg/L) SOLUBLE METALS COLUMN A Incubation 5A August 9 - 16, 2002 August 9, 2002 2000 00 —A— Soluble Iron ' -O'-Soluble Chromium ' " 'C00809 Cr — 'C00809 Fe 1500.00 1000.00 500.00 0.00 15.24 29.21 42.55 56.52 71 .76 86.36 100.97 Port Position (cm) August 16, 2002 2000.00 —fi— Soluble Iron -0-Soluble Chromium ' " 'C00816 Cr 1500.00 --'- C00813 Fe ”m 1000.00 \ //3 500.00 0.00 — 15.24 29.21 42.55 56.52 71 .76 86.36 100.97 Port Position (cm) 179 Metals Concentration (mg/L) SOLUBLE METALS COLUMN A Incubation Period 6A August 16 - 24, 2002 August 16, 2002 1200.00 -&— Soluble Iron 100000 +SOlllblC Chromium ' " C008l6 Cr g — - - — (‘00816 Fe 5 800.00 E o E g 600.00 8 o L) '5 400.00 0 2 0.00 - 15.24 29.21 42.55 56.52 71.76 86.36 100.9 Port Position (cm) August 24, 2002 1200.00 !...————-—-A— 1000.00 ‘ .3 —fi— Soluble Iron 300.00 "0-Soluble Chromium A " " C00820 Cr - ° - - C00820 Fe 600.00 K 400.00 200.00 (100 C 1 M I 3f 15.24 42.55 71 .76 100.97 Port Position (cm) 180 Metals Concentration (mg/L) Metals Concentration (mg/L) SOLUBLE METALS COLUMN A Incubation Period 7A August 24 - 30, 2002 August 24, 2002 l200.00 —A— Soluble Iron 1000-00 -0—Soluble Chromium ' ' 'C00824 Cr - - - - C00824 Fe 800.00 600.00 A 400.00 200.00 Y 0.00 l 15.24 29.21 42.55 56.52 71.76 86.36 100.97 Port Position (cm) August 30. 2002 1200.00 + Soluble Iron -0-Soluble Chromium 1000.00 I I 'C00826 Cr _ . - - C00826 Fe / 600.00 1 400.00 2m.00 - - - - - - - - - - - - - - - - - I - - I 15.24 42.55 71.76 100.97 Port Position (cm) 181 Metals Concentration (mg/L) Metals Concentration (mg/L) SOLUBLE METALS COLUMN A Incubation Period 8A August 30 - Septmeber 3, 2002 August 30, 2002 1200.00 —-A— Soluble Iron 1000.00 .0-Soluble Chromium " ' 'C00830 Cr "' ' ' - C00830 Fe 800.00 600.00 /A\ // 400.00 200.00 [A 1 0.00 -___.(m I F I W“ 15.24 42.55 71.76 100.97 Port Position (cm) September 3, 2002 1200.00 —A-— Soluble Iron 1000.00 "0-Soluble Chromium ' ' 'C00830 Cr - ' ' - C00830 Fe 800.00 600.00 (J 400.00 200.00 9. ---------------I 0'00 b ' fl? I fi’f . C 15.24 42.55 71.76 100.97 Port Position (cm) 182 APPENDIX C-4 Chlorinated Aliphatics 183 Solute Concentration (ug/L) Solute Concentration (ug/L) 300.00 250.00 8’ 8 l 50.00 8 8 50.00 0.00 300.00 250.00 200.00 150.00 fl .8 8 50.00 0.00 CHLORINATED VOC: COLUMN A June 27 - 28, 2002 (Pu-Inoculation) June 27, 2002 WVinyl Chloride *— -A— Cis-1,2-dichloroethene -O-Trichloroethene \ - - C00627 A L A H v v 15.24 29.21 42.55 56.52 71.76 86.36 100.97 Port Position (cm) June 28, 2002 "'0‘” Vinyl Chloride —o— Cis-l ,2-dichloroethene -°-Trichloroethene ' ' C00628 "O —-<‘r—— Q____ i 1 . 2 r 3 15.24 56.52 100.97 Port Position (cm) 184 Solute Concentration (ug/L) Solute Concentration (ug/L) Solute Concentration (ug/L) CHLORINATED VOCs COLUMN A June 28 - July 11, 2002 (Incubation Period 1A) June 28. 2002 300.00 , — —7-- - - I_ ~ ——- -— ——» — — —- - -- —-— ---—-—-—§ ”,0... Vinyl Chloride 250.00 +Cta-l.2-drchloroethcner__§ -°-Tnchloroethene ' ' 'C00628 3 200.00 — — : 150.00 100.00 0.00 :5 . £1 . c L“ 15.24 56.52 100.97 Port Position (cm) July 5. 2002 300.00 18— ———- —— -—‘—-——— —- —--v—--—-~—-- —-—- — —-—— m —— I 250.00 ”-0" Vinyl Chloride i +Cis-12-dichloroethene 20000 -°-Trichloroethene “— - - 'C00703 3 150.“) 4: 10000 W. 50.00 000 T r r 4 263 263 263 Port Position (cm) July 12. 2002 300.00 1 l w-<>~-- Vinyl Chloride 3 250.00 + C is-l .Z-dichloroethenc -°—Trichloroetherre ' ’ 'C00712 200.00 ”0'00 \\ '°°'°° \ so-m - - - . . - . - - - .- . . . . . . . . - .3 o.m : I M 15.24 56.52 100.97 Port Position (cm) 185 Solute Concentration (ug/L) Solute Concentration (ug/L) > Solute Concentration (ug/L) 2500.00 2000.00 1500.00 1000.00 500.00 0.00 2500.00 2000.00 1500.00 1000.00 500.00 0.00 2500.00 2000.00 1500.00 1000.00 500.00 0.00 CHLORINA'I'ED VOCs COLUMN A July 12 - 26, 2002 (Incubation Period 2A) July 12, 2002 l '0— Vinyl Chloride um —A— Cis- l ,2-dichloroethene -0- Trichloroethene H ' ' 'C00712 A A U i U 29.21 C? J 15.24 Port Position (cm) July 19,2002 “0"“ Vinyl Chloride -A- Cis-lJ-dichloroethene ——J -0-Trichloroethene ' ' 'C00715 56.52 Port Position (cm) July 26, 2002 a / , V -o-— Vinyl Chloride —A— Cis—l ,2-dichloroethene -0-Trichloroethene ' ' °C00723 15.24 29.21 56.52 71 .76 86.36 100.97 Port Position (cm) 186 CHLORINATED VOCs COLUMN A July 26 - August 2, 2002 (Incubation Period 3A) July 26, 2002 4500.00 400000 wow Vinyl Chloride +Cis-l,2—dichloroethene .0—Trichloroethene 3000.00 - - 'C00726 3500.00 2500.00 2000.00 1500.00 Solute Concentration (ug/L) 1000.00 500.00 0.00 15.24 Port Position (cm) August 2, 2002 4500. 00 ““0““ Vinyl Chloride 4000.00 —A— Cis-l ,2-dichloroethene ...-------...----0-Trichlorocthenc A 350000 - - -c00729 3000.00 2500.00 2000.00 1500.00 Solute Concentration (ug/L 1000.00 500.00 0.00 15.24 29.21 42.55 56.52 71.76 86.36 100.97 Port Position (cm) 187 CHLORINATED VOCs COLUMN A August 2 - 9, 2002 (Incubation Period 4A) August 2, 2002 4500.00 “W . . 