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Graduate College
12-2008
Evaluation of Electron Donor Materials Used to
Create Subsurface Permeable Reactive Barriers for
Enhanced Reductive Dechlorination of
Chlorinated Ethenes
Elizabeth S. Semkiw
Western Michigan University
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Semkiw, Elizabeth S., "Evaluation of Electron Donor Materials Used to Create Subsurface Permeable Reactive Barriers for Enhanced
Reductive Dechlorination of Chlorinated Ethenes" (2008). Dissertations. 815.
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EVALUATION OF ELECTRON DONOR MATERIALS USED TO CREATE
SUBSURFACE PERMEABLE REACTIVE BARRIERS FOR ENHANCED
REDUCTIVE DECHLORINATION OF CHLORINATED ETHENES
by
Elizabeth S. Semkiw
A Dissertation
Submitted to the
Faculty of The Graduate College
in partial fulfillment of the
requirements for the
Degree of Doctor of Philosophy
Department of Chemistry
Dr. Michael J. Barecelona, Advisor
Western Michigan University
Kalamazoo, Michigan
December 2008
UMI Number: 3340202
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Copyright by
Elizabeth S. Semkiw
2008
ACKNOWLEDGEMENTS
I wish, first of all, to thank Dr. Michael Barcelona for the generous support and
guidance he's given me as a professor, research advisor, and mentor. Thanks to his
encouragement, abundance of ideas, and insightful comments, I was able to continue
moving along a positive direction in this research. His faith and wry sense of humor
illuminated the days when I wasn't seeing the forest for the trees.
I very much appreciate the efforts of my research committee members to guide
my progress. Thanks to Dr. Michael Dybas for sharing his knowledge at every turn and
for his very kind attention and assistance with the collection and handling of sediment
and water samples and the preparation of microcosms in his anaerobic glove chamber at
Michigan State University. I am grateful, too, for his generosity in discussing and
sharing the results of his own microcosm experiments. Thanks to Dr. James Kiddle, Dr.
R. V. Krishnamurthy, Dr. Sherine Obare, and Dr. David Reinhold for their questions,
comments, and suggestions.
Thanks to Dr. Susan Stapleton for access to the resources of her lab and for being
wonderfully supportive every step of the way.
I am grateful to Dr. Moonkoo Kim for his friendship and guidance with the
laboratory work, particularly the instrumental analyses. Thanks to the time he took to
revive the purge-and-trap GC/MS and to do the initial VOC calibration, I was able to get
off to a good start in the research.
ii
Acknowledgements - Continued
I'd like to thank the staff of Ground Water Solutions, Inc. of Lansing, Michigan,
most especially Joel Parker and Bruce Beltman, for their kind assistance with the
collection and handling of sediment and ground water samples and sharing of field data.
I soon came to appreciate their dedication to hard work in often adverse conditions - the
cold in winter, tornados in spring, and swarms of bees in summer.
I'd like to thank SiRem Labs of Ontario, Canada for kindly providing the KB1
and KB1/LV1 culture prior to each experiment. Thanks very much to Phil Dennis of
SiRem for his recommendations on the effective use of the organisms for these
experiments.
Thanks to the many friends I've met in and around the lab: Pete Stuurwold,
Elissa Nourey, Evan Garrett, Matt VanNess, Bill Lizik, Elizabeth Delaney, Chad
Lawrence, Katie Anderson, Brendan Sanchez, Doug Mandrick, other chemistry graduate
students, and faculty. Thanks to Dr. Raymond Sung, Sean Bashaw, Pam McCartney, and
Annie Dobbs for all of their help.
Finally, I wish to thank my husband, Carl, and children, Thomas and Tess, for
being my emotional support, from beginning to end.
Elizabeth S. Semkiw
in
TABLE OF CONTENTS
ACKNOWLEDGMENTS
ii
LIST OF TABLES
viii
LIST OF FIGURES
ix
LIST OF ACRONYMS AND ABBREVIATIONS
xi
CHAPTER
I. INTRODUCTION
1
History and Scope of the Problem
1
In-Situ Biodegradation of Chlorinated Ethenes
4
Redox Conditions
4
Chlorinated Ethene Biodegradation Processes
6
Biostimulation
7
Permeable Reactive Barriers
9
Dairy Whey as a Biostimulant
11
Scope of this Study
12
II. MATERIALS AND METHODS
14
Introduction
14
Site Characterization
15
PRB Creation and Maintenance
16
Sampling Procedures
19
iv
Table of Contents - Continued
CHAPTER
Microcosm Preparation
20
Whey Degradation Pathway Experiment
20
Electron Donor Comparison Experiments
22
Analytical Methods
24
Chemicals
24
Chlorinated Ethenes
24
Dissolved Gasses
26
Organic Acids
27
III. PRELIMINARY MICROCOSM RESULTS
.
34
Native Dechlorinator Identity and Growth
34
Whey Degradation Pathway
36
IV. RESULTS OF THE LABORATORY COMPARISON OF ELECTRONDONOR MATERIAL EFFICIENCY
38
Introduction
38
Dechlorination Rates
39
Upgradient Source Zone Microcosms
39
Treatment Zone Microcosms
39
Carbon Flow in Whey-amended Microcosms
46
Carbon Flow in Lactate and HRC Amended Microcosms
52
H2 and Methane Production
53
v
Table of Contents - Continued
CHAPTER
Control Results
57
V. RESULTS OF THE FIELD STUDY OF ENHANCED TCE
BIODEGRADATION WITHIN A FULL-SCALE WHEY PRB
60
Introduction
60
Long-term CE and Ethene+Ethane Trends
60
Pilot Phase Trends
61
Operational Phase Trends
62
Effects of Whey Fermentation
67
VI. DISCUSSION
74
Electron Donor Material Comparison
74
Field Study
78
Electron Donor Material Cost Comparison
79
Conclusion
81
APPENDICES
A. Field Design Microcosm Study
83
B. TOC and TIC of Sediment Samples from Whey Injection and PRB
Monitoring Well Cores
86
C. Comparison of Organic Acid Concentrations Determined by GC/MS
andHPLC
91
D. Microbial Contamination of Whey Injection Wells
94
E. Redox Manipulation Capability of Whey
99
vi
Table of Contents - Continued
BIBLIOGRAPHY
103
vii
LIST OF TABLES
1.
H2 Concentrations and Standard Gibbs Free Energy Changes of
Terminal Electron Accepting Processes
6
2.
GC/MS Organic Acid - TMS Derivative Retention Times
29
3.
4.
GC/MS TMS-Organic Acid Derivative Ions and Retention Times
Percent Recoveries of Organic Acids Following Anion Exchange,
Lyophilization, andTMSI Derivatization
30
5.
6.
7.
8.
9.
31
Percent Recoveries of Organic Acids Following Lyophilization, BSTFATMCS Derivatization, and Dissolution in Hexane
32
Average Dehalococcoides DNA Concentrations and Yields in Source
Zone Aquifer Microcosms
35
Organic Acid Carbon Concentrations in Source Zone and Treatment
Zone Microcosms
49
Upgradient Source Zone Chlorinated Ethene and Ethene Average
Concentrations
67
Comparison of Estimated Material Costs of Whey, Lactate Syrup, and
HRC
81
vin
LIST OF FIGURES
1985-2001 National Water Quality Assessment (NWQA) Detection
Frequency of Select VOCs (A) and NWQA Study Units (B)
2
Permeable Reactive Barrier
9
Anaerobic Metabolic Pathways of Simple Sugars
15
Map of Field Site Injection and Monitoring Wells
17
Loading Masses and In-Situ Concentrations of 10 Whey Slurry Injections.
18
Preliminary Experiment Total Chloroethene Percentages (A) and
Total Organic Acid Carbon Percentages (B)
Total Chloroethene Percentages in Donor-amended Upgradient Source
Zone Microcosms (A) and Treatment Zone Microcosms (B)
41
Chlorinated Ethenes and Ethene Concentrations in Whey-amended
Upgradient Source Zone Microcosms (A) and Treatment Zone
Microcosms (B)
42
Chlorinated Ethenes and Ethene Concentrations in Whey+KBl/LVl
Amended Upgradient Source Zone Microcosms (A) and Treament Zone
Microcosms (B)
43
Chlorinated Ethenes and Ethene Concentrations in Lactate+KBl/LVl
Amended Upgradient Source Zone Microcosms (A) and Treatment
Zone Microcosms (B)
44
Chlorinated Ethenes and Ethene Concentrations in HRC+KB1/LV1
Amended Upgradient Source Zone Microcosms (A) and Treatment
Zone Microcosms (B)
45
Organic Acid Carbon Percentages in Donor-amended Source Zone
Microcosms (A) and Treatment Zone Microcosms (B)
48
Acetate Equivalent Consumption in Whey-amended Treatment Zone
Microcosms
52
ix
37
List of Figures - Continued
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
H2 Partial Pressures in Donor-amended Source Zone Microcosms (A) and
Treatment Zone Microcosms (B)
56
Methane in Donor-amended Source Zone Microcosms (A) and Treatment
Zone Microcosms (B)
57
Chlorinated Ethene Concentrations in Source Zone Bioaugmented Control
Microcosms (A) and Pasteurized Control Microcosms (B)
58
Chlorinated Ethene Concentrations in Treatment Zone Bioaugmented
Control Microcosms (A) and Pasteurized Control Microcosms (B)
59
Chlorinated Ethene and Ethene+Ethane Distributions in Injection Well
BIP3 (A) and Downgradient Wells NMW1A (B) and NMW1B (C)
64
Chlorinated Ethene and Ethene+Ethane Distributions in Injection Well
BIP7 (A) and Downgradient Well NMW2B (B)
65
Average Chlorinated Ethenes During Treatment Periods 5 - 10 in Injection
Wells BIP3 (A) and BIP7 (B)
66
COD and TCE Flux During Treatment Periods 5 - 10 in Injection Wells
BIP3 (A) and BIP7 (B)
70
Organic Acid Carbon Distribution in Treatment Period 10 in Injection
Well BIP3 (A) and Organic Acid Carbon Percentages during Treatment
Period 10 in Wells BIP3 and NMW1A (B)
71
Chlorinated Ethene Distributions in BIP 3 (A), NMW1A (B), and NMW1B
(C) During Treatment Period 10
72
Chlorinated Ethene Distributions in BIP7 (A) and NMW2B (B) During
Treatment Period 10
73
x
LIST OF ACRONYMS AND ABBREVIATIONS
BCA
BGS
BIP
BOD
BSA
BSTFA
CE
COD
cDCE
DNA
DNAPL
EPA
FID
GC
HPLC
HRC
IS
KB1
KB1/LV1
MCL
MS
MTBE
NMW
NOM
NPL
NWQA
OA
PCE
PCR
PRB
PRP
P/T
PVC
REDOX
rRNA
RGA
SAX
SDWA
bicinchoninic acid
below ground surface
bioamendment injection port
biological oxygen demand
bovine serum albumin
N,0-bis(trimethylsilyl)trifluoroacetamide
chlorinated ethene
chemical oxygen demand
cis-dichloroethene
deoxyribonucleic acid
dense non-aqueous phase liquid
Environmental Protection Agency
flame ionization detector
gas chromotographer
high performance liquid chromotographer
hydrogen release compound
internal standard
Dehalococcoides-containing bacterial culture
Dehalococcoides-containing bacterial culture
maximum contaminant level
mass spectrometer or mass spectrometry
methyl tert-butyl ether
nutrient monitoring well
natural organic matter
national priority list
national water quality assessment
organic acid
tetrachloroethene
polymerase chain reaction
permeable reactive barrier
polymeric reverse phase
purge and trap
polyvinyl chloride
oxidation-reduction
ribosomal ribonucleic acid
reduction gas analyzer
strong anion exchange
Safe Drinking Water Act
XI
List of Acronyms and Abbreviations - Continued
SIM
TBAOH
TDTMABr
TCE
TEA
TEAP
TIC
TMCS
TMS
TMSI
TOC
WAX
VC
VOC
select ion monitoring
tetrabutylammonium hydroxide
tetradecyltrimethylammonium bromide
trichloroethene
terminal electron acceptor
terminal electron accepting process
total inorganic carbon
trimethylchlorosilane
trimethylsilyl
trimethylsilylimidazole
total organic carbon
weak anion exchange
vinyl chloride
volatile organic compound
xii
CHAPTER I
INTRODUCTION
History and Scope of the Problem
The adequate assessment and protection of ground water quality are important for
maintaining a safe water supply for human consumption, as approximately half of the
United States population drinks water withdrawn from ground water systems (Shapiro et
al., 2004). Rural populations may be particularly susceptible to exposure to toxic water
contaminants, as a vast majority (97%) obtain drinking water from domestic wells which
aren't covered by the Safe Drinking Water Act (SDWA) and aren't systematically
monitored (Shapiro et al., 2004). Of further concern are the potentially deleterious
effects of ground water contamination on surface water quality as well as subsurface
microbial ecology, the importance and natural complexity of which have yet to be fully
deciphered and generally appreciated.
Recent national surveys of major aquifer and public and domestic well water
quality confirm the widespread presence of volatile organic compounds (VOCs) in
ground water (Moran et al., 2007; Moran et al., 2005). Prominent ground water
contaminants include chlorinated ethenes (CEs) and methyl t-butyl ether (MTBE) (Fig.l),
apparently due to their extensive migration from numerous contamination source points
(Moran et al., 2007; USEPA, 2002). CEs are persistent contaminants of National Priority
List (NPL) sites (ATSDR, 2007a). In 2007, tetrachloroethene (PCE) and trichloroethene
(TCE) were ranked the 3 rd and 4th most common and hazardous contaminants,
respectively, at waste sites where human exposure has been documented (ATSDR,
1
2007b). Meanwhile, numerous source point sites may have yet to be identified and
characterized, and a lack of funding for active treatment may be preventing adequate
contaminant migration control at contaminated sites of lower priority.
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EXPLANATION
B.
«
•
'
r
(T lHW>>Ma>IMari«ln<^IWMarSldlr
Figure 1. 1985-2001 National Water Quality Assessment (NWQA) Detection Frequency of Select VOCs
(A) and NWQA Study Units (B). Chloroform is a byproduct of water chlorination. www.usgs.gov.
circular 1292.
2
CEs are toxic industrial and commercial solvents and components of household
and agricultural applications. Used commonly as a dry cleaning solvent at approximately
30,000 sites nationwide, PCE can affect the nervous and reproductive systems upon high
or prolonged exposure and has caused liver and kidney damage or cancer in laboratory
animals (ATSDR, 1997). TCE is used primarily as a degreasing agent in engine-parts
manufacturing and can cause nervous, fetal, liver, and lung damage and abnormal
heartbeat upon high or prolonged exposure, and studies show evidence of cancer risk in
humans (ATSDR, 2003). Vinyl chloride (VC) is a reactant in the preparation of PVC.
The presence of VC in ground water results from spills, leaks, or discharge of mixtures
containing either VC or halogenated compounds which undergo in-situ degradation to
VC (e.g. higher chlorinated ethenes). VC is carcinogenic, and long-term exposure to VC
may result in damage to the liver, immune and nervous systems (ATSDR, 2006). U.S.
EPA drinking water standards (i.e. Maximum Contaminant Levels (MCL)) are 5 ug/L
(PCE and TCE) and 2 ug/L (VC).
Conventional pump-and-treat strategies were initiated in the 1980s to contain and
remediate contaminated ground water. VOCs, initially believed to be recalcitrant to
biodegradation, were physically removed by ex-situ treatment methods (e.g. air stripping,
activated carbon filtration) after contaminated ground water was pumped to the surface.
Pump-and-treat is costly, however, and can be prolonged as contaminants isolated in pore
spaces or as globules of dense non-aqueous phase liquid (DNAPL) or adsorbed to
sediment gradually dissolve in ground water (U.S.EPA, 1996a).
In the mid-1980s, CEs were shown to undergo microbially-mediated reductive
dechlorination under anaerobic conditions which may predominate in contaminant
3
plumes (Vogel and McCarty, 1985). Passive monitoring and remediation strategies
which rely on the natural attenuation/intrinsic bioremediation of contaminants may,
however, be insufficient at sites containing large contaminant plumes, complex site
hydrogeologies, and/or sparse monitoring well grids.
The persistence, mobility, and widespread presence of pollutants in the subsurface
warrant the implementation of more stringent control measures, requiring, in part,
focused attention on the improvement of ground water remediation strategies. To
increase the efficiency and lower the cost of site remediation, treatment strategies which
enhance the rates of in-situ contaminant degradation are currently being investigated to
either replace or supplement conventional pump-and-treat strategies (U.S.EPA, 2000).
Alternative or complementary ground water remediation strategies include the addition of
carbon substrate (biostimulation) and/or biomass (bioaugmentation) to the subsurface for
the enhancement of rates of in-situ biodegradation of contaminant plumes.
In-situ Biodegradation of Chlorinated Ethenes
Redox Conditions
Oxidation-reduction (redox) conditions are a primary determining factor of in-situ
CE biodegradation efficiency. Redox zones of natural and contaminated ground water
systems have been characterized according to the dominant terminal electron accepting
processes (TEAPs) by which H2 is oxidized (Chapelle, 2001). Changes in Gibbs free
energy and minimum H2 threshold levels for TEAPs are shown in Table 1. In the
dominant TEAP, H2 is consumed more efficiently (i.e., the half-velocity constant, Ks, is
lower) and at lower concentrations. Microorganisms which mediate the dominant TEAP
4
apparently thrive by maintaining H2 concentrations below the minimum threshold levels
of competing hydrogenotrophic microorganisms.
