PB90-199514
Innovative Processes for Reclamation of Contaminated
Subsurface Environments
Oklahoma Univ., Norman
Prepared for:
Robert S. Kerr Environmental Research Lab., Ada, OK
1989
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
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PB90-199514
INNOVATIVE PROCESSES FOR RECLAMATION
OF CONTAMINATED SUBSURFACE ENVIRONMENTS
by
Larry W. Canter, Leale E. Streebin, M. Carlota Arquiaga, Francisco E. Carranza,
Dennis E. Miller, and Barbara H. Wilson
School of Civil Engineering and Environmental Science
University of Oklahoma
Norman, Oklahoma 73019
CR-813672
Project Officer
William J. Dunlap
Processes and Systems Research Division
Robert S. Kerr Environmental Research Laboratory
Ada, Oklahoma 74820
ROBERT S. KERR ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
ADA, OKLAHOMA 74820
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TECHNICAL REPORT DATA
(Pleate react Instructions on the reverse before completing} 1
EPA/600/2-90/017
4. TITLE AND SUBTITLE
INNOVATIVE PROCESSES FOR RECLAMATION OF CONTAMINATED
SUBSURFACE ENVIRONMENTS ....
7. AUTHOR(S)
Larry W. Canter, Leale E. Streebin, M. Carlota Arquiaga
Francisco E. Carranza, Dennis E. Miller & Barbara H. Wilsor
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Civil Engineering and Environmental Science
University of Oklahoma
Norman, Oklahoma 73019
12. SPONSORING AGENCY NAME AND ADDRESS
Robert S. Kerr Environmental Research Lab. - Ada, OK
U.S. Environmental Protection Agency
Post Office Box 1198
Ada, OK 74820
HI 90 19 95 14 /AS
5.-Wtr«fMT OATE
6. PERFORMING ORGANIZATION COOE
8. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
CBPC1A
11. CONTRACT/GRANT NO.
CR-813672
13. TYPE OF REPORT AND PERIOD COVERED
Final Report - 8/86-8/89
14. SPONSORING AGENCY CODE
EPA/600/15
15. SUPPLEMENTARY NOTES
PROJECT OFFICER: William J. Dunlap, FTS: 743-2314
fixed-film wi «. £etter assess the capabilities and limitations of
Jround ii?J «r f S I °r • removin9 selected organic contaminants from
of ™H + * fJ0in contannnated vapor streams produced by air stripping
grouna treatment of contaminated ground water and vadose zone gases.
'?• KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
•
RELEASE TO THE PUBLIC
b.lDENTIFIERS/OPEN ENDED TERMS
19, SECURITY CLASS iTIus Rrpnrtl
UNCLASSIFIED
2O. SECURITY CLASS iTIiit paff;
UNCLASSIFIED
c. COSATl Field, Group
21. NO. OF^PAGES
22. PRICE
*0S
EPA Form 2220-1 (R.v. 4-77) PMCVIOUS EDITION is OBSOLCTC
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DISCLAIMER NOTICE
The information in this document has been funded wholly or in part by the United
States Environmental Protection Agency under Cooperative Agreement CR-813672 to the
University of Oklahoma. It has been subjected to the Agency's peer and administrative review,
and it has been approved for publication as an EPA document. Mention of trade names or
commercial products does not necessarily constitute endorsement or recommendation for use.
ti
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FOREWORD
EPA is charged by Congress to protect the Nation's land, air, and water systems.
Under a mandate of national environmental laws focused on air and water quality, solid waste
management and the control of toxic substances, pesticides, noise and radiation, the Agency
strives to formulate and implement actions which lead to a compatible balance between human
activities and the ability of natural systems to support and nurture life.
The Robert S. Kerr Environmental Research Laboratory is the Agency's center of
expertise for investigation of the soil and subsurface environment. Personnel at the Laboratory
are responsible for management of research programs to: (a) determine the fate, transport and
transformation rates of pollutants in the soil, the unsaturated and the saturated zones of the
subsurface environment; (b) define the processes to be used in characterizing the soil and
subsurface environment as a receptor of pollutants; (c) develop techniques for predicting the
effect of pollutants on ground water, soil, and indigenous organisms; and (d) define and
demonstrate the applicability and limitations of using natural processes indigenous to the soil and
subsurface environment for the protection of this resource.
This report describes research conducted to evaluate the feasibility of innovative
biological treatment of chlorinated hydrocarbons and alkylbenzenes in engineered systems.
Cometabolism of trichloroethylene and 1,1,1-trichloroethane by hydrocarbon-utilizing organisms
is demonstrated. Also shown is the ability of soil-bed reactors to treat benzene, toluene,
ethylbenzene, and o-xylene in the vapor phase.
Clinton W. Hall
Director
Robert S. Kerr Environmental Research Laboratory
111
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ABSTRACT
Research to better assess the capabilities and limitations of fixed-film bioreactors for
removing selected organic contaminants from ground water or from contaminated vapor streams
produced by air stripping of polluted ground water and by soil venting operations is described.
Work was focused on volatile chlorinated aliphatic hydrocarbons and light aromatic constituents
of distilled petroleum products, two groups of compounds which have been identified in polluted
ground water more frequently and usually in higher concentration than other organic pollutants.
Laboratory scale fixed-film bioreactors containing soil or diatomaceous earth materials were
employed to study the cometabolic removal of trichloroethylene and related compounds from
aqueous and vapor steams by biofilms sustained by primary substrates consisting of gaseous
aliphatic hydrocarbons; the removal of alkylbenzenes from air streams by biofilms utilizing these
compounds as primary substrate was examined in laboratory scale soil bioreactors. The
biodegradation processes involved and the effects of bioreactor operating parameters and systems
configurations on contaminant removal were evaluated. Results obtained indicate a significant
potential for employment of fixed-film bioreactors in systems for above ground treatment of
contaminated ground water and vadose zone gases.
IV
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TABLE OF CONTENTS
SECTION 1. INTRODUCTION
Conventional Treatment
Innovative Biological Treatment
Environmental Fate
Aerobic Biotransformation of Trichloroethylene
Previous Studies on Biotransformation of Trichloroethylene
Vapor Phase Treatment of Alkylbenzenes
Research Objectives
SECTION 2. CONCLUSIONS AND RECOMMENDATION
Conclusions
Recommendations
SECTION 3. LIQUID PHASE TREATMENT OF TRICHLOROETHYLENE
Materials and Methods
Results and Discussion
SECTION 4. VAPOR PHASE TREATMENT OF TRICHLOROETHYLENE
AND 1,1,1-TRICHLOROETHANE
Methods and Materials
Results and Discussion
SECTION 5. VAPOR PHASE TREATMENT OF ALKYLBENZENES
Methods and Procedures
Results and Discussion
PAGE
1
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2
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3
5
7
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19
38
38
40
45
45
47
REFERENCES
60
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LIST OF FIGURES
PAGE
Figure 1.1 Proposed Mechanism of TCE Breakdown by Methanotrophic Bacteria 4
Figure 1.2 Biotransformation of Chlorinated Ethylenes and Their
Breakdown Products 4
Figure 3.1 Schematic of Fixed-Film B ioreactor 16
Figure 3.2 Schematic of Bioreactors in Series 16
Figure 3.3 Schematic of Bioreactor with Multiport Methane Injection 18
Figure 3.4 Schematic of Bioreactor with Intermediate Methane Sampling Ports 18
Figure 3.5 Variation of Dry Cell Mass Per Unit Volume 22
Figure 3.6 Normalized Breakthrough Response of Sodium Chloride 22
Figure 3.7 Effect of Microbial Growth on Pressure Drop 24
Figure 3.8 Relation Between Basic Column Air-to-Water Ratio and Ratio of
TCE Mass in the Air and Water Phases of the Effluent for Sand 2 25
Figure 3.9 Relation Between Basic Column Air-to-Water Ratio and Ratio of
TCE Mass in the Air and Water Phases of the Effluent for Sand 3 25
Figure 3.10 Effect of Influent Methane Concentration on TCE Biodegradation 27
Figure 3.11 Effect of Influent Methane Concentration on TCE B iodegradation 27
Figure 3.12 Effect of Influent TCE Concentration on TCE Biodegradation 29
Figure 3.13 Effect of Influent TCE Concentration on TCE Biodegradation 29
Figure 3.14 Effect of Influent TCE Concentration on TCE Biodegradation 30
Figure 3.15 Effect of Influent TCE Concentration on TCE Biodegradation 30
Figure 3.16 Effect of Influent TCE Concentration on TCE Biodegradation 31
Figure 3.17 Effect of Influent TCE Concentration on TCE Biodegradation 31
Figure 3.18 Inhibition of Methanotrophs by TCE for an Influent Methane
Concentration of 0.8 mg/1 33
Figure 3.19 Inhibition of Methanotrophs by TCE for an Influent Methane
Concentration of 3.2 mg/1 33
Figure 4.1 Schematic of Bioreactors Treating Air Streams 39
