The Pennsylvania State University

The Graduate School

College of Engineering

Treatability Study of In Situ Bioremediation of Perchlorate in Vadose Zone Soil Using Gaseous Electron Donors

A Thesis in

Environmental Engineering

by

Hua Cai

© 2009 Hua Cai

Submitted in Partial Fulfillment of the Requirements

for the Degree of

Master of Science

May 2009

I grant The Pennsylvania State University the nonexclusive right to use this work for the University's own purposes and to make single copies of the work available to the public on a not-for-profit basis if copies are not otherwise available.

Hua Cai

ii

The thesis of Hua Cai was reviewed and approved* by the following:

Rachel A. Brennan Assistant Professor of Environmental Engineering Thesis Advisor

Bruce E. Logan Kappe Professor of Environmental Engineering

Brian A. Dempsey Professor of Environmental Engineering

Peggy Johnson Professor of Civil Engineering Head of the Department of Civil and Environmental Engineering

*Signatures are on file in the Graduate School

iii

ABSTRACT

Perchlorate (ClO4-), which has been used as oxidizer in solid rocket fuels since the

1950s, has become a widespread contaminant which may affect the drinking water

supplies of at least 15 million people in the United States. The health impact of

perchlorate is caused by its ability to block the uptake of iodide by the thyroid which

causes a reduction in hormone production. Perchlorate in vadose zone soil can serve as

a substantial source of groundwater contamination through rainwater infiltration.

Although some technologies have been developed to treat perchlorate-contaminated

water, the treatment of perchlorate in vadose soil is more problematic.

The Gaseous Electron Donor Injection Technology (GEDIT), which was invented

and developed by the consulting company, Camp Dresser and McKee, Inc. (CDM), is a

new in situ bioremediation technology for treating perchlorate in vadose-zone soils

(patent pending). This process involves the injection of electron donors as a gas into the

vadose zone in order to stimulate the anaerobic biodegradation of perchlorate. The

technology can be thought of as the reverse of bioventing - a process commonly used for

bioremediation of hydrocarbons in vadose soil. This technology is being demonstrated

and validated by CDM and The Pennsylvania State University through the Department of

Defense Environmental Security Technology Certification Program (ESTCP).

This research, including soil microcosm tests and column transport and

biodegradation experiments, is a treatability study for the site demonstration of this

technology which will be implemented at an Aerojet site in California in the summer of

2007. The microcosm studies were used to rapidly access the ability of candidate electron

iv

donors to effectively reduce perchlorate and to identify the appropriate electron donor to

be used at the site. Column studies were conducted to evaluate the transport of gaseous

electron donors through site soil and also to estimate the rate of perchlorate degradation.

Results from this research show that moisture is the key factor in perchlorate

bioremediation. It appears that 7% soil moisture content was not high enough to support

perchlorate bioremediation at the Aerojet site, and under 16% moisture content, hydrogen

was the most promising electron donor among those tested. When treated with hydrogen,

complete perchlorate reduction was achieved within 35-42 days, with a perchlorate

reduction rate of 0.13-0.19 d-1. Under high soil moisture content (16%), LPG and

1-hexene also facilitated perchlorate degradation, but with lower perchlorate reduction

rates and longer lag periods. Higher perchlorate reduction rates were achieved by

supplying a higher concentration of electron donor. Positive perchlorate degradation in a

negative control (having no external electron donor) was validated in supplemental

hydrogen production experiments, which indicated the possible presence of

H2-photoproducing microorganisms in the site soil. Column tests supplied with a 20%

hydrogen / 80% nitrogen gas mixture in soil with 10% moisture content did not produce

any perchlorate reduction after being incubated for 4-10 weeks; however, complete

denitrification was achieved within 4 weeks of incubation.

v

TABLE OF CONTENTS

LIST OF FIGURES ..................................................................................................... viii

LIST OF TABLES ....................................................................................................... x

ACKNOWLEDGEMENTS ......................................................................................... xii

1 INTRODUCTION .................................................................................................... 1

2 LITERATURE REVIEW ......................................................................................... 4

2.1 Properties and Use of Perchlorate ................................................................... 4 2.2 Risk and Health Effects of Perchlorate Exposure ........................................... 6 2.3 Contamination and Regulation of Perchlorate in the U.S.A. .......................... 6 2.4 Treatment of Perchlorate Contamination ........................................................ 8 2.5 Perchlorate Reducing Bacteria ....................................................................... 11 2.6 Treatment Options for Perchlorate in Vadose Zone Soil ................................ 13 2.7 GEDIT Technology ........................................................................................ 15 REFERENCES ..................................................................................................... 17

3 TRIAL MICROCOSM TESTS.............................................................................. 22

3.1 Abstract ........................................................................................................... 22 3.2 Materials and Methods ................................................................................... 22

3.2.1 Soil Samples ......................................................................................... 22 3.2.1.1 Aberdeen soil .............................................................................. 22 3.2.1.2 Grove Soil .................................................................................. 23 3.2.1.3 Fertilized Garden Soil ................................................................ 24 3.2.1.4 Organic Farm Soil ...................................................................... 24

3.2.2 Trial Tests Setup ................................................................................... 25 3.2.2.1 Trial Test #1 Setup: Aberdeen Soil with High Perchlorate

Concentration .................................................................................. 25 3.2.2.2 Trial Test #2 Setup: Freshly Collected Grove Soil vs.

Cold-Stored Aberdeen Soil at Low Perchlorate Concentration and Low pH ..................................................................................... 26

3.2.2.3 Trial Test #3 Setup: Fertilized Garden Soil at Neutral pH ......... 28 3.2.2.4 Trial Test #4 Setup: Organic Farm Soil with Slurry and

Activated Sludge Inoculum ............................................................. 29 3.2.3 Chemical Analysis ................................................................................ 31

3.3 Results ............................................................................................................. 32 3.3.1 Trial Test #1 Results: Aberdeen Soil with High Perchlorate

Concentration ......................................................................................... 32 3.3.2 Trial Test #2 Results: Freshly Collected Grove Soil vs. Cold-Stored

Aberdeen Soil at Low Perchlorate Concentration and Low pH ............. 33

vi

3.3.3 Trial Test #3 Results: Fertilized Garden Soil at Neutral pH ................ 34 3.3.4 Trial Test #4 Results: Organic Farm Soil with Slurry and Activated

Sludge Inoculum .................................................................................... 36 3.4 Discussion ....................................................................................................... 37

3.4.1 Trial Test #1 Discussion: Aberdeen Soil at High Perchlorate Concentration ......................................................................................... 37

3.4.2 Trial Test #2 Discussion: Freshly Collected Grove Soil vs. Cold-Stored Aberdeen Soil at Low Perchlorate Concentration and Low pH ................................................................................................... 38

3.4.3 Trial Test #3 Discussion: Fertilized Garden Soil at Neutral pH .......... 39 3.4.4 Trial Test #4 Discussion: Organic Farm Soil with Slurry and

Activated Sludge Inoculum .................................................................... 40 3.5 Conclusions ..................................................................................................... 41 ACKNOWLEDGEMENTS .................................................................................. 42 REFERENCES ..................................................................................................... 42

4 MICROCOSM TESTS ............................................................................................. 44

4.1 Abstract ........................................................................................................... 44 4.2 Materials and Methods ................................................................................... 45

4.2.1 Soil Characterization ............................................................................ 45 4.2.2 Experimental Design and Setup ........................................................... 45 4.2.3 Chemical Analyses ............................................................................... 49

4.3 Results ............................................................................................................. 50 4.4 Discussion ....................................................................................................... 55 ACKNOWLEDGEMENTS .................................................................................. 62 REFERENCES ..................................................................................................... 63

5 COLUMN STUDIES ................................................................................................ 65

5.1 Abstract ........................................................................................................... 65 5.2 Material and Methods ..................................................................................... 65

5.2.1 Soil Characterization ............................................................................ 65 5.2.2 Experimental Design and Setup ........................................................... 66

Hydrogen Column Setup ........................................................................ 68 5.2.3 Chemical Analysis ................................................................................ 69

5.3 Results of Hydrogen Columns ........................................................................ 71 5.4 Discussion ....................................................................................................... 74 ACKNOWLEDGEMENTS .................................................................................. 77 REFERENCES ..................................................................................................... 77

6 CONCLUSIONS, ENGINEERING SIGNIFICANCE, AND FUTURE WORK .... 78

6.1 Conclusions and Engineering Significance .................................................... 78 6.2 Future Work .................................................................................................... 81 REFERENCES ..................................................................................................... 82

vii

Appendix A ACRONYMS ....................................................................................... 84

Appendix B B-1 REACTIONS OF ELECTRON DONORS WITH PERCHLORATE .................................................................................................. 85

B-2 PROPERTIES OF ELECTRON DNORS ............................................................ 85

Appendix C TRIAL MICROCOSM TESTS DATA ................................................ 86

C.1 Trial Test #3 Setup ......................................................................................... 86 C.2 Trial Test #4 Setup ......................................................................................... 88

Appendix D MICROCOSM TESTS DATA............................................................. 90

D.1 Microcosm Setup Details ............................................................................... 91 D.2 Microcosm Tests Data ................................................................................... 92

D.2.1 Test 1: 7% moisture, 34 mg/kg H2 ...................................................... 92 D.2.2 Test 2: 7% moisture, 150 mg/kg ethyl acetate ..................................... 94 D.2.3 Test 3: 7% Moisture, 80 mg/kg 1-hexene ............................................ 96 D.2.4 Test 4: 7% Moisture, 75 mg/kg LPG ................................................... 98 D.2.5 Test 5: 7% Moisture, 114 mg/kg H2 .................................................... 100 D.2.6 Test 6: 7% Moisture, 501 mg/kg ethyl acetate .................................... 102 D.2.7 Test 7: 7% Moisture, 265 mg/kg 1-hexene .......................................... 104 D.2.8 Test 8: 7% Moisture, 250 mg/kg LPG ................................................. 106 D.2.9 Test 9: 16% Moisture, 34 mg/kg H2 .................................................... 108 D.2.10 Test 10: 16% Moisture, 150 mg/kg ethyl acetate .............................. 110 D.2.11 Test 11: 16% Moisture, 80 mg/kg 1-hexene ...................................... 112 D.2.12 Test 12: 16% Moisture, 75 mg/kg LPG ............................................. 114 D.2.13 Test 13: 16% Moisture, 114 mg/kg H2 .............................................. 116 D.2.14 Test 14: 16% Moisture, 501 mg/kg ethyl acetate .............................. 118 D.2.15 Test 15: 16% Moisture, 265 mg/kg 1-hexene .................................... 120 D.2.16 Test 16: 16% moisture, 250 mg/kg LPG ........................................... 122 D.2.17 Test 17: Negative control. 16% Moisture, no external electron

donor. ...................................................................................................... 124 D.2.Test 18 Positive control. 16% Moisture, 436 mg/kg ethanol. ................ 126

Appendix E COLUMN TESTS DATA .................................................................... 128

E.1 H2 Column Study Procedure and Calculation ................................................ 128 E.2 Breakthrough Time Calculation ..................................................................... 131 E.3 Dispersion number calculation ....................................................................... 133

viii

LIST OF FIGURES

Figure 2-1: Structure of the perchlorate ion (from Urbansky and Schock, 1999). ...... 5

Figure 2-2: National perchlorate detections as of September 2004 ( USEPA,2004). .. 7

Figure 2-3: Pathway of perchlorate biodegradation (Deitsch, 2005). .......................... 10

Figure 2-4: Energy profile for the rate-limiting step in perchlorate reduction (from Urbansky and Schock, 1999). The kinetic barrier resulted from the high activation energy Ea controls the reaction rate. .................................................... 10

Figure 2-3: Sketch of a cross-section of vadose zone and saturated zone. .................. 13

Figure 2-4: Sketch of Gaseous Electron Donor Injection Technology (GEDIT) (courtesy of CDM). ............................................................................................... 16

Figure 3-1: Nitrate concentration change over time during perchlorate bioremediation in Trial Test #3 with fertilized garden soil. ................................. 34

Figure 3-2: Perchlorate concentration change over time during bioremediation in Trial Test #3. ..................................................................................................... 35

Figure 3-3: Perchlorate concentration change over time during bioremediation in Trial Test #4. ..................................................................................................... 36

Figure 4-1: Perchlorate degradation in microcosm tests with different electron donors at 16% soil moisture. ................................................................................ 53

Figure 4-2: Relative change in perchlorate concentration over time used to estimate first order rate constants. ........................................................................ 54

Figure 4-3: Perchlorate and hydrogen concentration change over incubation time in negative control microcosms containing no external electron donor at 16% soil moisture. ......................................................................................................... 60

Figure 5-1: Schematic of the column setup and the columns in the laboratory. .......... 67

Figure 5-2: Hydrogen breakthrough curves for Column #1 and #2 with 10% soil moisture. ............................................................................................................... 72

Figure 5-3: Perchlorate (ppm), chloride (ppm), hydrogen (mg/kg) and soil moisture (%) along column length in Column #1 after 4 weeks of incubation. ... 73

ix

Figure 5-4: Perchlorate (ppm), chloride (ppm), hydrogen (mg/kg) and soil moisture (%) along column length in Column #2 after 10 weeks of incubation. ............................................................................................................ 74

Figure 5-5: Perchlorate and chlorate concentration in hydrogen columns with 10% soil moisture after 4 and 10 weeks of incubation. ................................................ 76

Figure 6-1: Perchlorate and moisture change along with the change of depth at the Aerojet site. ........................................................................................................... 80

x

LIST OF TABLES

Table 2-1: Properties of common perchlorate compounds (Adapted from ITRC, 2005). .................................................................................................................... 5

Table 2-2: Reported perchlorate reducing microorganism isolates and the compounds successfully tested as growth substrates. ........................................... 12

Table 3-1: Initial conditions of the different soils used in the trial microcosm tests. .. 25

Table 3-3: Setup matrix of Trial Test #2. Perchlorate reduction was tested in freshly collected grove soil (with either ethanol or ethyl acetate treated) and cold-stored Aberdeen soil (with ethanol treated). All of the soil samples were adjusted to 18% soil moisture and 500 ppb perchlorate. ...................................... 28

Table 3-5: Setup matrix of Trial Test #4. Perchlorate reduction was tested in Organic Farm soil treated with either ethyl lactate or 1-hexene under different soil moisture conditions and microbial inoculum. ................................................ 30

Table 3-6: Initial and final concentrations of perchlorate, nitrate and electron donors in Trial Test #2, in which ethyl acetate and ethanol were tested in grove soil and Aberdeen soil under 18% soil moisture and 500-ppb perchlorate. ........................................................................................................... 33

Table 3-7: Initial and final concentrations of nitrate and perchlorate in Trial Test #3. ......................................................................................................................... 35

Table 4-1: Matrix of experimental conditions tested in the microcosm experiments. .......................................................................................................... 46

Table 4-2: Properties of tested electron donors in microcosm tests ......................... 47

Table 4-3: Original and final conditions of the Aerojet site soil after 125-187 days of treatment using different electron donors at 16% soil moisture. (Table shows duplicate averages except where noted.) ................................................... 51

Table 4-4: First order perchlorate degradation rate constants, lag periods, and final perchlorate concentrations for the electron donors tested in the microcosm tests at 16% soil moisture. .................................................................................... 54

Table 4-5: First order perchlorate reduction rates observed in the literature and their experimental conditions. .............................................................................. 57

Table 4-6: Reaction equations of tested electron donors with perchlorate and the corresponding Gibbs free energies under standard and experimental conditions at low and high electron donor concentrations. .................................. 58

xi

Table 4-7: Setup and results of the 1-day hydrogen production test with the Aerojet soil at 16% soil moisture. ......................................................................... 61

xii

ACKNOWLEDGEMENTS

I would like to thank my advisor Dr. Rachel A. Brennan, for her advice,

encouragement, and sincere assistance during this research. I would also like to thank the

other committee members, Dr. Bruce Logan and Dr. Brian A. Dempsey.

I am also very thankful to Dr. Patrick J. Evans of Camp Dresser and McKee, Inc.,

for recommending testing hydrogen, LPG, 1-hexene, and ethyl acetate as electron donors

in these experiments, and for his useful advice throughout this research. Also, thanks to

my fellow graduate students and the staff of the Department of Civil and Environmental

Engineering.

I want to express my gratitude to my family, especially my mother who gave me

amazing encouragement and made me who I am.

This project was a collaboration with Camp Dresser and McKee, Inc. (CDM),

with funding provided by Department of Defense Environmental Security Technology

Certification Program (ESTCP). This cooperation and support is gratefully

acknowledged.

1

1 INTRODUCTION

Perchlorate (ClO4-), which has been used as oxidizer in solid rocket fuels since the

1950s, has become a widespread contaminant which may affect the drinking water

supplies of at least 15 million people in the United States. The health impact of

perchlorate is caused by its ability to block the uptake of iodide by the thyroid which

causes a reduction in hormone production. Perchlorate in vadose zone soil can serve as

a substantial source of groundwater contamination through rainwater infiltration.

Although some technologies have been developed to treat perchlorate-contaminated

water, the treatment of perchlorate in vadose soil is more problematic.

The Gaseous Electron Donor Injection Technology (GEDIT), which was invented

and developed by the consulting company, Camp Dresser and McKee, Inc. (CDM), is a

new in situ bioremediation technology for treating perchlorate in vadose-zone soils

(patent pending). This process involves the injection of electron donors as a gas into the

vadose zone in order to stimulate the anaerobic biodegradation of perchlorate. The

technology can be thought of as the reverse of bioventing - a process commonly used for

bioremediation of hydrocarbons in vadose soil. This technology is being demonstrated

and validated by CDM and The Pennsylvania State University through the Department of

Defense Environmental Security Technology Certification Program (ESTCP).

My hypothesis is that hydrogen, ethyl acetate, Liquefied Petroleum Gas (LPG),

and 1-hexene, can serve as electron donors and stimulate biological perchlorate reduction

in vadose zone soils.

2

Hydrogen has been shown by others to be an excellent electron donor capable of

supporting the activity of perchlorate reducing bacteria (Miller and Logan, 2000, Zhang

et al., 2002, Nerenberg et al., 2002, Kroon and van Ginkel, 2004). Ethyl acetate promoted

complete nitrate reduction and 10% perchlorate removal after 34 days of incubation in a

previous column study (Evans and Trute, 2006). LPG is a mixture of hydrocarbon gases,

the primary component of which is propane. Propane has been previously tested for

perchlorate degradation (Hoponick, 2006), however, it only supported denitrification, not

perchlorate reduction. Kniemeyer et al. (2006) reported anaerobic oxidation of propane

by novel sulfate-reducing bacteria, so propane theoretically has the capacity for

perchlorate reduction. Commercially available, LPG has the potential to be a

cost-effective electron donor. Even though to the best of our knowledge there is no other

research reported to use 1-hexene as an electron donor for perchlorate biodegradation, its

high vapor pressure and Henry’s constant make it a good electron donor candidate.

In this thesis, a literature review of perchlorate contamination and treatment is

introduced in Chapter 2. Chapter 3, 4 and 5 presented and discussed the results from a

series of trial microcosm tests, the microcosm tests conducted with the Aerojet site soil,

and the column tests. In Chapter 6 included the conclusion and engineering significance

of this research, and also the recommended future work.

3

REFERENCES

Evans, P. J., and Trute, M. M., 2006. In Situ Bioremediation of Nitrate and Perchlorate in

Vadose Zone Soil for Groundwater Protection Using Gaseous Electron Donor Injection

Technology. Water Environment Research 78(13):2436-2446.

Kniemeyer, O., Musat, F., Sievert, S. M., Knittel, K., Wilkes, H., Blumenberg, M.,

Michaelis, W., Classen, A., Bolm, C., Joye, S. B., and Widdel, F., 2006. Anaerobic

oxidation of propane and ethane by novel marine sulphate-reducing bacteria. Nature, in

press.

Kroon A. G. M. and van Ginkel, C. G., 2004. Biological Reduction of Chlorate in a

Gas-Lift Reactor Using Hydrogen as an Energy Source. J. Environ. Qual 33:2026-2029

Miller, J. P. and Logan, B. E., 2000. Sustained Perchlorate Degradation in an

Autotrophic, Gas-Phase, Packed-Bed Bioreactor. Environ. Sci. Technol. 34, 3018-3022.

Nerenberg, R., Rittmann, B.E., Najm, I., 2002. Perchlorate Reduction in a

Hydrogen-based Membrane-biofilm Reactor. J. AWWA. 94, 103-114.

Zhang, H., Bruns M. A., and Logan, B. E., 2002. Perchlorate Reduction by a Novel

Chemolithoautotrophic, Hydrogen-Oxidizing Bacterium. Environmental Microbiology

4(10):570-576.

4

2 LITERATURE REVIEW

Perchlorate is a widespread contaminant that is very persistent in the environment due to

its stable physical and chemical properties. Ammonium perchlorate, has been broadly

used in United States as an oxidizing additive in solid rocket propellant since the 1950s.

About 44 states in United States have been identified as having perchlorate users and

manufacturers, and the drinking water of 15 million people is potentially affected by

perchlorate contamination. Perchlorate in the vadose zone has a special significance

because it can serve as a contamination source to groundwater, but vadose zone

remediation is difficult. New technologies are in great need to address this problem.

Gaseous Electron Donor Injection Technology (GEDIT) is a newly invented technology

for in situ perchlorate bioremediation in vadose zone soil. This research, including soil

microcosm tests and column transport and biodegradation experiments, is a treatability

study for a site demonstration of this technology which will be implemented in California

in summer, 2007. In this chapter, the properties, use, health effects, and contamination of

perchlorate will be discussed, and the new GEDIT technology introduced.

2.1 Properties and Use of Perchlorate

Perchlorate is an anion consisting of a chloride ion combined with four oxygen

ions (ClO4-, Figure 2-1, (Urbansky and Schock, 1999)). It is the most oxidized form of

chlorine that exists in water with an oxidation state +7. The most common forms of

5

perchlorate are perchloric acid and perchlorate salts such as ammonium perchlorate,

potassium perchlorate, and sodium perchlorate, which all share the characteristics of high

solubility and mobility. Table 2-1 shows the physical properties of these perchlorate salts

and perchloric acid.

Figure 2-1: Structure of the perchlorate ion (from Urbansky and Schock, 1999).

Table 2-1: Properties of common perchlorate compounds (Adapted from ITRC, 2005).