4000.00 Vinyl Chlorlde _ —A— 05-] ,2-dlchloroethene A 3500.00 .0-Trichloroethene —« ...l h, "' ' 'C00802 3 3000.00 0- .8 \ E 2500.00 \ I: 0 g ZM-m - - - - - - - - - - - - - - - - - - - - A U \ / 3 1500.00 2% \3 43/ 1000.00 500.00 0‘00 a 4‘3 fi 45}— l5.24 42.55 56.52 100.97 Port Position (cm) August 9, 2002 4500.00 . Wr Vinyl Chlonde 4000.00 —A-— Cis- l ,2-dichloroethene -O-Trichloroethene g 3500.00 _ _ 'C00806 3 3000.00 a - - - - - - - - - - - - - - - - - - - - - - - - l .o 5 2500.00 8 § 2000.00 CL 3 1500.00 5 3 Y 1000.00 500.00 0.00 -W 56.52 71 .76 86.36 100.97 Port Position (cm) 15.24 29.21 42.55 188 CHLORINATED VOCs COLUMN A August 9 - 16, 2002 (Incubation Period 5A) August 9, 2002 4500.00 4000.00 3500.00 ""'"'"""------------. 3000.00 2500.00 2000.00 1500-00 wO-m Vinyl Chloride —-A— Cis- l ,2-dichloroethene Solute Concentration (ug/L) 1000.00 Trichloroethene 500.00 " 'C00809 0.00 15.24 29.21 42.55 56.52 71 .76 86.36 100.97 Port Position (cm) August 16, 2002 4500.00 4000.00 ----------------------- “'0'“ Vinyl Chloride A 3500.00 +Cis-1,2-dichloroethene g, -0-Trichloroethene ,5, 3000.00 _ _ 'C00809 _0 5 2500.00 - 8 8 2000.00 8 3 1500.00 :2 1000.00 500.00 0.00- a y . at . W— 15.24 29.21 42.55 56.52 71.76 86.36 100.97 Port Position (cm) 189 Solute Concentration (ug/L) Solute Concentration (ug/L) CHLORINATED VOCs COLUMN A August 16 - 24, 2002 (Incubation Period 6A) August 16, 2002 4500.00 4000.00 3500.00 3000.00 2500.00 2000.00 1500.00 ”‘0'"- Vinyl Chloride —A- Cis—l ,2-dichloroethene ] 00000 -0-Trichloroethene 500,00 " 'C008l6 0.00 15.24 29.21 42.55 56.52 71.76 86.36 100.97 Port Position (cm) August 24, 2002 4500.00 4000.00 3500.00 3000.00 2500.00 .g O\{/ 2000.00 v WVinyl Chloride 1500'00 —¢— Cis-l ,2-dichlonoethene 1 000.00 .0-Trtchloroethene ' ' 'C00820 500.00 0.00 . ...—...... ’éfi . if ~ . <8 15.24 42.55 71.76 100.97 Port Position (cm) 190 Solute Concentration (ug/L) Solute Concentration (ug/L) CHLORINATED VOCs COLUMN A August 24 - 30, 2002 (Incubation Period 7A) August 24, 2002 4500.00 4000.00 3500.00 3000.00 2500.00 2000.00 150000 “WOW" Vinyl Chloride 1000 00 —b-— Cis-l,2-dichloroethene . “-O-Tn'chloroethene 0.00 15.24 29.21 42.55 56.52 71.76 86.36 100.97 Port Position (cm) August 30, 2002 3500.00 3000.00 2500.00 0_ 2000.00 ”0“ o 0 1500.00 Vinyl Chloride __ —A- Cts-1,2-dtchloroethene -0-Trichloroetllene 1000.00 _ _ 'C00826 —— 500.00 15.24 42.55 71.76 100.97 Port Position (cm) 191 Solute Concentration (ug/L) Solute Concentration (ug/ L) CHLORINATED VOCs COLUMN A August 30 - September 1, 2002 (Incubation Period 8A) August 30, 2002 3500.00 7 3000.00 \ 2500.00 ¥ fir) 2000.00 1 500.00 “WOW“ ' Vinyl Chloride _ —A— C is-l ,2—dichloroethene 1 000.00 —0- Trichloroethene M ' " 'C00830 500.00 15.24 42.55 71.76 100.97 Port Position (cm) Septmeber 3,2002 3500.00 3000.00 2500.00 2000.00 -—————(7/% N— 1 500.00 “'0'" Vinyl Chloride I +Cis-i,2-dichloroethene 1000.00 .0-Trichloroethene +8 " ' 'C00830 500.00 A A 000 -W , j"; .. . . , fie 15.24 42.55 71.76 100.97 Port Position (cm) 192 APPENDIX D Large Column Study Analytical Results — Operational Phase A 193 APPENDIX D-l Fatty Acids 194 FATTY ACIDS COLUMN A Incubation Period 1B September 3 - 10, 2002 September 3, 2002 400.00 350 00 "’W‘O‘“ Propionic Acid ' —A— Acetic Acid i— "0-Lactic Acid 300.00 - - £00903 __ 250.00 200.00 150.00 Substrate Concentration (mg/L) 100.00 50.00 15.24 29.21 42.55 56.52 71.76 86.36 100.97 Port Position (cm) September 10, 2002 400.00 350.00 ......0... Pmplomc acid +Acetic Ac1d 300 00 .0-Lactic Acid g ' - - mom 5 5 250.00 2:: 8 3 20000 G 8 8 l50.00 8 .D :3 w 100.00 50.00 0-00 $ T W 15.24 42.55 100.97 Port Position (cm) 195 FATTY ACIDS COLUMN A Incubation Period 2B Septmeber 10 - 17, 2002 September 10, 2002 400.00 ““0"“ Propionic Acid 350.00 —A- Acetic Acid .0—Lactic Acid 300.00 - (mg/L) 250.00 tron 8’ 8 Substrate Concentra 15.24 29.21 42.55 56.52 71 .76 86.36 100.97 Port Position (cm) September 17, 2002 *W‘O‘“ Propionic Acid /A ”00°00 -a—— Acetic Acid -0—Lactic Acid ' ' 'C00910 1000.00 / 800.00 / 600.00 / Substrate Concentration (mg/L) 400.00 / 200.00 0.00 1r . 15.24 42.55 71 .76 100.97 Port Position (cm) 196 FATTY ACIDS COLUMN A Incubation Period 3B September 17 - October 1, 2002 September 17, 2002 400.00 A 0* Propionic Acid g ”0'00 -o—Acetic Acid 5 300.00 -o-Lactic Acid : I I I § 250.00 . ..... - C009” § 200.00 8 150.00 2 s 100.00 .8 :3 50.00 0.00 15.24 42.55 100.97 Port Position (cm) September 24, 2002 400.00 :3 35000 ”‘0‘"‘PI'OplOIIIC Add Eu +Acetic Acid 1; 30°00 -o-Lcctic Acid § 250.00 ' ' 'C009‘7 § 200.00 8 150.00 2 2 100.00 .‘8 63 50.00 0.00 15.24 42.55 71.76 100.97 Port Position (cm) Octoberl,2002 400.00 a 350.00 WOWPropionic Acid E —A—-Acetic Acid ‘5’ 30°00 -0-Lactic Acid E 250.00 - - -c00917 § 200.00 8 150.00 g .00... '8 m 50.00 0.00 15.24 29.21 42.55 56.52 71 .76 86.36 100.97 Port Position (cm) 197 400.00 350.00 300.00 250.00 200.00 150.00 100.00 50.00 0.00 mg/L 400.00 350.00 300.00 250.00 200.00 150.00 100.00 50.00 0.00 mg/L mg/L N o o o :3 400.00 350.00 300.00 250.00 200.00 150.00 100.00 50.00 0.00 mg/L FATTY ACIDS COLUMN A Incubation Period 4B October 1 - 8, 2002 October 1, 2002 d —_ -I_ _ ......o. Propionic Acid ~—_——i + Acetic Acid _j '0‘ Lactic Acid __i """"""""" - - 'C01001 ___.3 i e . t , \2~ . Lg . 