Figure 5.1 Soil Column Apparatus 46
Figure 5.2 Solvent Vapor Generator 46
Figure 5.3 Toluene Removal in Rubicon Sand Microcosm 55
Figure 5.4 Benzene Removal in Durant Loam Microcosm 55
Figure 5.5 Moisture Effect on Benzene Removal in Dougherty Sand Microcosm 58
Figure 5.6 Moisture Effect on Benzene Removal in Durant Loam Microcosm 58
VI
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LIST OF TABLES
PAGE
Table 3.1 Characteristics of Coarse Sands
Table 3.2 Analysis of Major Constituents of Ground Water
Table 3.3 Hydraulic Characteristics of Systems with Sand Media 2 and 3
Table 3 4 Pressure Drop for Unsaturated Packed Columns
Table 3.5 Removal of Methane at Different Depths for Several Influent Methane
Concentrations
Table 3.6 Elapsed Time for Complete Acclimation After Increasing the Influent
Methane Concentration
Table 3.7 Decrease of TCE Removal Resulting from Inhibition of Methanotrophs
Table 3.8 Specific TCE Utilization Rate Per Unit Mass of Methane Consumed
for Sand
Table 3.9 Removal of TCE and Methane
Table 3.10 Summary of Results of Bioreactors Fed with Natural Gas
Table 4.1 Removal of TCE from B ioreactor Packed with R63 5
Table 4.2 Removal of TCA from Bioreactor Packed with R635
Table 4.3 Removal of TCE from Bioreactor Packed with R630
Table 4.4 Removal of TCA from Bioreactor Packed with R630
Table 4.5 Gas Removals in Bioreactor Packed with R635
Table 4.6 Gas Removals in Bioreactor Packed with R630
Table 5.1 Hydrocarbon Removal in Duplicate Soil Columns
Table 5.2 Hydrocarbon Removal at Varying Inlet Concentrations
Table 5.3 Hydrocarbon Removal at Varying Flow Rates
Table 5.4 Hydrocarbon Removal by Different Soils
Table 5.5 Soil Column Moisture Contents
Table 5.6 Soil Characteristics
Table 5.7 Removal Rate Constants For Hydrocarbons in Soil Microcosms
Table 5.8 Microbial Densities
20
21
21
24
26
26
32
34
36
36
41
41
42
42
43
43
49
50
51
52
53
53
56
57
VII
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ACKNOWLEDGEMENTS
This research was funded in part by the United States Air Force Engineering and
Services Center, Engineering and Services Laboratory, Tyndall Air Force Base, Florida, through
an Interagency Agreement with the U.S. Environmental Protection Agency. The project would
not have been possible without this support, which is gratefully acknowledged.
Special thanks are extended to Mr. Robert L. Smith of the Robert S. Ken-
Environmental Research Laboratory who conducted studies to identify and enumerate protozoa
in the bioreactor systems. His expert assistance is greatly appreciated.
vui
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SECTION 1
INTRODUCTION
Ground water is the primary source of drinking water in the United States, with
estimates of approximately 80 percent of all drinking water supplies obtained from groundwater
sources (Tchobanoglous and Schroeder, 1985). Historically, ground water has been used for
drinking water without major treatment other than removal of minerals and final disinfection.
During the past few years, however, hundreds of synthetic chemicals have been detected in a
significant number of drinking water supplies, thus raising concerns about the potability and
purity of such waters.
In 1985, the total industrial production of the four most used chlorinated solvents -
trichloroethylene (TCE), tetrachloroethylene (PCE), 1,1,1-trichloroethane (TCA), and methylene
chloride - was 1.64 billion pounds (Storck, 1987). These large productions result in extensive
release to the environment through spilling, industrial wastewater discharge, landfilling, and
volatilization. A review of American and European drinking-water sources conducted by
Folkard (1986) revealed that the compounds most likely to be identified in contaminated ground
water are halogenated hydrocarbons of low molecular weight. TCE, PCE, and TCA were the
most prevalent because of their extensive use. Westrick et al. (1984) reported similar results
based on a U.S. Environmental Protection Agency survey in the United States. In this study, the
compounds found most commonly in finished ground water supplies were TCE, PCE, cw-1,2-
dichloroethylene (cis-DCE), rraw,y-l,2-dichloroethylene (trans-DCE) and 1,2-dichloroethane
with benzene and toluene also frequently found. The most common sources of these
contaminants were accidental spills, failure of underground storage tanks and associated
plumbing, and inappropriate disposal to landfills, evaporation pits, and sludge disposal lagoons.
CONVENTIONAL TREATMENT
Currently air stripping is the most widely used method of removing TCE and other
low-molecular-weight chlorinated compounds from contaminated ground water because of its
significantly lower capital and operating costs compared with other methods. Frequent use in the
treatment of waters containing the soluble constituents of petroleum products is also seen. Air
stripping achieves approximately 99 percent removal of TCE and offers a high degree of
flexibility in response to prevailing operating conditions (Folkard, 1986). The countercurrent
packed towers appears to be the most appropriate system in that it provides the most liquid
interfacial area and allows for high air-to-water ratios (Canter and Knox, 1985).
Carbon adsorption is less widely used than air stripping because of the high cost
associated with the disposal or thermal regeneration of the spent carbon. Activated carbon
adsorption can be up to four times as expensive as air stripping due to costs associated with the
landfilling at a hazardous waste disposal site or thermal regeneration of the used carbon (Knox
and Canter, 1988). However, adsorption is a well-established technology for reducing the
concentrations of TCE and related compounds to less than 5 u,g/l (Love and Eilers, 1982).
Carbon adsorption is efficient but costly; air stripping is both efficient and inexpensive. Both
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treatments have the disadvantage of transferral to another medium without ultimate destruction of
the compounds. Biological treatment has the potential to be a final treatment by metabolizing the
contaminants of interest to carbon dioxide, water, and cellular constituents.
INNOVATIVE BIOLOGICAL TREATMENT
Although biological treatment of easily metabolizable organic compounds has been
used for decades to treat municipal and industrial wastes, only in the past ten years has biological
treatment of recalcitrant compounds or innovative treatment of easily degraded compounds been
attempted. This report will focus on innovative treatment of water or air streams contaminated
with TCE and/or TCA using engineered systems and soil bed reactors to treat waste air streams
containing hydrocarbons. The biological treatment of the chlorinated compounds will rely on the
cometabolism of the contaminant using methane or butane as the primary substrate. The
hydrocarbon waste streams will be remediated using a soil bed containing bacteria capable of
metabolizing the compounds to water, carbon dioxide, and trace inorganic salts.
The contaminated water stream may come from pumping polluted ground water to
control plume migration or water from product recovery wells. Contaminated air streams are
produced during soil remediation procedures, air stripping of contaminated ground water, or
vapor collection from manufacturing or treatment processes.
In addition to being an ultimate treatment for contaminated air or water, biological
treatment may also be more economical than many alternative processes. Knox and Canter
(1988) have presented an economic evaluation of the cost of conventional treatment for the low
molecular-weight halogenated organics. In an initial economic analysis, the biological treatment
of TCE using propane as a primary substrate was found to be more cost effective than carbon
adsorption but less so than air stripping (Wilson and White, 1986). Treatment of waste
hydrocarbon vapor streams using biofilters has shown to be the most economical technology
available when compared to conventional treatments such as thermal degradation or activated
carbon (Kosky and Neff, 1988).
ENVIRONMENTAL FATE
In oxygenated subsurface materials, TCE and TCA are not ordinarily biodegradable
and tend to persist in those environments (Ghiorse and Wilson, 1988; Wilson et a/., 1981). TCE
and TCA are biodegraded only in subsurface materials where oxygen is not available by
undergoing a reductive dechlorination. The daughter products of TCE are the dichloroethylenes
and vinyl chloride (VC) and those of TCA are 1,1-dichloroethane and chloroethane (Barrio-Lage
et al, 1986; Barrio-Lage et al, 1987; Vogel and McCarty, 1985). TCA may also be
nonbiologically transformed to 1,1-dichloroethylene (1,1-DCE) by elimination and to acetic acid
by hydrolysis (Haag et al., 1986) with the potential of further biodegradation of 1,1-DCE to VC.