Redox zones become established as inorganic terminal electron acceptors (TEAs)
which yield higher free energy upon reduction are successively depleted: nitrate>Mn (IV)
> Fe(III) > sulfate > HCO3". In a pristine, confined aquifer, conditions become more
highly reducing with distance from an oxygen source, as TEAs are successively depleted
(Lovley et al., 1994). Redox zones develop in reverse order in contaminated conditions
with distance from a source of readily oxidized contaminant, due to the rapid depletion of
TEAs near the source zone (Lovley et al., 1994).
Enriched microcosm and fixed-bed column studies have shown that PCE and
TCE undergo microbially-mediated, sequential reductive dechlorination to ethene and
ethane under anaerobic conditions, with and without methanogenesis (DiStefano et al.,
1991; Freedman and Gossett, 1989; deBruin et al., 1992). Reductive dechlorination of the
more highly chlorinated (more oxidized) ethenes PCE and TCE is energetically favorable
and may occur rapidly under suboxic (nitrate-reducing or Fe(III)-reducing) and sulfatereducing conditions (Vogel et al., 1987; McCarty, 1997; Bagley and Gossett, 1990),
while complete reductive dechlorination appears to require methanogenic conditions
characterized by higher levels of H2.
5
Table 1. H2 Concentrations and Standard Gibbs Free Energy Changes of Terminal Electron Accepting
Processes. Note that TEAPs are not in thermodynamic equilibrium and the H2 concentrations are transient
at the nM level.
TEAPa
Characteristic H?
concentrations (nM)a
0.01 - 0.05
AG0 (kJ/H2)a
Mn(IV)-Reduction
MnC-2 + H2 -*Mn(OH)2
0.1-0.3
163
Fe(III)-Reduction
2Fe(OH)3 42Fe(OH)2 + 2H 2 0
0.2-0.8
50
Sulfate-Reduction
S042" + 4H2 + H+ ->HS" + 4H 2 0
1.0-4.0
38
Methanogenesis
HC03" + 4H2 +H+ ^CH 4 + 3H 2 0
5.0-15.0
34
Nitrate-Reduction
2N03" + 5H2 + 2H+ -*N2 + 6H 2 0
224
a. Chapelle, 2001
Chlorinated Ethene Biodegradation Processes
Chlororespiration, the metabolic process in which microorganisms use chlorinated
organic compounds as TEAs for energy production and growth, is likely the dominant
CE-biodegradation process at CE-contaminated sites (Loffler et al., 1999). Reductive
dechlorination of CEs may also occur by cometabolic processes, mediated by sulfate
reducers, methogens, or acetogens (ref. in Loffler et al, 1999). Dehalococcoides
ethenogenes strain 195, the first isolate shown to dechlorinate PCE to ethene, respires
PCE to VC but dechlorinates VC to ethene by a slower, cometabolic process (MaymoGatell et al., 1997; Maymo-Gatell et al., 1999). Dehalococcoides sp. strain FL2 also
dechlorinates VC to ethene cometabolically (He et al., 2005), while strains BAV1, GT,
6
VS, and KB1 are capable of the metabolic dechlorination of VC to ethene (He et al.,
2003; Duhamel et al., 2004; Muller et al., 2004; Sung et al., 2006). Dehalococcoides sp.
are a widely distributed bacterial species, native to European and North American sites
where complete CE dechlorination to ethene has been observed (Hendrickson et al,
2002). At 3 of the 24 CE-contaminated sites surveyed by Hendrickson et al. (2002),
partial dechlorination was apparently mediated by microbial species other than
Dehalococcoides.
Several bacterial populations capable of PCE respiration, including
Dehalococcoides sp., Dehalospirillum multivorans, and Dehalobacter restrictus, use H2
as a direct electron donor (Fetzner, 1998) (Eq. 1). Dehalococcoides and Dehalobacter
restrictus are obligate hydrogenotrophs (Fetzner, 1998). Dehalospirillum multivorans
may use pyruvate, lactate, ethanol, formate and glycerol as direct electron donors, in
addition to H2 (Fennell et al., 1997). Desulfuromonas chloroethenica can use acetate as a
direct electron donor (He et al., 2002).
C2CI4 + H2 ^C2HC13 + H4" + CI"
PCE
Eq. 1
TCE
Biostimulation
Intrinsic (i.e. passive) bioremediation strategies require the presence of native CEdegrading microorganisms and either bioavailable natural organic matter (NOM) or
anthropogenic organic compounds which may act as either direct or indirect electron
donors. The fermentation of electron-donor species may also serve to establish
sufficiently reducing conditions favorable for reductive dechlorination. At sites
7
characterized by low or depleted levels or low reactivity of electron donor species (e.g.
co-contaminants, NOM), bioremediation by reductive dechlorination requires the
addition of a carbon substrate stimulant. Excess electron donor may be needed to create
appropriate redox conditions and to drive complete dechlorination. Indeed, high levels of
direct electron donor (i.e. H2) have been shown to support improved long-term PCE
dechlorination in the presence of an active methanogenic community (Carr and Hughes,
1998). Overwhelming a system with carbon substrate, however, may stimulate primarily
the fast growth of alternate hydrogenotrophic populations (e.g. sulfate-reducers or
methanogens) resulting in the diversion of reducing equivalents away from
dechlorination.
Dechlorination efficiency may be improved by establishing conditions that favor
reductive dechlorination which, relative to methanogenesis, may be characterized by a
lower half-velocity constant and minimum H2 threshold concentration (Smatlak et al.,
1996). A mixed culture study by Fennell et al. (1997) indicated that slow organic acid
fermentation at low H2 partial pressures gives selective advantage to dechlorinators over
methanogens and that managing H2 delivery may be an efficient biostimulation strategy.
While the majority of isolated dechlorinating microbial species use H2 as a direct electron
donor, aquifer microcosm experiments by He et al. (2002) indicated an improved strategy
might increase the flux of H2 and acetate to stimulate both hydrogenotrophic and
acetotrophic dechlorinating populations (i.e. Desulfuromonas spp. which use acetate as a
direct electron donor in the conversion of PCE to cDCE).
Results of several pilot and full-scale field investigations suggest that stimulated
in-situ bioremediation is a potentially less expensive, more efficient technology for the
8
treatment of chlorinated hydrocarbons (USEPA, 2000). Understanding the fate of carbon
substrates, which may consist of a mixture of primary components and biodegrade to an
array of intermediate products, is, indeed, key to the development of efficient
biostimulation strategies.
Permeable Reactive Barriers
Alternative or complementary remediation strategies include the emplacement of
subsurface permeable reactive barriers (PRBs), zones of enhanced reactivity through
which ground water flows (Fig. 2). PRBs have been designed to intercept and treat
contaminated ground water to either contain contaminant plumes within site boundaries
or diminish the impact of contaminant release from source zones (Kalin, 2004; Striegel,
2001). The microbial ecology of PRBs is of particular importance, as a wide range of
ground water and sediment contaminants may undergo degradation by microbiallymediated processes (Kalin, 2004).
PerftMNble Rwcthw Barr ier
Figure 2. Permeable Reactive Barrier, www.powellassociates.com
9
Zero-valent iron (Fe°), the most common PRB material, has been shown to
enhance rates of abiotic reduction of halogenated hydrocarbons. The performance and
feasibility of Fe° technology are contingent upon the long-term reactivity and
permeability of the barrier and whether renewal frequency for the material is required
(Powell et al., 2002; Zhang and Gillham, 2005). A wide variety of organic carbon
substrates have also been employed in PRBs which may create and strengthen reducing
conditions favorable for the anaerobic biodegradation of CEs and serve as electron
donors for reductive dechlorination. Carbon substrate materials include lactate syrup,
molasses, emulsified vegetable oil, dairy whey, mulch, and Hydrogen Release
Compound® (HRC). Early field investigations of bioreactive barriers have demonstrated
rapid complete dechlorination in conjunction with the stimulated growth of
chlororespirers (Lendvay et al., 2003) that may thrive in symbiotic relationship with
fermentative microbes. Yet, performance and cost evaluations of carbon substrate
biobarriers await long-term studies of substrate efficiencies, application frequency, and
treatment design aimed at complete contaminant conversion to nontoxic products,
including ethane, ethene and CO2 (Barcelona, 2005; Barcelona and Xie, 2001).
The application of electron donor within the contaminant source zone is a
potentially efficient in-situ bioremediation strategy. An initial feasibility study indicated
that dechlorinating microbial populations may thrive at very high concentrations of CEs,
including saturating PCE concentrations (0.9 mM, or approximately 150 ppm) and TCE
concentrations up to 2.26 mM (297.2 ppm) (Yang and McCarty, 2000). Source-zone
treatment efforts may be particularly cost-effective if the CE concentrations are inhibitory
to competitive hydrogenotrophic processes, i.e. methanogenesis (Yang and McCarty,
10
2000). Complete dechlorination to ethene may, however, require treatment downgradient
of DNAPL source zones, due to the relatively slow rates of anaerobic degradation of
cDCE and VC and their high aqueous solubilities, as well as potential inhibitory effects
of high PCE or TCE concentrations on complete dechlorination (Adamson et al., 2003).
Indeed, the efficiency of in-situ bioremediation strategies may be maximized by
the placement of PRBs at the downgradient edge or in close proximity to a source zone
for the interception of high concentrations of CEs. As VC may undergo reductive
dechlorination slowly under anaerobic conditions (i.e., via anaerobic cometabolism), and
frequently drives remediation efforts if detected at property boundaries, it is important
that PRB design studies focus on the occurrence of complete dechlorination either within
or downgradient of the treatment zone.
Dairy Whey as a Biostimulant
Dairy whey is an abundant, sustainable, food-grade electron-donor material and,
thus, has been used in several studies of stimulated bioremediation of common pollutants.
Fermented whey was shown in a recent microcosm study to stimulate the aerobic
biodegradation of petroleum hydrocarbon n-hexadecane as an organic growth supplement
(Ostberg et al., 2007). Laboratory studies have demonstrated the immobilization of
selenium in mine wastes by whey-enhanced Se reduction (Knotek-Smith et al., 2006;
Bledsoe et al., 1999). Enhanced sulfate reduction, shown to occur in batches amended
with inexpensive organic by-products including whey, suggested neutralization of acidic
mine lakes may be achieved by addition of organic carbon (Fauville et al., 2004).
Dried dairy whey can be slurried and then pressure grouted (Barcelona, 2005;
Barcelona and Xie, 2001) and thus may be considered a prime candidate for creating and
maintaining a well-distributed, active biobarrier at low cost. Prior field-scale research
has demonstrated whey's value as a redox adjustment barrier material (Barcelona and
Xie, 2001). In a laboratory-based comparison of the efficiencies of food-grade electron
donors used to support the anaerobic dechlorination of PCE, dairy whey was found to
effectively support the dechlorination process at a material cost ($0.04/lb. PCE converted
to dichloroethene (DCE)) that would hardly approach the estimated cost of reducing
compounds such as sodium benzoate ($37/lb. PCE converted to ethene) (DiStefano et al.,
2001).
Scope of this Study
This study combines a long-term field investigation and laboratory aquifer
microcosm experiments to better evaluate the effectiveness and efficiency of complex
electron donor materials used to create bioreactive barriers for the enhanced
biodegradation of CEs ~ long-term and widespread ground water contaminants which
may serve as terminal electron acceptors in chlororespiration. Dairy whey, lactate syrup
(lactate), and HRC are common electron donor materials used to create and maintain
permeable bioreactive barriers. They are water-soluble and thus may be used to create a
PRB which extends the depth and the width of a contaminant plume. The laboratory
component of the study describes the first side-by-side comparison of the efficiencies of
these three biobarrier carbon substrates, as determined by comparison of dechlorination
rates, substrate degradation pathways, and H2 production. The field component of the
12
study examines for the first time the long-term efficacy of a full-scale whey PRB
designed to dechlorinate high concentrations of CEs (102-103 ug/L) migrating from a
source zone. The field results combined with laboratory analyses of whey anaerobic
degradation pathways and intermediate lifetimes allowed completion of a whey materialcost analysis.
In-depth analyses of PRB carbon substrate efficiencies and cost comparisons may
facilitate more efficient use of PRB technology and more effective site remediation.
Such investigative efforts may encourage more frequent applications of alternative
technologies and benefit mitigation of the impact of industrial and commercial site
contamination on national ground water quality.
13
CHAPTER II
MATERIALS AND METHODS
Introduction
The field work was performed at a TCE-contaminated industrial site located near
Battle Creek, Michigan. The field study spanned 5 years, during which time a full-scale
bioreactive barrier was created and maintained by 10 biannual whey injections.
Preliminary aquifer microcosm experiments were performed to investigate the identity of
native dechlorinating populations and their growth on whey and TCE (Appendix A) and
to design and test the field whey addition loads and frequency. Prior to the 6 whey
injection, an aquifer microcosm experiment was performed to determine whey's
anaerobic degradation pathway. Following the 8 injection, aquifer microcosms were
prepared with ground water and sediment collected within the source zone, upgradient of
the whey PRB for the comparison of electron donor material efficiencies (whey, lactate,
and HRC) and within the treatment zone for more in-depth investigation of the
effectiveness of the whey barrier and to more fully evaluate the efficiency of whey as a
donor material.
Dairy whey is composed of approximately 70% a-lactose (C12H22O11), lactate
syrup contains approximately 60% sodium lactate, and HRC, a proprietary mixture,
contains a polylactate ester which reportedly releases lactate upon hydrolysis. Organic
acids were hypothesized, according to known metabolic pathways (Fig. 3), to be primary
degradation products of whey lactose and lactate. Efforts to develop an analytical
method using GC/MS for the improved detection of an array of low-level organic acids
14
preceded performance of the microcosm experiments described in this study. Organic
acid concentrations in samples from the whey degradation pathway experiment were
detected using both GC/MS and HPLC methods. HPLC was used for organic acid
determinations during the electron donor comparison study and in the field study.
Simple sugars Amino nclds
X /
COOH
H2
Hydrogen
Prop Honks
acid
V
jr"~~' HCOOH
J0*
Pomifa
ac
Carbon
dioxide
Methane
Figure 3. Anaerobic Metabolic Pathways of Simple Sugars, www.soils.umn.edu
Site Characterization
For approximately 60 years, chlorinated solvent solutions were released into a
disposal pond at an industrial site located near Battle Creek, Michigan. A site map of
wells located within the disposal area is shown in Figure 4. Multiple borings at various
depths were completed in the 1980s and through 1998 (MW wells) to characterize the
local hydrogeology and to determine and monitor the dimensions of the contaminant
plume, which discharges to the Kalamazoo River located approximately 0.5 miles north
of the site. A 7-65 ft.-thick layer of glacial deposits, composed predominantly of sand
15
and gravel, overlies the Marshall aquifer, a regional sandstone aquifer. The water table
elevation is typically 10-15 ft. below ground surface (bgs). Beneath the former disposal
pond is a 10-15 ft.-thick calcareous silt layer embedded with peat, which partially
confines the upper (unconsolidated) saturated zone and may cause local perching of
ground water. The ground water flow direction was determined by mapping of
potentiometric surface contours. Ground water flow rates were estimated to be 0.5 - 1
ft/day.
PRB Creation and Maintenance
Early treatment efforts, including conventional pump-and-treat, were complicated
by the biofouling of pumping wells and were determined to be inefficient relative to
natural attenuation processes within the plume. In 2002, following microcosm studies
which suggested chlororespiration could be stimulated by addition of whey (Appendix
A), additional wells were installed to create, maintain, and monitor a full-scale dairy
whey PRB to be located within the downgradient region of the source zone,
approximately bordering the former disposal pond. Nine injection wells (BIP wells) were
installed approximately 30 feet apart to form a ~300 ft. transect of the contaminant plume
at the northern boundary of the source area. Wells were installed downgradient of the
PRB (NMW wells) and upgradient within the source zone (not shown on map) to monitor
the effectiveness of the PRB. PRB monitoring wells NMW1A and NMW IB were
aligned with BIP3, NMW1B and NMW2B were aligned with BIP7, and BMW4/2 was
aligned with BIP4, according to estimates of ground water flow direction. PRB injection
and monitoring wells were installed to the depth of the upper bedrock, approximately 4055 ft. bgs. The lengths of the well screens, located in the saturated zone, were 20-30 ft.
16
N
BiP-6
BiP-7
Prod
Well
NMW-2B
NMW-2A
MW-250*
«W-29Si
PW_41
^
MW-S
_MW-!9S
PW-2
MW-190
^
I
HI
MW-500W
MW-50QW-5QS
MW-500W-150S
Figure 4. Map of Field Site Injection and Monitoring Wells, www.gwsi.biz MW wells are plume
monitoring wells. BIP wells are PRB injection wells. NMW are PRB monitoring wells. The Kalamazoo
River is located approximately 0.5 miles north of the site. The source area is bounded by monitoring wells
MW-500 to the south, MW-3 to the northeast, and MW-8 to the north-west.