2—fii ..« . ___. . ..- .. ___‘_._ ___ 15.24 29.21 56.52 86.36 100.97 October 3, 2002 J I | i ”0“- Propionic Acid -&- Acetic Acid '0- Lactic Acid - - °C01001 I_____1_J J '1 15.24 29.21 42.55 56.52 71.76 86.36 100.97 v October 5, 2002 _._._ . ...—...... _- ___. _ ___, ....0- Propionic Acid __i + Acetic Acid '0' Lactic Acid - - ~C01001 _i l i 15.24 42.55 71 .76 100.97 October 8, 2002 *0“ Propionic Acid + Acetic Acid _— -°-I.actic Acid """""" - - -C01001 15.24 42.55 71 .76 100.97 Pon Position (cm) 198 Substrate Concentration (ms/L) (mg/L) (mg/L) (mg/L) FATTY ACIDS COLUMN A Incubation Period SB October 8 - 15, 2002 October 8, 2002 400.00 -—— ---- 4- 4 ~— 4 4 — -~ 4 35000 ~<>~Propionic Acid _1‘ .5 300,00 -&- Acetic Acid e.) § 250.00 -o-Loctic Acid .8; § A 200.00 K - - -C01003 _; 5 § 150.00 ' 3 V 100.00 \ § 50.00 \4 i 3 0.00 ‘A' l V I V l 3 i 15.24 42.55 71.76 100.97 Port Position (cm) October 10. 2002 400.00 - ---~- ~- — -- ~— -—- ~—-~---—- —- ~- ,- 350.00 “0 Propionic Acid 0 ‘5 300.00 -¢- Acetic Acid .; § 250.00 -°-I.actic Acid § A 200.00 - - £01003 r: 5 En 150.00 ‘ 3 ... 100.00 /5\ 4 g 50.00 4 :r \ A ; m 000 __épm—g 1 .. I 3 a 15.24 42.55 71.76 100.97 Port Position (cm) October 12. 2002 400.00 « 7'" f -- — -- ——~~~---—--~ -- r; 350.00 “-0-“ Propionic Acid _§ _5 300 00 -¢- Acetic Acid H? g ' . . , a 250.00 ............ ‘ -°-Lacttc Acrd .3} g A 2mm ' ' ‘C01008 3 8 ED 150.00 “E? "’ 100.00 4 ... 50 m A i 'g 0.00 ———8:F ”Ml" 8 r c r ‘15—, 15.24 42.55 71.76 100.97 Port Position (cm) October 14. 2002 400.00 - --- - -- -—~ 350.00 “0" Propionic Acid _. g 300.00 --6- Acetic Acid __ g ' - - ---------- - . . 8 200.00 C01008 8 150.00 _ «B 100.00 1 g 50.00 % m 0.00 W g 15.24 42.55 71.76 100.97 Port Position (cm) 199 October 9, 2002 400.00 T —- — -—— —— — -....- -, ___. ,_ -. 3,, _7 350.00 ""0““ Propionic Acid 300.00 -a- Acetic Acid H 250.00 ........... ‘ ‘O'Lactic Acid i— 200.00 ' ' 'C01008 _J 150.00 ; 100.00 i 50.00 ;\\ 0.00 ‘ . m2? , 3 , 3 in 15.24 42.55 71.76 100.97 Port Position (cm) October 11, 2002 400.00 . --.8. --_- ..., _______ _ 350.00 “°WPropionic Acid 5 30000 + Acetic Acid H 250.00 ‘°'Lactic Acid H 200.00 """""" - “C01008 _1 150.00 fi' 100.00 A J 15.24 42.55 71.76 100.97 Port Position (cm) October 13, 2002 400.00 *r—- _-__ — — - __1 350,00 "0* Propionic Acid ___5 -¢- Acetic Acid ? 223% -o- lactic Acid :: -------- ° - 'C01008 200.00 150.00 100.00 i 50.00 j; 15.24 56.52 100,97 Port Position (cm) October 15, 2002 400.00 _. 350.00 Whopionic Acid 300.00 + Acetic Acid H A 250.00 ............ '0' Lactic Acid _4 ED 200.00 ' ' ‘00'008 "’ 150.00 100.00 50.00 0.00 if . 3 , f , a 15.24 42.55 71.76 100.97 Port Position (cm) FATTY ACIDS COLUMN A Incubation Period GB October 15 - 22, 2002 400.00 350.00 300.00 250.00 Eb 200.00 150.00 100.00 50.00 15.24 29.21 42.55 56.52 71.76 86.36 100.97 Port Position (cm) October 17, 2002 400.00 350.00 300.00 250.00 .4 3200.00 150.00 100.00 50.00 0.00 15.24 29.2] 42.55 71.76 86.36 100.97 Port Position (cm) October I9, 2002 300.00« ———— — — — —— ~— -—- —*--r--——-~-——- 25000 f/“\ ....... 200.00 / \ 4 En 150.00 / \ b 100.00 so 00 / HOW-"we Q“ \ . f "It... ox. \ I 0.004,-r", icii'civé 15.24 29.2] 42.55 56.52 71.76 86.36 100.97 Port Position (cm) October 21, 2002 300.00 ~ -— ~w ---———~——~—- —~ ----———- ———-~———» *0” Propionic Acid 250.00-~— -o—AceticAcid -------/\---- 200.00 "—‘t *MC ACld / X $ 15000 - ° 'C01015 5 / \ 100.00 / \ 50.00 /o/.,FA\ X 000 'W‘t 15.24 29.21 42.55 56.52 71.76 86.36 100.97 Port Position (cm) October 16, 2002 400.00 - --- --—— ., --—-~—- —--# 350.00 300.00 250.00 200.00 150.00 100.00 50.00 0.00 15.24 42.55 Port Position (cm) 71.76 100.97 October 18, 2002 300.00 «h -- ——~ -——~____._-.---- ___ 250.00 200.00 150.00 / 100.00 ,_/’ ‘\.. E 0.00 I T A I A I J I v T e I 42.55 56.52 7|.76 86.36 100.97 Port Position (cm) m8"- 15.24 29.21 October20,2002 300.00 - - ~ —--—~4—-— — — .--._._ ___. ”I. b 250.00 /\ 200.00 150.00 l00.00 50.00 0.00 5 15.24 mg/L Mot.“ : 1 V I 1 v 42.55 71.76 100.97 Port Position (cm) October 22, 2002 300.00 250.00 200.00 “5's 1 0.00 e 5 100.00 50.00 0.00 l 5.24 42.55 71.76 Port Position (cm) 100.97 200 APPENDIX D-2 Major Anions 201 AN IONS COLUMN A Incubation Period 1B September 3 - 10, 2002 September 3, 2002 400.00 ““0“" Bromide 3 50.00 —A— Nitrite -0-Chloride 30000 -D_ Acetate + Nitrate —0— Phosphorus H 250.00 - - + - - Sulfate 200.00 150.00 Anion Concentration (mg/L) 100.00 50.00 0.00 - 15.24 29.21 42.55 56.52 71.76 Port Position (cm) September 10, 2002 86.36 100.97 400.00 350.00 “‘0'“ Bromide —A— Nitrite .0-Chloride -D'- Acetate 300.00 —III-— Nitrate —0— Phosphorus 250.00 - - + - - Sulfate s—i 200.00 150.00 Anion Concentration (mg/L) 100.00 50.00 0.00 15.24 42.55 71 .76 Port Position (cm) 202 Anion Concentration (mg/L) Anion Concentration (mg/L) ANIONS COLUMN A Incubation Period 2B September 10 -l7, 2002 September 10, 2002 400.00 ““0““ Bromide 350.00 —A— Nitrite — -0-Chloride 300.00 +Acetate ~— —iII— Nitrate 250.00 + Phosphorus _. - - + - - Sulfate 200.00 5 l*\[\ 150.00 \ 100.00 \ 50.00 ‘ Q 15.24 29.21 42.55 56.52 71.76 86.36 100.97 Port Position (cm) September 17, 2002 400.00 “0”“ Bromide 350.00 —A— Nitrite H .0-Chloride 300.00 +Acetate - + Nitrate 250.00 + Phosphorus H - - + - - Sulfate 200.00 150.00 100.00 "‘ O 3 H 50.00 + """""""""" i- / : ' o_oo ___—M , ---- * Lg 15.24 42.55 71.76 100.97 Port Position (cm) 203 400.00 350.00 fé’ 8 250.00 200.00 150.00 l00.00 Anion Concentration (mg/L) 50.00 0.00 400.00 350.00 é’ 8 250.00 Anion Concentration (mg/L) 3 N o 8 8 8 100.00 50.00 0.00 ANIONS COLUMN A Incubation Period 38 - September 17 - October I, 2002 September 17. 