These products of transformation of TCE and TCA are more mobile in ground waters than the
parent compounds, and, in the case of VC, more carcinogenic. TCE is resistant to hydrolysis,
with an estimated half-life between 0.9 to 2.5 years (Vogel et al., 1987). Most abiotic
transformations are very slow in contrast with biotic transformation (Bitton et al.t 1986).
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Because they are soluble in water, benzene, toluene, ethylbenzene, and the xylenes
are the indicators of groundwater contamination from petroleum products. These aromatic
compounds are readily biotransformed in oxygenated environments, with recent work also
indicating a biological fate in the absence of molecular oxygen (Grbit-Galic and Vogel, 1987;
Vogel and Grbifc-Galifc, 1986; Wilson etal, 1986).
AEROBIC BIOTRANSFORMATION OF TRICHLOROETHYLENE
Although aerobic biodegradation of TCE and related compounds does not occur
under ordinary aerobic conditions (Bouwer and McCarty, 1982; Wilson et at., 1981;) recent work
has shown that bacteria that oxidize gaseous hydrocarbons such as methane or propane are also
able to cometabolically oxidize TCE and other low molecular weight halogenated compounds
(Wilson and Wilson, 1985; Wilson and White, 1986; Fogel et al, 1986; Henson et al, 1988,
Wacketteftf/., 1989).
The biochemistry of methanotrophs to oxidize methane and cometabolize TCE has
been described by Horvath, 1972; Atlas, 1981; Fogel etal., 1987; Wilson et al., 1987; and Little
et al., 1988. Methane-utilizing bacteria oxidize methane first to methanol and then to
formaldehyde, which can be converted to biomass or to CO as follows:
CH -
43 2 — > b i o ma ss
The first step in methane oxidation is performed by the enzyme methane
monooxygenase (MMO), which obtains oxygen directly from molecular oxygen. MMO is a
highly non-specific enzyme able to insert an oxygen into a wide variety of nongrowth
compounds. MMO may exist as either a soluble or paniculate form; the soluble MMO has been
isolated from both type I and type II methanotrophs and is associated with a broader substrate
specificity than the paniculate form (Burrows et al., 1984). Oxygenations by the enzyme include
hydroxylation on n-alkanes such as ethane, the expoxidation of alkenes, and dechlorination of
aliphatic and aromatic substances.
The first step in degradation of TCE by methanotrophs is expoxidation to form TCE
epoxide, which is unstable and hydrolyzes rapidly in water at neutral pH with half-lives on the
order of seconds. The products of hydroxylation are dichloroacetic acid, glyoxilic acid, and
formic acid (Fogel et al, 1987), all of which can be further degraded by methanotrophs and other
heterotrophic organisms. These reactions are shown in Figures 1.1 and 1.2 (Wilson et al, 1987;
and Little etal, 1988).
The use of alkanes such as methane, propane, or butane as primary substrates for
cooxidation of chlorinated compounds has advantages. First, the alkane serves as the primary
source of carbon and energy needed to sustain a stable microbial community when the target
pollutant is present in trace amounts, as in the case of TCE in ground waters. An advantage of
butane over methane is its increased solubility in water (60 mg/1 versus 24 mg/1) and thus a
potentially increased concentration in a biofilm. Next, these alkanes are commonly used
industrial chemicals that are easily found and inexpensive. They are nontoxic to humans, and
therefore not considered pollutants, and easily biodegraded.
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Cl
TCEEPOXIDE
INTERMEDIATE
H CARBON
FORMATE MONOXIDE
OH'
"H>
DICHLOROACETIC
ACID **
r
GLYOXYLIC
x ACID
ORGANISMS
- ^ "HETEROTROPHIC
ORGANISMS
Figure 1.1. Proposed Mechanism of TCE Breakdown by Methanotrophic Bacteria
(Little, et al., 1988).
PCE
Cl
Cl
TCE
H
Cl
trans-
DCE
O
as-
DCE
Vinyl
Chloride
No
Product
half life
12 sees.
H OH
O
half life
1 min.
\ I J /
Biodegradable Hydrolysis Products
Figure 1.2. Biotransformation of Chlorinated Ethylenes and Their Breakdown
Products (Wilson, et al., 1987).
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PREVIOUS STUDIES ON BIOTRANSFORMATION OF TRICHLOROETHYLENE
The earliest study to evaluate the feasibility of TCE oxidation by methane-utilizing
bacteria was conducted with unsaturated soil at the U.S. Environmental Protection Agency's R.S.
Kerr Environmental Research Laboratory in Ada, Oklahoma (Wilson and Wilson, 1985; Wilson
and Wilson, 1987). Trichloroethylene was shown to degrade aerobically to carbon dioxide in an
unsaturated soil column exposed to a mixture of natural gas in air (0.6 percent). A removal of
one order of magnitude was observed during the 2-day residence time of water in the column.
In a second study (Wilson and White, 1986), a glass column 152 cm tall by 5 cm in
diameter was packed with 127 cm of coarse sand followed by 10 cm of a mixture on Lincoln plus
coarse sand at the top. A mixture of propane in air was supplied to the top of the column, and a
solution of 800 \ig/l of TCE and 760 ug/1 of TCA was continuously pumped to the head of the
column concurrently with the air. After 13 days of acclimation, removals of TCE from 80
percent to 95 percent were observed.
Recent work with a fixed-film, packed-bed bioreactor with a residence time of
approximately 50 minutes and using methane as the primary substrate treated synthetic ground
water containing TCE and DCE (Strandberg et a I., 1989). The initial concentrations of 1 mg/1
each were reduced >50 percent for TCE and >90 percent for DCE.
A field demonstration project recently completed by Battelle Columbus at Tinker Air
Force Base in Oklahoma has shown reductions of 260 ug/1 TCE in influent ground water to
approximately 110 ug/1 in effluent from fixed-film bioreactors using natural gas as the primary
substrate (U.S. Department of the Air Force, Progress Report). Mannville's Celite biocatalyst
carrier was used as the solid support. This is the first known field demonstration of remediation
of TCE-contaminated ground water in a surface bioreactor using the cooxidation process. The
substantial removal of TCE during one retention time in the bioreactor indicates the potential for
this remediation technique.
Stanford University conducted a pilot study of in-situ biodegradation of TCE by
stimulating the native bacterial population capable of degrading the contaminant within the
aquifer under saturated conditions (Roberts et al, 1989). The study was conducted on a shallow
confined sand and gravel aquifer at Moffett Naval Air Station. An extraction well and injection
wells were installed six meters apart, with three intermediate monitoring wells. Bromide was
used as a conservative tracer, with TCE continuously injected at average concentrations of 100
ug/1 during the initial stage and 60 ug/1 during the later stages.
Complete methane utilization was observed within a few weeks, confirming the
presence of indigenous methanotrophic bacteria. Mass balances indicated that under the
influence of active biostimulation with methane and oxygen, approximately 20 to 30 percent of
the TCE was degraded within 2 meters of travel in the test zone. The limited degree of TCE
transformation was attributed to the high degree of chlorination of the TCE molecule, resulting in
a slow rate of oxidation, limited methane-oxidizing population, and possible competitive
inhibition of TCE degradation by methane. No intermediate products were found, suggesting
completely aerobic conditions and treatment. Another interesting finding was the relatively high
sorption of TCE on the aquifer material (Roberts era/., 1989).
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As part of the Moffett Station in-situ biodegradation project, batch studies with
mixed cultures from the site were conducted to determine the effects of changing the methane,
oxygen, TCE, and biomass concentrations (Henry and Grbic-Galifc, 1987). Three mixed cultures
were enriched from the Moffett Field aquifer or from effluent from a saturated column. In
general, TCE transformation was rapid. All three cultures transformed TCE in the absence of
methane. One mixed culture transformed TCE significantly faster when no methane was present;
however, the rate of transformation for the other two mixed cultures when no methane was
present was significantly slower than when methane was provided. The removal of TCE was
found to be highly dependent on the types of methanotrophs used and the relative effects of the
main variables on the metabolic capacity of the mixed cultures. The degradation of TCE when
no methane was present was an interesting finding, as was the incorporation of C into the cell.
Recent work by Tsien et al, (1989), has observed degradation of TCE by the
Methylosinus trichosporium OB3b, a type II methanotroph, in both continuous and batch cultures
following the appearance of soluble MMO. In cultures with suppressed MMO, the oxidization
of TCE increased with increasing soluble MMO concentrations.