6

Perchlorate has been widely used by industry in the production of matches, air

bag initiators for vehicles, fireworks, etc. because it is an exceptional oxidizer (Logan,

2001, Motzer, 2001, ITRC, 2005). However, it is estimated that approximately 90% by

weight of all perchlorate is used to make the oxidizing agent for solid rocket propellant,

in the form of ammonium perchlorate (ITRC, 2005). More than 164 million pounds of

perchlorate-containing rocket propellant was expected to be disposed of by the United

States military by 2005 (Wallace et al., 1998).

2.2 Risk and Health Effects of Perchlorate Exposure

Perchlorate is of concern because it can interfere with the uptake of iodide in the

thyroid and may consequently result in a dose-dependent decrease in thyroid hormone

production (ITRC, 2005). Thyroid hormones are essential to the regulation of oxygen

consumption and metabolism throughout the body (Greer et al., 2002). Competitive

inhibition of iodide uptake by perchlorate may lead to both neurodevelopmental and

neoplastic related problems (USEPA, 2002).

2.3 Contamination and Regulation of Perchlorate in the U.S.A.

Perchlorate was an unregulated compound before 1997 (Chaudhuri et al., 2002).

Due to advancements in analytical methodology, a new ion chromatography (IC) method

that achieved a method detection limit of approximately 1 ppb and a reporting limit of 4

7

ppb was developed in 1997 (USEPA, 2002). Following this development, perchlorate

was discovered at various manufacturing sites, well water, and drinking water supplies in

44 states, potentially affecting the drinking water of 15 million people in the United Sates

(Logan, 2001). Perchlorate was placed on the Contaminant Candidate List (CCL) in

March 1998 as a contaminant that required additional research and occurrence

information before regulatory determination could be considered. The United States

Environmental Protection Agency (USEPA) started monitoring for perchlorate in public

drinking water systems through the Unregulated Contaminant Monitoring Rule (UCMR)

in 1999. The locations with the highest perchlorate contamination are southern

California, west central Texas, along the east coast between New Jersey and Long Island,

and in Massachusetts. Figure 2-2 shows the perchlorate contamination currently known to

the EPA as reported from various sources ( USEPA, 2004).

Figure 2-2: National perchlorate detections as of September 2004 (USEPA, 2004).

8

In 1997, the USEPA recommended a provisional reference dose (RfD) range of

0.0001 to 0.0005 mg/kg-day, which can be converted to preliminary clean-up levels of

4-18 ppb by using a standard default body weight of 70 kg and 2 L tap water

consumption per day over a lifetime (USEPA, 1999). In 2005, the EPA established an

official RfD of 0.0007 mg/kg-day of perchlorate (USEPA 5a), which can be converted to

24.5 ppb using the same method of calculation. The California Department of Health

Services (CA DHS) adopted 18 ppb as its provisional action level. Since then, several

states have adopted more stringent provisional levels ranging from 1 ppb to 18 ppb,

including Arizona, California, Maryland, Massachusetts, Nevada, New Mexico, New

York, and Texas (USEPA, 2005b).

2.4 Treatment of Perchlorate Contamination

Although perchlorate is a strong oxidizing agent, it is very stable. The high

strength of the chlorine-oxygen bonds causes its chemical reaction with most reducing

agents to be slower than observable. Because perchlorate is nonlabile (very slow to

react), it is not reduced by common reducing agents or precipitated by common cations,

which makes it difficult to be removed physically or chemically (Urbansky,1998). There

are some physical and chemical technologies that can be used to treat perchlorate

contaminated water, for example anion exchange, membrane filtration, and

electrodialysis, but they are relatively too expensive for large sites.

Bioremediation appears to be a promising method to treat perchlorate, in which

the molecule is sequentially reduced from perchlorate to chlorate to chlorite and finally to

9

chloride (Figure 2-3 , Deitsch, 2005). The first step of perchlorate reduction (perchlorate

reduction to chlorate) is the rate-limiting step of the pathway. Even though the reaction is

thermodynamically favored as shown by ΔE < 0, the reaction rate is controlled by the

kinetic barrier of the high activation energy Ea (Figure 2-4) (Urbansky and Schock,

1999).

Several microorganisms have been reported as having the ability to reduce

perchlorate under anaerobic conditions by using perchlorate as an electron acceptor. A

reductase enzyme is produced by these microbes under anaerobic conditions in the

presence of perchlorate (Chaudhuri et al., 2002) to lower the activation energy of

perchlorate reduction and reduce perchlorate (Urbansky and Schock, 1999). The

degradation of perchlorate appears to follow first order kinetics (Logan et al., 2001).

Perchlorate reducing microorganisms are ubiquitous in nature (Coates et al., 1999),

including the sites having no previous perchlorate exposure, although their numbers are

highly variable depending on the source (soil or water) (Wu et al., 2001).

10

Figure 2-3: Pathway of perchlorate biodegradation (Deitsch, 2005).

Figure 2-4: Energy profile for the rate-limiting step in perchlorate reduction (from Urbansky and Schock, 1999). The kinetic barrier resulted from the high activation energy Ea controls the reaction rate.

11

2.5 Perchlorate Reducing Bacteria

To date, all isolated perchlorate reducing microorganisms are members of

Proteobacteria. Most isolates in earlier studies belong to the β-Proteobacteria

(Dechloromonas or Dechlorosoma), but a recent study suggests that the genus

Azospirillum, a group of α-Proteobacteria, may be more prevalent at contaminated sites

than the current record of isolates suggests (Waller et al., 2004). All perchlorate reducing

bacteria isolated to date are facultative anaerobes (can use oxygen as an electron acceptor

if it is present), capable of reducing perchlorate, chlorate, in most cases nitrate (ITRC,

2005), and sometimes sulfate (Waller et al., 2004).

A variety of organic and inorganic compounds can be used by perchlorate

reducing bacteria as electron donors. Acetate has been used extensively in laboratory

studies of perchlorate bioremediation, however, other electron donor candidates including

ethanol, methanol, hydrogen gas, lactate, volatile fatty acids, pyruvate, emulsified

vegetable oil, sulfur, and iron have also been used. Table 2-2 summarizes reported

perchlorate reducing microorganism isolates and the electron donors and acceptors that

have been successfully tested.

12

Table 2-2: Reported perchlorate reducing microorganism isolates and the compounds successfully tested as growth substrates.

Culture Genus Electron donor Electron acceptor Reference Vibrio dechloraticans Cuznesove B-1168

Acetate, ethanol, glucose and sugars

Perchlorate, chlorate, oxygen

Korenkov et al., 1976

HAP-1 W. succinogenes

Hydrogen, aspartate, fumarate, malate, mixture of hydrogen and pyruvate, succinate, acetate, whey powder, peptone, yeast extract, brewers’ yeast, casamino acids, cottonseed protein

Perchlorate, chlorate

Wallace et al., 1996

GR-1 Proteobacteria acetate Perchlorate, chlorate, oxygen, nitrate, Mn(IV)

Rikken et al., 1996

CKB Dechloromonas agitata

Acetate, propionate, butyrate, lactate, succinate, fumarate, malate or yeast extract

Perchlorate, nitrate

Chaudhuri et. Al, 2002; Bruce et Al.,1999

JM Dechlorimona hydrogen Perchlorate, oxygen, nitrate, chlorate

Miller & Logan, 2000

KJ and PDX Dechlorosoma acetate Perchlorate, oxygen, chlorate

Logan et al, 2001

PS Dechlorosoma suillum Acetate Perchlorate, nitrate Chaudhuri et.

al, 2002

HZ Dechloromonas, Hydrogen, acetate, zero-valent iron

Perchlorate, chlorate, nitrate, oxygen

Zhang et al., 2002; Yu et. al, 2006

EAB1, EAB2, EAB3, RC1, RC2, PMC, PR, INS, ABL2

β-Proteobacteria Acetate, molasses, oleate, canola oil

Perchlorate, nitrate, sulfate

Waller et al., 2004

PMS1, PMS2, SN1A, SN1B, ABL1, AJ2, SN2

Azospirillum Acetate, molasses, oleate, canola oil

Perchlorate, nitrate, sulfate

Waller et al., 2004

PC1 Dechloromona Hydrogen, acetate Perchlorate, chlorate

Nerenberg et al, 2006

13

2.6 Treatment Options for Perchlorate in Vadose Zone Soil

The vadose zone, also called the unsaturated zone, extends from the top of the

ground surface down to the water table (Figure 2-3). This zone has special significance

because pollutants in the vadose zone can serve as a source of contamination to the

groundwater. When water passes through the vadose zone to the water table, for example

during rainfall or irrigation, contaminants will be brought to groundwater, especially if

the contaminant is very soluble, like perchlorate. An important way to prevent

groundwater pollution by perchlorate is to keep the water from contacting the

contaminated soil in the first place. Since it is difficult to prevent water infiltration into

the vadose zone, the treatment of contamination in vadose zone soil before it reaches the

groundwater is very important.

Figure 2-3: Sketch of a cross-section of vadose zone and saturated zone.

( from http://geology.er.usgs.gov/eespteam/brass/ground/groundintro.htm)

14

Current technologies to treat vadose zone soil include ex situ and in situ

remediation. Anaerobic composting was demonstrated in pilot tests as an effective ex situ

method for treating heavily perchlorate-impacted soils. Highly contaminated surface soil

(with a perchlorate concentration of approximately 42 ppm) was excavated from the field

and transported to an above ground biocell. After about twelve months of anaerobic

composting, complete perchlorate reduction was achieved. An infiltration gallery was

used at the same site as an in situ remediation technology to treat deeper but less

contaminated soil. Perchlorate was flushed out from the soil with extracted groundwater

and an in situ anaerobic treatment zone was created in shallow groundwater. Overall,

83% reduction in perchlorate was observed after a treatment period of 16 months (Smith

et al., 2002). Another site in Santa Clara Valley, California, will be treated using similar

technologies (Deitsch, 2005). Soils containing perchlorate at concentrations greater than

7.8 mg/kg (surficial soil) will be excavated and treated on site via ex situ anaerobic

composting, and soils containing perchlorate less than 7.8 mg/kg (deep soil) will be

treated in situ by irrigating the vadose zone with treated groundwater amended with

additional electron donors. A treatability study estimated a treatment time of two years to

achieve the desired soil remediation objectives (Deitsch, 2005).

Although the technologies described above have been effectively demonstrated,

they can only be used in a combined soil-groundwater treatment system where the

groundwater has already been contaminated by perchlorate. If the groundwater has not

been contaminated, for example in newly polluted area or when the water table is very

low, the infiltration method would not be suitable if we do not want to contaminate the

groundwater.

15

Only a few studies have been done for the in situ treatment of

perchlorate-contaminated vadose-zone soil. A pilot scale in situ vadose-zone

bioremediation study was conducted at a site in Karnack, Texas. A

Composting-Biotreatment technology was shown to be effective both in laboratory

experiments and a pilot study. Ethanol, horse manure, and chicken manure were used as

external carbon sources and mixed with the site surface soil. Water was applied to

achieve complete saturation only down to the desired treatment depths but above the

groundwater table. After 120-days of treatment, perchlorate concentration in the soil was

reduced from initial values ranging from 8.4 to 295.3 mg/kg down to 0 to 223.4 mg/kg

(Nzengung et al., 2003). Another study was performed by the consulting company

ARCADIS for the in situ remediation of perchlorate-impacted vadose zone soil at a site

located in northern California using In-Situ Reactive Zone (IRZ) technology. Dilute

solutions of corn syrup and ethanol were injected under high pressure throughout the

study area to deliver organic carbon and saturate the soil. Within eight months of

implementation, 81-93 percent of the perchlorate was reduced from an initial

concentration of 200-500 mg/kg (Frankel et al).

2.7 GEDIT Technology

The Gaseous Electron Donor Injection Technology (GEDIT) is an innovative in

situ bioremediation technology for the treatment of perchlorate in vadose zone soil

(patent pending) invented by the consulting company, Camp Dresser and McKee, Inc.

(CDM). Gaseous electron donors such as hydrogen/carbon dioxide or volatile organic

16

compounds are injected into the soil using injection wells in combination with optional

soil vapor extraction wells (Figure 2-4). Perchlorate reducing bacteria then use the

injected electron donors to reduce perchlorate after the electron donor has partitioned into

soil moisture. This technology is being demonstrated and validated by CDM and The

Pennsylvania State University through the Department of Defense Environmental

Security Technology Certification Program (ESTCP). Compared with the other vadose

zone perchlorate treatment technologies described above, this technology is less

disturbing and expected to be very cost-effective, and is best applied to sites that contain

perchlorate at depths greater than 5 feet.

This research, including soil microcosm tests and column transport and

biodegradation experiments, is a treatability study for the site demonstration of this

technology which will be implemented at an Aerojet site in California in the summer of

Injection Well

Groundwater

VadoseVadoseZoneZoneGED

GED

GED

GED

GED

GED

Electron Acceptor(perchlorate)

Electron Acceptor(perchlorate)

Gaseous Electron Donor InjectionGaseous Electron Donor Injection

GED = Gaseous Electron DonorGED = Gaseous Electron Donor

Injection Well

Groundwater

VadoseVadoseZoneZoneGEDGED

GEDGED

GEDGED

GEDGED

GEDGED

GEDGED

Electron Acceptor(perchlorate)

Electron Acceptor(perchlorate)

Gaseous Electron Donor InjectionGaseous Electron Donor Injection

GED = Gaseous Electron DonorGED = Gaseous Electron Donor

Figure 2-4: Sketch of Gaseous Electron Donor Injection Technology (GEDIT) (courtesy of CDM).

17

2007. The microcosm studies are used to rapidly access the ability of candidate electron

donors to effectively reduce perchlorate and to identify the appropriate electron donor to

be used at the site. Column studies are conducted to evaluate the transport of gaseous

electron donors through site soil and also to estimate the rate of perchlorate degradation.

REFERENCES

Bruce, R. A., Achenbach, L. A., and Coates, J. D., 1999. Reduction of (per)chlorate by a

novel organism isolated from paper mill waste. Environmental Microbiology

1(4):319-329.

Chaudhuri, S. K., O’Connor, S. M., Gustavson, R. L., Achenbach, L. A., and Coates, J.

D. 2002. Environmental Factors That Control Microbial Perchlorate Reduction. Applied

and Environmental Microbiology. 68(9):4425-4430.

Coates, J. D., Michaelidou, U., Bruce, R. A., O’Connor, S. M., Crespi, J. N., and

Achenbach, L. A. 1999. Ubiquity and Diversity of Dissimilatory (per)chlorate-Reducing

Bacteria. Applied and Environmental Microbiology Dec, 1999, 5234-5241.

Deitsch, J., Cox, E., Griffin, L., Gokmen, C., Borch, R., Monteleone, M., and McClure,

R. W. 2005. In-Situ Bioremediation of Perchlorate in Soil. GSP 142 Waste

Contaminment and Remediation. ASCE.

Frankel, A. J., Owsianiak, L. M., Wuerl, B. J., and Horst, J. F. In-Situ Anaerobic

Remediation of Perchlorate-Impacted Soils. ARCADIS.

18

Greer, M. A., Goodman, G., Pleus, R. C., and Greer, S. E., 2002. Health Effects

Assessment for Environmental Perchlorate Contamination: the Dose Response for

Inhibition of Thyroidal Radioiodine Uptake in Humans. Environ health Perspect.

110(9):927-937.

ITRC (Interstate Technology & Regulatory Council), 2005. Perchlorate: Overview of

Issues, Status, and Remedial Options. PERCHLORATE-1. Washington, D.C.: Interstate

Technology & Regulatory Council, Perchlorate Team.

Korenkov, V. N., Ivanovich, V., Kuznetsov, S. I., and Vorenov, J. V., 1976. Process for

Purification of Industrial Waste Waters From Perchlorates and Chlorates. U.S. Patent

No.3,943,055, March 9.

Logan, Bruce E., 1998. A Review of Chlorate- and Perchlorate-Respiring

Microorganisms. Bioremediation Journal 2(2):69-79.

Logan, B.E. 2001. Assessing the Outlook for Perchlorate Remediation. Environmental

Science & Technology 35(2001):482A-487A.

Logan, B. E., Zhang, H., Mulvaney, P., Milner, M. G., Head, I. M., and Unz, R. F., 2001

Kinetics of Perchlorate- and Chlorate-Respiring Bacteria. Applied and Environmental

Microbiology, June 2001, p.2499-2506.

Motzer, W. E., 2001. Perchlorate: Problems, Detection, and Solutions. Environmental

Forensics 2,301-311

Miller, J. P., and Logan, B. E., 2000. Sustained Perchlorate Degradation in an

Autotrophic, Gas-Phase, Packed-Bed Bioreactor. Environ. Sci. Technol.

2000,34,3018-3022.

19

Nzengung, V. A., Das, K. C., and Kastner, J. R. 2003. Pilot Scale In-Situ Bioremediation

Of Perchlorate-Contaminated Soils At The Longhorn Army Ammunition Plant.

Department of Geology. And Department of Biological and Agricultural Engineering.

The University of Georgia, Athens, GA 30602-4435.

Nerenberg, R., Kawagoshi, Y., Rittmann, B. E. Kinetics of a Hydrogen-Oxidizing,

Perchlorate-reducing Bacterium. Water Research 40(2006):3290-3296.

Rikken, G. B., Kroon, A. G., and van Ginkel, C. G., 1996. Transformation of

(per)chlorate into chloride by a newly isolated bacterium: Reduction and dismutation.

Appl. Microbial. Biotechnol. 45:420-426.

Smith, W., Morris, K. A., and Underwood, C. 2002 In Situ/Ex Situ Accelerated

Anaerobic Reduction of Perchlorate. Environmental Alliance Conference Presentations.

Available online at http://www.envalliance.com/Publications/Battelle_perchlolate_03.pdf

Urbansky, E.T. 1998. Perchlorate Chemistry: Implications for Analysis and

Remediation. Biorem. J. 2:81-95.

Urbansky E. T. and Schock, M. R. 1999. Issues in Managing the Risks Associated With

Perchlorate In Drinking Water. Journal of Environmental Management 56: 79-95.

U.S. Environmental Protection Agency. 1999. ORD Interim Guidance for Perchlorate.

U.S. Environmental Protection Agency. January 16, 2002. Perchlorate Environmental

Contamination: Toxicological Review and Risk Characterization. Office of Research and

Development, Washington, DC 20460.

20

U.S. Environmental Protection Agency. 2004. National Perchlorate Detections as of

September 23, 2004. Federal Facilities Restoration and Reuse Office. Available

at http://www.epa.gov/swerffrr/documents/perchlorate_map/nationalmap.htm.

U.S. Environmental Protection Agency, 2005a. Perchlorate and Perchlorate salts

Integrated Risk Information System. Available online at

http://www.epa.gov/iris/subst/1007.htm.

U.S. Environmental Protection Agency. 2005b. State Perchlorate Advisory Levels. Available at http://www.epa.gov/fedfac/pdf/stateadvisorylevels.pdf

Wallace, W. T., Ward, A. B., and Attaway, H., 1996. Identification of an anaerobic

bacterium which reduces perchlorate and chlorate as Wolinella succinogenes. J. Ind.

Microbiol. 16:68-72.

Wallace, W., Beshear, S., Williams, D., Hospadar, S., and Owens, M. 1998 Perchlorate

Reduction by a Mixed Culture in an Up-flow Anaerobic Fixed Bed Reactor. J. Ind.

Microbiol. Biotechnol. 20:126-131.

Waller, A. S., Cox, E. E., and Edwards, E. A., 2004. Perchlorate-reducing

Microorganisms isolated from contaminated sites. Environmental Microbiology

6(5):517-527.

Wu, J., Unz, R. F., Zhang, H., and Logan, B. E. 2001. Persistence of Perchlorate and the

Relative Numbers of Perchlorate- and Chlorate-Respiring Microorganisms in Natural

Waters, Soils, and Wastewater. Bioremediation Journal 5(2):119-130.

Yu, X., Amrhein, C., Deshusses, M. A., and Matsumoto, M. R., 2006. Perchlorate

Reduction by Autotrophic Bacteria in the Presence of Zero-Valent Iron. Enriron. Sci.

Technol. 2006, 40, 1328-1334.

21

Zhang, H., Bruns, M. A., and Logan, B. E. 2002. Perchlorate Reduction by a Novel

Chemolithoautotrophic, Hydrogen-oxidizing Bacterium. Environmental Microbiology

4(10), 570-576.

22

3 TRIAL MICROCOSM TESTS

3.1 Abstract

A series of trial microcosm tests were conducted to elucidate important factors

that could potentially affect the results of microcosm tests, including electron donor type

and concentration, soil pH and moisture, perchlorate contamination history and

contaminant concentration, indigenous microbial population, nitrate concentration, and

effect of cold storage of soil samples. Uncontaminated soils from three different locations

on The Pennsylvania State University campus in State College, Pennsylvania, and

perchlorate-contaminated soil from Aberdeen Proving Ground in Aberdeen, Maryland,

were used in a series of four trial tests. The setup, analysis, and results of these tests are

described in detail in this chapter.

3.2 Materials and Methods

3.2.1 Soil Samples

3.2.1.1 Aberdeen soil

The Aberdeen Proving Ground in Aberdeen, Maryland was a potential site for

GEDIT field demonstration. The Aberdeen soil was shipped from Aberdeen Proving

Ground, in Aberdeen, Maryland to The Pennsylvania State University (PSU) on Oct.15,

23

2005. The soil was collected via direct push drilling technology from three former drum

locations in the Field Training Exercise area at the site: Drum 3, Drum 6, and Drum 7.

Continuous soil cores were collected to 30-ft below ground surface (bgs) from each

location, and soil samples from 2, 5, 10, 20, and 30-ft bgs were bagged and sent to PSU.

The soil was tested by GPL Laboratories, LLLP (Frederick, MD) who found that the

Drum 3 area had no detectable perchlorate contamination and the soil from the other two

locations had perchlorate concentrations ranging from 0-5900 ppb, depending on the

depth. Nitrate concentrations were below detection. After arrival at PSU, the soil from the

three locations was well mixed in a big plastic container, covered, and stored at 4°C for

67 days until the experiments were performed. After mixing, the soil had a perchlorate

concentration of 566 ppb, and the soil moisture was 7.7±0.5% (average of three

replicates).

3.2.1.2 Grove Soil

The grove soil was collected from the grove behind the Beam Business

Administration building (near the water tower) on the PSU campus in State College

Pennsylvania. The site was covered by leaves, and the soil had a black color. The surface

soil (0-10cm) was collected into a plastic bag, sealed, and stored at room temperature for

two days until the experiments were performed. No perchlorate contamination was

detected in this soil and the soil moisture was 20.96±0.06% (duplicate average). The

initial nitrate concentration was not tested for this soil.