2002 0'“ Bromide —A— Nitrite Acetate + Nitrate + Phosphorus 15.24 42.55 7|.76 Port Position (cm) September 24. 2002 15.24 42.55 71.76 100.97 Port Position (cm) 400.00 350.00 Anion Concentration (mg/L) ... N N u .‘6 .8 .‘6 .8 8 8 8 8 100.00 50.00 0.00 October I , 2002 "'0‘“ Bromide + Nitrite .0-Chlon'de -D- Acetate + Nitrate -—-1 --0— Phosphorus - - + ' Sulfate W + ........... + ........... + ........... +. ........... + ........... ... ........... + 15.24 29.2] 42.55 56.52 71.76 86.36 100.97 Port Position (cm) 204 ANIONS COLUMN A Incubation Period 43 - October - 8, 2002 October 1, 2002 300.00-~——-——*—————— — “0* Bromide 3 250.00 +Nitrite Hf 1'1 '0-Chloride 9 200.00 \a‘ -t:—A_Cetatc L4 __1 \ -—Nttrate .3 R 150.00 -°-Phosphorus a; E \ ~-+-- Sulfate 100.00 \ A 0———ci .. - -. 44> 50.00 4... .. .\ "..th- 9 ; ............ +-' \ '.‘+__.-' 000 _W l5.24 29.21 56.52 86.36 100.97 Port Position (cm) October 3, 2002 300.00 - i~——~ — -— - ~———- ——~ —— —— ——— —— § 25000 “'0" Bromide -6- Nitrite ; .1 200.00 -0-Chloride -3- Acetate rd; \ -- Nitrate -°- Phosphorus 7 E" ”0'00 ~+~~ Sulfate ”J 100.00 , — . i 50 00 j : i o 00 ‘e = r4. . 4. . e‘ ' 15.24 29.21 42.55 56.52 7|.76 86.36 100.97 Port Position (cm) October 5, 2002 300.00 ~ ~—» —— 777777 ~———-~———-— -~—- -- »— —---- -- —~ ~ 3; 0* Bromide —&- Nitrite f 25000 '0' Chloride -'!f*- Acetate ‘—‘: —-— Nitrate —0— Phosphorus __i ..J 200.00 "4“ Sulfate g) 150.00 100.00 M C —O 50.00 / + ....................... 0.00 *——Ih=:——:;V/T”- ; I i fl 15.24 42.55 71.76 100.97 Port Position (cm) October 8, 2002 300.00 « - -———-———— 7~-———~- "t “0* Bromide -o— Nitrite i 250.00 '0- Chloride -'—'t- Acetate *— --Nitrate + Phosphorus 3'0 200-00 ~+-- Sulfate —f 150.00 " 8 100.00 50.00 0.00 _ 15.24 42.55 71.76 100.97 Port Position (cm) 205 ANIONS COLUMN A Incubation Period SB October 8 - 15, 2002 October 8, 2002 October 9. 2002 300.00 44 4 44 44 4 4 4 44- 4 4 4 4 300.00 T 4 -—--— 44» 4 44 4 4- 4 4 4 4 4] 2 0 00 \ 4+4 Bromide 4c— Nitrite --<>- Bromide -°- Nitrite i 5 ' \ -0-Chloride —o— Acetate *— 250'00 -0-Chloride ~3- Acetate ”—1 200.00 .... Nitrate .... Phosphorus ' 20000 -*- Nitrate -+- Phosphorus __j _J \ ..+.. Sulfate _1 "+'- Sulfate i 30 150.00 En [50.00 ‘1 E \ E f 100.00 % 100.00 ‘ i e .,.\--z-- c o .= 50.00 44-” \ _ 50.00 i "F .................. + . 0.00 53.!» . e . ; i 0.00 41 15. 4 42.55 71.76 100.97 l5.24 42.55 71.76 100.97 Port Position (cm) Port Position (cm) October 10. 2002 October ll, 2002 300.00 - — 4 — 4 4 —- 4 44 44 44-- 4 ~.. 300”" "" “ “" "’" i ...o... Bromide _¢__ Nitrite 2 "0" Bromide -o- Nitrite 3 25000 -0- Chloride -’4‘- Acetate : 25000 "'°' Chloride "D“ Acetate H 200 00 -— Nitrate —°- Phosphorushaf 200,00 -— Nitrate -°- Phosphorus—sf __J ' --+-4 Sulfate § ~+-- Sulfate i En 150.00 4 é ”000 i 100.00 . 4’? 100.00 4 i Cr 4\ 0‘0 ; C A ... 4‘) ; 50.00 / \ J. 50.00 / \ 1' 32/ .......... \f ................. + i t/ \ . 0.00 e r """" 4:" . 44 i “1 i 0.00 .. , """"" : . 15.24 42.55 71.76 100.97 15.24 42.55 71.76 100.97 Port Position (cm) Port Position (cm) October l2, 2002 October 13, 2002 300.00 T... - - -4— — 4—4— ~--- *4 7 300.00 4>--~- -—4— — "—4-- 4—4-4; 25000 -0— Cirigiid: 4':- Aceiaete 250'00 ‘°' gh'oridfi "-3" 3:0“: *7 ... - _._ . + ttrate -°- osp orus 3 200.00 ..+.. 1:33;: Phosphomse 200.00 ..+.. Sulfate 44 E0 150.00 in 150.00 l00.00 100.00 ‘, P #7 A) l 50.00 50.00 g l 0.00 i 0.00 4' 15.24 42.55 71 .76 100.97 15.24 56.52 100.97 Port Position (cm) Port Position (cm) October I4, 2002 October 15, 2002 300.00 4 ~—--——---'- -————— —- ——--——~-~ 300.00 I -— ——‘—_7 “0M Bromide + Nitrite -~<>-- Bromide -¢- Nitrite 250.00 -o-§iiloridc +91%: *—'; 25000 H -0-Chloride -0-Acetate / + “me + 03p 0m —Q-— ' —o— 200.00 ~+-- Sulfate ———; 200.00 4..—4+..gi'fi'f2fi Pmphmmi/ $ 150.00 $ 150.00 E E / 100.00 100.00 L 50.00 50.00 . 0.00 . 0.00 15.24 42.55 71.76 100.97 Port Position (cm) 206 42.55 71.76 100.97 Port Position (cm) October 15, 2002 ANIONS COLUMN A Incubation Period 63 October 15 - 22, 2002 ---04 Bromide ‘6'" Nitrite 40- Chloride i -. - - -- _ \___......___ \ ~0— Acetate —-— Nitrate + Phosphonts --+-4 Sulfate O l5.24 29.2I 300.00 4 250.00 - 200.00 42.55 56.52 71.76 86.36 Port Position (cm) October 17, 2002 ........ 4. ‘4 I 100.97 Srsooo E . 4o— Nitrite -0- Chloride -O- Acetate + Nitrate -°- Phosphorus ~+~ Sulfate 100.00 I 42.55 71.76 Port Position (cm) 50.00 {:7 Ao/ 0.00 3 """ r e . 