In a study by Little et al. (1988) two pure strains of TCE-degrading type I
methanotrophic bacteria were isolated from mixed ground water cultures enriched from ground
and surface water at a site contaminated by chlorinated alkenes. Pure cultures converted 1 to 2
percent of radiolabeled TCE to cell biomass, 10 to 16 percent to CO , and 15 to 22 percent to
water-soluble breakdown products. This accounts for approximately 2b to 40 percent of the TCE
added, from which one-third was converted to CO . This study confirmed the incorporation of
some carbon into cell biomass even for pure cultures, and showed that pure cultures
accumulated more water-soluble breakdown products, suggesting the need for mixed cultures for
complete mineralization.
Fliermans et al. (1988) searched for heterotrophic enrichment cultures able to
degrade TCE aerobically at high concentrations (i.e., 50-300 mg/1). More than 400 enrichments
and incubation mixtures obtained from subsurface sediments which had been heavily
contaminated with short-chain chlorinated solvents were examined for TCE utilization after a
one-month incubation period. Concentrations from 50-300 mg/1 TCE were used, and the studies
included radioisotope activity measurements and isolation enrichments. There was a significant
removal of TCE by the enriched cultures using several substrates including methane, propane,
methanol, acetate, and trypticose plus yeast extract. The enrichment cultures were stable and
used a variety of energy sources for growth but could not use methane as a sole source of carbon
and energy. Moreover, they did not have the phospholipid biomarker typical of methanotrophs
(Ringelberg et al, 1989).
The toxicity of TCE to methanotrophs has been studied by Janssen et al. (1987). The
authors reported that when methane is used as a sole carbon source, complete inhibition occurs at
concentration levels of 30-35 |im for most chlorinated aliphatic hydrocarbons and 45 u.m
specifically for TCE, which is equivalent to 6 mg/1. The addition of methanol caused a relief
from growth inhibition of some specific steps in the assimilation of methane, rather than a
general inhibitory effect. Lee et al. (1988) stated that inhibition of TCE in certain mixed cultures
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starts at a concentration of about 1 mg/1, and pointed out the importance of defining this toxicity
threshold limit.
Strand et af. (1988) studied the kinetics of TCE degradation by suspended cultures
on methane-oxidizing bacteria using a closed system reactor. It was found that methane
oxidation followed Michaelis-Menton kinetics, with K = 0.67 mg methane/1 and r = 47.2 ug
methane/ing VSS-hr. TCE removal followed first orde/kinetics with a rate constant of 3.7 x 10
1/mg VSS-hr for concentrations less than 3000 ug/1. TCE biodegradation was not inhibited by
the presence of dissolved methane concentrations in excess of 0.25 mg/1. In the absence of
methane, the culture continued to degrade TCE, but the degradation rate gradually decreased
until it ceased after 104 hours.
A more recent discovery is the cometabolism of TCE by bacteria that degrade
aromatic compounds, specifically phenol, toluene, and cresol (Wackett and Gibson, 1988).
Nelson et al. (1986) isolated a bacteria designated G4 from a number of soil and water samples
that were screened for the biological capacity to metabolize TCE. Further investigation by
Nelson et al. (1987) revealed that strain G4 degrades TCE only when preinduced with phenol,
toluene, o-cresol, or n-cresol. Recent studies have shown TCE degradation by Escherichia coli
containing the toluene dioxygenase genes cloned from Pseudomonas putida Fl (Zylstra et al.
1989).
VAPOR PHASE TREATMENT OF ALKYLBENZENES
Use of soil-bed bioreactors has been shown to be effective for a number of different
applications, and has the advantage of completely destroying the contaminant. Soil bed systems
have been shown to be effective for controlling such air waste streams as rendering plant
emissions (Prokop and Bohn, 1985) and wood and coal flue gases (Duncan eta!., 1982). Natural
gas has been shown to be readily degradable by soil microbes (Hoeks, 1972; Bohn, 1977).
Propane, isobutane, and n-butane have been effectively removed using a soil bioreactor system
(Kampbell et a/., 1987). Ethylene and acetylene have also been shown to be removed by soils
(Bohn, 1977). Additionally, soil-bed bioreactors allow a substantial savings in investment and
maintenance cost. Soil beds are estimated to cost about $8 per cubic foot per minute (cfm)
compared to $30 - $100/cfm for activated carbon systems (Bohn and Bohn, 1986).
Work has been done which indicates that some hydrocarbon streams may be
effectively controlled with a soil-bed bioreactor (Kampbell et al., 1987). A volume of son is
excavated and a piping network is placed within a layer of gravel. The purpose of the gravel
layer is to help distribute the injected gases. The excavated soil is then treated (if necessary) and
back filled over the gravel layer and distribution pipe. Peat or compost could also be substituted
for the soil, although soil is reported to have the greatest removal efficiency (Bohn and Bohn
1986).
A minimum residence time of one minute for gases within a soil filter has been
proposed (Bohn, 1975), but actual contact times probably need to be much longer depending on
the desired amount of removal. Residence time can be controlled by varying the inlet volumetric
flow rate or by planning for a larger bioreactor with either greater surface area or greater depth.
-------
Factors such as sou type, moisture content, temperature, and the particular compound
of interest greatly influence the biodegradation potential. In general, the type of soil which
rndnionCCOmni *" *"*** miCr°biaI P°pnlafinn under a Siven set of environmental
^"Tr W^ P™bably Produce the greatest biodegradation activity (Schoen and Winterlin,
1987, Abou-Assaf and Coats, 1987). The type of soil (sandy, loamy, or clayey) will determine
the internal pore structure which in turn will determine the adsorptive capacity of the soil and the
magnitude of the microbial population (Prokop and Bohn, 1985). Typically, population increases
of several orders of magnitude have been observed for hydrocarbon-utilizing microorganisms
after hydrocarbon spills (Atlas, 1981). Increases in the total biomass after hydrocarbon exposure
are sometimes not observed because exposure to hydrocarbons leads to an increase in the number
of hydrocarbon degrading microbes but not the overall population present in the sediment may
not increase (Heiticamp * */, 1987). As the waste gas passes through the soil bed, hydrocarbon
compounds are adsorbed and may be retained for an extended length of time before degradation
by microbial action (Prokop and Bohn, 1985). Soil beds may be designed on the basis of upflow
°f 'PP^-ately 10 cm of water
h A u ^ Cnvir0nnient which favors microbial growth and activity should also favor
hydrocarbon degradation. Soil moisture content has a large influence on microbial activity The
optunum moisture content of a soil is difficult to specify. It is generally thought that maximum
hydrocarbon oxidation occurs in the same moisture range which satisfies higher plants (Bohn
1977). More specific studies have shown that increasing the moisture content from 4 to 11 to 20
percent increases CO removal fa a soil column from ?4 ^ ^ ^ 95
(Duncan Bohn, and rfurr, 1982). Other studies specify that soil moisture contents of 9 to 20
percent (wet mass basis) are adequate for removal of components of waste gases containing
organic sulfides, aldehydes, amines and organic acids (Prokop and Bohn, 1985) SignifZt
™
ct UAh A 0iran WCre °SerVed " SOU m°iStUreS ™ -creased fom o
percent (Abou-Assaf and Coats, 1987).
OnHm, , d° ^ dWayS ensure faster hydrocarbon removals.
Optimum removal rates have been reported to occur at moderate moistures, 22 percent when
So n ' ' ou-ssa d
Coats 1987). Sods which are at or near the water holding capacity are reported to result in near
anaerobic condmons (oxygen transport limiting) which would inhibit degradation by aerobic
ZTT mi H ^ C°atS' 198?; SCh°en "ld WintCrlin' 1987)' Excess ™" - also
bought to fill up the soil pore spaces and prevent adsorption of the hydrocarbon compounds
(Prokop and Bohn, 1985). In one example of restricted mobility, propane was reported to be
removed more rapidly by soils having 10 percent moisture that the same soils at 20 percen
moisture (Edinger et a,., 1987). It has been reported that the greatest aerobic degradation",
simple or complex organic material can be expected to occur at 50 to 70 percent of a soil's water
holding capacity (Dibble and Bartha, 1979).