24

3.2.1.3 Fertilized Garden Soil

The fertilized garden soil sample was collected from a residence area about 3-4

miles north of the PSU campus in State College, Pennsylvania. It is a partially wooded

area surrounding apartment complexes. So unlike the thickly covered grove soil, the soil

from this site was exposed to the atmosphere due to frequent grounds maintenance. The

color of the soil from this site was brown. The surface soil (0-10cm) was collected into a

plastic bag, sealed, and stored at room temperature for 8 days until the experiments were

performed. No perchlorate contamination was detected in the soil, and this soil contained

7.54±0.04 ppm nitrate. The pH of this soil was 9.06 and the soil moisture was

10.96±0.03% (duplicate average).

3.2.1.4 Organic Farm Soil

Organic farm soil was collected from the Sustainability Center Organic Farm on

the PSU campus in State College, Pennsylvania. The surface soil (0-10cm) was collected

into a plastic bag and stored at room temperature for 30 days until the experiments were

performed. No perchlorate contamination was detected in the soil and the nitrate

concentration was found to be 60±3.1 ppm. The pH of this soil was 7.5±0.13 and the soil

moisture was 10.2±0.5%.

Table 3-1 Summary of the initial conditions of soils used in the trial microcosm tests.

25

3.2.2 Trial Tests Setup

3.2.2.1 Trial Test #1 Setup: Aberdeen Soil with High Perchlorate Concentration

The efficiency of ethanol, ethyl acetate, and 1-hexene as electron donors was

examined in this trial test with Aberdeen soil adjusted to 12% soil moisture and 500 ppm

perchlorate. A negative control containing no external electron donor was also set up.

For each of the electron donor candidates, duplicate 25-mL glass vials were

packed with 1-g of Aberdeen soil, purged with 10 psi lab grade nitrogen gas for 15

minutes to achieve anoxic conditions, and sealed with grey rubber stoppers and

aluminum crimp tops. A 250-mL gas-tight sample lock syringe (Hamilton) was used to

add 90-uL of degassed sodium perchlorate solution (0.062-g NaClO4 in 9-g DI water)

into each vial to adjust the perchlorate concentration to 500 ppm and the soil moisture to

12%. The electron donors, ethanol (2-uL), ethyl acetate (1.3-uL) or 1-hexene (1.4-uL)

were then injected onto the soil in the vials with a 5-uL syringe to achieve final

concentrations in the soil of 1575, 1130, and 959 mg/kg, respectively. The concentration

of each donor was ten times the stoichiometric amount required to reduce all of the

Table 3-1: Initial conditions of the different soils used in the trial microcosm tests. Soil Sources Perchlorate (ppb) Nitrate (ppm) pH Soil Moisture (%)

Aberdeen Soil 566 N/D 5.34 7.7±0.5

Grove Soil ND N/A 4.79 20.96±0.06

Fertilized Garden Soil ND 7.54±0.04 9.06 10.96±0.03

Organic Farm Soil ND 60±3.1 7.5±0.13 10.2±0.5 ND: None Detectable

N/A: The concentration was not tested.

26

perchlorate. The setup matrix of Trial Test #1 is shown in Table 3-2. After the setup, all

vials were incubated on a flat shaker and then moved to a rolling shaker after 17 days of

incubation to improve soil-headspace contact. During incubation, electron donor

concentrations were measured every 4-5 days, and at the end of the experiment (after one

month of incubation), all of the vials were sacrificed to test perchlorate concentrations in

the soil.

3.2.2.2 Trial Test #2 Setup: Freshly Collected Grove Soil vs. Cold-Stored Aberdeen Soil at Low Perchlorate Concentration and Low pH

To compare the perchlorate reduction ability of microorganisms from freshly

collected grove soil with the Aberdeen soil stored at 4°C for two months, and also to

retest the Aberdeen soil at a lower perchlorate concentration, Trial Test #2 was designed.

The grove soil moisture (21%) was higher than the designed soil moisture (18%),

so the soil was air dried for two days on lab bench to decrease the soil moisture to the

Table 3-2: Setup matrix of Trial Test #1. Perchlorate reduction was tested in Aberdeensoil augmented with either ethanol, ethyl acetate, or 1-hexene adjusted to 12% soil moisture and 500 ppm perchlorate.

Soil (g)

Vial volume (mL)

Soil moisture (%) Perchlorate (ppm) Electron donor

Original Adjusted Original Adjusted added (uL/vial)

Final conc. (mg/kg)

Negative Control 1 25 7 12 0.566 500 0 0

Ethanol 1 25 7 12 0.566 500 2 1575.1

Ethyl Acetate 1 25 7 12 0.566 500 1.3 1129.7

1-Hexene 1 25 7 12 0.566 500 1.4 959.1

27

desired level before the experiments. For each microcosm test bottle, 10-g of either grove

soil or Aberdeen soil was added to a 150-mL serum bottle which was then purged with

10-psi nitrogen gas for 15 minutes and sealed with a thick butyl rubber stopper and an

aluminum crimp top. The perchlorate concentration was adjusted to 500-ppb by adding

5-uL of sodium perchlorate solution (0.062-g NaClO4 in 50-mL DI water) to each grove

soil bottle and 1.1-uL to each Aberdeen soil bottle. To obtain the desired 18% soil

moisture in Aberdeen soil bottles, 1.58-mL degassed DI water was also injected.

After the adjustment of soil moisture and perchlorate concentration, the electron

donors ethanol (3.5-uL) or ethyl acetate (4.5-uL) were injected onto the soil in the bottles

to achieve final concentrations of 443 and 500 mg/kg, respectively. The final

concentrations of electron donors were ten times of the stoichiometric amount required to

completely reduce all the perchlorate added to the soil. No external electron donors were

added to the “Negative Control” bottles. Several replicates were setup for each test

condition (Table 3-3).

After setup, the bottles were shaken vigorously by hand to facilitate

headspace-soil contact, and then incubated in the dark at room temperature. The bottles

were shaken every 3 days to improve the contact of soil and headspace. For the grove soil

bottles, two duplicate ethanol and two duplicate ethyl acetate bottles were sacrificed

every week, and two duplicate negative control bottles were sacrificed every other week

to test perchlorate concentration. For the Aberdeen soil, it was anticipated that the

reaction would be slower due to extended cold storage of the soil prior to the experiment,

so two duplicate ethanol bottles and one negative control bottle were sacrificed every two

weeks. Before sacrificing, the concentrations of oxygen, carbon dioxide, and electron

28

donor in headspace were measured. After the second week, nitrate and pH were also

measured at the time of sacrifice.

3.2.2.3 Trial Test #3 Setup: Fertilized Garden Soil at Neutral pH

To eliminate the effect of pH to perchlorate bioremediation, fertilized garden soil

with a neutral pH was collected to conduct trial test #3. This trial test was performed in a

standard statistical factorial design with two variables: soil moisture content and electron

donor (Table 3-4). The soil moisture contents tested in this experiment were 10.8% which

was the natural moisture of the garden soil, and 15%. The electron donor candidates were

acetic acid and ethyl acetate, both with a concentration of 1000 mg/kg soil. The starting

perchlorate concentration was designed to be 10 ppm. A detailed description of the setup

is provided in Appendix C.1.

Table 3-3: Setup matrix of Trial Test #2. Perchlorate reduction was tested in freshlycollected grove soil (with either ethanol or ethyl acetate treated) and cold-stored Aberdeen soil (with ethanol treated). All of the soil samples were adjusted to 18% soil moisture and 500 ppb perchlorate.

Electron donor Mass of soil (g) Soil Source Perchlorate

conc. (ppb) Moisture Electron Donor (mg/kg) Replicates

Negative Control 10 Grove 500 18% 0 6 Ethanol 10 Grove 500 18% 443 10

Ethyl Acetate 10 Grove 500 18% 500 10 Ethanol 10 Aberdeen 500 18% 443 6

Negative control 10 Aberdeen 500 18% 0 3

29

All of the bottles were incubated in the dark at room temperature. One bottle of

every test was sacrificed every one or two weeks to test soil pH, moisture, perchlorate,

and nitrate concentration.

3.2.2.4 Trial Test #4 Setup: Organic Farm Soil with Slurry and Activated Sludge Inoculum

This trial test was performed in a standard statistical factorial design containing

three variables: soil moisture, electron donor type, and microbial inoculum (Table 3-5).

The electron donor candidates tested in this experiment were 1-hexene and ethyl lactate.

Soil moisture was adjusted from the original 10% to either “low moisture” (15%) or the

“slurry moisture” (50%). In “positive bacteria” groups, external microorganisms were

introduced by adding 5-mL activated sludge gathered from the Penn State Wastewater

Table 3-4: Setup matrix of Trial Test #3. Perchlorate reduction was tested in fertilizedgarden soil treated with either acetic acid or ethyl acetate under different soil moistureconditions.

Test No.

Mass per bottle (g)

Number of replicates

Moisture (%)

Perchlorate (mg/kg)

Electron Donor

E.D. Conc (mg/kg)

1 20 5 10.8% 10 Acetic acid 1000 2 20 5 15.0% 10 Acetic acid 1000 3 20 5 10.8% 10 Ethyl acetate 1000 4 20 5 15.0% 10 Ethyl acetate 1000

30

Treatment Plant. The starting perchlorate concentration was adjusted to 10 ppm. A

detailed description of the setup is provided in Appendix C.2.

One bottle of each test was sacrificed right after setup to test the electron donor,

pH, soil moisture, nitrate, and perchlorate concentrations as time zero samples. Other

Table 3-5: Setup matrix of Trial Test #4. Perchlorate reduction was tested in Organic Farm soil treated with either ethyl lactate or 1-hexene under different soil moisture conditions and microbial inoculum.

Test Description # of bottles

Final moisture (%)

Activated sludge

(inoculum) added per batch (uL)

Perchlorate conc. (mg/kg)

1-Hexene

1000 mg/kg

1 low moisture, negative bacteria 5 15% 0 10

2 low moisture, positive bacteria 5 15% 5000 10

3 slurry, negative bacteria 5 50% 0 10

4 slurry , positive bacteria 5 50% 5000 10

5 slurry , negative bacteria with autoclaved sludge 5 50% 5000 (autoclaved) 10

6 slurry, positive bacteria, autoclave whole bottle

5 50% 5000 10

Ethyl Lactate

1000 mg/kg

7 low moisture, negative bacteria 5 15% 0 10

8 low moisture, positive bacteria 5 15% 5000 10

9 slurry , negative bacteria 5 50% 0 10

10 slurry , positive bacteria 5 50% 5000 10

11 slurry , negative bacteria with autoclaved sludge 5 50% 5000 (autoclaved) 10

12 slurry, positive bacteria, autoclave whole bottle

5 50% 5000 10

31

bottles were incubated in the dark at room temperature, and every week, one bottle from

each test was sacrificed to analyze pH, nitrate, and perchlorate concentrations.

3.2.3 Chemical Analysis

An Agilent model 6890N gas chromatograph (GC) equipped with a DB-624

column and a flame ionization detector (FID) was used to test the electron donors.

Headspace samples (100 µL) were transferred from the microcosm bottles in a gas-tight

locking syringe to the injector which was held at a temperature of 150°C. Helium was

used as the carrier gas at a flow rate of 0.2 mL/min. The oven temperature was held at

45°C for 4 minutes, and then ramped to 60°C at a rate of 10°C /min, ramped to 100°C at

a rate of 20°C /min and then held at 100°C for 1 minute, giving a total run time of 8.5

minutes. The detector was held at 240°C where hydrogen, air, and nitrogen (as make up

gas) supplied the flame at a flow rates of 32, 400, and 30.7 mL/min, respectively.

Oxygen concentrations were quantified using a SRI 8610 B gas chromatograph

(GC) equipped with a thermal conductivity detector (TCD) and a Molesieve 5A

molecular sieve column (Alltech). Argon was used as the carrier gas with pressure set up

at 20 psi and the oven was held isothermally at 73°C.

Perchlorate and nitrate were extracted from 5-g soil by vortexing for 1 minute in

a 50-mL centrifuge vial containing 20-mL deionized water. A preliminary experiment

conducted in triplicate demonstrated that 106.6±6.1% of perchlorate was recovered from

the soil after only 0.5 minutes of vortexing. After vortexing, the extracts were centrifuged

at 5000 rpm for 15 minutes and the supernatant filtered through a 0.2-um-pore-diameter

32

filter to remove soil particles. The anion concentrations were measured using a DX-500

ion chromatograph (Dionex), equipped with an AS-11 column, and a ED40

Electrochemical Detector. A sodium hydroxide solution eluent with a flow rate of 1

mL/min was used to separate the species over a 30 minute run time. The eluent was

composed of 98.7% DI water and 1.3% 200mM sodium hydroxide at the beginning of

each run and held for 10 minutes, then ramped to 96.4% DI water and 3.6% 200 mM

sodium hydroxide and held until the time was 17.4 min, ramped to 65.5% DI water and

34.5% 200 mM sodium hydroxide and held from 18.8 min to 23 min, then ramped back

to 98.7% DI water and 1.3% 200 mM sodium hydroxide and held until the run ended.

The detection limit of nitrate was determined according to the procedure in USEPA

Definition and Method for MDL (USEPA, 1986) and was found to be 150 ppm.

Soil moisture content was determined gravimetrically according to D 2216-98

Standard Test Method for Laboratory Determination of Water (Moisture) Content of Soil

and Rock by Mass (ASTM, 1999), and the pH of the extracts after centrifuging was

measured with a Fisher Accumet AB 15 pH meter equipped with an Orion Thermo

Electron combination pH electrode.

3.3 Results

3.3.1 Trial Test #1 Results: Aberdeen Soil with High Perchlorate Concentration

Trial Test #1 was conducted for 31 days, from Dec.21, 2005 to Jan.21, 2006. No

perchlorate reduction was detected in any of the vials. At the end of the experiment, the

33

concentration of perchlorate was 528, 510, 530 and 510 ppm in the ethanol, 1-hexene,

ethyl acetate, and negative control bottles, respectively. An oxygen leak test was then

conducted and oxygen was detected in all vials with concentrations varying from 22

mg/L to 149 mg/L.

3.3.2 Trial Test #2 Results: Freshly Collected Grove Soil vs. Cold-Stored Aberdeen Soil at Low Perchlorate Concentration and Low pH

Trial Test #2 was run for 22 days, from Feb.12 to Mar. 05, 2006. The starting

point of perchlorate concentration was from 232 to 300ppb, which is lower than the

designed level (500-ppb) (Table 3-6). After 22 days of incubation, the perchlorate

concentration in garden soil microcosms containing ethyl acetate and ethanol was slightly

decreased, but the change was inconclusive due to the large deviations between samples.

No perchlorate reduction was observed after 22 days. The electron donor concentrations

decreased in garden soil bottles but not in Aberdeen soil bottles. No oxygen leaking was

detected in the headspace. The average concentration of nitrate decreased from the

second week to the third week, but again, the large deviations make it difficult to confirm

nitrate reduction.

Table 3-6: Initial and final concentrations of perchlorate, nitrate and electron donors inTrial Test #2, in which ethyl acetate and ethanol were tested in grove soil and Aberdeensoil under 18% soil moisture and 500-ppb perchlorate.

34

To help determine the reason for poor perchlorate degradation, the pH of pure DI

water and both soil samples were tested. The pH of DI water was 5.67; and adding 4.5-uL

pure ethyl acetate and 3.5-ul pure ethanol into 30-mL DI water only changed the pH to

5.77 and 5.68, respectively. The pH was 5.34 for the Aberdeen soil and 4.79 for grove

soil before the experiments.

3.3.3 Trial Test #3 Results: Fertilized Garden Soil at Neutral pH

This trial test was conducted for 42 days, from April 04 to May 16, 2006. The soil

pH stayed within the range of 8-8.5 throughout the test. Nitrate, with an initial

concentration of 7.5 ppm, was partially reduced in all but the low moisture ethyl acetate

bottles at time zero, and complete nitrate degradation was achieved in all bottles within 6

days (Figure 3-1).

Perchlorate (ppb) Nitrate (ppb) Electron donor (mg/kg)

Day 0 Day 22 Day 14 Day 22 Day 0 Day 22

Grove Soil

Negative Control 232±7 345±85 - 267±253 0 0 Ethyl

Acetate 255±37 210±26 126±48 101±26 5.6±0.4 0.5±0.04 Ethanol 259±42 154±3 203±146 30±41 14.2±0.16 1.25±0.1

Aberdeen Soil

Negative Control 263 443 76.6 - 0 0 Ethanol 300±11 345±22 271±16 - 14.4±0.08 16.4±0.8

35

Complete perchlorate degradation only occurred with acetic acid at 15% soil

moisture (Figure 3-2). The perchlorate concentration was reduced to below detection

within 28 days with acetic acid under the 15% moisture condition, but showed little

change even after 42 days of treatment in the other bottles. The initial and final

concentrations of nitrate and perchlorate at all conditions are summarized in Table 3-7.

0

2

4

6

8

0 10 20 30 40Time (days)

Soil

nitra

te (

ppm

) Acetic acid, 10.8% moisture

Acetic acid, 15% moisture

Ethyl aceate, 10.8%

Ethyl acetate, 15%

Figure 3-1: Nitrate concentration change over time during perchlorate bioremediation inTrial Test #3 with fertilized garden soil.

0

2

4

6

8

10

12

0 5 10 15 20 25 30 35 40 45Time (days)

Soil

perc

hlor

ate

(ppm

)

Acetic acid, 10.8% moisture

Acetic acid, 15.0% moisture

Ethyl acetate, 10.8% moisture

Ethyl acetate, 15.0% moisture

36

3.3.4 Trial Test #4 Results: Organic Farm Soil with Slurry and Activated Sludge Inoculum

This test was performed for 13 days, from July 13-26, 2006. At time zero, within

the same day the experiment was setup, perchlorate reduction could be detected in the

ethyl lactate-15%-native microorganism bottles, ethyl lactate-15%-activated sludge and

ethyl lactate-slurry-activated sludge bottles (Figure 3-3). Within a week, complete

perchlorate degradation was achieved in the bottles mentioned above, and also in all of

the 1-hexene-slurry bottles, except the “autoclaved whole bottle” control. Almost all of

the bottles achieved complete nitrate reduction, including the “autoclaved whole bottle”

controls.

Figure 3-2: Perchlorate concentration change over time during bioremediation in Trial Test #3.

Table 3-7: Initial and final concentrations of nitrate and perchlorate in Trial Test #3.

Electron Donor

Soil Moisture

Nitrate (ppm) Perchlorate (ppm) T=0 T=42 days T=0 T=42 days

Acetic acid 10.8% 6.75 0 11.16 8.2 15% 4.75 0 9.2 0

Ethyl acetate 10.8% 7.95 0 11.31 8.3 15% 5.9 0 7.96 6.8

37

3.4 Discussion

3.4.1 Trial Test #1 Discussion: Aberdeen Soil at High Perchlorate Concentration

There are many reasons that may have contributed to the lack of perchlorate

reduction in Trial Test #1. First of all, oxygen leaking into vials likely inhibited

perchlorate biodegradation. The grey stoppers used in this experiment were not gas tight

after a few punctures (the stoppers were punctured during setup and during the

measurement of electron donor every 4-5 days). Oxygen is a competitor for electron

donor with perchlorate, and only under anoxic conditions will perchlorate be used as

electron acceptor by perchlorate reducing bacteria. It is recommended that thick butyl

0

2

4

6

8

10

12

0 5 10Time (days)

ppm

1-hexene, 15%, native bacteria

1-hexene, 15%, w/ activated sludge

1-hexene, slurry, native bacteria

1-hexene, slurry, w/ activated sludge

1-hexene, slurry, w/ killed sludge

1-hexene, slurry, killed whole bottle

ethyl lactate, 15%, native bacteria

ethyl lactate, 15%, w/ activated sludge

ethyl lactate, slurry, native bacteria

ethyl lactate, slurry, w/ activate sludge

ethyl lactate, slurry, w/ killed sludge

ethyl lactate, slurry, killed whole bottle

Figure 3-3: Perchlorate concentration change over time during bioremediation in Trial Test #4.

38

rubber stoppers be used in subsequent microcosm tests. Second, compared with the

original 566-ppb perchlorate contamination in the Aberdeen soil, the adjusted 500-ppm

was one hundred times higher, potentially shocking the perchlorate reducing

microorganisms and forcing a lag period longer than a month. In the microcosm study

conducted by Wu at el. (2001), there was a lag period of about 20-50 days for perchlorate

degradation in the soil amended perchlorate concentration from 0 to 500-ppm. Another

study (Tan et al., 2004) reported lag times of perchlorate degradation ranging from 0 to

60 days depending on the soil source, initial perchlorate concentration, and nitrate

concentration. Also, the soil sample was stored at 4 °C for more than 2 months, which

may have caused the microorganisms to be less active and need longer time to regain

activity.

3.4.2 Trial Test #2 Discussion: Freshly Collected Grove Soil vs. Cold-Stored Aberdeen Soil at Low Perchlorate Concentration and Low pH

The low starting perchlorate concentration measured in this test may have been

due to the uneven distribution of perchlorate solution during injection onto the soil. This

unevenly distribution may have prevented the access of perchlorate for some of the

perchlorate reducing microorganisms and resulted in overall poor perchlorate

degradation. The fluctuation observed in the analytical measurements may also be due to

initial uneven distribution. More shaking could facilitate greater contact between the soil

and perchlorate and result in final soil concentrations closer to the designed

concentration. Alternatively, perchlorate could be mixed into the soil before aliquoting it

to the test bottles. From the data in Table 3-6 (section 3.3.2), the only bottle in which

39

perchlorate reduction was obvious was in the grove soil treated with ethanol as the

electron donor. Both nitrate and perchlorate concentrations decreased in this experiment.

Nitrate is a competitor with perchlorate for electron donor. Many researchers have

observed a preferential reduction of nitrate over perchlorate in soil, especially in soils

without prior exposure to perchlorate (Nozawa-Inoue et al., 2005; Tipton et al., 2003). So

it is expected that nitrate reduction occurred before perchlorate reduction in this

experiment.

Another factor that may have inhibited perchlorate reduction was the pH. The pH

of the DI water used in the perchlorate extraction was low (5.67) and so was the pH of

soil samples (5.34 for the Aberdeen soil and 4.79 for grove soil). The range of pH for

effective perchlorate degradation is around neutral pH (Coates and Achenbach, 2006). It

is recommended that the soil pH be checked and neutralized before conducting future

microcosm experiments, if necessary.