17
Dried whey masses and concentrations of the initial ten biannual injections are
shown in Figure 5. Conservative whey loading estimates, ranging from 0.2 to 1.7 kg per
m3 aquifer material, were used to avoid substrate-saturating conditions and limit the
consequent stimulation of methanogenesis. Treatment periods 1-7 constituted a low
whey mass, low slurry volume pilot phase. During the pilot phase of the study, whey
slurry volumes were estimated to fill 8.2m of aquifer volume, or approximately 0.9m at
each of the 9 injection wells. During the operational phase (treatment periods 8-10),
whey masses were increased between 17- and 135-fold and slurry volumes were
increased approximately 115-fold relative to pilot-phase injections. The increased whey
slurry volumes were estimated to fill approximately 1100-1700 m3 of aquifer volume.
Whey injections 1-10
1000
* ^ ^ ^ J> # ^ ^ jf
• mass
• concentration
^
Time (days after initial injection)
Figure 5. Loading Masses and In-Situ Concentrations of 10 Whey Slurry Injections. Concentration is
expressed as kg whey per m3 aquifer material. Whey was injected biannually for 5 years. A low-volume
pilot phase occurred through injection 7. The fully-operational phase occurred during treatment periods 810.
Through the 8 injection, dried whey slurried in extracted ground water was
injected into the nine injection wells. Beginning with treatment period 9, whey's
distribution was, furthermore, enhanced by a coupled injection/extraction delivery motif
(Dybas et al., 2002). Dried whey slurry and extracted ground water were combined ex-
18
situ in three ground water extraction and injection loops that connected three BIP wells
each: BIPs 1-3, BIPs 4-6, and BIPs 7-9. In each loop, ground water was extracted from a
BIP well at approximately 5.5 gallons/min. (20 L/min), mixed in-line with concentrated
whey slurry (prepared as 50 lbs/100 L per day), and then injected into the adjacent two
BIP wells. The final whey injection concentration was 50 lbs/ 28,900 L per loop per day,
or approximately 750 mg/L. BIPs 2, 5, and 8 were the extraction wells in the 9
injection event. BIPs 1,4, and 7 were the extraction wells in the 10th injection event.
Circulation was stopped when chemical oxygen demand (COD) increased in extraction
well samples, indicating migration of electron donor along the transect line. Total
injection times were 3-4 days.
Sampling Procedures
In the field study, ground water samples were collected using low-flow techniques
(Puis and Barcelona, 1996) from injection wells BIP3 and BIP7 and from the PRB
monitoring wells located approximately 5 feet (NMW1 A) and 20 feet (NMW1B &
NMW2B) downgradient and approximately 20 feet (BMW4/2) upgradient of the
injection well transect. Samples for organic acid determinations and, during treatment
period 10, for determinations of dissolved gases and VOCs were collected in duplicate
borosilicate 40mL vials. Vials were filled with minimal turbulence for dissolved gas and
VOC determinations and half-filled for organic acid determinations and kept inverted and
cool during immediate transport to the lab. The VOC and dissolved gas sample vials
were refrigerated (4°C), and organic acid samples were frozen. Long-term CE, ethene,
ethane, and COD data were obtained from Ground Water Solutions of Lansing,
19
Michigan. Samples for long-term analyses were collected monthly, except during the
final three months of treatment 9 when samples weren't collected.
For preparation of the microcosms, sediment cores were extracted by Geoprobe
techniques (Stearns Drilling Co., Dutton, Michigan), and ground water was collected by
the low-flow technique described by Puis and Barcelona (1996). Sediment samples were
quickly transferred onsite to open ziplock bags and sealed in clean paint cans, which were
then purged with N2 and stored under N2. Ground water was purged with N2 during
collection. Samples were immediately transported to the laboratory anaerobic glove
chamber (95% N2, l%-5% H2), equipped with O2 and H2 monitors (Coy Lab Products).
Microcosm Preparation
Whey Degradation Pathway Experiment
To determine the anaerobic degradation pathway of whey, a microcosm
experiment was performed prior to the 6 whey injection using aquifer materials
collected within the source zone, approximately 30 ft. upgradient of the whey barrier
transect at BIP3. Using anaerobic sampling techniques described above, sandy sediment
sample was obtained from one sediment core at 32-34 ft below ground surface (bgs).
Ground water sampled from the same bore hole was collected in clean Nalgene bottles.
Overnight storage of the aquifer samples and microcosm preparation materials in the
anaerobic glove chamber allowed remaining O2 to be purged from the chamber prior to
microcosm preparation on the following day.
Prior to sampling, clean 100-mL serum bottles, glassware, and utensils for
microcosm preparation were wrapped in foil and autoclaved at 121 °C for 30 minutes.
20
Glass syringes were sterilized in 50% ethanol overnight, rinsed with deionized water, and
air dried. Teflon-lined rubber septa and aluminum crimp caps were wrapped in foil and
heated in a 100°C convection oven for 1 hour.
Microcosms were prepared in 100 mL serum bottles, using 80 mL ground water
and 10±1 g (wet wt.) sandy sediment composite. Resazurin (CnHeNC^Na), prepared as a
2% aqueous solution and autoclaved, was used as a redox indicator (10 ppm, final
microcosm concentration). The reduction of resazurin to resorufin is indicated by a color
change from blue to pink. Under highly reducing conditions (i.e., < -110 mV), resorufin
is further reduced to dihydroresorufin, and the solution becomes colorless. Reoxidation
occurs with a shift to oxidizing conditions.
Twelve bottles received 800 mg/L dried dairy whey and approximately 10 ppb
KB1 culture (10 uL per 1 L of ground water), a mixed Dehalococcoides-containing
culture (kindly supplied by SiRem Labs, Ontario, Canada, www.siremlabs.com). Three
control series (twelve bottle each) were also prepared: a biostimulated control (whey
amendment only), a bioaugmented control (KB1 only), and a pasteurized control.
Controls were pasteurized by heating to 70°C in a convection oven for two consecutive
nights and cooling to room temperature in the intervening day. For the preparation of
abiotic microcosms, pasteurization is a more effective method than the addition of
pesticides (e.g. sodium azide), which selectively kill native microbes. All microcosms
were spiked with TCE (100% ACS-grade, J.T. Baker) (10 ppm, aqueous concentration)
following control pasteurization. Duplicate microcosms in each series were sacrificed on
days 3, 7, 14, 30, 60, and 90. Microcosms were stored in darkness, inverted to prevent
release of volatile gases through the septa, and continuously agitated.
21
Electron Donor Comparison Experiments
For laboratory examination of the efficiencies of electron donor materials,
sediment cores were extracted at two points within the plume following the 8th injection:
approximately 25 ft. upgradient and 5 ft. downgradient of the whey injection transect.
Using anaerobic sampling techniques described above, sandy sediment was obtained
from 29-35 ft below ground surface (bgs) (upgradient source zone) and 24-27 ft. bgs
(treatment zone). Microcosm preparation was done within 1 day (upgradient source
zone) or six weeks (treatment zone) of the sediment collection. Ground water, sampled
from adjacent treatment-zone injection and upgradient monitoring wells, was collected in
clean glass jars 1-2 days prior to microcosm preparation.
Prior to sampling, clean 100-mL serum bottles, glassware, utensils, and glass
syringes used for microcosm preparation were sterilized as described above. Teflon-lined
rubber septa and aluminum crimp caps were heated in a 100°C convection oven for 1
hour. All microcosms were prepared with 70 mL ground water and 10±1 g (wet wt.)
sandy sediment composite in the anaerobic glove chamber containing N2 and trace levels
of H2 (generally below the detection limit of the H2 detector). Resazurin was used as a
redox indicator (lOppm, prepared as a 2% solution).
Microcosm series were amended with dried dairy whey, 60% sodium lactate
syrup (EM Science), or HRC (Regenesis, www.regenesis.com) and inoculated with
KB1/LV1, a mixed Dehalococcoides-containing
culture (kindly supplied by SiRem Labs,
Ontario, Canada, www.siremlabs.com). Microcosm series prepared with electron donor
and exogenous culture are referred to hereafter as whey+KBl/LVl, lactate+KBl/LVl,
and HRC+KB1/LV1. An additional microcosm series amended with whey contained
22
only native microbes (whey microcosms). Final concentrations were approximately 10
ppb (v/v) culture and 1.64 mM (11.5 mmoles/kg sediment) electron donor (whey lactose,
lactate, or glycerol tripolylactate (i.e. propanoic acid, 2-[2-[2-(2-hydroxy-1-oxopropoxy)l-oxopropoxy]-l-oxopropoxy]-l,2,3-propanetriyl ester)). Electron donor calculations
assumed 70% lactose in whey, 60% lactate in sodium lactate syrup, and 100% glycerol
tripolylactate (C39H56O27) in HRC. Electron donor concentrations approximately equaled
the molarity of whey lactose injected during the field-site applications of whey.
Exogenous dechlorinating microbes were added to microcosms to ensure achievement of
results within the timeframe of the experiments. Bioaugmented control microcosm series
were prepared that contained KB1/LV1 but no exogenous electron donor. Killed control
microcosm series containing only ground water, sediment, and resazurin were pasteurized
as described above. All bottles were spiked with TCE (100% ACS-grade, J.T. Baker) on
the day following completion of the control pasteurization process (10 ppm or 0.076 mM
aqueous concentration TCE). Microcosms were stored in darkness, inverted to prevent
release of volatile gases through the septa, and continuously agitated on an orbital shaker
(VWR) at 75 r.p.m.
In total, 144 bottles were prepared. Six duplicate series of upgradient source zone
microcosms and six duplicate series of treatment zone microcosms were prepared:
bioaugmented control, pasteurized control, whey-amended, whey+KBl/LVl-amended,
lactate+KBl/LVl-amended, and HRC+KB1/LV1-amended series. Six microcosms were
prepared per series, intended for sacrifice on sample days 1, 5,14, 30, 60, and 90. Day
123 and day 155 data of the upgradient microcosms were collected from microcosms
previously sampled. Chlorinated ethene "day 5" data from upgradient HRC and sodium
23
lactate microcosms and one upgradient whey+KBl/LVl microcosm were lost due to
instrument malfunction.
Analytical Methods
Chemicals
VOC external standards (PCE, TCE, cDCE, 1,1 -DCE, tDCE, benzene, toluene,
ethylbenzenes, and xylene) and internal standards (l,2-dichloroethane-d4 and toluene-d8)
were purchased from Restek Corp. (Philadelphia, PA). 99.9% purge-and-trap-grade
methanol used for VOC standard dilution was obtained from Omnisolv. Ethene and
ethane standards were prepared from pure gases purchased in 17L cylinders from Scott
Specialty Gases. Hydrogen and methane standards were prepared from a Micromat 4-5%
mixture obtained from Matheson Trigas. High purity (99+% to 99.5+%) organic acid
standards (oxalic, formic, glycolic, pyruvic, oxalacetic, malic, malonic, lactic, acetic,
maleic, fumaric, succinic, citric, isocitric, 3-hydroxy-2-methylbutyric, 2-ethyl-2hydroxybutyric, 2-methyl-2-hydroxybutyric, a-ketoglutaric, crotonic, 3-hydroxybutyric,
isobutyric, butyric acids) were obtained from Sigma-Aldrich, Supelco, Fluka, and EMD.
GC/MS, GC/FID, and RGA ultra-high pure carrier gases were purchased from Airgas
Great Lakes (Battle Creek, Michigan). HPLC mobile phase was prepared with ultrapure
KH2PO4 crystals obtained from J.T. Baker, HPLC-grade acetonitrile from EMD, and
ACS-grade phosphoric acid from EM Scientific.
Chlorinated Ethenes
Aqueous phase chorinated ethenes were analyzed by Varian 3400 CX/Saturn 4
GC/MS equipped with an 01 Analytical DPM-16 Purge and Trap (P/T) autosampler and
24
VICI ValcoBond VB-624 60m x 0.25mm x 1.4um capillary column. The P/T
concentrator program was as follows: purge time 11 minutes at 20°C, desorb time 4
minutes at 180°C, bake time 30 minutes at 200°C; valve, transfer line, and external heater
temperatures 100°C. The GC oven temperature was held at 40°C for 2 minutes, ramped
to 50°C at 4°C/min., ramped to 220°C at 10°C/min, and held at 220°C for 15 minutes.
The transfer line temperature was 260°C and the detector temperature was 200°C. The
method is described in detail elsewhere (USEPA, 1996b).
The original concentration of VOC external standard (PCE, TCE, cDCE, 1,1DCE, tDCE, benzene, toluene, ethylbenzenes, and xylene) and internal standard (1,2dichloroethane-d4 and toluene-d8) solutions (as purchased from Restek Corp.) was 2,000
ug/mL methanol. A 200,000 ug/L stock calibration standard solution was prepared by 10times dilution in P/T-grade methanol, and 0.5mL portions were sealed in lmL amber
ampules (Wheaton) and preserved in a freezer. Calibration standards were prepared by
serial dilution of stock solution in deionized water to a range of concentrations between
10 and 400 ppb. A 20 ug/mL internal standard working solution was prepared by lOOx
dilution in P/T-grade methanol, and 0.5mL portions were sealed in amber ampules and
preserved in the freezer. Calibration and internal standard check runs were done
periodically throughout the study. 250 uL aqueous samples collected by gas-tight syringe
from microcosms and field sample vials were diluted in 5 mL deionized water containing
40 ppb internal standard.
Aqueous chlorinated ethene concentrations were converted to total (microcosm)
concentrations according to Henry's Law using dimensionless constants calculated at
25
25°C from the following Henry's constant values (kPa m3 mol _1 ): 1.03 (TCE), 0.46
(cDCE), 2.68 (VC) (Lide, 2005).
Dissolved Gasses
Standards for analysis of headspace gases were prepared by serial dilution of
either pure gas (ethene and ethane) or a 4% gas mixture (methane and H2) in 20 mL
carrier-gas-purged glass vials sealed with aluminum caps and Teflon-faced septa.
Transfers were made using Hamilton SampleLock syringes. For analysis of dissolved
gasses in field samples, 20 mL ground water samples were transferred to clean 40 mL
glass vials and allowed to equilibrate with air headspace overnight in the inverted vials.
Headspace methane, ethene, and ethane were analyzed with a Buck Scientific 910
GC-FID equipped with a Domnick Hunter UHP-20H hydrogen generator.
IOOUL
standards and samples were injected directly onto a washed molecular sieve column (5A
80/100, 6ft x 1/8" x 0.085SS). The oven temperature was increased from 55°C to 140°C
at 10 mL/min, and the detector temperature was 150°C. Helium was the carrier gas, set at
6 psi. The detection limit was approximately 1 ppm.
Headspace H2 was determined with a Trace Analytical RGA3 Reduction Gas
Analyzer equipped with a carbon molecular sieve Spherocarb column (60/80 30 %" x
Ms"). H2 detection occurs by reduction of heated mercuric oxide, followed by quantitation
of mercury vapor by UV photometry at 254 nm. The oven temperature was 150°C and
the detector temperature was 265°C. The nitrogen carrier gas flow rate was 20 mL/min.
The injection volume was 3 mL. H2 reacts with mercuric oxide with approximately 10%
efficiency, and the detection limit was approximately 10 ppb.
26
Headspace methane and ethene concentrations were converted to total
(microcosm) concentrations according to Henry's Law using dimensionless constants
calculated at 25°C from the following Henry's constant values (kPa m3 mol _1 ): 21.7
(ethene), 67.4 (methane) (Lide, 2005). Headspace H2 concentrations were converted to
aqueous concentrations using a Henry's constant of 7.06 x 104 atm/mol fraction (Carr and
Hughes, 1998).
Organic Acids
GC/MS
Derivatization of organic acids
Low molecular weight organic acids are notoriously difficult analytes in aqueous
solution. Organic acid derivatization methods were investigated for detection with
GC/MS. Initial efforts involved BF3-propanol esterification of organic acids following
the guidelines provided by Supelco. In a 10 mL reaction vial, approximately 2 mg of an
organic acid standard was combined with 1 mL hexane and 160 uL BF3.propanol. The
vial was placed in a 60°C water bath for various lengths of time (10 to 60 minutes). 100
uL water and 100 uL hexane were then added to the cooled vial, followed by 40% KOH
for neutralization. Saturated NaCl solution was added to remove excess propanol. The
hexane layer was extracted after shaking and then dried over anhydrous sodium sulfate.
The final volume was adjusted by addition of hexane. Final derivative concentrations
ranged from 10 to 50 ppm.
Organic acid derivatives were analyzed with HP GC/MS equipped with an HP5MS (30m x 0.25 mm x 0.25 urn) capillary column. The inlet temperature was 250°C.
27
The oven temperature was held at 50°C for 2 minutes, ramped to 220°C at 4°C/min, and
held at 220°C for 2 minutes. The helium carrier gas flow rate was 1.5 mL/min.
Propyl lactate and propyl malonate prepared by derivatization of lactic and
malonic acids were not detected with GC/MS. An n-propyl lactate standard was
purchased and appeared to not be miscible with hexane. Dichloromethane was then used
as the solvent. The minimum and maximum oven temperatures were changed (40°C and
280°C), and the initial temperature hold time was increased to 5 minutes. The flow rate
was decreased to 1 mL/min to increase retention times. A pulse-split mode was used to
possibly narrow a wide solvent peak. Methyl lactate was detected, potentially due to
degradation of propyl lactate in the inlet.