Temperature also has a direct influence on microbial activity. Microbial activity can
"
foraoidl rp !" "P * "^ 35°C' * ^* ^ microbial
falls off rapidly (Prokop and Bohn, 1985). In some cases little or no increase of microbial
-------
activity is seen above 20°C (Dibble and Bartha, 1979). Alternatively, even greater increases are
H°ekS (1972) rePOIted ^ t0 5-f°ld fa™~ »
nt H - » °^e" — P<-
from natural gas degradation accompanying a temperature increase of 13° to 20°C Propane
removal rates have also been shown to increase 10 fold over die temperature range of 2 • to 25 »C
(Edinger et al., 1987). A minimum temperature limit of 10
-------
RESEARCH OBJECTIVES
-
methane-TCE concentrations, and flow rates was studied
10
ere use "
-------
SECTION 2
CONCLUSIONS AND RECOMMENDATIONS
CONCLUSIONS
1. Bioreactors consisting of short sand columns supporting fixed biofilms
utilizing methane as the primary substrate appear capable of removing as much as 55 to 60
percent of TCE from influent water containing approximately 500 ug/1 of this pollutant, provided
operating parameters are optimized.
2. If concentrations of TCE approach 1000 ug/1, removal of contaminant from
influent waters will probably be inhibited, although utilization of primary substrate by the
biofilm continues. When influent TCE concentrations are near 1500 jig/1, methanotrophic
organisms constituting the biofilm will likely be completely inhibited.
3. The percentage removal of TCE from aqueous streams by fixed-film
bioreactors appears to increase with increasing TCE concentrations until the inhibitory
concentration is approached.
4. Operating conditions such as water flow, flow rates for air and methane, and
required nutrient concentrations to attain maximum TCE removal from polluted water by fixed-
film bioreactors are dependent on the supporting media and geochemistry of the treated water.
Hence, determination of optimized operating parameters for specific situations will probably be
required, at least until a more comprehensive data base on the effects of operating variables is
developed.
5. Multiport injection of primary substrate (methane) at various depths within the
bed of bioreactors containing sand as supporting medium appears to result in increased removal
of TCE from influent water, probably because of better distribution and increased mass of the
biofilm. Increased TCE removal can also be achieved by operating two bioreactors in series.
6. Limited observations indicate that biofilms developed and sustained on natural
gas as primary substrate, are at least as effective in removing TCE from influent water as
biofilms developed and sustained on pure methane. The probable ability of the more complex
substrate to support a greater diversity of microorganisms may be advantageous in terms of
biofilm sensitivity to concentration inhibition by TCE.
7. Fixed-film bioreactors supplied with primary substrate on an intermittent basis
to encourage endogenous respiration exhibit decreased removal of TCE from influent water in
comparison to similar bioreactors provided with a continuous supply of primary substrate.
8. The potential utility of fixed-film bioreactors for on site, above ground
treatment of ground water contaminated with relatively low levels of TCE (probably 500 \ig/\ or
less) appears to be promising and worthy of further evaluation and development. Such systems
should not be considered for treatment of waters containing TCE in the neighborhood of 1000
\ig/l or higher.
9. Bioreactors consisting of fixed biofilms sustained by butane as primary
substrate and supported on two sizes of diatomaceous earth packing materials are capable of
removing more than 90 percent of TCE and TCA from influent air streams under optimum
11
-------
conditions. Removals were stable in a bioreactor with the smaller size solid support. However,
rates of removal of the chlorinated hydrocarbons in another bioreactor with the larger support
were observed to decline severely after several days of reactor operation, possibly as the result of
build up of inhibitory products in recirculated nutrient solution or predation by protozoa.
10. The potential utility of fixed-film bioreactors for removal of volatile
chlorinated hydrocarbons from contaminated air streams generated by air stripping of polluted
ground water or soil venting applications appears high, provided problems associated with
operation of bioreactor systems over extended periods of time can be overcome.
11. Soil bioreactors appear to be capable of removing at least 35 to 40 percent of
volatile aromatic hydrocarbons such as benzene, toluene, ethylbenzene, and o-xylene from air
streams containing mg/1 concentrations of these contaminants.
12. Biological removal of alkylbenzenes from air during passage through soil is
sensitive to the flux of alkylbenzenes. At a give flow rate, the biomass will adjust to remove the
same fraction of alkylbenzenes regardless of initial concentration (over the range of 1 to 35 mg/l
air). At a given concentration, the biomass will adjust to remove the same fraction as flow rates
are decreased (0.9 to 0.2 ml air/cm cross section/m).
13. In these studies, the extent of removal of the various volatile aromatic
hydrocarbons from contaminated vapor streams by soil bioreactors varied significantly with the
soil sample used to prepare the bioreactor. This probably reflects qualitative and quantitative
differences in the microbial ecosystems of the different soils.
14. Soil moisture content appears to be very important in determining the
effectiveness of soil bioreactors in removing volatile aromatic hydrocarbons from contaminated
air streams.
RECOMMENDATIONS
1. Bioreactors comprised of fixed biofilms utilizing light aliphatic hydrocarbons
primary substrate should be further developed and evaluated in laboratory and pilot-scale studies
for treatment of ground water containing low levels (500 ug/1 or less) of TCE. Particular
attention should be given to the following:
a. The effect of TCE removal of employing propane, butane, or mixtures of light
hydrocarbons, including natural gas, as primary substrate for biofilm organisms should be
investigated. Comparative susceptibility to TCE concentration inhibition effects of biofilms
utilizing various primary substrates should be evaluated.
b. Materials other than sandy soils, such as ceramic materials and diatomaceous
earth products, should be examined as support media for fixed biofilms capable of
cometabolizing TCE in aqueous streams.
c. Variations in bioreactor system configurations to achieve increased
contaminant removal from contaminated water streams should be rigorously examined. This
should include further studies of the use of columns in series and the use of multiport injection of
primary substrate, better distribution and increase in biomass throughout the depth of the
bioreactor.
12
-------
2. Bioreactors utilizing light aliphatic hydrocarbons as primary substrates to treat
contaminated air streams should be subjected to further laboratory and pilot-scale studies.
Particular attention should be directed to the following:
a. The effect of recirculation of the effluent fluids on long-term stability of TCE
and TCA removal should be addressed.
b. The ability of different mixed cultures from various soils on their abilities to
oxidize TCE and TCA should be better defined using propane or aviation gasoline as a primary
substrate.
c. The potential for inhibition of TCE oxidization by TCA should be described.
d. Determine any potential effects by protozoal predation of the biomass on the
efficiency of the removal of TCE and TCA.
3. Soil based bioreactors to treat air streams contaminated with petroleum
hydrocarbons are ready for pilot-scale evaluation. Successful demonstration will require clean
understanding of the following:
a. The effect of soil moisture on mass transport limitations and microbial
biodegradation rates will have to be determined for the particular soil used in the demonstration.
b. The effect of other hydrocarbons in the spill on removal of the aromatics must
be determined.
c. The influence of physical properties of the soil such as grain size distribution,
organic carbon content, and soil moisture release characteristics should be defined to identify the
most appropriate soils for use in bioreactors.
13
-------
SECTION 3
LIQUID PHASE TREATMENT OF TRICHLOROETJIYLENE
METHODS AND MATERIALS
Laboratory experiments were conducted to determine the effects of changing
environmental conditions in unsaturated fixed-film bioreactors using methane as the primary
substrate, the goal being to remove TCE as a secondary substrate by the cometabolism process.
Lincoln fine sand was used as the inoculum in completely mixed reactors in which
air and natural gas were bubbled to develop enrichment cultures. The enrichment cultures were
then used to promote growth in laboratory-scale packed columns using methane as the sole
source of carbon. Air and water were injected, mineral nutrients were added, and the percent
removal of methane monitored. Finally, TCE was injected as a solution in the influent water, and
the percent removal monitored. After the completion of the enrichment and acclimation process,
the columns were operated over a range of hydraulic and organic loadings during which the
system performance was assessed by monitoring the influent and effluent concentrations of
methane and TCE, as well as the flows of water, air, and methane.
Two silica sands were used in the study as packing media. The sands were obtained
from water filter supplies and prescreened and washed to remove any organic matter. The
saturated hydraulic permeability (Darcy's constant) was determined by a constant head
permeameter according to ASTM method C127. The sieve analysis was performed according to
ASTM D 422-63 using standard U.S. Sieve Series.
A schematic of the packed column bioreactor design is depicted in Figure 3.1. The
columns were designed so that all surfaces in contact with the feed water solution containing
TCE are either glass or teflon. The reactors were constructed of borosilicate glass columns 61
cm long by 6 cm in diameter, filled with 6 cm of glass beads at the bottom, 48 cm of coarse sand
as the main packing media, and 6 cm of glass beads at the top. The entire media was supported
by a porous aluminum plate. Two headspace air sections in both ends of the columns were left
for even distribution of both air and liquid flows as well as to separate both phases for sampling
purposes.