The starting concentrations of electron donors were also lower than expected.

Because the pure electron donors in liquid state were dropped directly onto the soil

particles, adsorption may have decreased the evaporation of the donors. In future tests it

is recommended that liquid electron donors be dropped onto the glass wall and allowed to

completely volatize before shaking the bottle. It appears that ethyl acetate hydrolyzed to

ethanol after 3 weeks of incubation; therefore, the decreasing ethyl acetate concentrations

observed in this experiment do not necessarily represent consumption of the electron

donor. It is surprising, however, that ethyl acetate did not stimulate perchlorate reduction,

considering that ethanol is frequently used as an electron donor for perchlorate reduction.

40

Because so many confounding factors were involved, it is hard to evaluate the

activity of perchlorate reducing microorganisms from the two soil samples in this

experiment.

3.4.3 Trial Test #3 Discussion: Fertilized Garden Soil at Neutral pH

Nitrate reduction occurred very quickly in this test. At time zero, when the bottles

had only been exposed to the electron donor for a few hours, 1/3 of nitrate was reduced

under the acetic acid-15% soil moisture condition. Higher soil moisture resulted in a

lower nitrate concentration at time zero, and acetic acid was favored as an electron donor

over ethyl acetate by nitrate reducing microorganisms.

By adjusting the perchlorate concentration before placing the soil into the bottles,

the starting points of perchlorate concentration were more accurate and reasonable than

those in Trial Test #2. No perchlorate reduction was observed before nitrate reduction

was finished, as would be expected (Figure 3-1 and 3-2). Acetic acid was also more

favored by perchlorate reducing microorganisms, but it would not be a good electron

donor candidate for GEDIT due to its low Henry’s constant (2.94E-07 atm-m3/mol).

3.4.4 Trial Test #4 Discussion: Organic Farm Soil with Slurry and Activated Sludge Inoculum

This experiment was designed to compare the influence of soil moisture, electron

donor, and inoculum on perchlorate bioremediation. The soil sample used in this test was

41

collected from an organic farm, which was free of pesticides and commercial fertilizers

according to the Center for Sustainability at PSU.

Abiotic perchlorate reduction appears to have occurred in this experiment, as

shown by the decrease in perchlorate concentration in the “autoclaved whole bottle”

controls (Figure 3-3). This Abiotic reaction was weak and slow, and may have resulted

from the existence of some reduced matter in either the organic soil itself or in the added

activated sludge.

Given the same soil moisture content and the same electron donor, an external

inoculum of microorganisms did help with perchlorate reduction, but the difference was

not obvious except in the ethyl lactate-slurry test.

Given the same microbial source and the same electron donor, the difference in

soil moisture caused a large difference. The slurry bottles (50% moisture) had much

faster perchlorate reduction than did the 15% moisture bottles. Complete perchlorate

biodegradation was achieved within 7 days in slurry bottles with one exception – the

ethyl lactate bottle with native microorganisms. Compared with the 15% moisture

condition, the slurry has the advantage of better access to electron donor/acceptor and

nutrients for microorganisms. Since this research targets the vadose zone, the slurry tests

conducted here were only used to confirm the inhibition of perchlorate reduction by low

soil moisture which had been observed in previous experiments.

42

3.5 Conclusions

These trial tests were conducted to explore a better way to perform the microcosm

tests with a future field sample from the Aerojet site in California. The results from these

tests indicate the advantages of using thick butyl rubber stoppers to seal the bottles,

flushing the bottles with nitrogen after they are sealed, dropping the liquid electron donor

onto the wall of the bottles to let it fully evaporate into headspace, and adjusting the soil

moisture and perchlorate concentration before placing the soil samples into the bottles.

Perchlorate was reduced by native microbial communities in Trial Tests #3 and

#4, in soils not known to be previously exposed to anthropogenic perchlorate by adding

external gaseous electron donor. This confirms the potential of treating vadose zone soil

by injecting gaseous electron donor.

Oxygen concentration, pH, and soil moisture were found to be the main factors

affecting the rate of perchlorate reduction in all four of the trial tests. Oxygen

concentrations should be monitored throughout the entire experiment to ensure anoxic

conditions. The soil pH should be tested before setting up experiments and adjusted to

neutral if necessary. Higher soil moisture appears to dramatically affect perchlorate

reduction.

ACKNOWLEDGEMENTS

The project described was performed in collaboration with Camp Dresser and

McKee, Inc. (CDM), with funding provided by Department of Defense Environmental

Security Technology Certification Program (DoD ESTCP) project number ER-0511.

43

REFERENCES

Coates, J.D. and Achenbach, L.A. (2006). The microbiology of perchlorate reduction and

its bioremediative application. In Gu, B. and Coates, J.D. (Eds) Perchlorate,

Environmental Occurrence, Interactions, and Treatment. Springer Publishers, MA

Nozawa-Inoue, M., Scow, K. M., and Rolston, D. E., 2005. Reduction of Perchlorate and

Nitrate by Microbial Communities in Vadose Soil. Applied and Environmental

Microbiology, 71(7):3928-3934.

Tan, K., Anderson, T. A., and Andrew Jackson, W., 2004. Degradation Kinetics of

Perchlorate in Sediments and Soils. Water, Air, and Soil Pollution 151:245-259.

Tipton, D. K., Rolston, D. E., and Scow, K. M., 2003. Bioremediation and

Biodegradation: Transport and Biodegradation of Perchlorate in Soils. J. Environ.

Qual.32:40-46.

Wu, J., Unz, R. F., Zhang, H., and Logan, B. E., 2001. Persistence of Perchlorate and the

Relative Numbers of Perchlorate- and Chlorate-Respiring Microorganisms in Natural

Waters, Soils, and Wastewater. Bioremediation Journal 5(2):119-130.

44

4 MICROCOSM TESTS

4.1 Abstract

Sacrificial batch microcosm tests were used to rapidly assess the ability of

gaseous electron donors and various moisture contents to achieve optimal perchlorate

remediation in vadose zone soil taken from the Aerojet Propellant Burn Area site in

California. The electron donor candidates tested were hydrogen, 1-hexene, ethyl acetate,

and liquefied petroleum gas (LPG). Each electron donor was tested at two different

concentrations under two different soil moisture contents that were representative of

minimum and maximum moisture contents at the site. No perchlorate reduction occurred

in low moisture (7%) bottles after an incubation time of 125-187 days, and all bottles

except ethyl acetate achieved complete or partial perchlorate reduction in high moisture

(16%) bottles. Results from these microcosm tests indicate that hydrogen is the most

promising of the tested electron donors for the treatment of perchlorate in vadose zone

soil, achieving complete perchlorate degradation within 35-42 days, with a perchlorate

reduction rate of 0.133-0.189 d-1. LPG promoted complete perchlorate reduction at the

high LPG dose and 1-hexene promoted partial perchlorate reduction at both doses;

however, when compared to hydrogen, these donors had more significant lag periods of

21 - 49 days and lower perchlorate reduction rates of 0.008-0.033 d-1 and 0.008-0.016 d-1,

respectively.

45

4.2 Materials and Methods

4.2.1 Soil Characterization

The soil used in this test was collected using sonic drilling methods from a

perchlorate-contaminated Aerojet site in northern California and shipped to The

Pennsylvania State University in six 5-gallon buckets in August 2006. The day after

arrival, the soil was processed as follows. After removing large stones by hand and

passing the soil through a ½ inch sieve, all of the soil was well mixed together in a large

container and then transferred to four buckets, sealed, and stored at room temperature.

The following day, duplicate grab samples were taken from each bucket and tested for

perchlorate, nitrate, pH, and soil moisture. The resulting standard deviation of perchlorate

concentrations between the buckets was approximately 4-ppm (41% of the average

concentration), so the soil was remixed and redistributed to four buckets again and

retested for perchlorate, nitrate, pH, and soil moisture, as well as for total nitrogen and

total carbon. The remaining soil was stored at room temperature in the sealed buckets for

10 days until the experiments were performed.

4.2.2 Experimental Design and Setup

The microcosm tests were performed in a standard statistical factorial design

(Table 3-2). Soil moisture content, electron donor type, and electron donor concentration

were the variables evaluated in the test. According to the lowest and highest moisture

level naturally present at the field site, the moisture contents tested were 7 and 16%. The

46

electron donors tested were hydrogen, ethyl acetate, 1-hexene, and commercial liquid

petroleum gas (LPG), the main component of which is propane. These electron donors

were selected because of their high vapor pressures and high Henry’s constants

(Table 4-2), making them well-suited to transport in the vadose zone. Low and high

electron donor concentrations were designed to be three and ten times the quantity

required to stoichiometrically reduce all of the oxygen, nitrate, and perchlorate present in

the soil. The concentrations listed in Table 3-2 reflect these stoichiometric calculations

based on the actual nitrate and perchlorate concentrations, and conservatively assume that

the entire headspace is air. A negative control containing no electron donor and a positive

control containing ethanol, which was previously shown by CDM to give positive

perchlorate degradation results, were also tested.

Table 4-1: Matrix of experimental conditions tested in the microcosm experiments.

Test Number Electron Donor Electron donor concentration

(mg/kg soil) Soil moisture

1 H2 (+CO2) 34 (+374) 7% 2 Ethyl acetate 150 7% 3 1-Hexene 80 7% 4 LPG 75 7% 5 H2 (+CO2) 114 (+1254) 7% 6 Ethyl acetate 501 7% 7 1-Hexene 165 7% 8 LPG 250 7% 9 H2 (+CO2) 34 (+374) 16% 10 Ethyl acetate 150 16% 11 1-Hexene 80 16% 12 LPG 75 16% 13 H2 (+CO2) 114 (+1254) 16% 14 Ethyl acetate 501 16% 15 1-Hexene 165 16% 16 LPG 250 16% 17 Negative Control 0 16% 18 Positive Control 436 (Ethanol) 16%

47

For each test condition shown in Table 3-2, nine replicate bottles were

established to enable periodic sacrificial analysis of the soil, and half of the active tests

(tests 1, 2, 6, 7, 11, 12, 13, and 16) were randomly selected to be run in duplicate. To

setup the 234 microcosms, soil from the field site was transferred in 10-gram (g) aliquots

to 150-mL glass serum bottles. After the bottles were sealed with thick butyl rubber

stoppers and aluminum crimp tops, the gas in the bottles was purged with 10-psi

ultra-high purity nitrogen gas for at least 15 minutes to remove oxygen and maintain

anoxic conditions. Ten percent (10%) of the bottles were randomly chosen for headspace

oxygen analysis. Greater than 1% oxygen was detected in one of the bottles in the Test 5

set, so all nine bottles in Test 5 were re-purged with nitrogen, retested for oxygen, and

passed. After degassing all of the bottles, one of the candidate electron donors and

de-ionized water were injected into the bottles to achieve the desired test conditions.

During the injection, liquid electron donors (ethyl acetate and 1-hexene) were dropped

onto the wall of the bottles and allowed to completely vaporize into the gaseous phase

Table 4-2: Properties of tested electron donors in microcosm tests

Electron donor Candidates Molecular formula Formula weight

(g/mol) H

(atm-m3/mol) Psat

(mm Hg) Hydrogen H2 2 1.28E+00 760

Ethyl Acetate CH3COOC4H9 88.11 1.34E-04 60

1-Hexene CH3(CH2)3CHCH2 84.16 4.17E-01 100 LPG

(Liquefied Petroleum Gas, 90% propane)

CH3CH2CH3 44.1 6.00E-01 5700

Ethanol C2H5OH 46.07 5.00E-06 40

48

rather than injecting the electron donor liquid directly onto the soil. Carbon dioxide at

748 and 2508 mg/kg was added as a carbon source to microcosms containing hydrogen.

The amount of carbon dioxide injected was also three to ten times of the quantity

required to achieve total perchlorate degradation in order to ensure that lack of carbon

would not be a limiting factor for bioremediation. Prior to injecting the carbon dioxide or

gaseous electron donors (hydrogen and LPG), an equivalent volume of nitrogen gas was

withdrawn from the bottles to avoid increasing pressure. The total setup time for all 234

bottles was 48 days (delayed due to a GC problem, and some bottles were set up earlier

than others) during which time the bottles were stored at room temperature on the open

bench. After shaking to facilitate homogeneous headspace-soil contact, the first bottle of

each test condition was sacrificed immediately after the setup as the time zero

measurement. Other bottles were incubated in the dark at room temperature for a total of

two to three months and were shaken about 3 times per week to help gaseous electron

donor distribution and increase headspace-soil contact. Appendix D.1 summarizes the

setup details.

During the incubation, one of the replicates of each test condition was analyzed

every one to four weeks, the frequency depending on the observed rate of perchlorate

degradation. During the analysis process, the headspace electron donor concentration, O2,

and CO2 were tested first, and then the bottles were sacrificed (i.e., opened) to test the

soil for perchlorate, nitrate, nitrite, chlorate, chlorite, and chloride concentration, moisture

content, and pH. Between every two sampling points, the concentration of electron donor

in the headspace was tested weekly.

49

4.2.3 Chemical Analyses

An Agilent model 6890N gas chromatograph (GC) equipped with a DB-624

column and a flame ionization detector (FID) was used to test the electron donors (ethyl

acetate, 1-hexene, propane, and ethanol). Headspace samples (1000 µL) were transferred

from the microcosm bottles in a gas-tight locking syringe to the injector which was held

at a temperature of 150°C. Helium was used as the carrier gas at a flow rate of 0.2

mL/min. The oven temperature was held at 45°C for 4 minutes, and then ramped to 60°C

at a rate of 10°C /min, ramped to 100°C at a rate of 20°C /min and then held at 100°C for

1 minute, giving a total run time of 8.5 minutes. The detector was held at 240°C where

hydrogen, air, and nitrogen (as make up gas) supplied the flame at a flow rates of 32, 400,

and 30.7 mL/min, respectively.

Hydrogen and oxygen concentrations were quantified using a SRI 8610 B gas

chromatograph (GC) equipped with a thermal conductivity detector (TCD) and a

Molesieve 5A molecular sieve column (Alltech). Argon was used as the carrier gas with

pressure set up at 20 psi and the oven was held isothermally at 73°C. Carbon dioxide

concentration in headspace of samples was measured using a SRI GC (Model 310)

equipped with a TCD and a Porapak Q column. Helium was used as the carrier gas with

pressure set up at 20 psi and the oven was held isothermally at 83°C.

Perchlorate, chlorate, chlorite, chloride, nitrate, and nitrite were extracted from

5-g soil by vortexing for 1 minute in a 50-mL centrifuge vial containing 20-mL deionized

water. A preliminary experiment conducted in triplicate demonstrated that 106.6±6.1% of

perchlorate was recovered from the soil after only 0.5 minutes of vortexing. After

50

vortexing, the extracts were centrifuged at 5000 rpm for 15 minutes and the supernatant

filtered through a 0.2-um-pore-diameter filter to remove soil particles. The anion

concentrations were measured using a DX-500 ion chromatograph (Dionex), equipped

with an AS-11 column, and a ED40 Electrochemical Detector. A sodium hydroxide

solution eluent with a flow rate of 1 mL/min was used to separate the species over a 30

minute run time. The eluent was composed of 98.7% DI water and 1.3% 200mM sodium

hydroxide at the beginning of each run and held for 10 minutes, then ramped to 96.4% DI

water and 3.6% 200 mM sodium hydroxide and held until the time was 17.4 min, ramped

to 65.5% DI water and 34.5% 200 mM sodium hydroxide and held from 18.8 min to 23

min, then ramped back to 98.7% DI water and 1.3% 200 mM sodium hydroxide and held

until the run ended. The detection limit of nitrate was determined according to the

procedure in USEPA Definition and Method for MDL (USEPA, 1986) and was found to

be 150 ppm.

Soil moisture content was determined gravimetrically according to D 2216-98

Standard Test Method for Laboratory Determination of Water (Moisture) Content of Soil

and Rock by Mass (ASTM, 1999), and the pH of the extracts after centrifuging was

measured with a Fisher Accumet AB 15 pH meter equipped with an Orion Thermo

Electron combination pH electrode.

4.3 Results

Before the microcosm tests were initiated, the soil from the Aerojet site was

chemically characterized. The results of the soil characterization are provided in the

51

column marked “original” in Table 3-3. At the Aerojet site, the surface soil had the

highest perchlorate concentration at 59 ppm and the concentration decreased with

increasing depth. At a depth of 70 ft, the perchlorate concentration was 4.1 ppm. The

percentage of total nitrogen of the soil sample was 0.016% ± 0.006% and total carbon

was 0.037% ± 0.021%, tested in triplicate by the Agricultural Analytical Services

Laboratory at The Pennsylvania State University.

During the microcosm tests, the soil moisture content remained relatively constant

in both the low and high soil moisture sets. The soil pH remained near 7. No intermediate

perchlorate reduction products (chlorate and chlorite) were detected during the treatment.

The concentration of chloride increased proportionally with perchlorate reduction. Nitrate

concentration was reduced below the detection limit (150 ppb) at the time zero sampling

point (data not shown). The average final conditions of the soil after 125-187 days of

treatment with the different electron donors under high soil moisture content are

summarized in Table 3-3 . All of the profiles for each test condition are provided in

Appendix D.2.

Table 4-3: Original and final conditions of the Aerojet site soil after 125-187 days of treatment using different electron donors at 16% soil moisture. (Table shows duplicate averages except where noted.)

Original Ethyl Acetate 1-hexene LPG Hydrogen mg/kg - 150 501 80 265 75 250 34 114

Soil moisture % 8±0.6* 15 15.4 14.72 15.6 12.9 13.85 15.21 15.31 Soil pH - 6.85±0.3* 6.82 6.58 6.97 6.97 7.84 7.56 7.15 6.38

perchlorate ppm 8.2±1.3* 8.53 9.52 5 1.96 2.71 ND ND ND chloride ppm - 2.93 3.36 6.21 4.26 6.85 6.03 7.27 8.12 nitrate ppm 2.1±0.3* ND ND ND ND ND ND ND ND

electron donor mg/kg - ND ND 68.07 121 142.5 491.35 56.981 83.33

* = average of soil from 4 buckets after the second time of mixing with two duplicate measurements each.

ND = non detect

52

Perchlorate reduction was not observed in any of the 7% soil moisture sets

(Appendix D.2), regardless of which electron donor was present. Under high soil

moisture (16%), the bioremediation of perchlorate was supported by all of the electron

donors tested except ethyl acetate (Figure 3-1). Complete perchlorate removal was

achieved in 35 and 42 days with hydrogen at high and low concentration, respectively.

After 184 days of incubation, perchlorate concentration was reduced to zero in high LPG

concentration bottles, but had a residual of 2.71 ppm in low LPG concentration bottles.

The concentration of perchlorate was reduced to 1.96 ppm and 5 ppm in high and low

1-hexene concentration bottles, respectively. The 1-hexene bottles were only incubated

for 125 days in total due to the higher frequency of sacrificing at the beginning of the test.

Complete perchlorate reduction occurred within 77 days in the positive control and 183

days in the negative control.

53

Although the rates of perchlorate degradation are difficult to accurately quantify

in this experiment due to the observed shouldering (lag) effect and relatively low

sampling frequency, it does appear that perchlorate reduction followed a first order decay

(Figure 4-2). First order perchlorate reduction has been observed by others (Logan et al.,

2001), so this result is not unexpected. First order rate constants for perchlorate reduction

were estimated based on the slopes of the curves of each profile past the shoulder in

Figure 4-2 (i.e., the slopes of negative ln([ClO4-]/[ClO4

-]0) vs. time), with the exception of

hydrogen and the positive control, which were determined based on the initial straight

portion of the curve past the shoulder. The resulting estimated first-order rates of

perchlorate degradation, kClO4- (average), for each electron donor are provided in

0

2

4

6

8

10

12

0 20 40 60 80 100 120 140 160 180 200Time (days)

Perc

hlor

ate

conc

entra

tion

(ppm

34mg/kg H2 114mg/kg H2150mg/kg Ethyl Acetate 501mg/kg Ethyl Acetate80mg/kg 1-Hexene 265mg/kg 1-Hexene75mg/kg LPG 250mg/kg LPGNegative Control, no donor. Positive control. 436mg/kg Ethanol.

Figure 4-1: Perchlorate degradation in microcosm tests with different electron donors at16% soil moisture.

54

Table 4-4. The highest localized rates of perchlorate reduction, kClO4- (maximum), were

found for the positive control (ethanol), followed by hydrogen, LPG (high concentration),

negative control, 1-hexene (high concentration), LPG (low concentration), 1-hexene (low

concentration), and ethyl acetate.

-6

-5-4

-3-2

-10

1

0 25 50 75 100 125 150 175 200Time (days)

ln(C

/C0)

34mg/kg H2 114mg/kg H2150mg/kg Ethyl Acetate 501mg/kg Ethyl Acetate80mg/kg 1-Hexene 265mg/kg 1-HexeneNegative Control, no donor. Positive control. 436mg/kg Ethanol.75mg/kg LPG 250mg/kg LPG

Figure 4-2: Relative change in perchlorate concentration over time used to estimate firstorder rate constants.

Table 4-4: First order perchlorate degradation rate constants, lag periods, and final

55

4.4 Discussion

From the results of the microcosm test, it is obvious that high soil moisture is

critical to perchlorate bioremediation. Another study which tested the GEDIT technology

also concluded that soil moisture is the key factor (Evans and Trute, 2006). A similar

conclusion was obtained in a pilot study of in situ perchlorate bioremediation at The

Longhorn Army Ammunition Plant (LAAP) (Nzengung et al., 2003), which that the best

perchlorate concentrations for the electron donors tested in the microcosm tests at 16%soil moisture.

kClO4- (average)#

(day-1)

kClO4- (maximum)&

(day-1)

Lag period (days)

CCIO4- final

(ppm)

H2 Low 0.1327 0.1866 7 0.04*

High 0.1894 0.2 7 0.04*

LPG Low 0.0083 0.0366 49 2.71

High 0.0326 0.0552 21 0.04*

1-hexene Low 0.0079 0.0448 28 2.54

High 0.0161 0.1708 28 1.96

Ethyl acetate Low 0 0 >125 8.53

High 0 0 >184 9.52

Negative Control 0.0027 0.1022 21 2.12

Positive Control 0.1973 0.1973 49 0.04*

# Rates were estimated based on the slopes of the whole curves past lag periods except for H2 and Positive control. The rates of H2 were determined from the data of 7-42 days for low concentration and 7-35 days for high concentration. The rate for the positive control was determined by the data of 49-77 days.