Trimethylsilyl (TMS) derivatives of organic acids were also prepared, first using
trimethylsilylimidazole (TMSI) as the derivatizing agent. In lOmL reaction vials, organic
acid standards were derivatized by addition of excess TMSI in a nonpolar solvent
(dichloromethane, pentane, or hexane) and heating to 60°C -70°C for 20 min. If
necessary, volumes were reduced under N2. Products were analyzed using GC/MS
operated in splitless mode. Prepared organic acid-TMS derivative retention times are
shown in Table 2.
28
Table 2. GC/MS Organic acid -TMS Derivative Retention Times.
Trimethylsilyl derivative
Retention Time (minutes)
TMS-acetate
-proprionate
-isobutyrate
-butyrate
-oxalate
-lactate
-2-hydroxy-3-methylbutyrate
-2-ethyl-2-hydroxybutyrate
-malonate
-succinate
-malate
-adipate
-tridecanoate ~ IS
1.45
1.8
2.05
2.5
4.02
5.84
8.55
9.71
9.77
12.51
16.23
16.77
19.96
Trimethylsilyl derivatives of organic acids were also prepared by addition of
excess BSTFA-TMCS and heating at 70°C for 2 hours. Derivatives were dissolved in
either hexane, pentane, or isooctane. Calibration curves were constructed following
BSTFA-TMCS derivatization of acetate, lactate, propionate, butyrate, and pyruvate
standards, using tridecanoic acid internal standard as an indicator of derivatization
efficiency. Anisole was added to derivatized standard solutions as a GC/MS internal
standard. 2 uL were introduced into an HP GC/MS by splitless injection. The inlet
temperature was 250°C, and the oven temperature was 60 °C for 4 min. initially, ramped
at 6° C/min to 160°C, ramped at 20°C/min to 290°C, and was held at 290°C for 10 min.
Helium carrier gas flow rate was 1.1 mL/min. The solvent delay was 1.5 min. The
GC/MS was operated in selective ion monitoring (SIM) mode, for the detection of ions
with mass-to-charge ratios 73, 75, 78,108, 117, 128, 131,136, 145, 147,191, 234, and
271. Retentions times and ions are shown in Table 3. R values of five-point calibration
29
curves constructed for organic acid concentration ranges between 20 and 900 ppm were
0.9988-0.9990. The RF mean was 2.4 ± 11.2 % rsd.
Table 3. GC/MS TMS-Organic Acid Derivative Ions and Retention Times.
Retention Times (minutes)
Compound
Ions (m/z)
2.13
3.07
4.81
5.61
9.87
10.5
11.87
23.34
acetate
propionate
butyrate
anisole-IS
lactate
pyruvate
oxalate
tridecanoate-IS
75,117
75,131
75, 145
78,108
73,117,147,191
73, 147
73, 147
271
Separation of organic acids by ion exhange
Sugars present in high concentrations in microcosm samples contaminated the
GC/MS column. Organic acids were therefore separated, prior to GC/MS analysis, by
anion exchange using strong anion exchange (SAX) cartridges. In preparation, 5 mL of
5mM methanol was manually pushed through the SAX cartridge, followed by 5 mL of
deionized water. KOH was added to deprotonate organic acids in standards and samples
which were then injected into the cartridge at approximately Vi mL/min. Several
potential eluents, i.e. solutions which might bind more strongly to SAX and thereby
remove the adsorbed organic acids, were tested: 0.5 M HC1, 0.5 M HC1 in 50% methanol,
0.5 M KI, 0.5 M H 2 S0 4 , 0.5 M NaCl, 0.5 M benzoate, and 0.1 M citrate. In preparation
for the derivatization of the protonated organic acids, eluted samples were re-acidified by
addition of HC1 and then freeze-dried.
30
Shown below are percent recoveries of adipic, lactic, malonic, and succinic acids
following anion exchange, lyophilization, derivatization with TMSI, and dissolution in
hexane. Selected ion peak areas were compared with peak areas of standards that were
only derivatized. 0.1 M citrate was found to be the most effective eluent tested.
Recoveries of organic acids using 0.5 M KI, 0.5 M NaCl, and 0.5 M benzoate SAX
eluents were essentially zero.
Table 4. Percent Recoveries of Organic Acids Following Anion Exchange, Lyophilization, and TMSI
Derivatization.
0.5 M H2SQ4
0.1 M Citrate
lactic: 2.4%
malonic: 23%
succinic: 18%
adipic: 7.6%
lactic: 97.4%
malonic: 87.3%
succinic: 95.3%
adipic: 131.8%
Because citrate could be an analyte of interest, a weak anion exchange (WAX)
cartridge containing functionalized NH2 was also tested. Low percent recoveries
indicated organic acids may not be sufficiently retained, however, by the NH2 resin.
Lyophilization
During the whey degradation pathway experiment, organic acid standards
dissolved in MilliQ water and aqueous samples were freeze-dried prior to derivatization
with BSTFA-TMCS, dissolution in hexane, and detection by GC/MS, as described above.
Standard recovery percentages were determined by comparison to standards which had
been derivatized only (i.e., hadn't been dissolved in water and then freeze-dried).
Recovery percentages of standards are shown below.
31
Table 5. Percent Recoveries of Organic Acids Following Lyophilization, BSTFA-TMCS Derivatization,
and Dissolution in Hexane.
acetate
40.5% -- 67.3%
propionate
33.5% •• 59.5%
butyrate
41.0%-• 70.8%
lactate
27.3% •- 47.4%
Lactate losses were significantly higher than during the earlier testing of SAX
cartridges for separation of organic acids from sugars. The inclusion of acetate,
propionate, and butyrate in the standard solution may have affected lactate yields, or the
lyophilizer may have not been working as efficiently, leaving traces of water. In the
whey degradation pathway experiment, organic acid concentrations in aqueous samples
were corrected for apparent losses, as indicated by losses of standards which were
prepared and analyzed concurrently. Comparison of organic acid concentrations
determined using this method and using an HPLC method (described below) is shown in
Appendix B.
HPLC
Organic acids (oxalic, formic, glycolic, pyruvic, oxalacetic, malic, malonic, lactic,
acetic, maleic, fumaric, succinic, citric, isocitric, 3-hydroxy-2-methylbutyric, 2-ethyl-2hydroxybutyric, 2-methyl-2-hydroxybutyric, a-ketoglutaric, crotonic, 3-hydroxybutyric,
isobutyric, and butyric) were analyzed with a Waters HPLC (Waters 515 pumps
connected to 2487 dual wavelength absorbance UV/VIS detector) using a NovaPak CI8
reversed-phase guard cartridge and 250mm x 4.6mm CI 8 column. For preparation of the
32
mobile phase, 20mM KH2PO4 and 1% acetonitrile solution was filtered through a 0.45
urn membrane (Millipore) and adjusted to pH 2.2 using phosphoric acid. The isocratic
flow rate was 1 mL/min. Details of the RF-HPLC method are described elsewhere
(Tormo and Izco, 2004). Samples were filtered using a 0.45 urn syringe filter disc, frozen
until analysis, and run in triplicate.
Low-level organic acid determinations were done by preparation of organic acid
2-nitrophenylhydrazide derivatives and subsequent in-line concentration and analysis
with a Waters HPLC equipped with a dual absorbance detector, a 1.5cm polymeric
reversed-phase (PRP) guard cartridge placed in the sample loop as a concentrator, a
Waters NovaPak 1.5 cm C8 guard cartridge and 220cm x 4.6mm 4um C8 column. The
mobile phase was composed of 2mM tetrabutylammonium hydroxide (TBAOH), 2mM
tetradecyltetrimethylammonium hydroxide (TDTMABr), 50mM sodium acetate, and
2.5% n-butanol (buffer A) and 2mM TBAOH, 50mM TDTMABr, 50mM sodium acetate,
and 2.5% n-butanol (buffer B). Buffers were filtered through 0.45um membrane, and the
pH was adjusted by addition of phosphoric acid. The gradient program was 100% buffer
A from 0-5 minutes, 100% buffer A to 100% buffer B from 5-10 minutes, and 100%
buffer B from 10-60 minutes. Sample injection volumes were 0.5 mL or 1 mL. The
detailed analytical method has been described elsewhere (Albert and Martens, 1997).
Derivatized standards and samples were stored at 4°C and analyzed within 1 day of
derivatization.
33
CHAPTER III
PRELIMINARY MICROCOSM RESULTS
Native Dechlorinator Identity and Growth
Dehalococcoides was identified as a native dechlorinating genus of the site
(Appendix A). Detection of Dehalococcoides in source-zone samples indicated the
presence of native microbial consortia able to completely dechlorinate TCE to ethene
(Hendrickson et al., 2002). Average Dehalococcoides DNA concentrations and yields
are shown in Table 6. Conversion of pg DNA to RNA gene copies, assuming an average
molecular weight of 660 for a base pair in double-stranded DNA and one gene copy per
Dehalococcoides genome, is described elsewhere (Ritalahti et al., 2006). Yields of
native Dehalococcoides observed in whey-amended microcosms significantly exceeded
the yield in unpasteurized controls (Table 6). The Dehalococcoides yield in wheyamended microcosms spiked with lOppm TCE was of similar order of magnitude to the
yield previously observed in a biostimulation test plot in which lactate was continuously
recirculated for 101 days (Lendvay et al., 2003).
34
Dehalococcoides DNA
(pg/mL of slurry)*
Day
I 111 1
Table 6. Average Dehalococcoides DNA Concentrations and Yields in Source-Zone Aquifer Microcosms.
Yield
gene copies/
g sediment
0
32
113
221
Wheyamended
(Wppm TCE)
0.9002
3.7340
1.6127
1.6830
Wheyamended
(WOppm
TCE)
0.0685
0
1.874
0.097
1.16x10'
2.34 x 102
Unpasteurized
control
0
0.0006
0.0400
0.0004
1.78
3.18
6.48 x 103
* Dybas, M.J., unpublished
35
Whey Degradation Pathway
A preliminary microcosm experiment was performed to evaluate the anaerobic
whey degradation pathway. Organic acids accounted for approximately 100% of whey
lactose carbon added to the microcosms (Figure 6B). Within 10 days, lactose had been
converted to organic acids, and sufficient levels of organic acids were maintained through
the course of the study to support extensive dechlorination. Major organic acids detected
were acetate, lactate, butyrate and propionate. Acetate was produced in highest quantity
and degraded after a lag period of about 60 days in all but one of the donor-amended
microcosms (i.e. one whey+KBl microcosm) (Figure 6B). Lactate degraded rapidly,
while butyrate and propionate degradation (and H2 release) occurred more slowly.
Notably, propionate did not degrade in the time frame of the study, evidence that
propionate may act as a carbon sink and the diversion of high percentages of electron
donor material carbon to propionate may be undesirable. Propionate concentrations
were, however, relatively low.
Within 2 weeks, TCE dechlorination occurred in all but the abiotic controls
including the bioaugmented (KB-1 amended) control microcosms, indicative of either the
consumption by dechlorinating populations of H2 originally present in the microcosm
headspace or of the presence of readily oxidized organic compounds within the source
zone. However, levels of electron donor were apparently not high enough to support
dechlorination of cDCE in the bioaugmented controls. From 60 to 100 or 120 days, cDCE
dechlorination did occur in whey-amended microcosms, apparently correlating with the
release of H2 from the degradation of butyrate and perhaps acetate. Complete
dechlorination occurred only in microcosms amended with whey and KB1 (Figure 6A).
36
Results of this experiment confirmed that native microbial populations capable of
TCE dechlorination are present at the field site and that dairy whey supports microbiallymediated dechlorination of TCE, DCE and VC under strictly anaerobic conditions. Both
fast and slow H2-releasing organic acids were identified as whey degradation products.
-Whey
- Unpasteurized Control
-*—Whey+KB1
-a— Pasteurized Control
I
!
I
50
Time (days)
B.
_*—Whey+KB1
-Whey
100.0
8
80.0
U
60.0
*
40.0
Orga
bon
120.0
20.0
0.0
50
100
Time (days)
Figure 6. Preliminary Experiment Total Chloroethene Percentages (A) and Total Organic Acid Carbon
Percentages (B). Microcosms were prepared with source-zone aquifer materials collected upgradient of the
whey PRB. Chlorinated ethene percentages were calculated relative to values of the initial microcosm
sacrifice. Organic acid carbon percentages were calculated relative to the total carbon of the added whey.
37
CHAPTER IV
RESULTS OF THE LABORATORY COMPARISON OF
ELECTRON-DONOR MATERIAL EFFICIENCY
Introduction
For evaluation of the efficiencies of three electron donor materials (whey, lactate,
and HRC), donor-amended microcosms were prepared with sediment and ground water
collected either within the source zone upgradient of the whey PRB or within the whey
treatment zone and were spiked with TCE. Dechlorination rates, fermentation product
distributions, and H2 production were monitored as measures of substrate efficiency in
donor-amended aquifer microcosms.
Headspace H2 levels in controls decreased from uM to nM levels within the first
month, apparently due to leakage through the septa. H2 levels in electron-donor-amended
microcosms generally far exceeded control levels and were assumed to be transient levels
produced from electron donor degradation.
pH values were 6.8-6.9 in the field and newly prepared microcosms. During the
course of the experiments, pH values were 7-8. Final pH values were 7.6 (pasteurized
control), 7.8 (whey), 7.5 (whey+KBl/LVl), 7.2 (HRC®+KB1/LV1), and 8.0
(lactate+KBl/LVl). The buffering capacity of the aquifer sediment and water appeared
to be sufficient for the stabilization of pH. The average alkalinity of aqueous field
samples collected within 5 months of microcosm preparation was 329 mg/L as CaC03
±10.8% CV. The average TIC of sediment 24-27 ft bgs prior to whey treatment was
2.95% ±15.5% CV.
38
Dechlorination Rates
Upgradient Source Zone Microcosms
CE molarities were converted to equivalent percents to compare extents of
dechlorination in upgradient source zone microcosms, as shown in Figure 7A. Figures 811 show average CE distributions in the donor-amended microcosms. In the upgradient
microcosms, the rates of TCE dechlorination to ethene observed in whey+KBl/LVl and
lactate+KBl/LVl microcosms were comparable (4.02 ueq/L/day and 3.53 ueq/L/day,
respectively, as calculated from data shown in Fig. 7A). A long cDCE lag phase (day 14
to day 123) occurred in HRC+KB1/LV1 microcosms, which failed to show complete
dechlorination in the experimental time frame (Fig. 11 A). Dechlorination rates in whey
microcosms that contained only native microbes were similar until day 155, when only
one of the final duplicate microcosms showed complete dechlorination (Fig. 8A). Longer
TCE dechlorination lag times and complete dechlorination rates in whey than in
whey+KBl/LVl microcosms indicate either that native populations able to completely
dechlorinate TCE (e.g. Dehalococcoid.es) were low and/or had less competitive
advantage or that populations capable of chlororespiration of TCE to ethene were not
present.
Treatment Zone Microcosms
CE molarities were converted to equivalent percents to compare extents of
dechlorination in treatment zone microcosms, as shown in Figure 7B. Figures 8-11 show
average CE distributions in the donor-amended microcosms. Relatively rapid complete
39
dechlorination occurred in whey-amended treatment zone microcosms, irrespective of
microcosm KB1/LV1 augmentation (4.18 ueq/L/day and 4.13 ueq/L/day in whey and
whey+KBl/LVl, respectively, as calculated from data shown in Fig. 7B). Increased
rates in the treatment zone relative to source zone microcosms may be attributed to the
prior adaptation of the native dechlorinating community to whey, the establishment of
chlororespirers or higher native dechlorinating populations within the PRB, and/or
alteration of redox conditions due to exposure to whey in-situ. The effects of the whey
PRB installation were particularly evident in whey microcosms unaffected by the
exogenous culture, which exhibited the greatest increase in rates (corresponding to a
-79% increase) (Fig. 7B). Rapid VC dechlorination in whey-amended treatment zone
microcosms (Figs. 8B, 9B) and comparable complete dechlorination rates in treatment
zone microcosms regardless of microcosm bioaugmentation (Fig. 7B) suggest microbial
populations similar to KB1/LV1 were established in the treatment zone.
Interestingly, complete dechlorination rates did not increase in lactate+KBl/LVl
treatment zone relative to source zone microcosms (Fig. 7). As native microbes collected
within the treatment zone may have been previously exposed to whey, HRC+KB1/LV1
and lactate+KBl/LVl treatment zone results weren't directly comparable to
whey+KBl/LVl results; however, the data may serve as points of reference to examine
whey's improved efficiency.
40
z> o o o o
o
120
CE Equivalents (%)
A
0
50
Time (days)
100
150
B
100 ->s
80 c
a>
IQ
>
3
\,
60 \ k
or
Ul
40 -
UJ
O
20
0 —,
,—_
X
"—^:r i "
50
100
- - T —
150
Time (days)
-A— Whey
-Whey+KB1/LV1
-»—Lactate+KB1/LV1
-HRC+KB1/LV1
Figure 7. Total Chloroethene Percentages in Donor-amended Upgradient Source Zone Microcosms (A)
and Treatment Zone Microcosms (B). Total chloroethene percentages were calculated relative to the initial
measured values in microcosms.
41
Whey, Source Zone
120
100
si
Is
80
x> t£
C c
"'
IS
w
o
40
60
111 IE
° i
20
0 4+-+
Time (days)
B.