15.24 29.21 86.36 100.97 October 19. 2002 °"“ BP'fn'dc 300.004 ___ .----- ______ _i-O—Nltme -<>-Chroride 250.00 ‘ _fi 4 )- Acetate l5.24 29.2] 42.55 56.52 71.76 86.36 100.97 Port Position (cm) October 2|. 2002 300.00 i -<> Bromide 3 250.00 4 + Nitrite . . . *CW’ /\ 200400 ' —3-Aeetate —-—- Nitrate 15.24 29.21 Port Position (cm) 42.55 56.52 71.76 86.36 100.97 'S. F = SE S October 16. 2002 -*~<>~‘Bromide ”000 T'—-':' “r" — —_‘ —'——— —" “who +Nitrite 250.00 ‘ \4._. -°-Chloridc \ -O-Acctate 20°00 +Nitrate 150.00 \K ... Phosphorus ~-+-- Sult‘ te 100.00 a 50.00 I ............... +~'"""“"--4- 0.00 2 “4 ---- : ---- , \; . #1.. l5.24 42.55 71.76 100.97 Port Position (cm) October 18, 2002 "“9” Bromide 300.00 ...- --. ___ __ -_ ._-.- - - e- A +Nitn'te -°-Chloride 1*" 250.00 [4— ‘\ .40— Acetate 200.00 4: --Niuate 150 00 / \ ... Phosphonrs ' / \ --+--Sulfate 100.00 I I 50.00 4. lay/H: y ........ + 0.00 4 i —" r 4: r e . : 4 w ,#: 15.24 29.2l 42.55 56.52 71.76 86.36 100.97 Port Position (cm) October 20, 2002 300.00 ~— -4——-4 4444— 4—4 ——4—4 15.24 42.55 71.76 100.97 Port Position (cm) October 22. 2002 30000 . ~<>- Bromide _- 250.oo .n + Nitrite d b‘ hm. _ 200.00 4 -0- Acetate }\\ -°- Nitrate i 15.24 29.21 42.55 56.52 7|.76 86.36 l00.97 Port Position (cm) APPENDIX D-3 Soluble Metals 208 SOLUBLE METALS COLUMN A Incubation Period 1B September 3 - 10, 2002 September 3, 2002 500.00 450 00 -A- Soluble Iron ' -'0-Soluble Chromium 400.00 ' ' 'C00903 Cr A A - - - - C00903 Fe S, 350.00 ~54 / \ 5 300.00 is / \ 5 250.00 8 200.00 / \ 3 150.00 / \ 50.00 0.00 - 15.24 29.21 42.55 56.52 71 .76 86.36 100.97 Port Position (cm) September 10, 2002 500.00 -A— Soluble Iron L—4 45000 -O-Sotubrc Chromium 400,00 ' "' 'C00906 Cr A - - - - C00906 Fe Si 350 00 E 8 300.00 .E - - - - - - - - C - I - - - - - - - - - - - 5 250.00 8 8 200.00 ..“3 § 150.00 2 100.00 50.00 0.00 Q—H ‘ #1 l5.24 42.55 71.76 100.97 Port Position (cm) 209 Metals Concentration (mg/L) Metals Concentration (mg/L) SOLUBLE METALS COLUMN A Incubation Period 2B September 10 - 17, 2002 September 10, 2002 ”0000 —A— Soluble Iron ” A "0—Soluble Chromium l200.00 .- - 'C00910 Cr H /\ -'°- C00910Fe 1000.00 / \ 800.00 /A\ / \ 600.00 / \ / \ 400.00 2/ \\ / \ 200.00 ... 0.00 4 15.24 29.21 42.55 56.52 71 .76 86.36 100.97 Port Position (cm) September l7, 2002 1400.00 —-A— Soluble Iron -0—Soluble Chromium 1200.00 ' ' 'C00913 Cr - ‘ ° '- C00913 Fe 1000.00 800.00 //A 600.00 / 200.00 0.00 ___h I C I fl I (\fi 1524 42 55 71.76 100 97 Port Position (cm) 210 Metals Concentration (mg/L) Metals Concentration (mg/L) Metals Concentration (mg/L) SOLUBLE METALS COLUMN A Incubation Period 3B September 17 - August I, 2002 September l7, 2002 100000 i + SOlUblC Iron -0-Soluble Chromium 800.00 " ' 'C00917 Cr --'--C009l7 Fe 600.00 400.00 200.00 0.00 15.24 42.55 100.97 Port Position (cm) September 24, 2002 1000.00 44—4 —-A— Soluble Iron .0- Soluble Chromium 800.00 T“ - - -c00920 Cr /A -----C00920Fe / 600.00 / 400.00 / 200.00 A/A, 0.00 : ........ ' ....... ;: ....... ; ....... 5; ...... , ........ ‘ 15.24 42.55 71.76 100.97 Port Position (cm) October 1, 2002 1000400 —o— Soluble Iron -0- Soluble Chromium 300,00 - " 'C00924 Cr -----C00924 Fe 600.00 400.00 A / A..-./.-.\-..,/..--..\. \/ \/ t 0.00 - 15.24 29.21 42.55 56.52 7| .76 86.36 100.97 Port Position (cm) 211 l Metals Concentration (mg/ L) Metals Concentration (mg/L) SOLUBLE METALS COLUMN A Incubation Period 4B October 1 - 8, 2002 October 1, 2002 000.00 -0— Soluble Iron "0—Soluble Chromium ' " 'C01001 Cr 800.00 —--- coroot Fe A 600.00 400.00 \ 200.00 - - - - - - - - - - - - - - - - - - - - - - - I 4: T fie . 4} 15.24 29.21 56.52 86.36 100.97 Port Position (cm) October 8. 2002 1000.00 —A-— Soluble Iron -0-Soluble Chromium ' " 'C01004 Cr /A $00-00 - - - — (201004 Fe 600.00 400.00 A/{Y/ _ 2m.m _ - - - - - - - - - - - - - - - - - - - - - - 0.00 ...................... i ..... fl .......... 4 ..... fifi .. 7‘ 15.24 42.55 71 .76 100.97 Port Position (cm) 212 Metals Concentration (mg/L) Metals Concentration (mg/L) SOLUBLE METALS COLUMN A Incubation Period 5B October 8 - 15, 2002 October 8, 2002 1000.00 —A- Soluble Iron .0—Soluble Chromium " ' C01008 Cr 800-00 - - - - C01008 Fe 600.00 400.00 200.00 A’ . \/A C; 0.00 r $ r WA 1 3— 15.24 42.55 71.76 100.97 Port Position (cm) October 15, 2002 —A— Soluble Iron WOO-00 -0—So1ub1e Chromium ' ' COIOll Cr 800.00 -'-- C0101] Fe 600.00 400.00 200.00 ' ' ' ' ' 4 4 \A 0.00 — , j)— , $ 15.24 42.55 71.76 100.97 Port Position (cm) 213 Metals Concentration (mg/L) Metals Concentration (mg/L) SOLUBLE METALS COLUMN A Incubation Period 63 October 15 - 22, 2002 October 15, 2002 1200.00 —A— Soluble Iron 1000-00 -0-Sotubrc Chromium " ' 'C01015 Cr 800.00 -'--C01015Fe 600.00 400.00 0.00 U """""""""" 15.24 29.21 42.55 56.52 71.76 86.36 100.97 Port Position (cm) October 22, 2002 1200.00 —A-— Soluble Iron 1000.00 -'0-Soluble Chromium ' ' 'C01018 Cr - - - - C01018 Fe 800.00 600.00 400.00 /\\ 2m.m - - - - - - - - - - - - - - - - - - - - - - - 