The TCE solution was fed into one of the three top ports of the column by means of a
peristaltic pump with variable speed drives (Masterflex Pumps), using pump head number 14
(Masterflex Heads). The same water flow was withdrawn from one of the three bottom ports of
the columns using the same head size so that a continuous water flow will be established.
A similar peristaltic pump was used to pull a slight vacuum from one of the three
ports to establish an effluent air flow equal to the influent mixture of methane and air flow. To
achieve desired concentrations of methane in the influent, an air cylinder was connected by tygon
tubing to one of the top ports, with the pressure set to atmospheric by a PVC cylinder with an
open orifice that allowed excess air to bleed off. Methane was then injected in the influent
tubing. Sufficient tubing length was provided for proper mixing prior to the sampling port. The
control column was constructed identically to the other columns, but without the primary
substrate added.
14
-------
A concentrated aqueous solution (250 mg/1) of TCE was prepared in a 50 gal. HOPE
container with a floating lid and a sealed cover. A pump was installed for mixing, and several
ports with hall valves were placed in different locations of the container for adequate
recirculation. Tap water from a ground water supply, showing TCE concentrations below
detectable levels, was aerated for at least 244 hours before the solution was prepared. The
concentrated aqueous solution of TCE in the required volume was injected with a glass syringe
through a septum port installed in the discharge line of the pump. The solution was recirculated
for 30 minutes for proper mixing and then connected to the system.
A concentrated aqueous solution of ammonium sulfate and potassium phosphate
monobasic was prepared to produce a N/P ratio of 5/1. The concentrated solution contained 51
g/1 of ammonium sulfate equivalent to 10.6 g/1 of nitrogen, and 9.0 g/1 of potassium phosphate
monobasic equivalent to 2.0 g/1 of phosphorus. The amount of oxygen required for 100 percent
degradation of methane was estimated. The concentrated aqueous solution was diluted to
provide a ratio of 100/5/1 for oxygen, nitrogen, and phosphorus.
The flow of methane was regulated by a cartridge pump (ISMATEC) with speed
control. To achieve the desired mixture of air and methane, a peristaltic pump was used to
withdraw a constant flow of air from the bottom of the columns. The flows of air, methane and
water was changed by controlling the speed of the peristaltic pumps. Experimentation was made
with an air flow of 4 to 12 ml/min, a water flow of 1.8 to 8.5 ml/min, and a range of methane
flow of 0.017- 0.25 ml/min. Influent methane concentrations from 1 mg/1 to 16 mg/1 were
injected, and TCE concentrations from 80 u.g/1 to 1400 ug/1 were utilized.
The development of an enrichment culture was accomplished by bubbling air and
natural gas in three l-liter, completely mixed reactors seeded with Lincoln sand After two
weeks of operation, the reactor which showed the most extensive growth was selected as the
inoculum for the rest of the experiment. Next, an enriched biofilm was established in a packed
column bioreactor fed exclusively with methane as the sole source of carbon and energy with air
and water for aerobic conditions and a suitable environment for microbial growth.
As part of this experiment, two identical columns were assembled in series as
depicted in Figure 3.2. A known concentration of TCE was injected at one of the upper ports of
column 1, and the effluent water was pumped through Teflon tubing to column 2 by a peristaltic
pump. Air and methane were injected in a second port located at the top of column 1; additional
methane was injected in the effluent air line that interconnects both columns to insure an equal
influent methane concentration in both bioreactors. At the bottom of column 2, the air and water
effluents were withdrawn by peristaltic pumps. Sampling and flow measurements were
conducted at the locations shown in Figure 3.2. Prior to assembling the columns in series both
bioreactors were operated separately for at least 10 days to allow acclimation.
To observe how the distribution of microorganisms and contact times affect the
removal of TCE, a column was assembled as a replicate of another one in regard to influent TCE
concentration, water flow, air flow and total influent mass of methane; however, the methane was
injected at the top, one-third and two-thirds points of the multiport column by inserting 1/16 in
stainless steel tubing through ports located in the top and bottom caps of the column, as shown in
Figure 3.3. The total methane flow was monitored in the three lines by bubble
15
-------
METHANE
-S-,
ret
SOLUTION
G MET*. SAIIPUNG PORT
O TCt SAMPLING PORT
O rim MONITOIIINC
PERISTALTIC PUUP
Figure 3.1. Schematic of Fixed-film Bioreactor.
TAP
• ATCR
1 v -= —
]*• Q . o .
t
\
SJ^-^ SULU I t g*
4
O MCTN. SAMPLING POR
D TCC SAMPLING POT
O FLOW MONITORING
PCI1ISTJLTIC PUMP
Figure 3.2. Schematic of Bioreactors in Series.
16
-------
flowmeters to insure that the flow, and therefore the mass of methane, was the same as a parallel
control column with all the methane injected at the top.
Removal of methane under unsteady- and steady-state conditions was studied hy
utilizing a column with four sampling ports located at 0, 8, 15, and 61 cm, and monitoring the
remaining methane concentration at each port for several influent concentrations, keeping the
other variables constant; for example, the water flow, air flow, and TCE concentration. Figure
3.4 depicts this arrangement. Sampling points 1 and 4 were taken from the influent and effluent
air lines respectively; sampling points 2 and 3 were taken from two 1/16 in. stainless steel tubes
inserted in the column through two ports located at the top and bottom caps of the column. Prior
to sampling the columns a small amount of air was gently withdrawn with a gas tight syringe to
remove the stagnant air, then the samples were taken as described earlier. Methane and TCE
were monitored.
The unsteady-state study was performed by installing a new column that was be
seeded with microorganisms taken from an existing column. The removal of methane over time
was monitored hourly for the first day and twice a day the remaining period until steady state
conditions were reached at all the sampling points. The steady-state study was conducted by
changing the influent methane concentration and monitoring the methane concentration until no
further changes were observed at all sampling points. The information collected from these
studies, in conjunction with the estbnation of the dry cell mass per unit volume, was used to
explore the development of a model for methane removal.
The pressure drop through the packed column bioreactors was estimated at steady
state conditions using a sensitive differential manometer. Gage pressures were measured at the
influent and effluent lines and differential pressures were determined between both lines as a
double check.
To estimate the average retention time of a column for a given set of conditions, a
known concentration of sodium chloride was injected at the top of the column without disturbing
the normal operation of the flows. The change in electrical conductivity in the water effluent was
monitored with an electrical conductivity meter every 5 minutes until the concentration of
sodium chloride was constant for three consecutive readings. A normalized response curve
provided information not only about residence times, but also about the time span between the
first appearance of the tracer and the time when the effluent concentration equaled the influent
concentration.
At the end of the experiment, the columns were disassembled to estimate the dry
mass of cells per unit volume of media. Ten cubic centimeters of sand was collected at five
different depths (0 cm, 8 cm, 15 cm, 30 cm, and 61 cm) and weighted after drying at 103°C.
The residual sand was dried at 550 °C for two hours, then cooled in a desiccator and washed with
a solution of chromic and sulfuric acid. The samples were then dried at 550 °C., cooled and
weighed again. The same procedure was performed for the control column. The dry mass of
cells per unit volume was calculated as follows:
Where,
Drv mass of cells (A - B) - (C -
Vol ume of medi a V
17
-------
0 O
SOLUTION
,
J
t
(
*t°
IS
^$
P
i
I
£*£
•^
t
Cff
^LASS 6EAOS
~ (
? O
1
1
1
1
1
^
V\S
\\^
I
i
• •
S5
ks\^
i
1
M
^m
11
F~~^ Trr
^>-^ SULU1 1 ON
I
P-CLASS BEADS
— COARS: SIKO
0 METH. SAUPLINC PORT
D TCE SAMPLING PORT
O FLOW MONI TOR INC
P""^
\Oj PERISTALTIC PUMP
^
-------
A - weight of sample in column fed with methane after drying at 103 ° C
B = weight of sample in column fed with methane after drying at 550 °C
C = weight of sample from control column after drying at 103 °C
D = weight of sample from control column after drying at 550 °C
V = initial sample volume
Quick connectors were installed in the influent lines of the columns for sampling
purposes. Teflon plug valves were connected directly at the bottom caps of the columns to
temporarily divert the flow for sampling, allowing for rapid connection of the sample bottles
without disturbing the normal flow. Liquid samples were collected in 10 mi-bottles with screw-
cap lids and Teflon-faced septa. The bottles were left in place long enough for 10 to 20 flushings
before the samples were taken without headspace.