Whey, Treatment Zone
100
•
50
*
100
150
Time (days)
-TCE
-o—cDCE
-±_VC
-A— Ethene
Figure 8. Chloroethenes and Ethene Concentrations in Whey-amended Upgradient Source Zone
Microcosms (A) and Treatment Zone Microcosms (B).
42
CE & Ethene
Conceritration (uM)
A.
Whey+KB1/LV1, Source Zone
100 — - -
-
80 60 \
40 20 0 1
0
B.
100
Time (days)
150
Whey + KB1/LV1, Treatment Zone
100
CE & Ethene
Concentration (uM)
50
•- - -
-
- - -
80 60
40
20 n
0
50
100
150
Time (days)
-TCE
-a—cDCE
-VC
-A— Bhene
Figure 9. Chloroethenes and Ethene Concentations in Whey+KBl/LVl Amended, Upgradient Source
Zone Microcosms (A) and Treatment Zone Microcosms (B).
43
Lactate +KB1/LV1, Source Zone
50
100
150
Time (days)
B.
Lactate+KB1/LV1, T r e a t m e n t Z o n e
^
100 -,
5
* 3-
80 -
•c . 2
UJ n
60 -
£*
38
o
-
-
-
-
40 20 -
o
0
0
50
150
100
T i m e (days)
-TCE
- B — cDCE
-*—VC
. Bhene
Figure 10. Chloroethenes and Ethene Concentrations in Lactate+KBl/LVl Amended Upgradient Source
Zone Microcosms (A) and Treatment Zone Microcosms (B).
44
HRC+KB1/LV1, Source Zone
A.
CE & Ethene
Concentration (uM)
100
80 60
40 20 ,
0 i
1
„ 0
-20
50
-
100
Time (days)
*•
150
—
HRC+KB1/LV1, T r e a t m e n t Z o n e
B.
80
s
» 3.
•c . 2
UJ
13
08 ^
UJ <D
O
"
C
o
R0
40
?0
o
0
50
-20
-TCE
100
150
TTirTeiaays)
-B_cDCE
—+—VC
—A—Ethene
Figure 11. Chloroethenes and Ethene Concentrations in HRC+KB1/LV1 Amended Source Zone
Microcosms (A) and Treatment Zone Microcosms (B).
Carbon Flow in Whey-amended Microcosms
Figure 12 shows total organic acid carbon trends in donor-amended upgradient
source zone and treatment zone microcosms. Organic acid carbon distributions and totals
are shown in Table 7. Consumption of acetate reducing equivalents in whey-amended
treatment zone microcosms is shown in Figure 13.
According to carbon mass balance calculations, 100 ± 10 % of whey lactose
carbon was converted to organic acids (Fig. 12). 90.5% and 80.7% of whey lactose was
fermented to organic acids within 5 days in upgradient whey and whey+KBl/LVl
microcosms, respectively (Fig. 12A). Whey lactose may have undergone primary
fermentation somewhat more quickly in treatment zone microcosms (89-96% by day 2)
(Fig. 12B), as a result of the enhanced activity of fermenting populations within the
treatment zone. Relevant organic acid distributions were similar irrespective of
inoculation with KB1/LV1 culture (Table 7). Lactate, formate, and acetate appeared to
be the primary lactose fermentation products. Within ~30days, >70% of lactose carbon
was channeled to acetate, propionate and butyrate.
In upgradient microcosms, acetate concentrations and rates of organic acid carbon
flow to acetate were highest as a result of whey fermentation (Table 7). Whey underwent
a relatively high percent conversion to acetate and butyrate. TCE dechlorination to vinyl
chloride (VC) occurred between 32 and 90 days in whey microcosms (Fig. 8A), during a
period of slow propionate and more rapid butyrate and acetate metabolism (Table 7). In
whey+KBl/LVl microcosms, lactate, acetate, propionate, or butyrate metabolism (Table
7) may have contributed to the earlier TCE dechlorination to VC (between 14 and 60
days) (Fig. 9A). Acetotrophic activity was apparently higher in the absence of KB1/LV1
46
culture, and, following the near-depletion of butyrate and acetate by day 90, and low
electron donor supply may have contributed to slow VC dechlorination in whey
microcosms (Fig. 8A).
In treatment zone microcosms, the observed onset of acetate degradation (Table
7) and low-level methanogenesis (Fig. 13) occurred at 31 days, coinciding with extensive
dechlorination of cDCE to ethene between days 31 and 67 (Figs. 8B, 9B). Low
propionate levels remained constant during that period, and propionate fermentation
began only when dechlorination was complete (Table 7). 4.1±0.1% of acetate reducing
equivalents was directed to dechlorination between days 31 and 67 (Fig. 13). Apparently,
H2 produced by acetate degradation between days 31 and 67 was also directed to
conversion of acetate to butyrate. As dechlorination activity slowed, following day 67,
butyrate fermentation ensued and rates of acetotrophic and/or hydrogenotrophic
methanogenesis sharply increased, presumably as a result of decreased competition for
electron donor. Upon complete dechlorination by day 67, organic acid carbon accounted
for 70.5±0.7% and 76.8±4.2% of the lactose carbon in whey and whey+KBl/LBl
microcosms, respectively (Fig. 12B).
47
A. Upgradient Source Zone
120 ,
S
c
|
100 80 -
(0
«
"o
<
_o
'£
5.
O)
40 '
»
20 -
°
0
u.
60-
0
50
100
150
Time (days)
B. Treatment Zone
120
50
100
150
Time (days)
-A—Whey
• Whey+KB1/LV1
-«_Lactate+KB1/LV1
-HRC+KB1/LV1
Figure 12. Organic Acid Carbon Percentages in Donor-amended Source Zone Microcosms (A) and
Treatment Zone Microcosms (B). Percentages were calculated relative to total added carbon, assuming
70% lactose in whey, 60% lactate in sodium lactate syrup, and 100% propanoic acid, 2-[2-[2-(2-hydroxy-loxopropoxy)-l-oxopropoxy]-l-oxopropoxy]-l,2,3-propanetriyl ester (glycerol tripolylactate) in HRC®) in
microcosms prepared with contaminated ground water and sediment collected within the source zone
upgradient of the whey PRB and within the treatment zone.
48
Table 7. Organic Acid Carbon Concentrations in Source Zone and Treatment Zone Microcosms.
Average organic acid carbon (mM carbon per bottle) in duplicate aquifer microcosm series prepared with
sediment and ground water collected upgradient and within whey treatment zones. Whey microcosms
received 19.68 mM C assuming 70% lactose content in whey, lactate microcosms received 4.92 mM C
assuming 60% lactate content in lactate syrup, and HRC microcosms received 63.96 mM C assuming 100%
C39H56027 content in HRC.
Organic Acid Carbon Concentration(mM)
Upgradient Microcosms
Day
1
5
14
32
62
90
123
Whey-amended microcosms
Formate
0.56±0.4
<0.02
<0.02
<0.02
<0.02
<0.02
1.46±1.5
Lactate
2.62±0.7
1.51±1.3
0.33±0.0
3.46±0.5
1.20±0.1
0.74±0.4
0.70±0.4
Acetate
1.09±0.4
12.06±4.2
12.89±0.5
10.94±2.2
7.37±1.7
1.91±1.9
1.27±0.5
Propionate
<0.04
0.22±0.1
2.79±0.5
1.10±0.7
1.39±0.7
0.82±0.1
0.39±0.1
Butyrate
0.46±0.1
3.97±1.3
4.57±1.2
4.22±0.2
5.55±0.2
<0.04
<0.04
Total
4.92±0.6
17.77±1.5
20.58±1.2
19.73±0.8
15.55±1.3
3.48±1.5
3.83±1.3
Whey+KB 1/LV1-amended microcosms
Formate
0.56±0.2
4.06±0.1
<0.02
<0.02
<0.02
<0.02
<0.02
Lactate
3.46±0.0
2.58±0.3
1.94±0.2
1.43±0.1
0.88±0.5
0.66±0.66
0.99±0.99
Acetate
1.80±0.3
4.22±0.2
11.26±2.1
12.93±0.2
13.57±0.5
12.53±3.4
13.38±2.7
Propionate
<0.04
0.51±0.1
1.35±0.7
3.91±2.5
2.33±0.7
4.42±3.2
4.87±3.8
Butyrate
<0.04
4.46±1.2
5.33±1.4
3.64±2.6
3.95±0.3
0.89±0.3
1.41±0.8
Total
5.96±0.6
15.85±1.2
21.17±0.3
21.91±0.3
20.74±1.4
18.51±1.5
20.66±0.7
Sodium Lactate+KBl/LVl-amended microcosms
Lactate
4.99±0.3
<0.03
<0.03
<0.03
<0.03
<0.03
<0.03
Acetate
0.16±0.0
1.07±0.0
1.18±0.0
1.38±0.1
1.18±0.27
1.36±0.0
0.46±0.0
Propionate
<0.04
3.21±0.2
3.12±0.1
3.09±0.5
2.02±0.78
2.51±0.0
0.86±0.1
Butyrate
<0.04
<0.04
<0.04
<0.04
<0.04
0.27±0.3
<0.04
Total
5.15±0.3
4.28±0.1
5.67±0.2
4.46±0.6
3.2±1.05
4.16±0.2
1.32±0.1
49
Table 7 - continued
HRC®+KBl/LVl-amended microcosms
Lactate
15.07±2.3
<0.03
5.54±0.52
0.42±0.4
<0.03
<0.03
<0.03
Acetate
0.08±0.08
1.64±0.5
5.49±1.0
10.06±1.2
8.66±2.4
8.61±0.2
7.45±1.2
Ketoglut
0.17±0.1
0.10±0.03
<0.04
<0.04
<0.04
<0.04
<0.04
Propionate
5.09±5.09
11.19±2.5
16.15±1.7
28.80±5.7
19.96±4.2
29.24±0.1
27.22±4.3
Butyrate
5.61±1.5
3.40±3.40
3.26±0.8
1.24±0.1
<0.04
<0.04
<0.04
Isobutyrate
6.64±0.7
1.70±1.70
1.91±0.02
<0.04
<0.04
<0.04
<0.04
Total
32.65±2.0
18.02±4.8
32.43±1.2
40.53±4.2
28.63±6.6
38.11±0.5
34.67±5.5
Treatment Zoi
Day
2
Microcosms
5
16
31
67
94
136
Whey-amended microcosms
Formate
4.48±0.3
1.68±1.68
<0.02
<0.02
<0.02
<0.02
<0.02
Lactate
4.70±0.2
2.54±0.3
0.18±0.18
<0.03
<0.03
<0.03
<0.03
Acetate
4.02±0.4
8.51±1.0
11.79±0.1
12.55±0.4
11.37±0.0
6.53±4.6
0.23±0.1
Propionate
0.15±0.1
0.70±0.1
0.88±0.1
0.95±0.1
0.95±0.1
1.0±0.1
0.19±0.19
Butyrate
3.45±1.0
3.41±1.4
0.44±0.3
0.38±0.3
0.83±0.4
0.27±0.1
0.17±0.17
Total
17.55±0.2
17.34±2.0
14.3±0.6
14.94±0.4
13.83±0.1
8.39±4.1
1.12±0.4
Whey+KBl/LVl-amended microcosms
Formate
4.91±0.7
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
Lactate
4.41±0.1
2.46±0.2
0.57±0.3
<0.03
<0.03
<0.03
<0.03
Acetate
4.80±0.5
7.0±0.3
10.9±0.4
12.42±0.1
11.67±0.1
7.21±5.0
5.76±4.2
Propionate
0.09±0.09
0.62±0.1
0.82±0.0
1.06±0.1
1.04±0.0
1.07±0.1
0.46±0.4
Butyrate
4.21±1.1
2.95±1.0
0.74±0.1
0.76±0.1
1.17±0.6
0.31±0.2
0.13±0.1
Total
18.87±0.1
13.5±0.1
13.97±0.5
15.35±0.3
15.06±0.8
9.74±5.4
7.03±5.1
50
Table 7 - continued
Lactate+KBl/LVl-amended microcosms
Lactate
4.86±0.1
0.97±0.7
<0.03
<0.03
<0.03
<0.03
<0.03
Acetate
0.01±0.01
2.17±0.3
2.72±0.4
2.41±0.5
2.63±0.1
2.22±0.1
1.98±0.2
Propionate
<0.04
0.02±0.01
0.13±0.1
0.18±0.18
<0.04
<0.04
<0.04
Total
4.88±0.1
3.16±0.4
2.85±0.3
2.66±0.4
2.68±0.0
2.22±0.1
2.01±0.3
HRC®+KBl/LVl-amended microcosms
Formate
0.34±0.34
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
Lactate
12.28±0.9
12.23±1.1
3.76±2.2
<0.03
0.92±0.6
<0.03
<0.03
Acetate
1.24±0.01
3.19±0.1
22.05±1.5
26.11±5.9
18.14±0.3
27.24±11
7.97±5.9
Ketoglut
0.12±0.1
.08±0.06
<0.04
<0.04
<0.04
<0.04
<0.04
Propionate
0.73±0.5
2.96±0.2
12.64±1.7
17.13±1.8
19.93±0.8
20.41±1.2
6.59±5.2
Butyrate
4.92±0.2
1.81±0.3
<0.04
<0.04
<0.04
<0.04
<0.04
Isobutyrate
11.01±0.8
8.36±0.2
5.26±2.1
<0.04
<0.04
<0.04
<0.04
Total
30.66±2.8
28.64±1.9
43.71±7.5
45.94±9.3
41.06±2.0
49.27±14
14.59±11
51
Figure 13. Acetate Equivalent Consumption in Whey-amended Treatment Zone Microcosms. Acetate
reducing equivalents produced between days 31 and 67 and days 31 and 94 were diverted to
methanogenesis, butyrate production, and dechlorination during those time periods. Data points are
averages of treatment zone whey-amended and whey+KBl/LVl-amended values calculated from average
duplicate microcosm results.
Carbon Flow in Lactate and HRC Amended Microcosms
Within 5 days in source zone upgradient microcosms, sodium lactate was entirely
converted to propionate and acetate (approximate molar ratio 2:1) (Table 7). Propionate
degradation occurred after day 32 (following a ~2-week cDCE dechlorination lag phase)
and appeared to supply reducing equivalents for the dechlorination of cDCE and VC to
ethene (Fig. 10A). In treatment zone conditions, lactate carbon was primarily channeled
to acetate (Table 7). Acetate degradation occurred after 67 days (following ~50-day
cDCE lag phase) and appeared to provide reducing equivalents for the dechlorination of
cDCE to ethene (Fig. 10B). The efficiency of acetate, defined in terms of the percentage
of its reducing equivalents directed to dechlorination, compared to efficiencies observed
in whey treatment zone microcosms (data not shown). As complete dechlorination was
52
approached on day 94, organic acid carbon accounted for 45.3±1.2% of the total added
carbon.
In HRC-amended microcosms, lactate, propionate, butyrate, and isobutyrate
carbon was channeled to primarily acetate and propionate within ~30 days (Table 7),
during which time the total organic acid carbon percentage increased to 62.8%
(upgradient) and 67.8% (treatment zone) (Fig 12). Propionate carbon levels were highest
in HRC-amended microcosms, accounting for 71.0% (upgradient) and 39.6% (treatment
zone) of the measured total organic acid carbon at day 32. In upgradient conditions,
steady propionate levels (Table 7) and steady total organic acid carbon percentages (Fig.
12A) were maintained between days 32 and 123 (during a cDCE lag phase from day 14
to day 123 (Fig. 11 A)). In treatment zone conditions, percent conversion to propionate
was highest in HRC microcosms, and propionate continued to accumulate through day 94
(Table 7). Propionate thus appeared to serve as a carbon sink in upgradient and treatment
zone microcosms that may have prolonged dechlorination. In treatment zone
microcosms, the percentage of acetate reducing equivalents directed to dechlorination
was lower than in whey and lactate microcosms (0.38±0.2%) (data not shown) apparently
due to acetotrophic methanogenic activity during dechlorination. Following complete
dechlorination in treatment zone microcosms (day 136), organic acids accounted for
22.8±17.3% of assumed total added carbon (Fig. 12B).
H2 and Methane Production
Figure 14 shows H2 trends in donor-amended, upgradient source zone and
treatment zone microcosms. An initial rapid release of H2 occurred in whey-amended
microcosms during lactose degradation (556.2-563.7 nM H2, aqueous concentration
53
minus controls) and in HRC+KB1/LV1 microcosms (389.7 nM H2, aqueous
concentration minus controls). After approximately 2 weeks, H2 concentrations had
decreased to low levels Fennell et al. (1997) suggested may give competitive advantage
to dechlorinators over methanogens. Low H2 partial pressures were steadily maintained
in whey microcosms containing only native microbes (10"35 to 10"4'9 atm and 10"3'8 to 10"
45
atm in upgradient and treatment zone conditions) within approximately 2 weeks
through the end of the experiments. After approximately 2 weeks, similar H2 partial
pressures were steadily maintained in treatment zone whey+KBl/LVl microcosms (10"4
to 10"4'4 atm) but fluctuated in upgradient conditions between 10"3 2 atm andlO"65 atm.