0.00 — 15.24 29.21 42.55 56.52 71.76 86.36 100.97 Port Position (cm) 214 APPENDIX D44 Chlorinated Aliphatics 215 Solute Concentration (ug/L) Solute Concentration (u g/L) CHLORINATED VOCs COLUMN A September 3 - 10, 2002 (Incubation Period 13) September 3, 2002 1200.00 ““0““ Vinyl Chloride —A— Cis-l ,2-dichloroethene 1000.00 __ -0-Trichloroethene ' ' 'C00903 800.00 600.00 400.00 200.00 0.00 -—o——-4—-o-——-r-——o-——~i-—-¢.4 , a . M— 15.24 29.21 42.55 56.52 71 .76 86.36 100.97 Port Position (cm) September 10, 2002 1200.00 \ 1000.00 800.00 fi') 600.00 “Orr-Vinyl Chloride 400.00 -fl'-Cis-l,2-dichloroethene .0-Trichloroethene "' " C00906 200.00 0.00 M r 15.24 42.55 71 .76 100.97 Port Position (cm) 216 CHLORINATED VOCs COLUMN A September 10 - I7, 2002 (Incubation Period 2B) September 10, 2002 4000.00 WVinyl Chloride 350000 +Cis-1,2dich1oroethene A 3000 00 -'0‘-Trichloroethene ”g, - - C00910 § 2500.00 4E - - - . - - I - - I - - - - - - - - - - I - - - 3 2000.00 — 8 8 3 1500.00 2 «3 1000.00 500.00 0-00 -—W~MMWW 15.24 29.21 42.55 56.52 71 .76 86.36 100.97 Port Position (cm) September 17, 2002 4000.00 MW Vinyl Chloride 3500.00 —A-— Cis-l ,2-dichloroethene PT A ---.-----------*Tfichk)roethene :: 3 2500.00 0-—-='—C A 't: E 200000 \ o . a \0 U 8 1500.00 2 :3 1000.00 500.00 0.00 __M . , W— 15.24 42.55 71.76 100.97 Port Position (cm) 217 Solute Concentration (ug/ L) Solute Concentration (ug/L) Solute Concentration (ug/L) CHLORINA'I‘ED VOCs COLUMN A September 17 - October I, 2002 (Incubation Period 33) September 17, 2002 5000.00 4500.00 """'-""""' """" 4000.00 3500.00 3000.00 2500 --4~<>4~v1ny1Ch1oride 2000 +Cis-l.2-dichlorocthene Trichloroethene ’ 'C00917 1500 1000.00 500.00 0.00 15.24 42.55 71.76 100.97 Port Position (cm) September 24. 2002 $000.00 450000 ~-~-<>v-~ Vinyl Chloride —A— Cis-l .2-dichloroethene 400000 Trichloroethene 3500.00 " 'C00920 3000.00 2500.00 2000.00 1500.00 1000.00 500.00 0.00 15.24 42.55 71.76 100.97 Port Position (cm) October I, 2002 5000.00 4500.00 -<>-~Vinyl Chloride 4000.00 —A— Cis-l ,2-dichlomethcne Trichloroethene 3500.00 - 'C00927 3000.00 2500.00 2000.00 1500.00 1000.00 500.00 0.00 15.24 29.21 42.55 56.52 71.76 86.36 100.97 Port Position (cm) 218 CHLORINATED VOCs COLUMN A October I - 8, 2002 (Incubation Period 48) October 1, 2002 3000400 ““2“- . . . . - :1“. :1 .“:’2 .t‘: -T. '.—. 2500.00 X 2000.00 \4 i E)“ 150000 ‘”“ 4404 Vinyl Chloride :3 100000 a__ +Cis-l.2-dichloroethene 4 '0-Trichlomethene 50000 ‘ - - -C01001 000 3 1 5, 1 G4 r 4),. ‘ ;-4.,..,...'.Ae.e. worm. 15.24 29.2] 42.55 56.52 71.76 86.36 100.97 Port Position (cm) October 3. 2002 300000 ‘ ‘a‘ ‘ . . :77; . .".";. ::’. 1 _ i " ..,-__ —> “0 Vinyl Chloride 1 2500.00 +Cis-1.2dich1ometheric 4 -O-Trichloroethene $10 200000 c>______._—<>\- - -C01001 7 x 1 1000.00 " 500.00 0.00 v ,- 4 40 4 I .. T 15.24 42.55 71 .76 100.97 Port Position (cm) October 5, 2002 250000 “0* Vinyl Chloride —A— Cis- l .2-dichloroethene i '0-T ' hi the - - 'C01004 i 2000.00 "c ("0‘ “° i o-——-———°——'—°\ 1 1000.00 500.00 i 000 V r "I" r °"T' ‘ ' 15.24 42.55 71.76 100.97 Port Position (cm) October 8, 2002 3000.00 __ 250000 ~4<>—- Vinyl Chloride —a—- Cis-l 2-drchloroethene 2000_00 -°-Trichloroethene - - -C01004 _ '3}, 1500.00 W 1000.00 500.00 0.00“ N“ 1 v 1 15.24 29.21 42.55 56.52 71.76 86.36 100.97 Port Position (cm) 219 CHLORINATED VOCs COLUMN A October 8 - 15, 2002 (Incubation Period 5B) October 8. 2002 October 9. 2002 2500.00 4 — ‘ ___‘".'.—_’_“_ ________ ':‘_ . _ :1 _ ‘ T1 250000 “F“ .............. —;_._: - .:—. _ _' 1 _1 1500-00 M E$1500.00 4v 44 3 1000.00 5 1000.00 500.00 500.00 ‘ - _ . ’__..——o E 0.00 4: i 4 - i 0.00 ___3' ; 1 8,... W 15.24 42.55 71.76 100.97 1524 42.55 71.76 [0097 Port Position (cm) Port Position (cm) October 10. 2002 October 11. 2002 250000 1 ” ; .2 -‘IT’IL'T; . .‘f; -17.}- . I." ' ”'3 2500-00 “"' ' ""f.’ ." . .‘T‘.’ IT. 2 Li: . ." ‘. .'.“."‘;’”.' ‘- A ' "j 2000.00 °——o\o\n ; 200000 J: __J 1500.00 4 <1 1500.00 R) on = 1000.00 3 1000.00 500.00 500.00 1524 42-55 7|.76 10097 15.24 42.55 71.76 100.97 Port Position (cm) Port Position (cm) October 12. 2002 October 13, 2002 250000 , _ L- _ _ - 2500.00 4- 4—44 - — 44-- 200000 """"""""""" 2000.00 ., 1500-00 4 3 1500.00 fl 4, :- = 1000.00 4 3 1000.00 500.00 4 0.00 _ 1,. W19 15.24 42.55 71.76 100.97 15.24 56.52 100.97 Port Position (cm) Port Position (cm) October 14, 2002 October 15,2002 2500.00 44- -—— - —— -—~ — —— —— — - — 4 2500-00 20000) ...................... 2000.00 150000 uW 150000 9 1000.00 ” 1000.00 500.00 500.00 0 00 m 0.00 ' . 15.24 42.55 71.76 100.97 P0" Posrtion (cm) Port Position (cm) *0" Vinyl Chloride -¢- Cis-1,2-dichloroethene -°-Trichloroethene ' ' 'C01011 220 CHLORINATED VOCs COLUMN A October 15 - 22, 2002 (Incubation Period 68) October 15. 2002 October 16. 2002 2500-00 i " "‘ A '—**“ - -"- 7* -- --*- _ -1 2500.00 — — - ——— -—— - -— A o————\ -‘ """"""""""" L 2000.00 M : 2000.00 \ _, 1500.00 V A0 A; _J 1500.00 0 ED 2 in :1 l000.00 - 3 l000.00 500.00 500.00 000 a 1 3 i ; 1 v ......w ., A . . , 000 _ O-- T. .Y 7 ,, r“ ........o i 15.24 29.2l 42.55 56.52 71.76 86.36 100.97 15.24 42.55 7|.76 100.97 P0" Position (cm) Port Position (cm) October I7. 