& The maximum perchlorate reduction rate observed for each donor during incubation.

The calculation was based on the data from:

H2 (low): 7-21 days; H2 (high): 7-21 days;

LPG (low): 49-77 days; LPG (high):124-184 days;

1-hexene (low): 49-77 days; 1-hexene (high): 42-49 days;

56

treatment results were achieved in the wettest (saturated) soils. For GEDIT, however, the

soil moisture content can not be increased to saturation conditions because the transport

of gaseous electron donors is restricted in high moisture soil. In this microcosm test, 7%

was too low to support perchlorate bioremediation and 16% was successful at reducing

perchlorate in most bottles but it may not be the optimum moisture. In future research,

additional soil moisture contents should be tested to determine the lowest soil moisture

content that can support complete perchlorate reduction and also to determine the

optimum moisture for both perchlorate bioremediation and electron donor transport.

Of the electron donors tested, hydrogen appears to be the most promising for

several reasons. Compared to other electron donors, hydrogen has the highest Henry’s

constant which gives it a better mobility in the gaseous phase (Table 4-2). Although not

extremely different, the Gibbs free energy of hydrogen reacting with perchlorate is lower

than that of the other tested electron donors (Table 4-6), theoretically making its

utilization more favorable by microorganisms. In addition, the simple small hydrogen

molecule is readily utilized by microorganisms. Hydrogen has been widely used as an

electron donor for isolating perchlorate reducing bacteria (Table 1-2) and is also used in

the treatment of (per)chlorate contaminated water (Miller & Logan, 2000; Nerenberg et

al., 2002; Kroon & van Ginkel, 2004).

In this experiment there was a 7 day lag period in both high and low hydrogen

bottles before perchlorate degradation began. This lag period is similar to the 14 day lag

period observed in another study of perchlorate bioremediation in vadose zone soil with

hydrogen as the electron donor (Nozawa-Inoue et al., 2005). Shorter lag periods for

perchlorate degradation with hydrogen/carbon dioxide than other electron donors may

57

also imply that there are more autotrophic than heterotrophic perchlorate-reducing

microbial populations in the soil. The observed perchlorate reduction rate kClO4- was

almost the same in the low and high hydrogen concentration bottles. There is not

sufficient data, however, to imply a relationship between hydrogen concentration and

perchlorate reduction rate due to lack of sampling points between day 7 and day 21. To

study how higher hydrogen concentrations affect perchlorate reduction rate, higher

sacrificing frequency between 7 and 21 days of incubation is needed. Overall perchlorate

removal rate is a first order reaction (Logan et al., 2001). The highest observed

perchlorate reduction rate kClO4- (maximum) obtained from this research is similar to or

smaller than those obtained in other research conducted with slurry sediments/soils

(Table 4-5). Based on the importance of soil moisture for stimulating perchlorate

degradation, it is not unexpected that slurry microcosms would produce higher rates.

Table 4-5: First order perchlorate reduction rates observed in the literature and theirexperimental conditions.

Soil Electron Donor (mg/kg) Soil moisture Rate kClO4

- (d-1) Source

HW84 Sidestream TVS (115.9) Slurry 0.37±0.07

Tan, 2003

HW84 Mainstream TVS (84.5) Slurry 0.14±0.02

Longhorn TVS (43.3) Slurry 0.16±0.08

HW317 TVS (160.5) Slurry 1.42±0.67

HW317/MN TVS (70.6) Slurry 0.11±0.03

Aerojet

H2 (34 -114) 16% 0.187-0.189

This research

LPG (75 – 250) 16% 0.037-0.043

1-hexene (80-165) 16% 0.045-0.171

None (H2?) 16% 0.102

Ethanol 16% 0.197

TVS = Total volatile solids in the sediments/soils.

58

The observed favorability of electron donors in this experiment was H2 > ethanol

> 1-hexene > LPG > ethyl acetate. This order is in accord with the order of Gibbs free

energy in Table 4-6 except for ethyl acetate. The reason why ethyl acetate failed to serve

as electron donor for perchlorate remediation in this microcosm test and also in the trial

microcosm tests (discussed in Chapter 2) is not clear. Ethyl acetate was tested in another

study of GEDIT (Evans and Trute, 2006), in which approximately 10% perchlorate

removal was observed in the middle and end of a column containing 10% soil moisture

while no perchlorate reduction occurred at the first 1/3 of column after 34 days of

incubation. Ethanol was also tested in that research and showed promising perchlorate

reduction but poor transport in column tests. Propane has once been tested; however, it

only supported denitrification but not perchlorate reduction (Hoponick, 2006). To the

best of our knowledge there is no other research reported to use 1-hexene as an electron

donor for perchlorate bioremediation.

Table 4-6: Reaction equations of tested electron donors with perchlorate and the correspondingGibbs free energies under standard and experimental conditions at low and high electron donor concentrations.

Reaction Equation (1 e-) ∆ G 0 (kJ/mol)

∆ G (kJ/mol ) Low E.D.

High E.D.

0.5 H2/CO2 + 0.125 ClO4- → 0.125 Cl- + 0.5 H2O -133.9 -129.6 -131.1

0.03 Ethyl acetate + 0.125 ClO4

- → 0.125 Cl- + 0.19 H2O + 0.19 CO2 -123.5 -123.2 -123.2

0.028 1-Hexene + 0.125 ClO4- → 0.125 Cl- + 0.17 H2O + 0.17 CO2 -122.9 -122.67 -122.75

0.05 Propane + 0.125 ClO4- → 0.125 Cl- + 0.2 H2O + 0.15 CO2 -120.8 -120.23 -120.38

0.083 Ethanol + 0.125 ClO4- → 0.125 Cl- + 0.25 H2O + 0.17 CO2 -126.4 -125.77

59

Intermediate products of perchlorate reduction (chlorate and chlorite) were not

detected during these microcosm tests, similar to the results of other research which

documented that perchlorate reduction to chlorate is the rate limiting step. Chlorate

accumulation has been reported, however, in both mixed and pure cultures of

hydrogen-oxidizing, perchlorate-reducing bacteria (Nerenberg et al., 2002, Nerenberg et

al., 2006).

Complete perchlorate reduction occurred in the negative control which contained

no external electron donor, and the degradation rate was higher than that observed for

1-hexene (low concentration), LPG, and ethyl acetate. The only explanation for this

phenomenon would be that another electron donor was generated in the negative control

bottles and served as an electron donor. During the experimental setup, the bottles were

filled with soil, purged with nitrogen, and allowed to sit on the open lab bench for at least

one day (different for different bottles). After this period, a small amount of hydrogen

was detected in all of the bottles, before any electron donor was injected. This hydrogen

peak remained in all of the bottles throughout the entire incubation, except for the

negative control. In the negative control bottles, the change of perchlorate concentration

and hydrogen concentration seems related (Figure 4-3). During the first 14 days,

hydrogen was accumulating during the lag period of perchlorate biodegradation. Then,

perchlorate and hydrogen concentration dropped simultaneously. The measured hydrogen

concentration change (1.4 mg/kg) from day 14 to day 49 is approximately twice the

stoichiometric electron donor requirement for perchlorate reduction (with a 6.4 ppm

perchlorate concentration change). The kClO4- in the negative control, however, was only

1/5 of that for the low concentration hydrogen bottles (Table 4-4). There was also a

60

longer lag period. This may indicate the perchlorate degradation in the negative control

was limited by the concentration of hydrogen.

To prove that the hydrogen detected in non-hydrogen-injected bottles was

produced biologically, a small experiment was performed. For this experiment,

microcosm bottles were set up in duplicate in the same way as described in the Materials

and Methods section with a nitrogen gas (N2) headspace and Aerojet soil at 16%

moisture. The following four conditions were tested: 1) Empty Controls containing only

N2; 2) Autoclaved Controls containing autoclaved soil; 3) Active-Light containing soil

incubated in the light on the bench; and 4) Active-Dark containing soil incubated in dark

(Table 4-7). All bottles were incubated on the bench for one day except the Active-Dark

which were incubated in a dark drawer. No hydrogen accumulation was detected in either

the empty or autoclaved controls (kept on the open bench), which eliminated the

0

2

4

6

8

10

0 25 50 75 100 125 150 175 200

Time (days)

Perc

hlor

ate

(ppm

)

0

1

2

3

4

H2

(mg/

kg)

perchlorate

H2

Figure 4-3: Perchlorate and hydrogen concentration change over incubation time innegative control microcosms containing no external electron donor at 16% soil moisture.

61

possibility that the hydrogen was introduced with the nitrogen gas or was the result of an

abiotic reaction in the soil. For the Active ones, the two bottles left in the light on the

open lab bench (as what happened in the microcosm test) generated hydrogen which was

detected the next day, whereas the two bottles incubated in the dark immediately after

setup showed no hydrogen production (Table 4-7). So it seems that hydrogen producing

microorganisms are present in the Aerojet site soil and that they are photoautotrophic.

With light, and under anaerobic conditions, organic residuals in the soil can be

converted to hydrogen and carbon dioxide by H2-photoproducing microorganisms, such

as purple bacteria (Nandi and Sengupta, 1998). Assuming the simplest carbon source

carbon monoxide, the reaction equation would be:

CO + H2O + light → H2 + CO2

Many of the hydrogen-producing bacteria are ubiquitous in nature and are

spore-formers (Cheong and Hansen, 2006). It is possible that the H2-photoproducing

bacteria survived in the Aerojet soil in spore-form and after the soil moisture was

adjusted and anoxic conditions achieved, they germinated because the conditions in

microcosm bottles were favorable. In the negative control, the hydrogen produced by the

Table 4-7: Setup and results of the 1-day hydrogen production test with the Aerojet soil at16% soil moisture.

Soil Purged w/ N2

Autoclaved Incubate Initial H2 (uM)

Final H2 (uM)

Hydrogen Produced

Empty Control No Yes No Light 0 0 No

Autoclaved Control Yes Yes Yes Light 0 0 No

Active-Light Yes Yes No Light 0 3.06±0.34 Yes

Active-Dark Yes Yes No Dark 0 0 No

62

H2-photoproducing bacteria may have then been utilized by the

hydrogen-oxidizing-perchlorate-reducing bacteria to reduce perchlorate. Even though

hydrogen generation should be companied by carbon dioxide production as shown in the

equation above, no carbon dioxide was detected in the negative control bottles. It is

possible that the concentration of carbon dioxide was too low to be detected on the GC,

and it is also possible that part of the carbon dioxide produced was used as carbon source

for perchlorate biodegradation. In those bottles with external electron donors of ethyl

acetate, 1-hexene (low concentration), and LPG, even though small amount of hydrogen

was also detected, the perchlorate degradation rate was lower than that in the negative

control. This may indicate that these chemicals are toxic to perchlorate reducing

microorganisms and inhibited their activity, but no evidence has been found to support

this conjecture. To better explain the results, a study of the microbial community in the

Aerojet soil using molecular microbial technology, and a study of the possible toxicity of

the tested electron donors to perchlorate reducing bacteria, may be needed.

ACKNOWLEDGEMENTS

The project described was performed in collaboration with Camp Dresser and

McKee, Inc. (CDM), with funding provided by Department of Defense Environmental

Security Technology Certification Program (DoD ESTCP) project number ER-0511.

Bob Parette is thanked for his assistance in IC method development.

63

REFERENCES

American Society for Testing and Materials (ASTM), 1999. Standard Test Method for

Laboratory Determination of Water (Moisture) Content of Soil and Rock by Mass.

Designation: D 2216-98. The Annual Book of ASTM Standards.

Cheong, D-Y and Hansen, C. L., 2006. Bacterial Stress Enrichment Enhances Anaerobic

Hydrogen Production in Cattle Manure Sludge. Appl Microbiol Biotechnol 72:635-643.

Evans, P. J., and Trute, M. M., 2006. In Situ Bioremediation of Nitrate and Perchlorate in

Vadose Zone Soil for Groundwater Protection Using Gaseous Electron Donor Injection

Technology. Water Environment Research 78(13):2436-2446.

Logan, B. E., Zhang, H., Mulvaney, P., Milner, M. G., Head, I. M., and Unz, R. F., 2001.

Kinetics of Perchlorate- and Chlorate-Respiring Bacteria. Applied and Environmental

Microbiology 37(6):2499-2506.

Hoponick, J. R., 2006. Status Report on Innovative In Situ Remediation Technologies

Available to Treat Perchlorate-Contaminated Groundwater. National Network for

Environmental Management Studies Fellow.

Kroon, A. G. M. and van Ginkel, C. G., 2004. Biological Reduction of Chlorate in a

Gas-Lift Reactor Using Hydrogen as an Energy Source. J. Environ. Qual. 33:2026-2029.

Miller, J. P., and Logan, B. E., 2000. Sustained Perchlorate Degradation in an

Autotrophic, Gas-Phase, Packed-Bed Bioreactor. Environ. Sci. Technol. 34:3018-3022.

Nandi, R. and Sengupta, S., 1998. Microbial Production of Hydrogen: An Overview.

Critical Reviews in Microbiology, 24(1):61-84.

64

Nerenberg, R., Rittmann, B. E., and Najm, I., 2002. Perchlorate Reduction in a

Hydrogen-Based Membrane-Biofilm Reactor. Journal AWWA. November 2002, 103-114.

Nerenberg, R., Kawagoshi, Y., Rittmann, B.E. 2006. Kinetics of a hydrogen-oxidizing,

perchlorate-reducing bacterium. Water Research 40(2006):3290-3296.

Nozawa-Inoue, M., Scow, K. M., and Rolston, D. E., 2005. Reduction of Perchlorate and

Nitrate by Microbial Communities in Vadose Soil. Applied and Environmental

Microbiology, 71(7):3928-3934.

Nzengung, V. A., Das, K. C., and Kastner, J. R. 2003. Pilot Scale In-Situ Bioremediation

Of Perchlorate-Contaminated Soils At The Longhorn Army Ammunition Plant. Final

Report on Perchlorate Remediation at LHAAP. Department of Geology. And Department

of Biological and Agricultural Engineering. The University of Georgia, Athens, GA

30602-4435.

Tan, K., Anderson, T. A., and Andrew Jackson, W., 2003. Degradation Kinetics of

Perchlorate in Sediments and Soils. Water, Air, and Soil Pollution 151:245-259.

U.S. Environmental Protection Agency, 1986. Guidelines Establishing Test Procedures

for the Analysis of Pollutants (App. B, Part 136, Definition and Procedures for the

Determination of the Method Detection Limit): U. S. Code of Federal Regulations,

Title 40, CFR 51 FR 23703.

65

5 COLUMN STUDIES

5.1 Abstract

Column studies were conducted to quantify the transport rates of the best

performing electron donors from the microcosm study (Chapter 3) through vadose zone

soil from a perchlorate-contaminated site containing 10% soil moisture. After quantifying

the transport rates, the columns were incubated for one to two months to evaluate the

resulting extent of perchlorate degradation. In the first column study, 20% hydrogen (H2)

(balance nitrogen, N2) was tested as the sole electron donor, without any added carbon

source. Hydrogen breakthrough time from the columns #1 and #2 was 3.4 and 5.58 hours,

respectively. Complete nitrate reduction was achieved in both columns but no perchlorate

degradation was detected in column #1 after 4 weeks of incubation or in column #2 after

10 weeks of incubation.

5.2 Material and Methods

5.2.1 Soil Characterization

The soil sample used in this test was collected from a perchlorate contaminated

Aerojet site in California and shipped to The Pennsylvania State University in six 5-gallon

buckets in August 2006. After removing large stones by hand and passing the soil through

a ½ inch sieve, all of the soil was well mixed and stored in 4 buckets at room temperature

66

until the experiments were performed. The soil from each bucket was tested for

perchlorate, nitrate, pH, and soil moisture on the second day of arriving, and then remixed

and redistributed to four buckets again due to the deviation of soil from four buckets. After

remixing, the soils from each bucket was tested for perchlorate, nitrate, pH, and soil

moisture again, and also total nitrogen and total carbon.

5.2.2 Experimental Design and Setup

The column studies were conducted in columns made of clear polyvinylchloride

(PVC) pipes measuring 2 inches in diameter and 5 feet in length. The ends were capped

with 2-inch-diameter PVC caps and stainless steel Swagelok fittings. Sampling ports

consisting of drilled holes plugged with thick butyl rubber stoppers were placed every 2

inches along the length of the columns (29 in total) to enable the discrete measurement of

gaseous electron donor transport (Figure 5-1).

67

Site soil contaminated with

Mass Flow Controller Sampling Ports

Gas Mixture

68

Prior to packing the columns, the soil moisture was adjusted to 10% by adding

distilled water and mixing well. An attempt was made to run the columns at the average

soil moisture of the microcosm tests (12%), but at this level the monitoring of electron

donor concentrations along the column length was impeded due to the high-moisture soil

clogging the gas-tight syringe needle as soon as it was inserted into the column. Therefore,

10% soil moisture content was chosen as a compromise that enabled easier monitoring of

the soil gas in the columns. The columns were packed by adding 1-2” lifts of soil and

tapping the sides of the column between lifts to promote even soil distribution. Each

column was packed with a total of 4.94 kg soil to achieve a soil density of 1.6-g/ml to

imitate the soil conditions at the site. After packing, the column caps and the stoppers

placed in the sampling ports were sealed with Epoxy glue. Duplicate columns were made

for each test. Prior to injecting the electron donor, the tubing connections and column

sampling ports were leak-tested while the column was being purged with 0.1 cm/s nitrogen

gas.

Hydrogen Column Setup

Two duplicate columns (Column #1 and #2) were set up to test hydrogen as the

sole external electron donor. Before introducing hydrogen to each column, the columns

were purged with nitrogen gas at 0.01 cm/s for about 10 hours (2.4 pore volumes) until less

than 1% oxygen was detectable in the column effluent to ensure anoxic conditions. A gas

mixture consisting of 20% hydrogen and 80% nitrogen was then purged through the

Figure 5-1: Schematic of the column setup and the columns in the laboratory.

69

columns at a pressure of 10-psi and a flow rate of 0.01 cm/s via mass flow rate controllers

(AALBORG, Model# GFC17). The effluents of the columns were tested for hydrogen

concentration every half hour to capture breakthrough curves. After hydrogen was

observed to travel from the beginning to the end of the columns and reached the same

hydrogen concentration throughout (4-5 pore volumes), the gas injection was stopped and

the column ends capped. Headspace samples were taken with a 250-uL gas-tight locking

syringe (Hamilton) from seven ports spaced evenly along the column length to construct a

hydrogen profile. The columns were then incubated in the dark at room temperature for

4-10 weeks. During incubation, headspace samples were taken along the column length to

check hydrogen and oxygen concentration every 1-2 weeks. Columns were re-purged with

the 20% hydrogen / 80% nitrogen gas mixture every 2-3 weeks when >1% oxygen

concentration was detected in the column, or when the hydrogen concentration was

observed to significantly decrease.

After 4 weeks of incubation, Column #1 was sacrificed and the soil samples behind

every other sampling port analyzed for perchlorate, chlorate, chlorite, chloride, nitrate,

nitrite, pH and soil moisture. The other duplicate column (Column #2) was sacrificed after

10 weeks of incubation and the soil similarly analyzed.

5.2.3 Chemical Analysis

Hydrogen and oxygen concentrations were quantified using a SRI 8610 B gas

chromatograph (GC) equipped with a thermal conductivity detector (TCD) and a

Molesieve 5A molecular sieve column (Alltech). Argon was used as the carrier gas with

70

pressure set up at 20 psi and the oven was held isothermally at 73°C. Carbon dioxide

concentration in headspace of samples was measured using a SRI GC (Model 310)

equipped with a TCD and a Porapak Q column. Helium was used as the carrier gas with

pressure set up at 20 psi and the oven was held isothermally at 83°C.

Perchlorate, chlorate, chlorite, chloride, nitrate, and nitrite were extracted from 5-g

soil by vortexing for 1 minute in a 50-mL centrifuge vial containing 20-mL deionized

water. A preliminary experiment conducted in triplicate demonstrated that 106.58±6.1% of

perchlorate was recovered from the soil after only 0.5 minutes of vortexing. After

vortexing, the extracts were centrifuged at 5000 rpm for 15 minutes and the supernatant

filtered through a 0.2-um-pore-diameter filter to remove soil particles. The anion

concentrations were measured using a DX-500 ion chromatograph (Dionex), equipped

with an AS-11 column, and a ED40 Electrochemical Detector. A sodium hydroxide

solution eluent with a flow rate of 1 mL/min was used to separate the species over a 30

minute run time. The eluent was composed of 98.7% DI water and 1.3% 200mM sodium

hydroxide at the beginning of each run and held for 10 minutes, then ramped to 96.4% DI

water and 3.6% 200 mM sodium hydroxide and held until the time was 17.4 min, ramped

to 65.5% DI water and 34.5% 200 mM sodium hydroxide and held from 18.8 min to 23

min, then ramped back to 98.7% DI water and 1.3% 200 mM sodium hydroxide and held

until the run ended. The detection limit of nitrate was determined according to the

procedure in USEPA Definition and Method for MDL (USEPA, 1986) and was found to

be 150 ppm.

Soil moisture content was determined gravimetrically according to D 2216-98

Standard Test Method for Laboratory Determination of Water (Moisture) Content of Soil

71

and Rock by Mass (ASTM, 1999), and the pH of the extracts after centrifuging was

measured with a Fisher Accumet AB 15 pH meter equipped with an Orion Thermo

Electron combination pH electrode.

5.3 Results of Hydrogen Columns

In Column #1, hydrogen breakthrough time was calculated as 3.4 hours (0.81 pore

volumes) (Shackelford, 1994), while it took approximately 5.58 hours (1.32 pore volume)

for hydrogen to breakthrough in Column #2 (Figure 5-2, see Appendix E.2 for

breakthrough time and pore volume calculations). The hydrogen dispersion was calculated

based on the step tests of column flushing (Lee, 1993). The dispersion number (D/µL) of

column #1 and #2 was 0.019 and 0.015 (see Appendix E.3 for detailed dispersion number

calculations). With dispersion numbers less than 0.02, the columns can be considered to

plug flow. After 21 days of incubation, the hydrogen concentration in both Columns #1

and #2 were found to have decreased to 1% of the incubated concentration, and oxygen

was detected in Column #2. A leak at the column outlet cap was detected in Column #2

and repaired. Both columns were re-purged with 20% hydrogen / 80% nitrogen gas

mixture and then incubated. Before sacrificing, Column #2 was re-purged two additional

times at 42-days and 63-days of incubation to replenish the hydrogen concentration (no

oxygen was detected in column #2 during this period).