After approximately 2 weeks, H2 partial pressures were steadily maintained in upgradient
lactate+KBl/LVl microcosms (10"4'1 to 10"4'6 atm) but, in treatment zone conditions,
increased from 10~5'2-10"6'9 atm to 10~4'2 - 10"4'3 atm after 67 days as dechlorination neared
completion. In HRC+KB1/LV1 microcosms, H2 partial pressures in upgradient
T
1
T
1
conditions gradually decreased until day 123 (10"' to 10" atm) but were steadily
maintained in treatment zone conditions (10"3'6 to 10"4'5 atm).
In some of the microcosm series, H2 concentrations agreed with levels suggested
previously to be minimum threshold levels of dechlorination (0.1 nM). In
lactate+KBl/LVl treatment zone microcosms, H2 levels were steadily maintained during
the cDCE lag phase (1-5 nM H2 aqueous concentration, characteristic of sulfate-reducing
conditions), decreased at the onset of cDCE dechlorination (0.1 nM H2 aqueous
concentration), and then rebounded during dechlorination and the onset of
methanogenesis (>30nM H2 aqueous concentration, characteristic of methanogenic
conditions). H2 levels also decreased to low levels in upgradient HRC+KB1/LV1
54
microcosms at the onset of cDCE dechlorination (0.1 nM H2 aqueous concentration) and
in whey+KBl/LVl microcosms during cDCE dechlorination (0.3 nM H2 aqueous
concentration).
Figure 15 shows methane trends in donor-amended, upgradient source zone and
treatment zone microcosms. Methanogenesis in lactate+KBl/LVl upgradient and
treatment zone microcosms apparently occurred after complete dechlorination (~90 days)
and at similar rates. In upgradient lactate-amended microcosms, methane production
(322 uM, days 90 to 123) appeared to correspond to acetate consumption (449 uM, days
90 to 123) (i.e., appeared to be acetotrophic). Methanogenesis also occurred at similar
rates in treatment zone and upgradient whey+KBl/LVl microcosms, although somewhat
earlier in treatment zone microcosms apparently as a result of faster complete
dechlorination. In whey+KBl/LVl treatment zone microcosms, methanogenesis (2214
uM) also appeared to correspond to acetate consumption (2955 uM). In contrast, high
rates of methanogenesis occurred during dechlorination in HRC+KB1/LV1 treatment
zone microcosms and, inconsistently, in upgradient whey microcosms which contained
only native microbes. In HRC+KB1/LV1 treatment zone microcosms, methane
production (5280 uM) and acetate consumption (5086 uM) between days 67 and 136
seemed to correspond (i.e., the methanogenic activity during dechlorination appeared to
be acetotrophic).
55
A. Upgradient Source Zone
0
-1
50
100
150
Time (days)
B. Treatment Zone
o
-1 -
£§ -4^
-7 -8
0
^y
^v
i
>
50
100
150
Time (days)
-A—Whey
Whey+KB1/LV1
-«_Lactate+KB1/LV1
HRC+KB1/LV1
Figure 14. H2 Partial Pressures in Donor-amended Source Zone Microcosms (A) and Treatment Zone
Microcosms (B). Microcosms were prepared with contaminated ground water and sediment collected
within the source zone upgradient of the whey PRB and within the treatment zone.
56
Upgradient Source Zone
6000
5000
4000
3000
2000 1000 0
„,....-.• ,&*
50
100
Time (days)
150
B. Treatment Zone
6000
5000
4000
3000
2000
1000
0
50
100
Time (days)
.Whey
_±_Whey+KB1/LV1
-Lactate+KB1/LV1
-H— HRC+KB1/LV1
150
Figure 15. Methane in Donor-amended Source Zone Microcosms (A) and Treatment Zone Microcosms
(B). Microcosm series were prepared with contaminated aquifer water and sediment collected within the
source zone upgradient of the whey barrier and within the treatment zone.
Control Results
Concentrations of CEs in pasteurized controls did not significantly vary,
indicating low abiotic CE loss rates supported the integrity of experimental conditions
maintained throughout the study (Figs. 16B, 17B). TCE was dechlorinated to cDCE in
bioaugmented control microcosms containing sediment and water upgradient of the PRB,
57
suggesting that the organic content of aquifer materials contributed to dechlorination in
upgradient microcososms (Fig. 16A). TCE conversion was not observed in
bioaugmented treatment-zone controls (Fig. 17A).
A. Source Zone
Bioaugmented Control
120 -, —§
--
_#_TCE
-
- -
-
-B—cDCE
--
tr- VC
- - - - — -
100 -
I
80 -
§ |
60.
O
40 -
^--^-
UJ
O
20
n
0
B. Source! Zone
Pasteurizec Control
50
150
100
Time (days)
_«_TCE
-e—cDCE
A—VC
O
a> oo
O
O
*.
O
to
3
CE Concentration
(uM)
inn
3fts8=rS—
()
B
—i—x
50
B
*—i
100
Time (days)
a
m
1
r-i [
15C
Figure 16. Chlorinated Ethene Concentrations in Source Zone Bioaugmented Control Microcosms (A) and
Pasteurized Control Microcosms (B). Bioaugmented microcosm series were prepared with sediment and
ground water collected within the source zone upgradient of the whey PRB and amended with KB1/LV1
culture.
58
A. Treatment Zone
Bioaugmented Controls
.TCE
g
cDCE _ * _ V C
60 50 40
30
—11
20 10 0
•*—
B
• I
,
20
40
B. Treatment Zone
Pasteurized Controls
,
H
60
Time (days)
r
80
a—
100
.TCE - B — cDCE —*—VC
80
•
§
I
70
60 " ^
50 •^-s
S 40
V-
-so
20
10
0 -mm—
m
" "5—
» i
20
—m—,
1—m
40
60
Time (days)
r
80
m—
100
Figure 17. Chlorinated Ethene Concentrations in Treatment Zone Bioaugmented Control Microcosms (A)
and Pasteurized Control Microcosms (B). Bioaugmetned microcosm series were prepared with sediment
and ground water collected within the treatment zone and amended with KB1/LV1 culture.
59
CHAPTER V
RESULTS OF THE FIELD STUDY OF ENHANCED TCE BIODEGRADATION
WITHIN A FULL-SCALE WHEY PRB
Introduction
This field study was performed to evaluate the efficacy of a full-scale whey PRB,
designed to stimulate the dechlorination of high aqueous concentrations of CEs (102-103
ug/L) migrating from a source zone. The field study examines the effects of altering the
donor loading mass, loading frequency, and injection method on CE distributions. The
longevity of the applied whey was determined by comparison of COD, organic acid, and
CE trends. These field results combined with the laboratory determinations of whey
anaerobic degradation pathways and intermediate lifetimes allowed completion of a whey
material cost analysis.
Long-term CE and Ethene+Ethane Trends
Figures 18 and 19 show long-term CE and ethene+ethane trends in injection well
(BIP3, BIP7) and downgradient monitoring well (NMW1 A, NMW1B, NMW2B) samples
following the 5 whey injection. Injection well CE concentrations averaged to mean
treatment-period concentrations are shown in Figure 20. Long-term CE and ethene
average concentrations of ground water samples collected upgradient of the PRB (well
BMW4/2) are shown in Table 8.
In the long-term analysis of VOCs in injection well samples, TCE and cDCE
concentrations tended to decrease, while total CE levels tended to consist of higher VC
percentages (Fig. 20). TCE and cDCE levels (Figs. 18A, 19A) were consistently lower
than upgradient concentrations (Table 8), while BIP7 VC levels (Fig. 19A) periodically
exceeded upgradient concentrations. Total CE levels tended to decrease in injection well
BIP3 (Fig. 20 A). These trends are indicative of the growth of dechlorinating microbial
populations and possible favorable alteration of biogeochemical conditions within the
whey biobarrier.
Pilot Phase Trends
During the pilot phase of the field study (i.e., prior to the 8 injection), TCE
levels in injection well samples remained quite low and appeared to be slowly decreasing.
Average TCE values decreased from 17.4 ug/L to 14.4 ug/L in BIP3 samples and from
20.6 ug/L to 9.9 ug/L in BIP7 samples (Fig. 20). BIP3 cDCE levels also gradually
decreased (to 43 ug/L), while VC and ethene remained below detection (Fig. 20A).
cDCE and VC levels in BIP7 were, however, more variable. BIP7 CE and ethene+ethane
levels increased following the 6th injection, although apparently the result of injection of
contaminated extraction water (Fig. 19A). Within 145 days of the 7 injection, BIP7
cDCE, VC, and ethene+ethene levels again increased (to 410, 710, and 1060 ug/L,
respectively) (Fig. 19A), potentially the result of seasonal ground water flow fluctuations.
During the pilot phase, TCE averages downgradient of the PRB were lower, while
cDCE, VC, and ethene concentrations were generally higher, than averages upgradient of
the PRB. TCE concentrations increased, however, with distance downgradient of the
whey injection zone. NMW1A TCE values decreased to below the MCL within one
month of injection 6 but then rebounded and remained high during the 7th treatment
period (141.7 ug/L - 200 ug/L on average in 5^-7& treatment periods), and NMW1B TCE
averages were consistently higher than in NMW1A (387 ug/L -390.8 ^g/L in the 5th - 7th
61
treatment periods). cDCE concentrations averaged 5280 ug/L -6734.4 ug/L in NMW1A
and NMW1B and 701.6 ug/L -2121.4 ug/L in NMW 2B.
Operational Phase Trends
At injection well BIP3, VC and ethene levels tended to increase following
injection 9, and TCE levels fell below the MCL during the 10 treatment period (Fig.
18A). BIP7 cDCE levels decreased from 620 ug/L prior to injection 8 and remained <53
ug/L in treatment periods 8 through 10, while ethene+ethane concentrations increased
more sharply than VC concentrations (Fig. 19A).
Significant decreases in CE concentrations were observed downgradient of the
whey PRB during the operational phase of the study. TCE averages at monitoring points
downgradient of the PRB (NMW wells) were generally lower than upgradient averages
and decreased, following injection 9, to either <50 ug/L or below the MCL (Figs. 18B,
18C, 19B). Throughout the study, cDCE and VC averages at NMW1A and NMW1B
wells (downgradient of BIP3) (2000-6734 ug/L cDCE; 540-2100 ug/L VC) were higher
than the average concentrations at the upgradient well (Figs. 18B, 18C). Through the 9
treatment period, VC averages at NMW2B (downgradient of BIP7) were also higher (Fig.
19B). However, steady decreases were observed after the 8th injection. Relative to
averages in the 5th treatment period, cDCE averages decreased by 56% (NMW1 A) and
70% (NMW1B) by the 9th treatment period, and by the 10th treatment period NMW2B
cDCE and VC averages decreased by 90%. Downgradient VC trends, furthermore,
deviated from cDCE trends, as VC concentrations increased sharply to 2100 ug/L
(NMW1A) and 1600 ug/L (NMW1B) after injection 10 (Fig. 18B-C). Ethene averages
62
were much higher than the upgradient average and began to increase downgradient of
BIP3 during the 8th and 9th treatment periods.
3
UJ
u
750
A. BIP3
950
1150
1350
1550
Time (days after initial injection)
1750
2500
2000_
u
Q
4000
2000
750
B. NMW1A
950
1150 1350
1550
1750
Time (days after initial injection)
UJ
u
Q
750
1150 1350 1550
950
C. NMW1B
T i m e (days after initial injection)
-TCE
-S—cDCE
-VC
1750
. Bhene+Hhane
Figure 18. Chlorinated Ethene and Ethene+Ethane Distributions in Injection Well BIP3 (A) and
Downgradient Wells NMW1A (B) and NMW1B (C). Ground water samples were collected during
treatment periods 5-10.
500
inj 8
450
It
400 /i
/ i
350 300
1 I
250
/ /•lift \ i Til 1
200 1
• IIIV \\ ' • / /////
/ /1Van
150 / / 111 \
/ j
100 - 77/ IfT r \ X yr
50 0-
j7
v
Ul
o
* fa
i5nn
Kjnj 1 3
- 1000_^
S
"5>
- 800 3 -
n
ll
I1
*J 9/
UJ
I 1
i A
i
i
600 - 400 g
a
J
7\ - - 200
/'A
-0
950
1150
1350
1550
750
A. BIP7
Time (days after first injection)
1750
4000
TCE, DCE ug/L)
3500
*—*
3000
4000
2500
J
3
2000
UJ
1500
^2000 g
1000
500
B. NMW2B
-TCE
750
950
1150 1350 1550 1750
Time (days after initial injection)
-H—cDCE
-VC
- Ethene+Ethane
Figure 19. Chlorinated Ethene and Ethene+Ethane Distributions in Injection Well BIP7 (A) and
Downgradient Well NMW2B (B). Ground water samples collected during treatment periods 5-10.
65
• TCE
• cDCE
a VC
__
ORH
3" 200 o>
3
^
1
50
5
O)
2 100 >
<
ui 50 O
p-.
6
7
9
10
m
o
a.BIP3
\
\
Treatment
*&
-^
%
-0-
%
>,
^ .
'-•'a
%
\>
%
>•
<*3
<5\
Periods 5-10
«*
1?
^
.*o
Time (days after initial injection)
• TCE
o cDCE
.
?p;n
3"
°F
a VC
_
200 10
O)
3
^
^
<£,
150-
0
o>
2
100 -
1
<
LU
O
5
8
9
50
0-
b.BIP7
%
^
>,
*
*
W
Treatment
\fiU
>,
*,
<,
<*»
<»
Periods 5-10
'
<fr
%
%
%
\
Time (days after initial injection)
Figure 20. Average Chlorinated Ethenes During Treatment Periods 5 through 10 in Injection Wells BIP3
(A) and BIP7 (B).
Table 8. Upgradient Source Zone Chlorinated Ethene and Ethene Average Concentrations. Ground water
samples were collected from well BMW4/2, located within the source zone approximately 20ft. upgradient
of the whey PRB.
Upgradient Chlorinated Ethenes and Ethene
Average (\ie/D
CV (%)
TCE
620
13.0
cDCE
1650
16.3
VC
132.5
23.5
Ethene
3.1
1.7
Effects of Whey Fermentation
Long-term COD and TCE flux trends in injection well samples are shown in
Figure 21. Organic acid distributions in the 10l treatment period are shown in Figure 22.
Figures 23 and 24 show CE distributions during treatment period 10, which was initiated
by whey injection from days 1 to 3.
As COD levels increased, TCE fluxes within the injection zone generally
decreased (Fig. 21). Conversely, as observed during the 8th treatment period within 92
days of injection (particularly at BIP7), the TCE flux increased as COD levels fell (Fig.
21B). BIP3 cDCE levels, likewise, increased within 92 days of the 8th injection (Fig.
18 A).
The presence of organic acid intermediates and increased COD levels were
sustained in injection wells through day 73. The detection of organic acids within one
day of injection at NMW1A suggests the immediate onset of whey degradation and the
slight downgradient migration of electron donor (Fig. 22B). In the microcosm study,
96.2 ± 0.4 % whey carbon was recovered as organic acid carbon, while no more than
67
22% of whey carbon was detected in field analyses, due likely to dilution, advection, and
dispersion processes.
Organic acid carbon distributions in BIP3 (Fig. 22A) compared favorably with the
distributions of whey metabolite carbon observed in the anaerobic microcosm
experiments (Table 7). The fermentation of lactate and butyrate apparently provided
immediate reducing equivalents for dechlorination within the injection zone. Acetate and
propionate then appeared to stimulate dechlorination through day 73 of the 10 injection
event. In BIP3, cDCE and VC concentrations decreased significantly by day 73 (29.7
ug/L cDCE; 28.3 ug/L VC) and further by day 133 (10.4 ug/L cDCE; 4.5 ug/L VC) (Fig.
23 A). In BIP 7, cDCE and VC levels decreased within 70 days (from 51.0 ug/L and
535.0 ug/L to 11.1 ug/L and 190.4 ug/L, respectively) (Fig.24A). Ethene+ethane levels,
furthermore, increased through day 70 within the treatment zone (Figs. 18A, 19A).
Continued dechlorination observed within the injection zone through day 133 may be
attributed to the undetected presence of whey metabolites.
Downgradient (NMW) CE trends observed within 70-73 days of the 10 whey
injection reflected improved conditions within the injection zone. TCE levels in
NMW1A samples decreased from 26.3 ug/L prior to injection to below the MCL during
the injection period and through day 73 (Fig. 23 A). In NMW1B samples, TCE
concentrations decreased from a maximum level of 219.6 ug/L observed during injection
to <51.3 ug/L between day 10 and day 73 (Fig. 23C). cDCE and VC levels in NMW1A
and NMW1B were significantly higher than in the injection zone (Fig. 23); however, in
NMW1B, cDCE was apparently being dechlorinated to VC, as cDCE levels decreased by
14.75 uM while VC levels increased by 18.0 uM. NMW2B cDCE and VC levels
68
decreased significantly by day 70 (from 1758.6 ug/L and 4309.4 ug/L during injection to
51.0 ug/L and 28.6 ug/L, respectively) (Fig. 24B).
While CE concentrations continued to decrease through 133 days (following the
10th injection) within the PRB, increases in CEs were observed in downgradient wells
after 100 days of injection (significantly, increases inNMWIA CEs, 116 days after
injection 8; NMW2B cDCE and VC, 130 days after injection 10; and NMW1A and
NMW1B TCE levels, 133 days after injection 10) (Figs. 23B-C, 24B). Increases in total
CEs were less apparent in treatment period 9, which was relatively short (138 days) (Figs.