2002 October 18, 2002 2500.00 * ' " ‘ - ‘ ---' -- 2500.00 A —- — —A~- - - ~- - A———- -———A—— AA 2000.00 Q 2000,00 """""""""""""""" 3 500”" 500.00 A : 0.00 fwr' T W 1 0 00 ~—C ' r" O r r O 1“”‘0 T M A r O fl 1524 2921 42.55 56.52 71.76 86.36 100.97 15.24 29.21 42.55 56.52 71.76 86.36 100.97 P0“ Position (cm) Port Position (cm) October [9, 2002 October 20. 2002 ZSOOOOA -, 7, ---——— ~ — —AA-; 250000 -- - -..--_ 2000.00 ’ W ' Q, 150000 R ‘0 oh 500 00 4 .. 0° _\o———o————¢ l5.24 29.21 42.55 56.52 71.76 86.36 100.97 1524 42,55 7|.76 10097 Port Position (cm) Port Position (cm) October 21, 2002 October 22, 2002 2500.00 - MW.-- . s--. --.- A_-_ “___--.s 250000 - - -- -- M—AAAAAA-A-u ————A— - A ---------------------------- : ; 2000.00 3 2000-00 j S 1500.00 \o—JN i i l500.00 Vi ’ 5 1000.00 3 1000.00 A 500.00 500.00 0.00 /:/o. ..o... 0&3 j 000 gw/ _ 15,24 42,55 71,76 10097 l5.24 42.55 71.76 100.97 Port Position (cm) Port Position (cm) "'0‘" Vinyl Chloride + Cis—l ,2-dichlorocthene -0- Trichloroethene ’ - 'C01018 221 APPENDIX E Daily Solute Concentrations within a Control Volume Conveyed through Column A Incubation Periods 4B-6B 222 TCE Concentration (ug/L) cis-DCE Concentration (ug/L) VC Concentration (uyL) TCE (a), eta-DCE (b), and VC (c) profllu over the length of Column A during Incubation Period 43 3000 2500 g 1500 g 1 1000 g j 1 , -o-10/1/2002 1 I i | --o-- 10/3/2002 500 ' r l 1 +10/5/2002 ‘ a. 1 3 ----- o ------ 10/8/2002 0 ' A r 4: r i i 0 15 3o 45 60 75 90 105 Port Position (cm) 10/1/2002 --o--10/3/2002 + 10/5/2002 --------o 10/8/2002 0 l 5 30 45 60 75 90 l 05 Port Position (cm) 500 300 1011/2002 no -- 10/3/2002 + 10/5/2002 ------ 0... 10/8/2002 200 ...................... ............ 0 15 30 45 60 75 90 105 Port Position (cm) 223 TC E Concentration (ug/L) cis-DC E Concentration (us/L) VC Concentration (us/L) 2500 TCE (a), cis-DCE (h), and VC (c) profiles over the length of Column A during incubation Period SB 105 2000 I 1500 f s 1 1 1 L ...... CO 1000 : 1 1 1 f -0- 10/8/2002 : "° " 10/9/2002 E - ~ + 10/10/2002 500 _ A L i -.- lonmooz . ; l . —+— l0/13/2002 a ' - " "’ lO/l4/2002 . ' 1.0..l0/15/2002 0 * A 1 if A if I 0 15 30 45 60 75 90 105 1000.00 T T I b. l i I _o- lo/mooz E i , , a _. 10/9/2002 300.00 i I + lone/2002 _fi 1 ' - r - 10/12/2002 600.00 J1 "- WWZOOZ _ [ i “.0...” 10/1 Samz i 1 200,00 1' P 1" i (if .................... 0.00 -.—. ------ I 0 15 30 45 60 75 90 1000.00 f t 1 T A I I 1. I . . a _ ‘ 10,9an 800.00 T + 10,10/2002 . —o— 10/11/‘2002 J - ° " 10/12/2002 600.00 —+— 10]] 3f2002 - ' ' 10/14/2002 "'°"10/15/2002 400.00 L i- 200.00 0.00 0” 0 15 30 45 60 75 90 105 P011 POSition (cm) 224 Solute Concentration (ug/L) cis-DCE Concentration (ug/L) VC Concentration (ug/L) TCE (a), cis-DCE (b) and VC (c) profiles over the length of Column A during incubation Period 68 2500 ................................................ 1 t ......... 2000 : -\ = \ : a 1500 -f " ‘ c 1000 I 1 E I I ------ C0 -o-10115/2002 500 » I 1 --¢~lO/lé/2002 +10/17/2002 E , I —o—10/13/2002 «4009/2002 E a 1 I —1—-10/20/2002 ---10/21/‘2002 : ° . , ~o—10/22/2002 0 ’ I e I I J. A; 0 15 3o 45 60 75 90 105 Port Position (cm) 1250 I 1 , . I ~-‘I I I -o-10/15/03 : b' I I ,l ‘1 I I --t--10/16/03 100° 1 " ° . 1 I +10/17/03~ I r X X I -°-10/18/03 750 ' I f . \ “A' -o-10/19/03_I 1 . ,' +~ ., —+—10/20/o3 C I ,’ \_ ”M‘x‘ 1 ---~10/21/03 500 ' P 1’ ‘ "‘5 ‘ ~o~10/22/o3 _I 250 0 . Port Position (cm) 105 1250 .. I T I . I l _ c. I I I I I -o-10/15/o3 _ I . 1 I I --o--10/16/03 1000 _ 1 1 1 +10/17/03T _ I -°-10/18/03 . 1 , -.- 10/19/03 750 I I I . —o—10/20/o3 F“ ; I I --- 10/21/03 . --D- 500 I I 10/22/03 250 I ,, ,_ - I. " ‘: Wt“! CL ”___ ..«..r33'. ...... ~ '\ m 0 15 3o 45 60 75 90 105 Port Position (cm) 225 APPENDIX F Daily Solute Concentration Profiles Over Column A Incubation Periods 43-63 226 Pore fluid concentrations of fatty acids, major anions, and VOCs in pore fluids passing through Column A during Incubation Period 48 4 —o—Lactate 3 +PropionateF—fi <\ ...Au Acetate E 2 l .. ..... fi'°""""°" . A ................ i‘ 0 tr” ' j I a 0 I 2 3 4 5 6 7 Days 0.4 A“ + Phosi’hate 0.3 ”VSUIfate §02 0.1 'A ......................... O I O I I I fl Days 24 l ..-A--ciS-DCE l6 \ +VC a 12 - g? 8 4 ......... A.‘ 00 -------- A " a T 4' I I 4 0 1 2 3 4 5 6 7 Days 227 uM uM Pore fluid concentrations of fatty acids, major anions, and VOCs in pore fluids passing through Column A during Incubation Period SB 4 —°— Lactate 3 + Propionate ---A - - Acetate 0 l 2 3 4 5 6 7 Days 0.4 , -<>—Nitrate + Phosphate 0'3 "-0“ Sulfate 0 l 2 3 4 5 6 7 Days 20 —°— TCE - - -A- - cis-DCE 15 + VC 10 228 Pore fluid concentrations of fatty acids, major anions, and VOCs in pore fluids passing through Column A during Incubation Period 63 -°— Lactate + Propionate - - -A' - Acetate 2 3 Days 0.4 A —°—Nitrate + Phosphate "-6" Sulfate 2 :5 A. O l 2 3 4 5 6 7 Days 20 1\ «m 16 "-4" C-DCE 12 2 :3 8 4 0 0 l 2 3 4 5 6 7 APPENDIX G Reactive Transport Modeling Results 230 APPENDIX G-l Calibration 231 CALIBRATED MODEL RESULTS 1.25-r _____- —————~——~A Simulated cis—DCE Concentrations l .4 0.75 E; E 0.5 O 5 0.25 -I Simulated TCE Concentrations . O f I l' I l 0 0 25 50 75 100 125 0 20 40 60 80 100 120 1.25 -I—--————-——~—— _~———-— “-- - ~ _w —~ 12 “T” __.