72

After 4 weeks of incubation, no appreciable perchlorate degradation was detected

in Column #1 (Figure 5-3). The concentration of hydrogen was decreased from the initial

conditions, but was approximately uniform with length at 3 mg/kg, indicating that no

hydrogen “floating” occurred. Soil moisture was retained at its original value of

approximately 10% along the column length, and pH was approximately 6.5. No nitrate

(NO3-) was detected in any of the soil samples from Column #1, compared to the original

background concentration in the soil of 2.1±0.3 ppm NO3-.

0.0

1.0

2.0

3.0

4.0

5.0

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Pore Volumns

Hyd

roge

n in

soil

(mg/

kg)

Column #1Column #2Column inlet

Column #1 Breakthrough, 0.81 pore volumes

Column #2 Breakthrough, 1.32 pore volumes

0.0

1.0

2.0

3.0

4.0

5.0

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Pore Volumns

Hyd

roge

n in

soil

(mg/

kg)

Column #1Column #2Column inlet

Column #1 Breakthrough, 0.81 pore volumes

Column #2 Breakthrough, 1.32 pore volumes

Figure 5-2: Hydrogen breakthrough curves for Column #1 and #2 with 10% soil moisture.

73

After another 6 weeks, Column #2 was sacrificed after being purged 2 more times

with the 20% H2 / 80% N2 gas mix, but still no perchlorate reduction was observed

(Figure 5-4). Along the column length, hydrogen concentrations were fairly uniform, at 2.5

mg/kg. Soil moisture was around 10% along the column length and the pH was around 6.6.

No nitrate was detected from any soil samples of Column #2.

0

2

4

6

8

10

12

14

0 50 100 150

column length (cm)

Perc

hlor

ate,

Chl

orid

e, N

itrat

(ppm

), an

d H

ydro

gen

(mg/

k

0%

4%

8%

12%

16%

20%

Soil

moi

stur

e (%

Perchlorate

Chloride

hydrogen

Nitrate

soilmoisture

Figure 5-3: Perchlorate (ppm), chloride (ppm), hydrogen (mg/kg) and soil moisture (%)along column length in Column #1 after 4 weeks of incubation.

74

5.4 Discussion

Even though the columns were packed in the same way and had approximately the

same density as the site soil, the breakthrough curves were variable between columns. This

variability may have been caused by different soil particle sizes and relative locations in

each column causing preferential pathways and therefore different retention times. Longer

sitting (i.e., settling) time may have changed the micro-distribution of soil in the columns

as well. Columns that were packed and then immediately purged (column #2), seemed to

have longer retention times than those that were allowed to settle before being purged

0

2

4

6

8

10

12

14

16

0 50 100 150Column length (cm)

Perc

hlor

ate,

chl

orid

e, n

itrat

e(p

pm),

and

hydr

ogen

(mg/

k

0%

4%

8%

12%

16%

20%

Soil

moi

sture

(%)

Perchlorate

Chloride

hydrogen

Nitrate

SoilMoisture

Figure 5-4: Perchlorate (ppm), chloride (ppm), hydrogen (mg/kg) and soil moisture (%) along column length in Column #2 after 10 weeks of incubation.

75

(column #1). Compared with the theoretical breakthrough time calculated as 4.23 hours

(Appendix E.1), the Column #1 breakthrough time was shorter than expected. This may be

because Column #1 was the “oldest” column had been packed the earliest and purged with

gas several times before conducting the column test. Also, although the soil density in the

column was controlled to simulate site condition, the excavated-mixed-and-repacked

column soil would be very different from the real site conditions. For future work, a

drilling core obtained directly from the site is recommended to be used for column studies,

in which the electron donor transport rate can be estimated more accurately.

Complete denitrification was achieved in both columns (#1 and #2), but no

perchlorate reduction was observed. Even though many researchers have reported

preferential nitrate reduction prior to the onset of perchlorate degradation (Nozawa-Inoue

et al., 2005), there was no change in perchlorate concentration between columns #1 and #2

even after incubating for more than 6 weeks (Figure 5-5). There are several reasons which

may be responsible for the lack of perchlorate degradation in the columns including

oxygen infiltration, low soil moisture, and/or lack of carbon source. The column was made

by clear PVC, which is an oxygen permeable material (Doyon et al., 2006). Therefore, it

is possible that the electron donor concentration was decreasing because it was being

consumed by oxygen infiltrating into the column, and perchlorate degradation was

inhibited by the presence of oxygen. Lack of moisture may have also impeded perchlorate

reduction in columns #1 and #2. Microcosm test results showed that 7% moisture content

is too low to support perchlorate biodegradation. Ten percent in this column test is higher

than 7%, but it is not clear that if it is high enough to support perchlorate reduction. In

future tests, a method for sampling the soil gas in the columns even in the presence of high

76

soil moisture should be developed. Another possible reason for the lack of perchlorate

degradation would be limited carbon source since no carbon dioxide was injected with the

hydrogen gas and the original total carbon in the soil was low (0.037% ± 0.021%). To find

out is carbon the limit factor of perchlorate reduction in columns #1 and #2, a

four-gas-mixture test will be conducted. Columns #3 and #4 will be purged with a gas

mixture of 2% propane, 10% carbon dioxide, 20% hydrogen, and 68% nitrogen and

incubated in the dark at room temperature. Column #3 will be sacrificed on April 25, 2007,

and Column #4 will be sacrificed on May 25, 2007, after being incubated for 4 and 8

weeks, respectively. Only after seeing the results of the four-gas-mixture columns, will it

be clear if columns #1 and #2 were limited in moisture or in carbon.

0

2

4

6

8

10

12

14

16

0 20 40 60 80 100 120 140 160

Column Length (cm)

ppm

Perchlorate, 4 weeks Perchlorate, 10 weeksChloride, 4 weeks Chloride, 10 weeks

Figure 5-5: Perchlorate and chlorate concentration in hydrogen columns with 10% soilmoisture after 4 and 10 weeks of incubation.

77

ACKNOWLEDGEMENTS

This project was performed in collaboration with Camp Dresser and McKee, Inc.

(CDM), with funding provided by Department of Defense Environmental Security

Technology Certification Program (DoD ESTCP) project number ER-0511.

Booki Min is thanked for his assistance in the column setup.

REFERENCES

Doyon, G., Gagnon, J., Toupin, C., and Castaigne, F., 2006. Gas Transmission Properties

of Polyvinyl Chloride (PVC) Films Studied Under Subambient and Ambient Conditions

for Modified Atmosphere Packaging Applications. Packaging Technology and Science

4(3):157-165.

Lee, S. R., 1993. The Role of Carbon Dioxide in the Combustion of Kraft Black Liquor

Char. Doctor’s Dissertation. The Institute of Paper Science and Technology, Atlanta,

Georgia.

Nozawa-Inoue, M., Scow, K. M., and Rolston D. E., 2005. Reduction of Perchlorate and

Nitrate by Microbial Communities in Vadose Soil. Applied and Environmental

Microbiology 71(7):3928-3934.

Shackelford, D. D., 1994. Critical Concepts for Column Testing. Journal of Geotechnical

Engineering 120(10):1804-1828.

78

6 CONCLUSIONS, ENGINEERING SIGNIFICANCE, AND FUTURE WORK

6.1 Conclusions and Engineering Significance

Hydrogen is the best of the tested electron donors for supporting perchlorate

remediation in vadose zone soil.

To effectively apply GEDIT to treat perchlorate-contaminated vadose zone soil,

picking a suitable electron donor is very important. Based on the results of this study, it is

suggested that hydrogen be used at the Aeroject site because it can promote perchlorate

and nitrate degradation at high rates and is easily and transported through vadose zone

soil.

LPG and 1-hexene can also serve as electron donors for perchlorate

bioremediation.

To the best of our knowledge, LPG and 1-hexene have not been previously

successfully tested as electron donors for supporting perchlorate degradation. They

should be considered as electron donors in future bioremediation studies. Especially

LPG, the cost of which is very low, would be a good donor to use at large sites.

Higher electron donor concentrations can promote faster perchlorate

degradation rates.

79

Assuming that perchlorate reduction is a first order reaction, the following

equations should apply: C/C0 = exp (-kt), from which k = (lnC0 – lnC)/t. The results of

this research indicated that with the same initial perchlorate concentration (C0),

microcosms with higher electron donor concentrations resulted in more complete

perchlorate reduction (lower C), in a shorter t time (t), which should result a higher first

order rate constant (k). This is accordance with the observed perchlorate reduction rate

calculated in this research. Therefore, in the bioremediation of perchlorate using GEDIT,

supplying a higher concentration of electron donor may increase the rate of perchlorate

reduction, decrease the cleanup time, and save operating costs, but the cost of electron

donor would be increased. A cost effective design should consider both alternatives.

Soil moisture is the key factor in stimulating perchlorate bioremediation in the

tested vadose zone soil.

Given the same electron donor, electron donor concentration, and soil properties,

higher moisture content microcosms in this research achieved complete or partial

perchlorate reduction (except for ethyl acetate) whereas no perchlorate degradation was

detected in low moisture content bottles. Soil moisture content is the key factor in

perchlorate bioremediation. During soil bioremediation, moisture should be monitored

and adjusted if necessary.

The soil moisture content of 7% is not enough to support perchlorate

bioremediation in the Aerojet site soil using the tested electron donors.

80

At the Aerojet site, increasing the soil moisture may be necessary to achieve the

perchlorate cleanup goal. At the Aerojet site, the shallow surface soil (0-20 ft below

ground surface (bgs)) has the highest perchlorate concentration (59 ppm maximum) as

well as the highest soil moisture content (34.3% maximum) (Figure 6-1). From

approximately 25 – 70 ft bgs, the soil moisture content appears to be lower than 16%.

Since the perchlorate reduction rates in soils with moisture contents between 7% and

16% have not yet been tested, the site soil is conservatively recommended to be

irrigated to increase the soil moisture throughout the lower portion of the contaminated

zone (i.e., to 70 ft bgs) to 16%. If an above ground irrigation system is used, when the

deepest soils reach the desired moisture content, the shallow soils may be too wet for

sufficient electron donor transport. Therefore, it is recommended that an underground

irrigation system be installed at 25 ft bgs.

1

10

100

0 10 20 30 40 50 60 70 80Depth (ft)

Con

cent

ratio

n.

Perchlorate (mg/kg)

Moisture (%)

Lab Perchlorate (mg/kg)

Lab NO3/NO2 (mg-N/kg)

Log. (Moisture (%))

Figure 6-1: Perchlorate and moisture change along with the change of depth at the

81

6.2 Future Work

More research is needed to study the relationship between soil moisture content

and perchlorate biodegradation. Additional soil moisture contents between 7 – 16 %

should be tested to determine the minimum requirement for perchlorate reduction and

also to find the optimal moisture content for supporting perchlorate bioremediation.

In the microcosm tests, a considerable amount of hydrogen was naturally

produced by native soil bacteria and used to reduce perchlorate, making

H2-photoproducing microorganisms worthy of further study. To confirm the activity of

H2-photoproducing microorganisms in the microcosm study, molecular microbial

analysis to target these microorganisms in the Aerojet soil is needed. Polymerase chain

reaction (PCR), reverse transcriptase PCR (RT-PCR) and fluorescence in situ

hybridization (FISH) have been used to target to the hydrogenase gene, which may

contribute to anaerobic biohydrogen production (Jen et al., 2007). The PCR primers and

PCR-generated probes used to identify photobiological hydrogen production strains are

also available (Schutz et al., 2003). If confirmed, this may bring another option to ex situ

perchlorate bioremediation: stimulating the intrinsic H2-producting bacteria in the

presence of light to supply electron donor for perchlorate reduction, instead of adding

external electron donors. Since there would be considerable value in facilitating

hydrogenesis for in situ perchlorate reduction, the microorganisms which can perform

“dark” hydrogen fermentation should also be investigated.

Aerojet site.

82

A study of the perchlorate reducing bacteria in the Aerojet soil would also be very

useful to help understand the results of this research. Who are the perchlorate reducing

bacteria stimulated in these tests and what is their population? What is their relationship

with the photoautotrophs? How is the community affected by injecting different electron

donors? Was the perchlorate reduction rate limited by the population? Would it be

helpful to supply nutrients?

To eliminate the possibility of carbon limited perchlorate reduction in columns #1

and #2, a mixture of 2% propane, 10% CO2, 20% H2, and 68% N2 are going to be tested

in the second column study. Column #3 will be sacrificed on April 25, 2007 and Column

#4 will be sacrificed on May 25, 2007, to determine the extent of nitrate and perchlorate

degradation.

Finally, a column study conducted with a drilling core obtained directly from the

site might be more accurate than a column packed in the lab. Even though the columns in

the lab were packed to the same density as the site soil, the micro-environment in the soil

was still very different. Conducting the electron donor transport study on a drilling core

could enable a better estimate of breakthrough time and transport rate of the electron

donor.

REFERENCES

Jen, C. J., Chou, C.-H., Hsu, P.-C., Yu, S.-J., Chen, W.-E., Lay, J.-J., Huang, C.-C., and

Wen, F.-S., 2007. Flow-FISH analysis and isolation of clostridial strains in an anaerobic

83

semi-solid bio-hydrogen producing system by hydrogenase gene target. Environmental

Biotechnology 74(8):1126-1134.

Schutz, K., Happe, T., Troshina, O., Lindblad, P., Leitao, E., Oliveira, P., and Tamagnini,

P., 2004. Cyanobacterial H2 Production - A Comparative Analysis. Planta

218(3):350-359.

Appendix A

ACRONYMS

bgs Below Ground Surface

CA DHS California Department of Health Services

CCL Contaminant Candidate List

CDM Camp Dresser and McKee, Inc.

DI De-Ionized

ESTCP Environmental Security Technology Certification Program

FID Flame Ionization Detector

GC Gas Chromatograph

GEDIT Gaseous Electron Donor Injection Technology

ITRC Interstate Technology & Regulatory Council

IC Ion Chromatography

IRZ In-Situ Reactive Zone

LAAP Longhorn Army Ammunition Plant

LPG Liquefied Petroleum Gas

ND None detectable

ppb Parts per billion

ppm Parts per million

PSU The Pennsylvania State University

PVC polyvinylchloride

RfD Provisional reference Dose

TCD Thermal Conductivity Detector

UCMR Unregulated Contaminant Monitoring Rule

UHP Ultra High Purity

USEPA United States Environmental Protection Agency

Appendix B

B-1 REACTIONS OF ELECTRON DONORS WITH PERCHLORATE

B-2 PROPERTIES OF ELECTRON DNORS

Electron donor Candidates Molecular formula Formula weight (g/mol)

H (atm-m3/mol)

Psat (mm Hg)

1-Hexene CH3(CH2)3CHCH2 84.16 4.17E-01 100

Acetic Acid CH3COOH 60.05 2.94E-07 10 Ethanol C2H5OH 46.07 5.00E-06 40

Ethyl Acetate CH3COOC4H9 88.11 1.34E-04 60

Ethyl Lactate CH3CH2OCOCHOHCH3 118.13 5.83E-07 3.75 Hydrogen H2 2 1.28E+00 760 LPG (Liquefied Petroleum Gas, 90% propane)

CH3CH2CH3 44.1 6.00E-01 5700

Reaction Equation (1 e-)

0.028 1-Hexene + 0.125 ClO4- → 0.125 Cl- + 0.17 H2O + 0.17 CO2

0.125 Acetic acid + 0.125 ClO4- → 0.125 Cl- + 0.25 H2O + 0.25 CO2

0.083 Ethanol + 0.125 ClO4- → 0.125 Cl- + 0.25 H2O + 0.17 CO2

0.03 Ethyl acetate + 0.125 ClO4- → 0.125 Cl- + 0.19 H2O + 0.19 CO2

0.042 Ethyl lactate + 0.125 ClO4- → 0.125 Cl- + 0.21 H2O + 0.21 CO2

0.5 H2/CO2 + 0.125 ClO4- → 0.125 Cl- + 0.5 H2O

0.05 propane + 0.125 ClO4- → 0.125 Cl- + 0.2 H2O + 0.15 CO2

Appendix C

TRIAL MICROCOSM TESTS DATA

C.1 Trial Test #3 Setup

A 1g/L sodium perchlorate solution and a 100g/L acetic acid solution were

prepared and degassed with nitrogen gas in advance. The soil sample was split into four

100-gram batches. For Test 1, 1 mL perchlorate solution and 1 mL acetic acid solution

were added to one 100-g batch of soil and mixed well. The soil was then split into five

20-g aliquots. Four of the 20-g batches were placed into four serum bottles, sealed with

thick butyl rubber stopper and aluminum crimp tops, and then flushed with ultra high

purity (UHP) nitrogen gas for 15 minutes to remove oxygen. The remaining 20-g aliquot

was analyzed for pH, moisture, nitrate, and perchlorate as the time zero sample. Test 2

was prepared in the same way as Test 1, but 5.1 mL water was added to the 100-g soil

batch and mixed well to increase the moisture to 15% prior to being aliquoted into the

serum bottles. For Test 3, 1 mL perchlorate solution was added into one 100-g batch of

soil and mixed well. After splitting the soil to five 20-g aliquots, placing them into five

serum bottles, flushing with nitrogen and sealing with stoppers, 22uL pure ethyl acetate

was injected into the headspace. The bottles were shaken vigorously to facilitate

soil-headspace contact. One of the serum bottles was sacrificed immediately for time zero

measurement in the same way as in Test 1. Test 4 was setup the same way as Test 3 but

6.0 mL water was added to the 100-g soil batch to adjust soil moisture.

The setup is also summarized in the table below:

Table C.1-1 Trial #3 Setup

Soil Moisture Perchlorate Electron Donor

Test

Mass per

bottle (g)

Time points

(weeks) Number bottles

Total Soil mass per batch (g)

Final (%)

Initial (%)

Added Water per soil batch (mL)

Perchlorate addition (mg/kg)

1 g/L ClO4

solution volume

per batch (mL) Name

Final Conc

(mg/kg)

Volume of 100 g/L electron

donor solution per soil batch

(mL)

Volume of pure electron

donor injected into each bottle

(µL)

1 20 0,1,2,3,4 5 100 10.8% 9% 0.0 10 1.0 Acetic acid 1000 1.0 0.00

2 20 0,1,2,3,4 5 100 15.0% 9% 5.1 10 1.0 Acetic acid 1000 1.0 0.00

3 20 0,1,2,3,4 5 100 10.8% 9% 1.0 10 1.0 Ethyl

acetate 1000 0.0 22

4 20 0,1,2,3,4 5 100 15.0% 9% 6.0 10 1.0 Ethyl

acetate 1000 0.0 22

C.2 Trial Test #4 Setup

The organic farm soil was split into four 100-gram batches and eight 55.6-gram

batches. The 100-gram batches were used for low moisture tests and 55.6-gram for slurry

tests. A 1g/L sodium perchlorate solution was prepared and degassed with nitrogen gas in

advance. Taking test 1 as an example, 1 mL perchlorate solution and 5 mL DI water were

added to one 100-g batch soil in a bowl and mixed well. The soil was then split into five

20-g aliquots, put into serum bottles, sealed with butyl rubber stoppers, and then flushed

with nitrogen gas. For each “hexane” bottle, 29.7-uL pure 1-hexene was injected and

dropped onto the glass wall. After the liquid 1-hexene was visually observed to fully

evaporate into the headspace, all of the bottles were shaken vigorously. For each “ethyl

lactate” bottle, 19.4-uL pure ethyl lactate was injected in the same way. For slurry tests,

38-mL DI water was mixed with the 55.6-g soil batch. For positive bacteria tests, 5-mL

activated sludge was mixed with the soil batch. In test 5 and 11, the activated sludge

added was autoclaved and in test 6 and 12, regular activated sludge was added but after

setup, the whole bottles were autoclaved.

The setup is also summarized in the table C.2-1 next page:

Table C.2-1 Trial #4 Setup

moisture Activated sludge perchlorate electron donor

Test Description # of

bottles

Total soil

mass per

batch (g)

Original soil

mass per

batch (g)

Final (%)

Initial (%)

Added water

vol per batch (ml)

Added vol of

activated sludge

per batch (ul)

Final conc

(mg/kg)

1g/L ClO4-

solution vol.

added per batch

(mL) Name

Final conc.

(mg/kg)

vol. of pure E.D. injected

into headspace

of eatch bottle (uL)

1 low moisture,

negative bacteria 5 100 100.0 15% 10% 5 0 10 1 1-hexene 1000 29.7

2 low moisture,

positive bacteria 5 100 100.0 15% 10% 0 5000 10 1 1-hexene 1000 29.7

3 high moisture,

negative bacteria 5 100 55.6 50% 10% 43 0 10 1 1-hexene 1000 29.7

4 high moisture,

positive bacteria 5 100 55.6 50% 10% 38 5000 10 1 1-hexene 1000 29.7

5 high moisture, killed control 5 100 55.6 50% 10% 38

5000 (autoclaved) 10 1 1-hexene 1000 29.7

6 autoclave whole

bottle 5 100 55.6 50% 10% 38 5000 10 1 1-hexene 1000 29.7

7 low moisture,

negative bacteria 5 100 100.0 15% 10% 5 0 10 1 ethyl

lactate 1000 19.4

8 low moisture,

positive bacteria 5 100 100.0 15% 10% 0 5000 10 1 ethyl

lactate 1000 19.4

9 high moisture,

negative bacteria 5 100 55.6 50% 10% 43 0 10 1 ethyl

lactate 1000 19.4

10 high moisture,

positive bacteria 5 100 55.6 50% 10% 38 5000 10 1 ethyl

lactate 1000 19.4

11 high moisture, killed control 5 100 55.6 50% 10% 38

5000 (autoclaved) 10 1

ethyl lactate 1000 19.4

12 autoclave whole

bottle 5 100 55.6 50% 10% 38 5000 10 1 ethyl

lactate 1000 19.4

Appendix D

MICROCOSM TESTS DATA

91

D.1 Microcosm Setup Details

Design soil moisture Electron Donor

Test #

Electron Donor

Electron donor conc.