18B-C, 19B). These results suggest that, after approximately four months, the whey PRB
may become less uniform and require renewal.
69
_»—COD
200 b
180 160 ^140 J
r*
^120i
-§•100 § 80 O
„„
60 - i
40 20 0750
BIP3
II J6
•
;
•
1
•'•
.i
i
i
1
i
1
1
1
i
i
i
1
1
1
1
•
n/
•
950
20
18
i n i o " 16_
i
i
i
T
H1
H
14f
12 j
'l\
! •
l\
1\
1 \
1
1
--
10I
x
3
r
\\ V
A
/•^B
//1'
/ i
/ i
A
/
•
1150
i
i
.:. Q
irjj9
ir}j8 j
>k
i
i
i
i
i
i
-G—TCE
1350
LU
u
\ 11
\ IA
»
'
1550
\\*\
i\
•' V *
Time (days after first injection)
1750
200
O)
E,
Q
O
O
750
B. BIP7
950
1150
1350
1550
1750
Time (days after first injection)
Figure 21. COD and TCE Flux During Treatment Periods 5 - 10 in Injection Wells BIP3 (A) BIP7(B).
70
-•—lactate
±
acetate _g—propionate
0
BIP3
50
&
butyrate
100
Time (days after injection 10)
-A— Injection Well BIP3
. Dow ngradient Well (NMW1 A)
^ 25
50
100
Time (days after injection 10)
Figure 22. Organic Acid Carbon Distribution in Treatment Period 10 in Injection Well BIP3 (A) and
Organic Acid Carbon Percentages During Treatment Period 10 in Wells BIP3 and NMW1A (B).
Percentages were calculated relative to the approximate whey lactose carbon mass in the whey slurry
injected in treatment period 10.
71
A. BIP3
700
600
_j
500
?
3
400
o
>
uf 300
200
o
au 100
0
» • »
50
100
Time (days)
B. NMW1A
-B-
• cDCE
• VC
150
-TCE
6000
o» 5000
£
4000
uf 3000
O
S 2000
50
100
T i m e (days)
C. NMW1B
• cDCE _ A _ V C
150
• TCE
8000
UJ
^
u
2000
50
100
150
Time (days)
Figure 23. Chlorinated Ethene Distributions in BIP3 (A), NMW1A (B), and NMW1B (C) During
Treatment Period 10.
72
50
100
Time (days)
B. NMW2B
' 2000
o> 1500
3
LU
O
ao 1000
111"
o
500
50
100
Time (days)
150
Figure 24. Chlorinated Ethene Distributions in BIP7 (A) and NMW2B (B) During Treatment Period 10.
73
CHAPTER VI
DISCUSSION
Electron Donor Material Comparison
Organic acids were significant degradation products of the three PRB materials
studied. Within -30 days, H2 partial pressures generally agreed with low levels that were
measured by Fennell et al. (1997) in their short-term test of cultures amended with
butyrate, a slow-degrading organic acid (10"4'2 to 10"50 atm. at 1:1 and 1:2 PCE:acceptor
donor ratios). Occasionally, H2 partial pressures agreed with or fell below the levels
produced in the propionate-amended culture test by Fennell et al. (1997) (10"5'1 atm). In
their short-term tests, the rapid primary fermentation of lactate produced higher levels of
H2 that was diverted to methanogenesis. In this study, low H2 partial pressures were
maintained in the long-term as a result of fermentation of the primary carbon substrates to
slow-degrading intermediate products. Competition from methanogens for available H2
was apparently minimized, as a result of slow release of H2, in the donor-amended
microcosms.
As observed in a long-term test by Fennell et al. (1997), significant
methanogenesis occurred after approximately 100 days and may be attributed to the
eventual establishment of acetotrophs in the microcosms. Acetotrophic methanogenesis
may have been inhibited in the presence of cDCE, as observed in a previous study (Yang
and McCarty, 1998), although methane did accumulate in the presence of cDCE in HRC
treatment-zone microcosms.
Interestingly, rapid VC dechlorination and comparable complete dechlorination
rates in whey and whey+KBl treatment zone microcosms indicates that native
74
dechlorinating species established within the PRB are similar to Dehalococcoides KB1, a
strain capable of chlororespiration of VC to ethene. Comparison of dechlorination rates
and carbon distributions furthermore indicates that native dechlorinating, acetogenic, and
acetotrophic populations were more prominent in treatment-zone sediment. This
suggests that whey, which primarily underwent fermentation to acetate and H2, may
support a diverse - and potentially more efficient - dechlorinating community,
comprised of PCE and TCE dechlorinators which preferentially use acetate as a direct
electron donor, syntrophic acetate oxidizers, and H2-utilizing dechlorinators. Whether
acetate serves as a direct electron donor at the contaminated site is unknown. If
acetotrophic methanogenesis is energetically coupled to the conversion of VC to acetate,
as observed previously in stream-bed sediment microcosms (Bradley and Chapelle,
1999), the stimulation of acetotrophic populations could hypothetically increase rates of
complete dechlorination by an alternative pathway. Reductive dechlorination to ethene
was the predominant pathway in these microcosms, although slightly low observed
ethene values, according to mass balance calculations, allow for the possibility of partial
conversion of VC to acetate.
The relatively slow dechlorination in HRC-amended microcosms appears to be
related to a slower rate of substrate degradation and slower release of H2 reducing
equivalents, as indicated by a gradual decrease in H2 partial pressure (upgradient) and the
persistence or accumulation of organic acid (particularly propionate) carbon. As a sole
donor amendment to aquifer microcosms, propionate has been shown previously to
support PCE dechlorination to DCE (Gibson and Sewell, 1992). Previously in aquifer
microcosms amended with an organic acid mixture, however, propionate persisted and
75
appeared to not contribute to dechlorination (Gibson et al., 1994). In that study,
propionate appeared to not degrade due to inhibition by high levels of acetate and/or H2
(Gibson et al., 1994). In an HRC-amended tank designed to simulate a field-scale
aquifer, propionate also appeared to serve as a H2 sink (Adamson et al., 2003).
Propionate may persist in the presence of organic acids which more readily undergo
anaerobic degradation and which, as a result, may maintain levels of acetate and/or H2
above the thermodynamic threshold of propionate oxidizers (Gibson et al., 1994).
Whether propionate contributes to complete dechlorination or serves as a carbon sink
under field conditions warrants further investigation, particularly in the field. Indeed,
HRC's slow fermentation and high carbon content may benefit more efficient
dechlorination in field conditions, where propionate may more readily degrade and where
contamination is a long-term problem.
The high dechlorination rates combined with high organic acid carbon
percentages observed during dechlorination in whey-amended microcosms are indicative
of whey's relative (molar) efficiency. Whether whey is relatively cost-efficient depends
on material longevities and required PRB renewal frequencies. Whey lactose carbon was
diverted primarily to acetate by the time dechlorination was complete in treatment zone
microcosms (-60 days). Particularly if acetotrophic methanogens are active during
dechlorination in field conditions, this suggests that whey reinjection may be necessary
on a quarterly or even bimonthly basis. More frequent injection of whey may be
required, as a result of more rapid dechlorination and depletion of primary and
intermediate electron donor species, than in applications of alternative materials that
76
ferment more slowly (i.e. HRC). Additional field study is needed to investigate the
dissolution rate of whey deployed as a thick slurry.
While the percent of acetate reducing equivalents directed to dechlorination was
quite low in all of the donor-amended microcosms, long-term field efficiency is of
paramount concern. Donor:acceptor ratio may be lower as a result of carbon substrate
dilution and a high CE flux, and reducing equivalents may, therefore, be more efficiently
directed to dechlorination in the field. Typically, the whey loading values used at the
Battle Creek site to create and sustain the whey PRB were ~0.2 kg/m . Under
sufficiently reducing conditions, this loading value may be considered appropriate for the
establishment of a more active dechlorinating community, as evidenced by whey's
increased efficiency in treatment zone microcosms.
More extensive field investigations are needed to determine the relative
efficiencies of PRB materials as reductive barriers. Side-by-side field investigations will
best compare the long-term efficiencies of PRB substrates as donor materials for
dechlorination. Different PRB materials may in long-term field studies stimulate
different fermentative populations, thus affecting fermentation pathways and
dechlorination rates. Substrate concentrations may also determine fermentation pathways.
Efficiency may be defined in terms of dechlorination time or chloride released over time
under amended conditions relative to unamended conditions:
Efficiency
= ^*amend
\f\mamend
unamend
[^unamend
77
Field Study
The purpose of the field study was to determine the effectiveness of a full-scale
whey bioreactive barrier designed to intercept and treat high concentrations of CEs within
the downgradient region of a source zone. In this long-term study, TCE concentrations
were consistently low within and downgradient of the PRB. Spatial and temporal CE and
ethene+ethane trends were indicative of enhanced rates of TCE and cDCE dechlorination
to VC and ethene. The steady long-term decline in CE concentrations within the PRB
(BIP3 well), to near or below 5 ug/L by the end of the study, was indicative of improved
dechlorination efficiency, apparently resulting from the growth of dechlorinating
microbial populations and possible favorable alteration of biogeochemical conditions
within the injection zone. As indicated by increased ethene concentrations, native
microbial species capable of complete TCE dechlorination, including Dehalococcoides
detected within the source zone, were stimulated within the PRB.
During the pilot phase of the study, high concentrations of CEs (particularly of
cDCE and VC) detected within 20 feet downgradient of the PRB, relative to
concentrations detected at injection wells, suggested that whey was not being well
distributed along the PRB transect, resulting in concentrations of microbial colonies near
the injection wells. Decreases in TCE and cDCE and sharp increases in VC
concentrations in the downgradient wells after the 8th or 9th injections, however, indicated
improved treatment within the barrier. CE and ethene+ethane trends indicate that, with
continuation of the treatment approach used in the operational phase of the study,
downgradient CE and ethene distributions may more closely reflect improved
dechlorination rates in the PRB.
78
Actual source-zone aqueous CE concentrations could very likely exceed average
levels detected within the one upgradient well sampled, resulting in higher than expected
cDCE and VC concentrations downgradient of the PRB. If DNAPL is contained within
the treatment zone, high downgradient cDCE and VC concentrations could also be the
result of increased rates of CE dissolution in ground water within the treatment zone.
Several mechanisms have been proposed which may affect the rate of dissolution of
DNAPL in ground water, including biodegradation (Hood et al., 2007). CE
biodegradation can increase rates of CE mass transfer into the aqueous phase, resulting in
increased concentrations of the less-chlorinated ethenes which have higher solubilities
than TCE (Cope and Hughes, 2001). Previously in a laboratory and field study of the
effects of whey, high whey concentrations (10% mass/mass) were shown to increase rates
of TCE dissolution in the aqueous phase, apparently due to the effects of whey's
hydrophobic protein fraction (Macbeth et al., 2006).
Comparison to upgradient CE concentrations suggests that, by placement of the
PRB within the downgradient region of a source zone, whey injection enhanced rates of
dechlorination by 20.7 umols/m2/day. Comparison of CE concentration decreases in the
three downgradient wells, between the 5* and 10 treatment periods, gives a comparable
average dechlorination rate of 23.8 umols/m2/day ± 32.6% CV.
Electron Donor Material Cost Comparison
Assuming from the laboratory experiments that ~96% of whey lactose carbon is
diverted to organic acid carbon, one may conclude from the laboratory and field
observations that, where -800 mg/L whey concentrations are used, organic acid
79
intermediates may be degraded within approximately four months. Within these
parameters, the material cost of a dairy whey PRB can be determined. Based on the
average market price of edible whey powder ($0.3348-$0.3361 / lb in 2006 (USDA,
2006)), the estimated material cost is $0.77 per kg whey. Considering that injection of
approximately 300 kg of whey sustained a 300-ft PRB in treatment periods 8-10, whey is
estimated to cost $0.77 per lateral foot. For the creation and maintenance of a 300-ft
whey PRB by tri-annual injections, the annual material costs were approximately $700.
Table 9 shows an estimated material cost comparison of whey, lactate syrup, and
HRC. Estimated costs in the middle column are based on the carbon content of the three
materials, as determined in the donor material comparison experiments: 28.0% (whey),
19.3% (lactate syrup), and 32.0% (HRC) by weight. The estimated material cost of
whey, based upon carbon content, is 34.9% and 6.7% of the estimated costs of lactate
syrup and HRC carbon, respectively. In the final column, substrate costs are based on
the bioavailability of their carbon content and the assumption that propionate is not
readily biodegradable. In the anaerobic microcosms prepared with upgradient source
zone sediment and water, <22.5% of whey lactose carbon, 51% of sodium lactate carbon,
and 65.2% of HRC carbon were converted to propionate carbon. The estimated material
cost of bioavailable whey carbon is 22.0% and 3.0% of the estimated costs of lactate
syrup and HRC carbon, respectively.
80
Table 9. Comparison of Estimated Material Costs of Whey, Lactate Syrup, and HRC.
Substrate Cost
Substrate Material
$ per Kg
$ per Kg carbon0
$ per Kg available carbon
Dried Whey
0.77a
2.75
3.55
Sodium Lactate
1.52"
7.88
16.10
HRC®
13.20"
41.25
118.50
a
U.S.D.A., 2006
Raymond et al., 2004
c
Calculations based on the measured total organic acid carbon content of sweet dried whey (70% <xlactose), sodium lactate syrup (60% concentration), and HRC®
d
Calculations based on measured available carbon content, assuming propionate isn't readily
biodegradable
b
Actual substrate material costs will depend on loading volumes and the frequency
of reapplication, if necessary. As a slowly degrading material, HRC may provide
reducing equivalents for dechlorination for a relatively long time period. Continued field
study, ideally involving side-by-side investigation, is needed to determine the relative
efficiencies of complex carbon substrates.
Conclusion
The results of this laboratory and field study indicate that complex electron donor
materials undergo anaerobic biodegradation to organic acids and that the biodegradation
of organic acids, in turn, may provide a slow release of H2 at levels below the minimum
threshold of hydrogenotrophic methanogens. Dairy whey was apparently the most
efficient material of the three materials studied. The complete dechlorination of TCE
occurred more rapidly in microcosms amended with whey or lactate, while wheyamended microcosms contained the highest percent carbon upon complete
81
dechlorination. In comparing carbon content or bioavailable carbon content, the material
cost of whey was determined to be significantly lower than lactate or HRC.
Results of the study indicate that dechlorinating populations similar to
Dehalococcoides sp. strain KB1 were established by periodic injection of whey and that,
with the establishment of microbial populations capable of complete dechlorination by a
respiratory process, a whey PRB may effectively and efficiently dechlorinate high
concentrations of CEs migrating from a source zone. With continued use of extractioninjection well loops, native dechlorinating populations which colonized volumes
surrounding injection wells during the pilot phase may become better established along
the PRB transect. Ultimately, if the emplacement of a PRB fails to effectively stimulate
complete dechlorination, methanotrophs may mediate the aerobic cometabolic
degradation of cDCE and VC downgradient of the barrier (Forrester et al., 2005).
Efficient treatment designs may also include emplacement of downgradient oxidative
barriers that more readily degrade the lower-chlorinated ethenes. In situations where
chlorinated contaminant sources persist, sequential active and passive technologies may
be necessary.
82
Appendix A
Field Design Microcosm Study
83
A microcosm experiment was performed by Dr. Michael Dybas at Michigan State
University to determine whether halorespiration processes were occurring at the site and
to examine the growth of native Dehalococcoides in the presence and absence of whey.
Microcosms were prepared using sediment and water collected within the source zone.
250 mL bottles, containing 200 mL anaerobic ground water and 15 g sediment, were
sealed with Mini-inert® valves and Teflon-lined rubber septa and spiked with either
1 Oppm or 1 OOppm TCE. Triplicate microcosms were amended with 750 mg/L whey.
Living controls were also prepared in triplicate. Triplicate killed controls were
pasteurized by heating to 70°C in a convection oven for two consecutive nights and
cooling to room temperature in the intervening day. For the preparation of abiotic
microcosms, pasteurization is a more effective method than the addition of pesticides
(e.g. sodium azide), which selectively kill native microbes.
Native Dehalococcoides DNA was quantified withl6S rRNA gene targeted realtime PCR (qPCR), using primers 5' CTGGAGCTAATCCCCAAAGCT 3' (forward) and
5' CAACTTCATGCAGGCGGG 3' (reverse). qPCR is an accurate and precise method
of quantifying DNA and is described in detail elsewhere (Griintzig et al., 2001). DNA
samples were analyzed in triplicate. UltraClean Soil DNA extraction kits (MO BIO
Laboratories, Inc, Solana Beach, CA) were used to extract DNA from sediment pellets.
To obtain sediment pellet samples, bottles were shaken vigorously to slurry the sediment
and water, and 1.5 mL slurry samples were centrifuged at 10,000 rpm for 6 minutes.
DNA concentrations were determined in pg/mL units.