TCE gas samples were collected by using a gas-tight syringe to withdraw a given
volume of air from a sampling port provided with a septum. The sample was then injected into a
packed resin trap (Tenax GC) using nitrogen as the carrier gas.
Duplicate gaseous samples for methane were collected in the influent and effluent
lines by using a gas-tight syringe with push-button valves to withdraw a volume of air from the
sampling port, then immediately injected into an FID gas chromatograph for analysis.
Trichloroethylene was analyzed by a Hewlett Packard 5880 gas chromatograph
equipped with a Hewlett Packard 7675A purge and trap analyzer. Modification of purge-and-trap
Method 624, modified for GC/ECD, found in "Methods for Organic Chemical Analysis of
Municipal and Industrial Wastewater," EPA-600/4-82-957, July, 1982, was used for the analyses.
After the purging was completed, the sorbent column was desorbed onto the gas chromatograph
with temperature-programming and electron capture detection (BCD). The limit of detection was
1 ug/l. Methane was analyzed on a Tracor 560 Gas Chromatograph using isothermal
programming and Flame lonization Detection (FID).
RESULTS AND DISCUSSION
The research program involved an acclimation period and several steady-state
phases. The program was designed to explore the following issues:
(a) Hydraulic performance of the sands used to construct bioreactors.
(b) Effect of biofilm growth on the pneumatic and hydraulic performance of the
bioreactors.
(c) Effect of air to liquid ratio on TCE removal.
(d) Effect of methane concentration on TCE removal.
(e) Effect of TCE concentration on TCE removal.
(f) Effect of delivery schedule of methane on TCE removal.
(g) Comparison of TCE removals with natural gas and methane.
To explore these issues, the fixed-film bioreactors were operated over a range of
TCE concentrations, methane concentrations, and air and water flows for two coarse sands used
as packing media. Different arrangements of the columns were made for one of the coarse sands
during the last phases of the program, with the aim being to improve the biodegradation of TCE
19
-------
System performance was assessed by monitoring the influent and effluent concentrations of
methane and TCE along with the hydraulic characteristics of the columns.
Two coarse silica sands (sands 2 and 3) were used as packing material. Roth sands
were prescreened and washed to remove organic matter. The characteristics of the sands used
during the experimentation are shown in Table 3.1. Adsorption tests for TCE were performed to
determine the Freundlich isothenn constants; however, no adsorption was detected even after
three days of contact for TCE concentrations of 465 ug/1. These results confirm the absence of
significant organic matter, clay or silt.
Table 3.1. Characteristics of Coarse
Coarse
Sand
2
Porosity
ij (%)
38.0
Density
(g/cm )
2.67
Saturated
Hydraulic
Permeability
(cm/day)
5.97 x 103
Coefficient
of Uniformity
(Cu, D60/D10)
1.66
Sands
Effective
Size
(mm)
0.75
Mean
Size
(mm)
0.72
Unified Soil
Classification
— — — -_^____^__
Pnnrlv OraH»<1
Sand
3 39'9 L61 1'50xl°4 1-74 1.60 1.40 Poorly Graded
Sand
Tap water from a local ground water source was used to prepare the TCE solution
The quality characteristics of the ground water are shown in Table 3.2. A summary of the
hydraulic characteristics of the system for the two sands is shown in Table 3.3. The selected
water flows were well below the saturated hydraulic conductivity (Table 3.3).
The unsaturated hydraulic retention time was estimated by using sodium chloride as
a tracer. The electrical conductivity in the effluent was measured over time and the data were
normalized to get the response curves. The results are summarized in Table 3.3. As expected
the unsaturated hydraulic retention times were greater for sand 2 than for sand 3 due to its'
smaller mean particle size.
Although a narrow range of grain sizes were selected to provide a uniform flow, the
elapsed time from the first appearance of the tracer to the time when the effluent concentration
equalled the influent concentration was remarkably longer than expected. Unsaturated packed
columns are not truly plug-flow reactors but a combination of plug-flow and mixed reactors due
ironfly to internal short circuiting, wall effects, the tortuosity of the channels and the mixing
effect produced by the air stream. Therefore, when a liquid is applied to the column, various
portions of he liquid follow different flow patterns. With lateral and longitudinal mixing
different restdence tunes result for different particles of fluid even though they were part ofThe
bToroVfh r M TPPlied ^ thC ^ Additi°nally' P« °f
-------
Table 3.2. Analysis of Major Constituents of Ground Water
Constituent Concentration
mg/1
Calcium 10.4
Magnesium 10.2
Sodium 153
Carbonate ion 5.4
Bicarbonate ion 310
Chloride ion 41
Hardness 68 as CaCO
Alkalinity 340 as CaCO
pH 8-8.25 units
TCE < I ug/1
The effect of microbial growth on the unsaturated hydraulic retention time was
studied by feeding three columns with methane at concentrations of 3.2,14, and 24 mg/l in air,
keeping all the other conditions constant.
Table 3.3. Hydraulic
Water Flow
(tnl/min)
1.85
4.2
6.0
4.2
4.2
6.0
8.4
Air Flow
(mg/min)
7.5
7.5
7.5
7.5
11.0
6.4
11.0
Media
Sand 2
Sand 2
Sand 2
Sand 3
Sand 3
Sand 3
Sand 3
Characteristics of Systems with Sand Media 2 and 3
Porosity
0.38
0.38
0.38
0.40
0.40
0.40
0.40
Saturated
Hydraulic
Retention Time
(min)
253.8
111.8
78.2
117.4
117.4
82.2
58.7
Average
Unsaturated
Hydraulic
Retention Time
(min)
125.0
75.0
57.5
45.0
47.5
37.5
27.5
Hydraulic
Loading.
(ft3/d/ft )
4.3
9.8
14.0
9.8
9.8
14.0
19.6
The dry density of the microorganisms per unit volume of sand was determined with
influent methane concentrations of 24, 14, 8.9, and 3.5 mg/1, respectively (Figure 3.5). Most of
the biomass developed in the upper 15 cm of the columns, with biomass decreasing sharply by
30 cm. Thereafter, the biomass remained low and fairly constant.
The observed methane removal was in excess of 98 percent at all four methane
concentrations. Although the observed microbial growth was significantly greater in the column
receiving the higher concentration of methane (Figure 3.5), little difference was observed in the
residence times of water (Figure 3.6). Because most of the biofilm developed in the top 8 to 15
cm of the columns, microbial growth did not significantly affect the residence tune in the
column.
21
-------
Figure 3.5. Variation of Dry Cell Mass Per Unit Volume of Sand as a
Function of the Influent Methane Concentration. (Water
Flow: 6.3 ml/min; Air Flow: 6.0 ml/min; Influent TCE
Concentration: 270 u.g/1; Media: Sand 3).
TIME(MINS)
C«TMiJ.JHC/L ••«•« CKCTHi III.OXC/l. •-•-• CHCTHiZU. IHG/t
Figure 3.6. Normalized Breakthrough Response of Sodium Chloride For
Several Influent Methane Concentrations. (Water Flow:
6.0 ml/min; Air Flow: 6.4 ml/min; Influent TCE Concentration:
270ug/l; Media: Sand 3; Average Residence Time: 37.5 minutes).
22
-------
Pressure drops were measured for some of the air-water combinations by means of a
precise differential water manometer (Table 3.4). The losses through the columns increased as a
result of either increasing the water or air flows; however, an increase in the water flow rate had
a greater impact on the system. The effect of microbial growth on pressure drop was assessed by
measuring the pressure drop in the five columns under steady state operations with regard to
methane; other measured conditions were nearly constant (see Table 3.4). Although the growth
of microorganisms did not affect the unsaturated hydraulic retention time, it affected the pressure
drop significantly, which increased from 0.19 in. of water/ft in a clean column to more than 0.44
in. of water/ft in the column acclimated to 25 mg/I methane (Figure 3.7, Table 3.4).
Since air stripping is to be expected in any packed tower fed with a volatile chemical,
the air-to-water ratio is a fundamental parameter in designing an aerobic bioreactor for treating
TCE. Air is provided as the source of oxygen; however, if the TCE is not completely removed
by the methanotrophs, some TCE will leave the system in the air stream.