(mg/kg)

Soil moisture

mass per

bottle (g)

No. of bottles

Total mass

soil per batch

(g)

Final (%)

Initial (%)

Water Added per

batch (mL)

Name Final Conc

(mg/kg)

Pure e.d. injected into each bottle

(µL)

CO2 injected into each

bottle (uL)

1* H2 34 7% 10 18 180 7% 7.0% 0.00 H2 34 4046.8 2023.4 2* Ethyl

acetate 150 7% 10 18 180 7% 7.0% 0.00 Ethyl

acetate 150 1.7

3 1-Hexene 80 7% 10 9 90 7% 7.0% 0.00 1-Hexene 80 1.2 4 LPG 75 7% 10 9 90 7% 7.0% 0.00 LPG 75 408.9 5 H2 114 7% 10 9 90 7% 7.0% 0.00 H2 114 13568.7 6784.3

6* Ethyl acetate

501 7% 10 18 180 7% 7.0% 0.00 Ethyl acetate 501 5.6

7* 1-Hexene 265 7% 10 18 180 7% 7.0% 0.00 1-Hexene 265 3.9 8 LPG 250 7% 10 9 90 7% 7.0% 0.00 LPG 250 1363.0 9 H2 34 16% 10 9 90 16% 7.0% 9.64 H2 34 4046.8 2023.4

10 Ethyl acetate

150 16% 10 9 90 16% 7.0% 9.64 Ethyl acetate 150 1.7

11* 1-Hexene 80 16% 10 18 180 16% 7.0% 19.29 LPG 80 1.2 12* LPG 75 16% 10 18 180 16% 7.0% 19.29 Propane 75 408.9 13* H2 114 16% 10 18 180 16% 7.0% 19.29 H2 114 13568.7 6784.3 14 Ethyl

acetate 501 16% 10 9 90 16% 7.0% 9.64 Ethyl

acetate 501 5.6

15 1-Hexene 265 16% 10 9 90 16% 7.0% 9.64 1-Hexene 265 3.9 16* LPG 250 16% 10 18 180 16% 7.0% 19.29 LPG 250 1363.0 17 Negative

control 0 16% 10 9 90 16% 7.0% 9.64 None 0 0.0

18 Positive control

436 16% 10 9 90 16% 7.0% 9.64 Ethanol 436 5.5

* = Tests that were randomly chosen to run duplicates.

92

D.2 Microcosm Tests Data

D.2.1 Test 1: 7% moisture, 34 mg/kg H2

Time (days) 0 7 14 21 28 35 42 49 56 63 91 144 203

Perchlorate average (ppm) 11.42 10.1 - 9.7 - 10.1 - 11.21 - 10.05 9.46 12.021 12.84 std. dev. 2.94 0.39 - 0.35 - 0.13 - 1.22 - 0.1 2.5 0.5215 1.265

Chloride average (ppm) 2.7 1.92 - 2.54 - 3.54 - 5.81 - 5.49 2.7 5.53 5.29 std. dev. 0.08 0.03 - 0.58 - 0.76 - 0.27 - 2.7 1.04 0.28 0.8

Nitrate average (ppb) J J - J - J - J - J J J 0 std. dev. - - - - - - - - - - - - 0

Hydrogen average (mg/kg) 32.91 31.28 - 33.86 - 62.63 67.7 73.6 62.44 99.28 78.931 76.9 80.106

std. dev. 0.31 4.62 - 0.16 - 0.67 2.75 2.5 1.18 2.04 4.93 0 1.48

CO2 average (ppmv) 31.49 21.638 - 24.315 - 67.3 - 20.92 - 25.85 61.29 63.26 58.62 std. dev. 0 0 - 0 - 0 - 0 - 0 0 0 0

moisture average (%) 6.56 7.79 - 6.98 - 6.49 - 6.39 - 7.12 6.87 8.71 6.64 std. dev. 0.15 0.119 - 1.37 - 1.1 - 0.25 - 0.31 0.56 0 0.57

pH average 7.24 6.45 - 6.69 - 7.2 - 7.19 - 7.2 7.19 7.14 7.02 std. dev. 0.05 0.05 - 0.01 - 0.08 - 0.04 - 0.15 0.08 0 0.08

J = below the detection limit (the substance in question was detected, but at levels below that which can be accurately characterized by the test method).

93

Test1: 7% moisture, 34mg/kg H2

0

2

4

6

8

10

12

14

16

0 50 100 150 200Time (days)

Perc

hora

te, c

hlor

ide

(ppm

); pH

(uni

ts); M

oistu

re (%

) .

0

20

40

60

80

100

120

H2

(mg/

kg);

CO

2 (p

pmv)

, nitr

at(p

pb)

Perchlorate Chloride moisture pH

Nitrate Hydrogen CO2

94

D.2.2 Test 2: 7% moisture, 150 mg/kg ethyl acetate

Time (days) 0 7 14 21 28 35 42 49 56 63 70 91 143 202

Perchlorate average (ppm) 11.06 11.14 - 10.68 - 10.72 - 10.29 - 10.45 - 10 12.404 11.68 std. dev. 0.26 1.05 - 0.14 - 0.55 - 1.94 - 0.19 - 1.68 0.2755 1.14

Chloride average (ppm) 1.75 2.33 - 2.83 - 1.62 - 5.06 - 2.79 - 3.18 4.99 5.8 std. dev. 1.17 0.86 - 0.17 - 0.29 - 0.61 - 0.14 - 0.81 0.77 0.32

Nitrate average (ppb) J J - J - J - J - J - J J 0 std. dev. - - - - - - - - - - - - - 0

Ethyl Acetate

average (mg/kg) 53.35 28.35 - 8.31 0.11 4.43 0.02 10.22 0 0 1.36 0 0 0 std. dev. 2.14 5.34 - 6.9 0.14 5.84 0 14.45 0 0 0.09 0 0 0

CO2 average (ppmv) 0 0 - 0 - 0 - 0 - 0 - 0 0 0 std. dev. 0 0 - 0 - 0 - 0 - 0 - 0 0 0

moisture average (%) 6.55 7.3 - 6.81 - 6.41 - 7.86 6.13 6.83 - 6.49 7.7 7.37 std. dev. 0.54 1.06 - 0.32 - 0.88 - 0.92 0.08 0.37 - 0.5 0.24 0.3

pH average 6.71 6.63 - 6.79 - 6.64 - 6.7 - 6.38 - 7.17 7.02 6.81 std. dev. 0.01 0.08 - 0.11 - 0.16 - 0.04 - 0.03 - 0.06 0.13 0.04

J = below the detection limit (the substance in question was detected, but at levels below that which can be accurately characterized by the test method).

95

Test 2: 7% moisture, 150mg/kg Ethyl Acetate

0

2

4

6

8

10

12

14

0 50 100 150 200Time (days)

Perc

hora

te, c

hlor

ide

(ppm

); pH

(uni

ts);

Moi

stur

e (%

) .

0

10

20

30

40

50

60

Ethy

l Ace

tate

(mg/

kg);

CO

2(p

pmv)

, nitr

ate

(ppb

)

Perchlorate Chloride moisture pH

Nitrate Ethyl Acetate CO2

96

D.2.3 Test 3: 7% Moisture, 80 mg/kg 1-hexene

Time (days) 0 7 14 21 28 35 42 49 77 125 185

Perchlorate average (ppm) 10.64 11.21 - 10.16 - 10.9 - 11.86 10.86 11.86 10.44 std. dev. 0.01 0.01 - 0.14 - 0.29 - 0 1.05 0.55 1.25

Chloride average (ppm) 1.37 3.02 - 3.99 - 4.67 - 4.87 3.22 7.64 3.59 std. dev. 1.93 0.02 - 0.07 - 0.29 - 0 0.54 0.8 0.62

Nitrate average (ppm) J J - J - J - J J J 0 std. dev. 0 - - - - - - - - - 0

1-Hexene average (mg/kg) 9.96 78.432 100.738 110.49 63.67 131.351 145.61 29.04 29.03 114.541 111.087

CO2 average (ppmv) 0 0 - 0 - 0 - 0 0 0 0

moisture average (%) 5.95 5.96 - 7.56 - 6.72 - 6.39 6.99 6.01 5.55 pH average 6.89 6.54 - 6.84 - 7.04 - 7.44 7.25 6.98 7.29

J = below the detection limit (the substance in question was detected, but at levels below that which can be accurately characterized by the test method)

97

Test 3: 7% moisture, 80mg/kg 1-Hexene

0

2

4

6

8

10

12

14

0 50 100 150 200Time (days)

Perc

hora

te, c

hlor

ide

(ppm

);pH

(uni

ts);

Moi

stur

e (%

) .

0

20

40

60

80

100

120

140

160

1-H

exen

e (m

g/kg

); C

O2

(ppm

v), n

itrat

e (p

pb)

Perchlorate Chloride moisture pHNitrate 1-Hexene CO2

98

D.2.4 Test 4: 7% Moisture, 75 mg/kg LPG

Time (days) 0 7 14 21 28 35 42 49 77 124 184

Perchlorate average (ppm) 9.73 10.81 - 11.01 - 8.03 - 11.81 10.36 12.4 11.29 std. dev. 0.52 0.01 - 0.71 - 5.23 - 0 1.3 0.03 0

Chloride average (ppm) 3.78 4.22 - 5.23 - 2.88 - 5.18 4.13 4.76 4.58 std. dev. 0.04 0.02 - 0.32 - 2.3 - 0 0.81 0.29 0

Nitrate average (ppb) J J - J - J - J J J 0 std. dev. - - - - - - - - - - 0

LPG average (mg/kg) 58.152 221.69 235.62 176.01 253.02 263.4 84.653 312.976 356.734 283.645 221.692

CO2 average (ppmv) 0 0 - 0 - 0 - 0 0 0 0

moisture average (%) 5.41 5.56 - 4.88 - 6.1 - 5.72 5.67 6.94 6.32 pH average 7.03 7.17 - 7.18 - 6.91 - 6.99 7.2 7.01 7.32

J = below the detection limit (the substance in question was detected, but at levels below that which can be accurately characterized by the test method)

99

Test 4: 7% moisture, 75mg/kg LPG

0

2

4

6

8

10

12

14

0 50 100 150 200Time (days)

Perc

hora

te, c

hlor

ide

(ppm

);pH

(uni

ts);

Moi

stur

e (%

) .

0

50

100

150

200

250

300

350

400

LPG

(mg/

kg);

CO

2 (p

pmv)

nitra

te (p

pb)

Perchlorate Chloride moisture pHNitrate LPG CO2

100

D.2.5 Test 5: 7% Moisture, 114 mg/kg H2

Time (days) 0 7 14 21 28 35 42 49 77 128 187

Perchlorate average (ppm) 10.76 11.7 - 9.48 - 11.3 - 10.91 9.82 12.4176 12.98 std. dev. 0.04 0.04 - 0.1 - 0.54 - 0.03 2.75 0.5 1.13

Chloride average (ppm) 2.79 3.17 - 2.53 - 5.28 - 3.93 3.68 4 5.56 std. dev. 0.05 0.03 - 0.6 - 0.58 - 0.09 1.57 0.16 0.1

Nitrate average (ppb) J J - J - J - J J J 0 std. dev. - - - - - - - - - - -

Hydrogen average (mg/kg) 91.43 109.8 - 152 122.56 104.38 93.36 75.198 142.173 116.498 123.574

CO2 average (ppmv) 75.79 82.6 - 196.24 - 66.83 - 36.73 59.31 61.33 96.8 moisture average (%) 8.39 5.49 - 4.45 - 6.05 - 5.25 4.54 5 5.57 pH average 7.11 7.23 - 7.05 - 6.92 - 6.54 7.31 6.85 7.3

J = below the detection limit (the substance in question was detected, but at levels below that which can be accurately characterized by the test method)

101

Test 5: 7% moisture, 114mg/kg H2

0

2

4

6

8

10

12

14

16

0 50 100 150 200Time (days)

Perc

hora

te, c

hlor

ide

(ppm

); pH

(uni

ts);

Moi

stur

e (%

) .

0

50

100

150

200

250

H2

(mg/

kg);

CO

2 (p

pmv)

.nitr

ate

(ppb

)

Perchlorate Chloride moisture pHNitrate Hydrogen CO2

102

D.2.6 Test 6: 7% Moisture, 501 mg/kg ethyl acetate

Time (days) 0 7 14 21 28 35 42 49 77 125 184

Perchlorate average (ppm) 9.8 10.35 - 9.04 - 10.87 - 11.48 10.18 12.11 11.1std. dev. 0.28 0.75 - 2.07 - 2.38 - 0.02 1.35 0.68 0.58

Chloride average (ppm) 2.68 3.16 - 4.18 - 3.92 - 4.95 3.21 4.7 4.62std. dev. 0.37 0.66 - 0.72 - 0.85 - 0.47 1.1 0.19 0.12

Nitrate average (ppb) J J - J - J - J J J J std. dev. - - - - - - - - - - 0

Ethyl Acetate

average (mg/kg) 373.7 289.14 3.83 101.12 2.48 73.22 63.87 21.3 11.7 0 0 std. dev. 110.1 75.95 0.26 7.65 1.29 50.65 40.6 9.34 11.9 0 0

CO2 average (ppmv) 0 0 - 0 - 0 - 0 0 0 0 std. dev. 0 0 - 0 - 0 - 0 0 0 0

moisture average (%) 5.86 6.45 - 4.96 - 6.13 - 6.48 6.65 6.71 6.39 std. dev. 0.23 0.61 - 0.06 - 0.08 - 0.17 0.04 0.62 0.24

pH average 6.84 6.39 - 6.4 - 6.08 - 6.24 6.32 7.07 6.18 std. dev. 0.01 0.06 - 0.07 - 0.1 - 0.014 0.05 0.06 0.23

J = below the detection limit (the substance in question was detected, but at levels below that which can be accurately characterized by the test method)

103

Test 6: 7% moisture, 501mg/kg Ethyl Acetate

0

2

4

6

8

10

12

14

0 50 100 150 200Time (days)

Perc

hora

te, c

hlor

ide

(ppm

); pH

(uni

ts);

Moi

stur

e (%

) .

0

100

200

300

400

500

600

Ethy

l Ace

tate

(mg/

kg);

CO

2(p

pmv)

, nitr

ate

(ppb

)

Perchlorate Chloride moisture pH

Nitrate Ethyl Acetate CO2

104

D.2.7 Test 7: 7% Moisture, 265 mg/kg 1-hexene

Time (days) 0 7 14 21 28 35 42 49 77 125 185

Perchlorate average (ppm) 9.55 10.57 - 9.96 - 11.07 - 11.14 10.68 13.09 10.62std. dev. 0.39 0.28 - 0.15 - 0.34 - 0.17 0.65 0.42 0.68

Chloride average (ppm) 2.28 3.12 - 5.16 - 4.92 - 4.76 3.93 6.17 5.52std. dev. 0.07 0.49 - 0.045 - 0.31 - 0.82 0.68 1.2 0.67

Nitrate average (ppb) J J - J - J - J J J 0std. dev. - - - - - - - - - - 0

1-Hexene average (mg/kg) 43.8 209.426 311.334 253.403 102.724 74.896 213.139 85.59 221.272 131.273 129.52 std. dev. 4.5 - - - -- - - - - -

CO2 average (ppmv) 0 0 - 0 - 0 - 0 0 0 0std. dev. 0 0 - 0 - 0 - 0 0 0 0

moisture average (%) 6.51 6.37 - 6.81 - 6.31 - 6.47 6.9 7.21 6.52 std. dev. 0.21 0.32 - 0.17 - 0.8 - 0.1 0.73 0.02 0.44

pH average 7.08 6.65 - 7.15 - 7.125 - 7.325 7.185 6.965 7.31 std. dev. 0.13 0 - 0.01 - 0.035 - 0.06 0.02 0.01 0.01

J = below the detection limit (the substance in question was detected, but at levels below that which can be accurately characterized by the test method)

105

Test 7: 7% moisture, 265mg/kg 1-Hexene

0

2

4

6

8

10

12

14

0 50 100 150 200Time (days)

Perc

hora

te, c

hlor

ide

(ppm

);pH

(uni

ts);

Moi

stur

e (%

) .

050100150200250300350400450500

1-H

exen

e (m

g/kg

); C

O2

(ppm

v), n

itrat

e (p

pb)

Perchlorate Chloride moisture pHNitrate 1-Hexene CO2

106

D.2.8 Test 8: 7% Moisture, 250 mg/kg LPG

Time (days) 0 7 14 21 28 35 42 49 77 124 184

Perchlorate average (ppm) 9.38 10.85 - 10.73 - 10.83 - 11.38 10.66 11.98 10.34std. dev. 0.17 0.11 - 0.19 - 0.22 - 0 1.28 0.04 2.25

Chloride average (ppm) 1.27 2.71 - 5.16 - 4.89 - 4.03 2.98 5.03 4.76std. dev. 0.02 0.06 - 0.05 - 0.2 - 0 0.5 0.32 1.26

Nitrate average (ppb) J J - J - J - J J J 0 std. dev. - - - - - - - - - - 0

LPG average (mg/kg) 198.05 628.76 705.87 798.79 638.8 506.9 248.261 600.656 826.349 914.157 680.5

CO2 average (ppmv) 0 0 - 0 - 0 - 0 0 0 0

moisture average (%) 7.48 6.51 - 6.46 - 6.44 - 6.56 6.05 7.03 7.01 pH average 6.77 7.27 - 7.18 - 6.89 - 6.91 7.22 6.98 7.25

J = below the detection limit (the substance in question was detected, but at levels below that which can be accurately characterized by the test method)

107

Test 8: 7% moisture, 250mg/kg LPG

0

2

4

6

8

10

12

14

0 50 100 150 200Time (days)

Perc

hora

te, c

hlor

ide

(ppm

);pH

(uni

ts);

Moi

stur

e (%

) .

01002003004005006007008009001000

LPG

(mg/

kg);

CO

2 (p

pmv)

nitra

te (p

pb)

Perchlorate Chloride moisture pHNitrate LPG CO2

108

D.2.9 Test 9: 16% Moisture, 34 mg/kg H2

Time (days) 0 7 14 21 28 35 42 49 77 128 187

Perchlorate average (ppm) 8.34 7.91 - 0.58 - 0.39 0 0 0 1.33 0 std. dev. 0.04 0.05 - 0.02 - 0.54 0 0 0 0.49 0

Chloride average (ppm) 1.8 3.57 - 6.77 - 13.26 4.75 5.35 4.65 6.3 7.27 std. dev. 0.08 0.61 - 0.1 - 2.07 0.26 0.2 1.77 0.8 0.31

Nitrate average (ppb) J J - J - J J J J J 0 std. dev. - - - - - - - - - - 0

Hydrogen average (mg/kg) 29.67 36.88 35.35 70.1 122.56 37.795 76.115 62.626 75.689 75.463 56.981

CO2 average (ppmv) 25.14 22.03 - 20.92 - 19.61 36.75 0 5.22 60.5 0 moisture average (%) 14.84 14.66 - 15.71 - 15.32 15.65 15.06 15.56 15.17 15.21 pH average 7.28 7.14 - 6.5 - 7.01 6.32 6.71 7.36 7.01 7.15

J = below the detection limit (the substance in question was detected, but at levels below that which can be accurately characterized by the test method)

109

Test 9: 16% moisture, 34mg/kg H2

02468

1012141618

0 50 100 150 200Time (days)

Perc

hora

te, c

hlor

ide

(ppm

);pH

(uni

ts);

Moi

stur

e (%

) .

0

20

40

60

80

100

120

140

H2

(mg/

kg);

CO

2 (p

pmv)

nitra

te (p

pb)

Perchlorate Chloride moisture pHNitrate Hydrogen CO2

110

D.2.10 Test 10: 16% Moisture, 150 mg/kg ethyl acetate

Time (days) 0 7 14 21 28 35 42 49 77 125 184

Perchlorate average (ppm) 7.96 9.12 - 9.47 - 7.75 - 9.64 8.71 8.28 8.53std. dev. 0.36 0.002 - 0.1 - 0.34 - 0 1.44 0.63 0

Chloride average (ppm) 1.41 2.46 - 5.53 - 4.67 - 3.64 3.46 3.85 2.93std. dev. 0.08 0.01 - 0.04 - 0.31 - 0 1.33 0.27 0

Nitrate average (ppb) J J - J - J - J J J 0std. dev. - - - - - - - - - - 0

Ethyl Acetate

average (mg/kg) 85 34.5 0.07 3.672 0.73 0.142 0 0 0 0 0

CO2 average (ppmv) 0 0 - 0 - 0 - 0 0 0 0

moisture average (%) 14.05 15.46 - 15.54 - 14.94 - 14.76 14.7 15.59 15 pH average 6.9 6.9 - 7 - 6.64 - 6.81 6.99 7.05 6.82

J = below the detection limit (the substance in question was detected, but at levels below that which can be accurately characterized by the test method)

111

Test 10: 16% moisture, 150mg/kg Ethyl Acetate

0

2

4

6

8

10

12

14

16

18

0 50 100 150 200Time (days)

Perc

hora

te, c

hlor

ide

(ppm

); pH

(uni

ts);

Moi

stur

e (%

) .

0

10

20

30

40

50

60

70

80

90

Ethy

l Ace

tate

(mg/

kg);

CO

2(p

pmv)

, nitr

ate

(ppb

)

Perchlorate Chloride moisture pHNitrate Ethyl Acetate CO2

112

D.2.11 Test 11: 16% Moisture, 80 mg/kg 1-hexene

Time (days) 0 7 14 21 28 35 42 49 77 125

Perchlorate average (ppm) 8.44 8.76 - 6.7 8.23 7.72 8.02 8.91 2.54 5 std. dev. 0.28 0.23 - 0.79 0.77 0.9 0.62 0.65 2.94 3.68

Chloride average (ppm) 0.66 2.59 - 2.38 7.87 4.55 3.65 3.74 4.14 6.21 std. dev. 0.74 0.43 - 0.38 4.83 0.42 0.78 0.53 0.68 1.11

Nitrate average (ppb) J J - J J J J J J J std. dev. - - - - - - - - - -

1-Hexene average (mg/kg) 27.4 74.89 70.99 90.9 59.96 73.87 83.11 51.536 24.262 68.065 std. dev. 6.42 1.36 3.3 5.89 25.8 4.88 15.61 28.79 6.2 8

CO2 average (ppmv) 0 0 - 0 0 0 0 0 0 0 std. dev. 0 0 - 0 0 0 0 0 0 0

moisture average (%) 14.32 14.48 - 14.9 15.05 15.02 15.05 14.96 14.65 14.72 std. dev. 1.38 0.34 - 0.38 0.95 0.36 0.6 0.3 0.69 0.18

pH average 7.08 6.91 - 7.26 7.28 7.34 7.16 7.415 7.27 6.97 std. dev. 0.01 0.08 - 0.09 0.04 0.11 0.04 0.12 0.04 0

J = below the detection limit (the substance in question was detected, but at levels below that which can be accurately characterized by the test method)

113

Test 11: 16% moisture, 80mg/kg 1-Hexene

02468

1012141618

0 20 40 60 80 100 120 140Time (days)

Perc

hora

te, c

hlor

ide

(ppm

);pH

(uni

ts);

Moi

stur

e (%

) .