At 105 and 175 days, whey-amended microcosms spiked with 10 ppm TCE
contained 38.0% and 27.8%, respectively, of chlorinated ethene equivalents added to the
84
microcosms. The rate of dechlorination was comparable to the rate observed in the
upgradient source zone whey-amended microcosms in the electron-donor comparison
study (40.8% at day 90; 23.1 % at day 155). Complete dechlorination was observed
within 420 days in whey-amended microcosms spiked with lOppm TCE. In wheyamended microcosms spiked with lOOppm TCE, dechlorination lagged at cDCE,
apparently as a result of the inhibition of the activity of cDCE-dechlorinating
populations. TCE concentrations in source zone aqueous samples were significantly
lower, although microbial populations within the source zone may encounter high TCE
concentrations due to DNAPL dissolution in ground water.
In unpasteurized control microcosms, TCE was dechlorinated to cDCE, indicative
of the presence of readily oxidized organic matter within the source zone. Within 595
days, dechlorination failed to extend past cDCE in unpasteurized contols.
85
Appendix B
TOC and TIC of Sediment SamplesfromWhey Injection
and PRB Monitoring Well Cores
86
Table A2-1. Total Organic Carbon (TOC) Content of Whey Injection Well (BIP) and Whey Barrier
Monitoring Well (NMW) Sediment Core Samples.
3
10
12
14
16
18
20
24
26
31
48
Average % Organic
Carbon
1.79048
3.126009
37.65021
43.19142
11.39602
8.99149
7.820125
1.264217
0.91226
0.022343
0.10858
10
13
17
24
47
1.540627
5.926332
8.05891
40.59621
10.34357
0.376948
0.07005
BIP-7-4
BIP-7-8
BIP-7-14
BIP-7-17
BIP-7-27
BIP-7-29
BIP-7-34
BIP-7-39
14
17
27
29
34
39
4.45727
16.31382
1.803948
1.162828
0.598589
0.025939
0.70376
0.311803
NMW-1B-10
NMW-1B-12
NMW-1B-14
NMW-1B-16
NMW-IB-18
NMW-1B-29
10
12
14
16
18
29
7.592778
6.195084
9.269311
7.973798
5.812682
1.260552
NMW-•2B-7
NMW- 2B-9
NMW- 2B-11
NMW- 2B-13
NMW- 2B-14
NMW- 2B-15
NMW-•2B-16
NMW- 2B-19
NMW-•2B-23
7
9
11
13
14
15
16
19
23
6.509953
4.970273
3.413456
1.600162
13.71319
0.733974
0.629982
0.21728
0.50369
Well sample
BIP-3-3
BIP-3-10
BIP-3-12
BIP-3-14
BIP-3-16
BIP-3-18
BIP-3-20
BIP-3-24
BIP-3-26
BIP-3-31
BIP-3-48
BIP-5-4
BIP-5-8
BIP-5-10
BIP-5-13
BIP-5-17
BIP-5-24
BIP-5-47
Depth (ft)
87
Table A2-1 - continued
NMW-2B-27
NMW-2B-33
NMW-2B-35
NMW-2B-40
27
33
35
40
0.266113
0.524544
0.753547
0.562683
88
Table A2-2. Total Inorganic Carbon (TIC) Content of Whey Injection Well (BIP) and Whey Barrier
Monitoring Well (NMW) Sediment Core Samples.
Well
BIP-3
BIP-3-10
BIP-3-12
BIP-3-14
BIP-3-16
BIP-3-18
BIP-3-20
BIP-3-24
BIP-3-26
BIP-3-31
BIP-3-48
Depth (ft)
3
10
12
14
16
18
20
24
26
31
48
Average % Inorganic Carbon
0.407739
1.904542
3.715582
0.395462
8.280483
3.17154
7.653447
2.475457
2.990435
0.01634
0.180814
BIP-5-4
BIP-5-8
BIP-5-10
BIP-5-13
BIP-5-17
BIP-5-24
BIP-5-47
4
8
10
13
17
24
47
0.554946
0.056733
6.755745
0.86984
7.613955
0.386232
0.148609
BIP-7-4
BIP-7-8
BIP-7-14
BIP-7-17
BIP-7-27
BIP-7-29
BIP-7-34
BIP-7-39
4
8
14
17
27
29
34
39
0.025689
5.944222
4.077364
2.586362
0.15641
0.081341
0.628567
1.092215
NMW-1B-10
NMW-1B-12
NMW-1B-14
NMW-1B-16
NMW-1B-18
NMW-1B-27
NMW-1B-29
10
12
14
16
18
27
29
2.934707
3.876496
6.673866
4.679785
5.238348
3.388218
0.299651
NMW-2B-7
NMW-2B-9
NMW-2B-11
NMW-2B-13
NMW-2B-14
NMW-2B-15
NMW-2B-16
7
9
11
13
14
15
16
1.383191
8.579492
7.700088
1.006599
0.310764
1.366179
0.399263
89
Table A2-2 - continued
NMW-2B-19
NMW-2B-23
NMW-2B-27
NMW-2B-33
NMW-2B-35
NMW-2B-40
19
23
27
33
35
40
2.038751
0.64501
0.452147
0.977068
0.821032
1.29573
90
Appendix C
Comparison of Organic Acid Concentrations Determined by
GC/MS and HPLC
91
Table A3. Comparison of organic acid concentrations in whey-amended microcosms as determined by
GC/MS and HPLC.
Sample
slcbl
slcb2
si sal
si sa2
s2cbl
s2cb2
s2 sal
s2sa2
s3cbl
s3cb2
s3 sal
s3 sa2
s4cbl
s4cb2
s4 sal
s4 sa2
s5cbl
s5cb2
s5 sal
s5 sa2
s6 cbl
s6cb2
s6 sal
s6 sa2
Sample
slcbl
slcb2
si sal
si sa2
s2cbl
s2cb2
s2 sal
s2sa2
s3cbl
s3 cb2
s3 sal
s3 sa2
s4cbl
s4cb2
s4 sal
s4 sa2
Acetate (ppm)
HPLC
GC/MS
10.29
86.42107
66.066
283.646
146.9531
136.4612
75.05101
133.089
239.3776
94.70638
125.1013
308.0582
409.8241
555.4321
480.4515
174.833
313.2024
389.4198
380.7227
7.017417
2.336414
452.1858
21.40311
335.98
230.04
192.43
157.65
347.19
382.37
340.95
354.63
376.23
359.82
465.56
357.7
208.93
325.76
480.18
518.37
25
6.9
480.21
15
Propionate (ppm)
HPLC
GC/MS
0
6.68
7
0
9.89
0
9.46
3.601992
7.4
11.1305
7
8.847719
14.21
13.38393
12.86
9.701687
21.39321
9.49
27.7467
8.82
25.19822
20.31
23.7
28.31128
9.49
17.74793
8.82
24.63198
48.50979
20.31
58.70148
23.7
Mean
309.813
188.4965
164.4456
116.3505
240.1395
310.8738
217.8282
239.8656
342.1441
384.822
510.4961
419.0757
191.8815
319.4812
434.7999
449.5464
16.00871
4.618207
466.1979
18.20156
mean
6.530996
9.265249
7.92386
13.79696
11.28084
15.4416
18.28335
22.75411
26.00564
13.61896
16.72599
34.40989
41.20074
Stdev
37.00571
58.75135
39.57593
58.40631
151.3923
101.1109
174.1205
162.3013
48.20474
35.35822
63.5492
86.7984
24.11025
8.87958
64.17718
97.33132
12.71561
3.226942
19.8161
4.527685
Stdev
4.142238
2.63786
1.306535
0.584123
2.233265
8.41684
13.3832
3.456491
3.260665
5.839237
11.18076
19.94026
24.74978
%rsd
11.94453
31.1684
24.06627
50.19858
63.04347
32.52473
79.9348
67.66344
14.08902
9.188201
12.44852
20.71186
12.56518
2.779375
14.76016
21.65101
79.42931
69.87435
4.250578
24.87526
%rsd
63.42429
28.47048
16.48861
4.233708
19.79697
54.50755
73.19882
15.19063
12.5383
42.87578
66.84662
57.94921
60.07121
92
Table A3 - continued
s5cbl
s5cb2
s5 sal
s5 sa2
s6cbl
s6cb2
s6 sal
s6 sa2
Sample
slcbl
si cb2
si sal
si sa2
s2cbl
s2cb2
s2 sal
s2sa2
s3cbl
s3cb2
s3 sal
s3 sa2
s4 cbl
s4cb2
s4 sal
s4sa2
s5cbl
s5cb2
s5 sal
s5 sa2
s6cbl
s6cb2
s6 sal
s6 sa2
15.9458
17.94742
29.33264
25.63821
14.10147
16.36122
30.51058
40.8566
10.4
10.7
19.18
18.35
15.01
13.47
17.79
23.05
Butvrate (ppm)
GC/MS
HPLC
0
0
0
0
0
0
0
0
94.69067
115.48
87.38948
114.16
127.5934
157.23
93.8943
153.18
179.3214
100.12
182.2083
62.3
267.4198
152.73
217.1385
128.08
87.36676
99.83
137.5679
104.38
138.6833
106.19
179.5995
142.84
91.34948
87
76.2404
65.96
32.82937
75.96
12.30375
28.28
13.1729
14.32371
24.25632
21.9941
14.55574
14.91561
24.15029
31.9533
mean
105.0853
100.7747
142.4117
123.5372
139.7207
122.2541
210.0749
172.6093
93.59838
120.974
122.4366
161.2197
89.17474
71.1002
54.39469
20.29188
3.921474
5.1247
7.179004
5.15354
0.642426
2.044398
8.994812
12.59117
std dev
14.70028
18.92962
20.95622
41.92132
56.00387
84.78796
81.09794
62.97389
8.81284
23.4674
22.97622
25.99288
3.075548
7.269343
30.49796
11.29691
29.76925
35.77774
29.59642
23.43146
4.413562
13.70643
37.24515
39.40491
%rsd
13.98889
18.78409
14.71524
33.93418
40.08272
69.35385
38.6043
36.4835
9.41559
19.39872
18.76581
16.12264
3.4489
10.22408
56.0679
55.67209
10.6253
cb= whey microcosms; sa= whey+KBl microcosms; s=sacrifice
93
Appendix D
Microbial Contamination of Whey Injection Wells
94
Introduction
Following the third whey injection, wells were tested for biofouling, i.e.
contamination of well screens as a result of high growth rates of native microbial
populations. Microbial contamination of injection wells is a common occurrence due to
high carbon concentrations and suboxic conditions in the vicinity of wells.
Materials and Methods
Sediment-ground water suspensions were collected using a bailer from whey
injection (BIP) wells and one upgradient (BMW4/2) well, near the top of the water table.
Samples were stored at 4°C until analysis, which was completed within one week of
sampling.
Fe (ID and Total Iron
For determination of Fe (II), sediment - ground water suspensions were analyzed
using a Chemetrics Fe (II) test kit, according to the test procedures (without addition of
the activator solution). Samples were diluted by 25-fold using meq water to bring Fe (II)
concentration within the range of the kit calibration.
For total iron determinations, sediment - ground water suspension were filtered
and dried overnight in a 100°C convection oven. Cooled samples were weighed and then
acidified to pH 2-3 by addition of HC1 in MilliQ water to reduce Fe(III) to Fe(II). Total
iron content was then determined using a Chemetrics Fe (II) test kit. For digestion of
very insoluble iron species (e.g. magnetite, ferrite), samples were pretreated by addition
of 5 drops of activator solution per 25 mL sample mixture and gently boiled until
95
volumes were reduced by approximately 50%. Samples were diluted to 25 mL, and then
by 22-fold, by addition of meq water to bring Fe(II) concentrations within the range of
the kit calibration. Test procedures for photometric analysis were then followed.
TOC and TIC
Sediment - ground water suspensions were filtered and dried overnight in a 40°C
convection oven. Samples for TOC analysis were then acidified overnight with 2 mL
sulfurous acid. CaCC>3 and KHP standards were prepared for TIC and TOC analysis,
respectively.
Protein
A BCA assay (Pierce), using the microplate procedure, was performed to
determine sediment - ground water sample protein concentrations. Bovine serum
albumin (BSA) was used for construction of the standard curve (Fig. A4).
BCA protein assay
0
500
1000
y = 0.0018x + 0.0573
R2 = 0.993
1500
2000
BSA concentration (ug/mL)
Figure A4. BCA Protein Assay Calibration Curve.
96
Results and Discussion
Table A4 shows the results of carbon, protein, and iron determinations. Injection
well BIP9 protein and TOC concentrations were relatively high. The total iron content
was higher in injection wells than in the upgradient well. The results suggested that
growth of bacteria which oxidize Fe (II) to Fe (III) and the precipitation of ferric iron
may have contaminated the injection wells.
Table A4. TOC, TIC, Protein Content and Iron Content of Sediment Samples Collected from Injection
(BIP) Wells and Upgradient (BMW4/2) Wells.
TIC analysis of filtered, weighed sediment
Well
BMW4
BIP3
BIP9
ug TIC /me sediment
26.91
36.85
14.09
% TIC
2.69
3.69
1.41
TOC analysis of filtered, weighed sediment
Well
BMW4
BIP3
BIP9
ug TOC/mg sediment
38.45
25.63
69.59
%TOC
3.85
2.56
6.96
Protein analysis of filtered, weighed sediment suspended in water
Well
BMW4
BIP3
BIP9
ug protein/mg sediment
24.99
18.1
53.55
Iron analysis of unfiltered suspended sample
Well
BMW4
BIP3
BIP9
Iron II (ppm)
23
41
29
Iron analysis of filtered, dried, weighed sample
Well
BMW4
BIP3
BIP9
Total iron (me iron/mg sediment)
0.172
0.2679
0.2546
% i ron
17.2
26.8
25.5
98
Appendix E
Redox Manipulation Capability of Whey
99
Introduction
Addition of reductants may create or strengthen reducing conditions that are
favorable for reductive treatment approaches, including the enhanced anaerobic
degradation of CEs. Whey's value as a redox adjustment barrier was demonstrated in
prior field-scale research in which oxygen levels were significantly, steadily depressed
downgradient of a whey PRB (Barcelona and Xie, 2001). In that field study, short-term
(11 months) monitoring of dissolved oxygen concentrations resulted in calculation of
pseudo-first-order rate constants of whey depletion and dissolution and allowed
investigators to predict, by mathematical modeling, the lifetime of a whey PRB. Prior to
beginning the field study described in this dissertation, respirometry experiments were
undertaken to more fully evaluate the redox manipulation capability of whey. The results
of a 5-day biological oxygen demand (BOD) experiment are discussed in comparison to
closed column study and prior field results.
Materials and Methods
Four BOD batch series (6 300-mL bottles per series) were prepared for the
determination of BOD of dried dairy whey, as described in detail elsewhere (Greenberg
et al., 1992). Microbial seed was obtained from water treatment plant sediment and the
seed solution was stirred continuously at room temperature. Dilution water was prepared
by addition of lmL each of phosphate buffer, 27.5 mg/L CaCb solution, 22.5 g/L MgS0 4
solution, and 0.25g/ L FeCl3 solution to 1L deionized water. Three control series
contained either dilution water only; 2% glutamic acid/glucose in dilution water; or seed
in dilution water. The fourth series contained 125 mg/L whey and seed in dilution water.
Volumes of seed were approximately 2mL per bottle and 0.1 mL per 1L in the first and
second trials, respectively. Bottles were filled to the brim with air-saturated solutions,
sealed with glass stoppers, and wrapped in foil. Bottles were stored in darkness at room
temperature (20°C). One BOD bottle per series was sacrificed each day for six days.
Aqueous O2 concentrations were measured with an Orion (model 97-08) O2 electrode
connected to a pH meter.
Results
In the first BOD trial, the depletion of O2 occurred within one day in seed control and
whey/seed batches. Results of the second trial in which seed volumes were decreased are
shown in table A5.
Table A5. 0 2 Levels in the 2nd Trial of a 5-day Biological Oxygen Demand (BOD) Study of Whey.
Whey & Seed
Seed Control
Glucose-Glutamic
Acid
Control
Dilution Water
Control
8.04
7.57
4.92
4.24
2.22
1.49
7.78
7.67
7.08
6.79
6.70
6.54
7.76
7.76
7.77
4.81
3.89
3.82
7.90
7.85
7.84
7.78
7.68
7.79
6.45
1.24
3.94
0.11
02 (mg/L)
Day
0
1
2
3
4
5
5-day DO uptake
(mg/L)
The BOD of whey depletion, expressed as mg of dissolved oxygen (DO) uptake per kg
whey, was calculated to be
101
(6A5mg/L-1.24mg/L)
*
r
2__i
Ar,Ann
=
..
42400mg / kg
(l25mg/LJ-^110 mg
Oxygen utilization rates for whey depletion were
6.45 mg 11 - 1.24 mg / L
•= 0.17 mMO 2 ISdays
32 mg I mmol
or, 0.034mM 0 2 / day or 1.4 x 10"6 moles 02/L/hr
The pseudo first-order rate contant k of oxygen uptake was
ln(8.04mg / L) - ln(l A9mg IL)
5days
0.336/day
or, 0.014/hr.
Discussion
Rates of oxygen utilization in the aerobic batch experiment (1.4 x 10"6 moles
02/L/hr) compared to initial rates observed in column experiments (1.87 x 10"6 - 2.47 x
10" moles 02/L/hr) in which ratios of whey to available O2 were varied from 1:10 to
10:1. The consumption rate constant determined from the batch experiment compared
with column results (0.012/hr - 0.014/hr) and the field constant of whey consumption in a
pristine aquifer (0.303/day) determined by Barcelona and Xie (2001).
102
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