__._,____.. .__,__., _ ‘ Simulated VC Concentrations 10 Emulated Hydrogen Concentrations 1 g _. ........ I 8 / AAA; A _J 0.75 i 2R 2"". ._‘, 3,, .. E 0.5 0.25 o 4 0.018 . - : 0.016 ‘ ET” C°“°°“"a"°"s , 000000016 Simulated Sorbed —~I 0.014 V 0.00000014 "ig TCE Concentrations—I 0.012 0le i", ...... . ..... t A a 0.01 \k - _. E 0.008 0.006 0.004 0.002 0 0 20 40 60 80 100 120 0 25 50 75 100 125 0.0018 ~~ . ~ 0.14 -~ — —~ : 0.0016 :1 Simulated Xm Concentrations 0 Simulated Xim Concentrations 0.0014 0.0012 g 0.001 e a 0.0008 f 0.0006 0.0004 0.0002 1 0 : r= * 1 100 120 Length (cm) -°-Dayl -~~--Day2 +Day3 --=--Day4 —°- Day 5 - + - Day 6 "“5- Day 7 232 APPENDIX G—2 Sensitivity Analyses 233 SENSITIVITY ANALYSIS N0 HYDROGEN IN SYSTEM Simulated TCE Concentrations r I 20 40 I I 60 80 I 1 100 120 Simulated VC Concentrations A 0.75 mg/L 0.5 0.25 100 120 Simulated ETH Concentrations"? mg/L 9 § 0.00045 0.0004 0.00035 0.0003 0.00025 0.0002 0.00015 0.0001 0.00005 20 40 Length (cm) 100 120 —°-Day5 --+--Day2 ---Day6 --Day3 WDay7 -...-. Day 4 Simulated C-DCE Concentrations 0.75 0.5 mam.“ 0.25 - .. . 0 20 40 60 80 100 120 l 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 20 40 60 80 100 l 20 1 .80E-07 1 .60E-07 l .4OE-07 1 JOE-07 1 DOE-07 8.0013-08 6.00E-08 4005-08 2008-08 0.00E+00 Simulated Sorbed TCE Concentrations “...- mg/mg 0 20 40 60 80 100 120 0.04 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0 0 20 40 60 80100120 LenstMcm) 234 SENSITIVITY ANALYSIS NO REACTION TERMS __-._._._ __ _ —. ~._——————-__ 5 Simulated C-DCE Concentrations Simulated TCE Concentrations 1 1 0 . . , , 1 1 o 3 v ; 1; 4' 1 T w .. . o 20 4o 60 so 100 12c 0 20 40 60 80 100 120 125 T “A 1* —" ...-_____-______fi_ fi 12 ~1~-~——-— —- S' lated Hydro en Concentratznsw. s lmu l Simulated VC Concentrations 1 10 k g l i .‘ ........ q 0.75 ~d’ 7e. 2": g i 6 ' ' ‘ E 0.5 1 4 0.25 2 0 = I 5' I: h: t l : "fig—j ‘1 o 0 20 40 60 80 100 120 0 Simulated Sorbed — TCE Concentrations—g E 0 20 40 60 80 100 120 0 20 40 60 80 100 120 0.0018 0.14 0.0016 0.12 Simulated Xim Concentrations 1 0.0014 0.0012 0-1 0.001 0.08 0.0008 0.06 0.0006 04 0.0004 °- 0.0002 0.02 0 0 0 2° 4° 5° 3° '00 12° 0 20 40 00 so 100 120 MR!!!) Length (cm) -°-Dayl ---~--Day2 -—Day3 -----Day4 + Day 5 -+-- Day 6 ...... Day 7 235 SENSITIVITY ANALYSIS NON-RATE LIMITED SORP'I'ION CONDITIONS 1.25 « Simulated VC Concentrations 1 0.75 mg/L 0.5 0.25 0.018 0.016 0.014 0.012 0.01 0.008 mg/L 20 40 60 80 100 120 . _ -.. _____.—-.-.D_.____ .._._..-...._1 - Simulated ETH Concentrations 5 I \ \ 20 40 6O 80 100 120 Simulated Xm Concentrations 0 20 40 60 80 100 120 LeastMcm) -°-Dayl --*-~Day2 -*-Day3 --‘-'Day4 +Day5 -*--Day6 -Day7 236 1.25 - -- Simulated C-DCE Concentrations ___- -J 0.75 0.5 - 0.25 - 100 120 60 80 100 120 000000018 1 -—~ --- j. Simulated Sorbed -—5I YTCE Concaltrations—i; 1 1 fl. 0 20 40 60 80 100 120 0.14 0 12 Simulated Xim Concentrations 0.1 0.08 0.06 0.04 0.02 0 Length (cm) 80 100 120 APPENDIX G-3 Predictions 237 MODEL PREDICTION DOUBLE LENGTH OF BAZ -m.________ —----—~A- -—---—— 1.25 Simulated TCE Concentrations Simulated C-DCE Concentrations 0.75 , ..J. mg/L ‘ ........... 0.5 , .. ’ 0.25 ~ 0 20 40 6O 80 100 120 0.25 4— 51mm“ ET" l“‘~e{‘\-. J Simmm W —1 Concentrations l,’ ‘4‘ ”N1. ‘ TCE Concentrations_ 0.2 ' \\\ \Sk . - \ E1 0.15 // \ :1“ a ‘ ‘ \M E h” \ \\ ‘1‘ - . ‘1‘~ . .. ‘ff‘r’1in 0-1 '5 .. i 1" xr~<>,x"”;‘fl ‘~ \ ‘ “152%".er 0.05 we» 0 4 . e . 0 20 40 60 so 100 120 60 so 100 120 0.0045 . _ 0.3 ~ —— ___- 0.004 S‘T‘th‘“ x‘“ mmnms 0 25 ,1 Simulated Xim 0.0035 ' , ’,\ Concentrations—T 0.003 0.2 if]! A‘ .3 0.0025 0 15 _,-,: ' 11 a 0.002 ° 1,1,7 \ 0.0015 0.1 1:? """" ‘. 0.001 .\ 0.0005 0.05 ~ 0 0 i u I - T = i: = 0 20 40 60 80 100 120 0 20 40 60 so 100 120 math (cm) Length(cm) -°-Day 1 -+- Day2 --Day3 --I--Day4 +Day5 --‘--Day6 WDay7 238 mg/L MODEL PREDICTION USE OF BAZ TREATED GROUNDWATER FOR FEED MAKE-UP (Recycle) A Simula—tiedTCE Concentrations mg/L 40 1.25 — —-- —_.- 0.75 1 mg/L 0.5 0.25 O 4 120 0.018 0.016 0.014 0.012 0.01 0.008 0.006 0.004 0.002 0.001 8 0.0016 0.0014 0.0012 0.001 0.0008 0.0006 0.0004 0.0002 239 1.25 -~-— Simulated C-DCE Concentrations 1 0.75 ' " ".1 “=“t:::-~\\ 05 \ \ \“ ......” -\. \“ K. 0.25 4 ‘2‘ L‘ a 'w R s 0 I I .... - 0 20 40 60 80 100 120 12 ~ - A~~- ~-'——+—————w—- ---—___“- ~ ‘1 Simulated Hydrogen Concentrations 1 10 K ' CNbO‘ Simulated Sorbed ‘ TCE Concentrations 0le En" 00000001 3 0.00000008 0.00000006 0.00000004 0.00000002 0 0 20 40 60 80100120 Simulated Xim Concentrations 1 40 60 80 100 120 Ingmar!) BIBLIOGRAPHY ASTDR. 2001. CERCLA List of Hazardous Substances. Ballapragada, B.S., H.D. Stensel, J.A. 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