0

20

40

60

80

100

120

1-H

exen

e (m

g/kg

); C

O2

(ppm

v), n

itrat

e (p

pb)

Perchlorate Chloride moisture pHNitrate 1-Hexene CO2

114

D.2.12 Test 12: 16% Moisture, 75 mg/kg LPG

Time (days) 0 7 14 21 28 35 42 49 77 124 184

Perchlorate average (ppm) 8.45 9.1 - 10 - 8.84 - 10.87 3.9 4.25 2.71std. dev. 0.42 0.4 - 0.99 - 0.57 - 0.32 4.53 2.92 0.35

Chloride average (ppm) 1.56 2.52 - 3.94 - 3.97 - 3.81 4.64 6.14 6.85std. dev. 0.79 0.25 - 0.52 - 0.34 - 0.13 1.28 1.18 1.69

Nitrate average (ppb) J J - J - J - J J J J std. dev. - - - - - - - - - - -

LPG average (mg/kg) 63.53 201.05 272.06 170.12 261.36 235.2 347.99 292.187 377.18 374.665 301.312 std. dev. 7.26 75.67 14.98 26.66 27.9 5.53 24.7 5.2 34.92 5.97 0

CO2 average (ppmv) 0 0 - 0 - - 0 0 0 0std. dev. 0 0 - 0 - - 0 0 0 0

moisture average (%) 13.83 15.07 - 13.31 - 14.36 - 14.28 13.62 14.46 12.87 std. dev. 0.44 0.92 - 0.15 - 0.24 - 1.89 0.81 1.2 0

pH average 6.96 7 - 7.13 - 6.62 - 7.11 7.2 6.96 7.84 std. dev. 0.07 0.06 - 0.03 - 0.33 - 0.11 0.02 0 0

J = below the detection limit (the substance in question was detected, but at levels below that which can be accurately characterized by the test method)

115

Test 12: 16% moisture, 75mg/kg LPG

02468

1012141618

0 50 100 150 200Time (days)

Perc

hora

te, c

hlor

ide

(ppm

);pH

(uni

ts);

Moi

stur

e (%

) .

0

50

100

150

200

250

300

350

400

LPG

(mg/

kg);

CO

2 (p

pmv)

nitra

te (p

pb)

Perchlorate Chloride moisture pHNitrate LPG CO2

116

D.2.13 Test 13: 16% Moisture, 114 mg/kg H2

Time (days) 0 7 14 21 28 35 42 49 77 128 187

Perchlorate average (ppm) 5.93 8.05 - 0.49 - 0 0.25 0.59 0 0.466 0 std. dev. 3.99 0.23 - 0.05 - 0 0.43 0.69 0 0.024 0

Chloride average (ppm) 2.16 3.42 - 4.76 - 8.26 6.22 4.87 5.52 6.73 8.12 std. dev. 0.61 0.45 - 0.12 - 2.88 0.36 3.44 1.65 0.59 1.96

Nitrate average (ppb) J J - J - J J J J J J std. dev. - - - - - - - - - - -

Hydrogen average (mg/kg) 111.77 112.7 118.12 159.82 128.91 97.22 126.455 126.96 112.945 72.85 83.326std. dev. 32.6 1.6 3.07 1.14 2.37 1.7 26.02 21.87 32.67 9.02 5.153

CO2 average (ppmv) 77.67 73.88 - 210.15 - 57.35 82.48 19.98 63.52 62.38 68.14

std. dev. 2.27 0.61 - 0.78 - 2.36 18.45 20.38 67.18 3.01 0.43

moisture average (%) 14.11 14.28 - 14.76 - 14.86 14.71 15.42 14.45 15.42 15.31 std. dev. 0.02 0.13 - 0.3 - 0.55 0.02 0.44 0.86 1.06 1.23

pH average 7.33 7.31 - 6.79 - 6.95 6.6 6.68 7.04 7.045 6.38 std. dev. 0.07 0.05 - 0.04 - 0.04 0.08 0.06 0.56 0.08 0.1

J = below the detection limit (the substance in question was detected, but at levels below that which can be accurately characterized by the test method)

117

Test 13: 16% moisture, 114mg/kg H2

02468

1012141618

0 50 100 150 200Time (days)

Perc

hora

te, c

hlor

ide

(ppm

);pH

(uni

ts);

Moi

stur

e (%

) .

0

50

100

150

200

250

H2

(mg/

kg);

CO

2 (p

pmv)

nitra

te

Perchlorate Chloride moisture pHNitrate Hydrogen CO2

118

D.2.14 Test 14: 16% Moisture, 501 mg/kg ethyl acetate

Time (days) 0 7 14 21 28 35 42 49 77 125 184

Perchlorate average (ppm) 9.04 8.97 - 9.52 - 9.42 - 10.15 9.29 9.67 9.52std. dev. 0.16 0.04 - 0.13 - 0.51 - 0 1.49 0.44 0

Chloride average (ppm) 2.14 2.16 - 5.11 - 3.59 - 3.48 2.62 3.42 3.36std. dev. 0.09 0.05 - 0.19 - 0.18 - 0 0.98 0.42 0

Nitrate average (ppb) J J - J - J - J J J 0std. dev. - - - - - 83 - - - - 0

Ethyl Acetate

average (mg/kg) 296.38 282.763 3.75 113.974 1.92 25.84 31.99 4.913 1.015 0 0

CO2 average (ppmv) 0 0 - 0 - 0 - 0 0 0 0

moisture average (%) 12.99 15.74 - 14.75 - 14.74 - 15.64 15.59 15.97 15.4pH average 6.75 6.56 - 6.55 - 6.25 - 6.3 6.45 6.99 6.58J = below the detection limit (the substance in question was detected, but at levels below that which can be accurately characterized by the test method)

119

Test 14: 16% moisture, 501mg/kg Ethyl Acetate

02468

1012141618

0 50 100 150 200Time (days)

Perc

hora

te, c

hlor

ide

(ppm

); pH

(uni

ts);

Moi

stur

e (%

) .

0

50

100

150

200

250

300

350

Ethy

l Ace

tate

(mg/

kg);

CO

2(p

pmv)

, nitr

ate

(ppb

)

Perchlorate Chloride moisture pHNitrate Ethyl Acetate CO2

120

D.2.15 Test 15: 16% Moisture, 265 mg/kg 1-hexene

Time (days) 0 7 14 21 28 35 42 49 77 125

Perchlorate average (ppm) 8.51 9.11 - 7.91 8.3 7.85 7.67 2.32 1.75 1.96 std. dev. 0.01 0.05 - 0.01 0.4 0.56 0.22 0 0.016 0.54

Chloride average (ppm) 2.05 2.68 - 1.97 4.05 4.36 2.69 6.75 4.48 4.26 std. dev. 0.04 0.03 - 0.06 0.01 0.46 0.14 0 0.4 0.34

Nitrate average (ppb) J J - J J J J J J J

std. dev. - - - - - - - - - -

1-Hexene average (mg/kg) 48.215 211.11 103.13 191.27 67.84 108.556 212.885 40.65 356.845 120.96

CO2 average (ppmv) 0 0 - 0 0 0 0 0 0 0

moisture average (%) 16.51 - - 12.76 14.23 13.78 13.83 13.83 15.58 15.57 pH average 7.22 6.88 - 7.18 7.08 7.22 7.3 7.33 7.24 6.97 J = below the detection limit (the substance in question was detected, but at levels below that which can be accurately characterized by the test method)

121

Test 15: 16% moisture, 265mg/kg 1-Hexene

02468

1012141618

0 20 40 60 80 100 120 140Time (days)

Perc

hora

te, c

hlor

ide

(ppm

);pH

(uni

ts);

Moi

stur

e (%

) .

050100150200250300350400

1-H

exen

e (m

g/kg

); C

O2

(ppm

v), n

itrat

e (p

pb)

Perchlorate Chloride moisture pHNitrate 1-Hexene CO2

122

D.2.16 Test 16: 16% moisture, 250 mg/kg LPG

Time (days) 0 7 14 21 28 35 42 49 77 124 184

Perchlorate average (ppm) 8.51 8.67 - 10.08 - 8.06 - 5.77 3.83 1.1 0std. dev. 0.57 0.31 - 0.52 - 4.32 - 2 2.33 0.13 0

Chloride average (ppm) 1.04 2.19 - 4.36 - 4.66 - 5.09 4.3 5.26 6.03

std. dev. 0.18 0.28 - 0.12 - 0.69 - 0.51 0.72 0.81 0.83

Nitrate average (ppb) J J - J - J - J J J 0

std. dev. - - - - - - - - - - 0

LPG average (mg/kg) 214.2 590.55 693.16 498.5 733.52 509.3 901.34 615.87 1022.21 830.391 589.22 std. dev. 0.25 46.56 52.29 0.07 176.48 69.4 180.8 21.3 82.93 158.8828 2.76

CO2 average (ppmv) 0 0 - 0 - 0 - 0 0 0 27.5 std. dev. 0 0 - 0 - 0 - 0 0 0 38.89

moisture average (%) 15.84 15.31 - 14.2 - 13.64 - 14.47 14.38 14.78 13.85 std. dev. 1.29 1.54 - 0.85 - 0.68 - 0.37 0.07 0.15 0.05

pH average 7.04 7.23 - 7.28 - 6.96 - 7.3 7.245 6.975 7.56 std. dev. 0.99 0.02 - 0.04 - 0.1 - 0.13 0.007 0.01 0.08

J = below the detection limit (the substance in question was detected, but at levels below that which can be accurately characterized by the test method)

123

Test 16: 16% moisture, 250mg/kg LPG

02468

1012141618

0 50 100 150 200Time (days)

Perc

hora

te, c

hlor

ide

(ppm

);pH

(uni

ts);

Moi

stur

e (%

) .

0

200

400

600

800

1000

1200

LPG

(mg/

kg);

CO

2 (p

pmv)

nitra

te (p

pb)

Perchlorate Chloride moisture pHNitrate LPG CO2

124

D.2.17 Test 17: Negative control. 16% Moisture, no external electron donor.

Time (days) 0 7 14 21 28 35 42 49 77 124 183

Perchlorate average (ppm) 8.59 9.47 - 8.87 - 2.12 - 2.47 0.85 1.26 0std. dev. 0.35 0.03 - 0.08 - 1.91 - 0 0.086 0.13 0

Chloride average (ppm) 1.16 4.12 - 4.34 - 6.28 - 1.3 5.09 7.7 5.5std. dev. 0.09 0.004 - 0.04 - 0.02 - 0 0.44 0.88 0.23

Nitrate average (ppb) J J - J - J - J J J 0 std. dev. - - - - - - - - - - 0

Electron Donor average (mg/kg) 0 0 0 0 0 0 0 0 0 0 0

CO2 average (ppmv) 0 0 - 0 - 0 - 0 0 0 0

moisture average (%) 15.41 15.67 - 14.36 - 14.87 - 15.7 14.36 16.25 14.95pH average 7.07 7.09 - 7.3 - 7.05 - 6.97 7.23 7.13 7.58

J = below the detection limit (the substance in question was detected, but at levels below that which can be accurately characterized by the test method)

125

Test 17: Negative control. 16% moisture, no electron donor.

02468

1012141618

0 50 100 150 200Time (days)

Perc

hora

te, c

hlor

ide

(ppm

); pH

(uni

ts);

Moi

stur

e (%

) .

00.10.20.30.40.50.60.70.80.91

CO

2 (p

pmv)

, nitr

ate

(ppb

)

Perchlorate Chloride moisturepH Nitrate CO2

126

D.2.18 Positive control. 16% Moisture, 436 mg/kg ethanol.

Time (days) 0 7 14 21 28 35 42 49 77 124 183

Perchlorate average (ppm) 8.49 9.2 - 8.5 - 6.29 - 10.04 0 1.17 0std. dev. 0.08 0.04 - 0.27 - 0.11 - 0 0 0.08 0

Chloride average (ppm) 1.07 4.06 - 2.96 - 4.2 - 3.8 4.99 4.77 6.64std. dev. 0.03 0.03 - 3.53 - 0 - 0 0.31 0.97 0.68

Nitrate average (ppb) J J - J - J - J J J 0std. dev. - - - - - - - - - - 0

Ethanol average (mg/kg) 14.23 5.034 3.32 3.184 3.04 3.412 3.964 3.183 5.93 1.742 2.257

CO2 average (ppmv) 0 0 - 0 - 0 - 0 0 0 0

moisture average (%) 14.63 15.34 - 14.65 - 14.69 - 15.14 15.67 14.27 15.8 pH average 7.11 7.15 - 7.14 - 6.86 - 6.89 7.15 7.05 7.22

J = below the detection limit (the substance in question was detected, but at levels below that which can be accurately characterized by the test method)

127

Test 18: Positive control. 16% moisture, 436mg/kg Ethanol.

02468

1012141618

0 50 100 150 200Time (days)

Perc

hora

te, c

hlor

ide

(ppm

);pH

(uni

ts);

Moi

stur

e (%

) .

0

2

4

6

8

10

12

14

16

Etha

nol (

mg/

kg);

CO

2(p

pmv)

, nitr

ate

(ppb

)

Perchlorate Chloride moisture pHNitrate Ethanol CO2

128

Appendix E

COLUMN TESTS DATA

E.1 H2 Column Study Procedure and Calculation

Electron Donor: Hydrogen

Soil moisture: 10%

Bulk gas velocity: 0.01 cm/s

1. Measure the moisture content of the stored soil in duplicate. Add DDI water if

necessary to raise the soil moisture to 10%.

2. Weigh out the mass of soil to be packed into the two duplicate columns to make the

soil density in each column similar to the site conditions (1.6 g/mL,

GEDIT_calc_Nov2005 spreadsheet).

Column Dimensions: D = 2 in = 2in × 2.54 cm/in = 5.08 cm

H = 5 ft = 5 × 30.48 cm/ft = 152.4 cm

Area = пD2/4 = п(5.08 cm)2/4 =20.27 cm2

V = Area × H = 152.4 cm × 20.27 cm2 = 3089.15 cm3 =

3089.15 mL

So the mass of soil that needs to be packed into each column is:

Soil mass = 3089.15 mL ×1.6 g/mL = 4942.64 g = 4.94 kg

3. Pack the soil into two columns made of clear polyvinylchloride (PVC) pipe. Pack the

columns by adding 1 – 2” lifts of soil and tapping the side of the column between lifts

to promote even soil distribution within the column.

129

4. Purge both of the columns with nitrogen gas until less than 1% oxygen is detectable in

the column effluent.

5. Inject 20% hydrogen and 80% nitrogen mixed gas at a bulk gas average linear velocity,

vave = 0.01 cm/s, assuming a soil porosity, n = 40%.

Flow rate, Q = (vave×n) ×A

= (0.01 cm/s × 0.4)×20.27 cm2 = 0.081 cm3/s×60 s/min = 4.86 cm3/min =

4.86 mL/min

The mass flow controllers will be set to 4.9 mL/min.

6. The effluent of the column will be tested for H2 concentration every 30 minutes for

the first 2 hours, and then after an increase in H2 has been observed, samples will be

taken approximately every 10 minutes to capture a breakthrough curve that contains a

minimum of five points for each column.

Time to breakthrough, t = H/v

= (152.4cm)/(0.01 cm/s) × (1 hour/3600 sec) = 4.23

hours

7. After hydrogen has been observed to travel from the beginning to the end of the

duplicate columns, gas injection will be stopped, and the column ends capped.

Headspace samples for hydrogen will then be taken (at t = 0) with a 250 uL gas-tight

syringe from seven sampling ports spaced evenly along the column length (i.e., out of

the 28 total ports on the column, every third port will be sampled). The columns will

then be incubated at room temperature in the dark for 2 – 4 weeks.

8. After 2 – 4 weeks, 200 uL headspace samples will again be withdrawn from every

third sampling port of both columns to measure hydrogen and oxygen concentrations

and test for “hydrogen floating”. If hydrogen levels have dropped below 2 mg/kg, the

columns will be repurged with the 20% H2 / 80% N2 gas mixture as before, and then

130

capped. The hydrogen concentrations along the length of the column will be

remeasured prior to continued incubation.

9. After approximately 2 months of total incubation, the headspace will again be sampled

for hydrogen and oxygen as before, and then one of the duplicate columns will be

sacrificed and the soil behind every other sampling port (i.e., 14 out of the total 28

ports on the column) will be analyzed for perchlorate, chlorate, chlorite, chloride,

nitrate, nitrite, pH, and soil moisture. If, at this time, perchlorate levels are below

detection, then the second column will also be sacrificed and analyzed; if not, it will be

allowed to incubate approximately one more month before being sacrificed and

analyzed as described above.

Appendix

Electron donor sufficiency calculation:

Void fraction of soil = 40% (GEDIT_Evans_calc_Nov2005 spreadsheet)

Volume of H2 in the column = 40% × Vcolumn ×10% = 40% × 3089.15 mL × 10% =

123.57 mL

Mass of H2 in the column = 1.013 ×105 Pa × 123.57mL × 1m3/1000mL = 5.10 mol 8.314472 [m3·Pa·K-1·mol-1] × 295 K

Perchlorate concentration in the soil is about 10ppm.

Perchlorate mass in the column = 4.94 kg soil × 10 mg/kg = 49.4 mg / 99.45g/mol

= 0.5 mmol

Degrade 1mol perchlorate needs 4 mol hydrogen,

So H2 mass needed to degrade all of the perchlorate in column = 0.5 mmol × 4

= 2 mmol.

Safety factor = 5.10 × 103 mmol / 2 mmol = 2550

The hydrogen is sufficient!

131

E.2 Breakthrough Time Calculation

Taking column #1 as an example, the breakthrough time was calculated as shown:

Inlet hydrogen concentration C0 = 4.96 mg/kg.

Time (hour)

Peak Area

H2 in Soil (C) (mg/kg) c/c0 Integration

(C2/C0-C1/C0)*t2

0 0.000 0.000 0 0 0.5 0 0.000 0 0 1 18.088 0.186 0.037402 0.037402 1.5 71.017 0.728 0.146847 0.164167 2 141.871 1.455 0.293356 0.293019 2.25 173.72 1.782 0.359212 0.148177 2.5 199.008 2.041 0.411502 0.130724 2.75 221.248 2.269 0.457489 0.126465 3 248.329 2.547 0.513486 0.167992 3.25 269.721 2.766 0.55772 0.143759 3.5 285.291 2.926 0.589915 0.112683 4 305.7185 3.135 0.632155 0.168957 4.25 329.778 3.382 0.681904 0.211435 4.5 350.768 3.598 0.725306 0.195311 4.75 369.112 3.786 0.763238 0.180173 5 388.053 3.980 0.802403 0.195828 5.25 409.748 4.202 0.847263 0.235516 5.5 425.169 4.361 0.87915 0.175379 5.75 444.397 4.558 0.918909 0.228614 6 461.248 4.731 0.953753 0.209064 6.25 474.469 4.866 0.981091 0.170862 6.5 480.1355 4.924 0.992808 0.076161 6.75 483.188 4.956 0.99912 0.042605 7 481.6182 4.940 0.995874 0 8 479.245 4.915 0.990967 0 10 480.799 4.931 0.99418 0 15 481.866 4.942 0.996387 0

SUM= 3.414292 (Breakthrough time)

132

Plot breakthrough curve with time as x-axis and C/C0 as y-axis. The area

surrounded by the breakthrough curve, y-axis and the C/C0=1 line (as darked area in the

plot below), equals the breakthrough time.

0.00.10.20.30.40.50.60.70.80.91.0

0 2 4 6 8 10 12 14 16

Time (hour)

C/C

0

The integrated area =

Column #1 Breakthrough time = 3.4 h

0.00.10.20.30.40.50.60.70.80.91.0

0 2 4 6 8 10 12 14 16

Time (hour)

C/C

0

The integrated area =

Column #1 Breakthrough time = 3.4 h

It has been calculated in Appendix E.1 that the breakthrough time for an empty

column is 4.23 hours. So the breakthrough time (hour) is divided by 4.23 to be converted

to the unit of pore volume.

133

E.3 Dispersion number calculation

Time (hour) Ci (mg/kg) Cidti Ei = C/Q tiEidti ti2Eidti

0 0.000 0.000 0 0 0

0.5 0.000 0.000 0 0 0

1 0.186 0.093 0.00315 0.001575 0.001575

1.5 0.728 0.364 0.012367 0.009276 0.013913

2 1.455 0.728 0.024706 0.024706 0.049413

2.25 1.782 0.445 0.030253 0.017017 0.038289

2.5 2.041 0.510 0.034657 0.02166 0.054151

2.75 2.269 0.567 0.03853 0.026489 0.072845

3 2.547 0.637 0.043246 0.032434 0.097303

3.25 2.766 0.692 0.046971 0.038164 0.124033

3.5 2.926 0.731 0.049683 0.043472 0.152153

4 3.135 1.568 0.05324 0.10648 0.42592

4.25 3.382 0.846 0.05743 0.061019 0.259332

4.5 3.598 0.899 0.061085 0.068721 0.309244

4.75 3.786 0.946 0.06428 0.076332 0.362578

5 3.980 0.995 0.067578 0.084473 0.422365

5.25 4.202 1.051 0.071356 0.093655 0.49169

5.5 4.361 1.090 0.074042 0.101808 0.559942

5.75 4.558 1.139 0.07739 0.111249 0.639681

6 4.731 1.183 0.080325 0.120488 0.722925

6.25 4.866 1.217 0.082627 0.129105 0.806908

6.5 4.924 1.231 0.083614 0.135873 0.883175

6.75 4.956 1.239 0.084146 0.141996 0.958473

7 4.940 1.235 0.083872 0.146777 1.027437

8 4.915 4.915 0.083459 0.667673 5.341386

10 4.931 9.862 0.08373 1.674595 16.74595

15 4.942 24.710 0.083916 6.293669 94.40504

SUM 58.893 10.229 124.966

Q = Σ Ci ti = 58.89

T = Σ ti Ei dti = 10.23

134

σ2 =Σ ti2 Ei dti – T2 = 20.34

σ0 = σ2 / T2 = 0.19

σ02 = 0.38

Dispersion number d =σ02 / 2 =0.019