An estimation of the effect of the air-to-water ratio on the stripping of TCE was
developed by plotting the TCE mass ratio in the air and water effluent versus the volumetric air
to water ratio. The results are shown in Figures 3.8 and 3.9. The following relations were
obtained by linear regression of the collected data:
TCE in effluent air fug) „ „„ Air
rrr:—— : , . = 0.728 — -0.092 (sand 2)
TCE in effluent water (ug) Wat er
correlation coefficient = 0.95
TCE in effluent air (ug^ Air n£AA f ._
IT—; — , = 1.068— -0.644 (sand 3)
TCE in effluent water (^g) Water
correlation coefficient = 0.94
These relations hold independent of the influent TCE concentration, the percent
removal of TCE, or the influent methane concentration, excepting for the special condition when
the TCE concentration was greater than 1400 ug/1 for an air-to-liquid ratio greater than 2.6.
These results may be due to the microbial growth developing in the upper 8 to 15 cm of the
columns. If TCE is biodegraded in the upper section, the remaining longer segment of the
column will allow a mass transfer equilibrium to develop between air and water. Based on the
information above, a system with a low air-to water ratio is recommended.
Pure methane was used as the primary substrate because it has been proven by others
to support cometabolism for the removal of TCE and related compounds. The removal of
methane without the addition of nutrients ranged from 30 to 70 percent (data not shown). After
the addition of nitrate and phosphate, in the ratio of 100/20/5 for COD/N/P, the removal of
methane was two orders of magnitude higher and independent of the influent methane
concentration (Table 3.5).
The removal of methane with depth in the column bioreactors was studied by taking
air samples at 8,15, 30,41, and 61 cm (Table 3.5). This was accomplished by installing a new
column seeded with microorganisms from one existing column, and increasing the methane
23
-------
0.6
0.5
8
2
a
§0.3
o:
a
0.1
0.0
21
u 3 6 3 12 15 YT
INF. METHANE CONC.(MG/L)
Figure 3.7. Effect of Microbial Growth on Pressure Drop at Several Steady
State Conditions: (Water Flow: 4 ml/min; Air Flow: 7 ml/mhr
% Methane Removal: 99+; Media: Sand 3).
27
Media
Sand 2
Sand 2
Sand 3
Sand 3
Sand 3
Sand 3
Sand 3
Sand 3
Sand 3
Sand 3
Sand 3
Water
Flow
(ml/min)
4.2
6.1
4.2
6.0
4.2
8.5
5.9
5.9
5.9
5.9
5.9
ire Drop for Unsaturated Packed Columns
Air
How
(ml/min)
7.5
7.7
7.5
7.5
11.0
11.0
6.7
6.6
6.6
6.6
6.6
Methane
Concentration
in Air
(mg/1)
0
0
0
0
0
0
0
3.0
8.3
14.1
24.9
Pressure Drop
(in water/ft
of column)
1.40
1.67
0.11
0.18
0.19
0.41
0.18
0.19
0.23
0.24
0.44
24
-------
0.5
1.0 1.5
2.0 2-5 3.0
AIR/WATER
Figure 3.8. Relation Between Basic Column Air-to-Water Ratio and Ratio of
TCE Mass in the Air and Water Phases of the Effluent for Sand 2.
2.0
Figure 3.9. Relation Between Basic Column Air-to-Water Ratio and Ratio of
TCE Mass in the Air and Water Phases of the Effluent for Sand 3.
25
-------
concentration stepwise from 3.7 to 24 mg/1 while keeping the water and air flow nearly constant.
After each methane increment, the methane concentration in the intermediate ports and effluent
were measured until steady state conditions were reached. Additionally, samples at 30 cm and
41 cm were taken in two columns with an influent methane concentration of 3.8 and 14 mg/1,
respectively. The influent TCE concentration for the study of methane removal with depth was
440 ug/1.
Table 3.5. Removal of Methane at Different Depths for Several Influent Methane Concentrations
Methane Concentrations and Removal
Influent
Cone.
(mg/1)
3.8
9.7
13.9
24.1
Cone.
(mg/1)
O.I
0.3
2.4
7.1
8cm
%
Removal
97.8
97.0
82.6
70.3
Cone.
(mg/1)
0.04
0.06
0.33
1.8
15cm
%
Removal
99.1
99.4
97.6
95.5
3° cm 41cm 61cm
Cone- % Cone, % Cone
(mg/1) Removal (mg/1) Removal (mg/|)
0.01 99.7 0.01
— — 0.03
0.05 99.6 — .... o.03
0.03
%
Removal
99.7
99.7
99.8
99.9
More than 95 percent of the methane was removed in the top 8 cm when the influent
methane concentration was less than 10 mg/1. The same removal was achieved in the top 15 cm
when the influent methane concentration was between 13 mg/1 and 25 mg/1. In all the cases more
than 99 percent of the methane was removed throughout the length of the column. The columns
responded to an increase in the influent methane concentration within a few days (Table 3.6).
The effects of influent methane concentrations were studied by keeping all the
variables constant while varying the influent methane concentration. At a water flow of 6.1
ml/min, an air flow of 6.2 ml/min and an influent TCE concentration of 430 ug/1, the percent
removal of TCE increased as the influent methane concentration increased (Figure 3.10). Even
though the percent removal increased with an increment in the influent methane concentration,
the specific mass of TCE removal per unit mass of methane consumed decreased (Tables 3.7,
3.8); this means that a large increment in the concentration of the primary substrate produced
only a relatively small increment in TCE removal.
Table 3.6. Elapsed Time for Complete Acclimation After Increasing the Influent Methane Concentration
Methane Elapsed Time
Methane Concentration for Complete
Concentration After Increment* Acclimation
(mgyi)
-------
40
30
UJ
o:
10
3 6 9 12
1NF1. METH. CONC.(MG/L)
Figure 3.10. Effect of Influent Methane Concentration on TCE
Biodegradation. (Water Flow: 6.1 ml/min; Air Flow:
6.2 ml/min; Influent TCE Concentration: 430 jig/1;
Media: Sand 3).
15
BOf
50
I
130
ui
o
20
10
5 10
INFL. METH. CONC.(MC/L)
15
Figure 3.11. Effect of Influent Methane Concentration on TCE
Biodegradation. (Water Flow: 4.1 ml/min; Air Flow:
7.6 ml/min; Influent TCE Concentration: 430 ug/1;
Media: Sand 3).
27
-------
In a second experiment, the conditions were kept similar in regard to air flow and
influent TCE concentration (air flow: 7.6 ml/min, influent TCE concentration: 430 ug/1), but the
water flow was reduced to 4 ml/min (Figure 3.11). The data obtained was in agreement with the
first experiment, but the percent removals were higher, and could be attributed to the increased
retention time.
The amount of TCE removed per unit of methane consumed ranged from 3.4 to 30
ug of TCE per gram of methane (Table 3.8), except for one isolated case when the methane
concentration was 1 mg/1. As was noticed before, although an increase in the influent methane
concentration resulted in a greater removal of TCE, the specific TCE utilization decreased
significantly. For example, when the influent methane concentration was reduced from 13 to 3
mg/l the specific utilization increased from 2 to 10 ug of TCE removed per mg of methane
consumed. Conversely, an increase in the influent TCE concentration increased the specific TCE
utilization.
Several models were tried to fit the data collected from the study of methane
consumption versus depth for sand number 3 (Table 3.5). The model that best fit the data
followed Monod kinetics. For the development of the model it was assumed that the inhibitory
effect of TCE was negligible. The model is somewhat limited because a constant air flow rate
was used during the study of methane removal. The model is as follows:
ln(S/So) = K (I/So - 1/S) - K H"
where
So = influent methane concentration (mg/1)
S = methane concentration at depth H (mg/1)
H = depth
K ,K = constants
As the model is not linear with regard to H, regression analysis was performed with an iterative
process to evaluate n, the results are shown below:
K = 0.022
K = 2.018
n=0.31
r =0.967
The effect of the influent TCE concentration was assessed by establishing a set of
conditions that were kept constant (i.e., water flow, air flow, methane inflow) while the TCE
concentration was changed. As the influent TCE concentration increases, the percent removal
increased (Figure 3.12 - Figure 3.17). This behavior has been reported by Grady and Lim (1980)
when they stated "...when the applied flow rate is low, an increase in flow rate will cause a
relatively large reduction in the resistance to mass transfer, consequently, the rate of removal can
increase. As further increases are made in the flow rate, however, additional reduction in the
mass transfer resistance becomes negligible so that no benefits accrue to offset the deleterious
effect of the decreased time of contact."
28
-------
100 110 1JO 130 uo ISO
200 210 220 230 :40
INFL. TCE CONC.(UC/L)
Figure 3.12. Effect of Influent TCE Concentration on TCE
Biodegradation. (Water Flow: 6.1 ml/min (Upper Curve,
4.1 ml/min (Lower Curve); Air Flow: 7.4 ml/min; Influent
Methane Concentration: 15 mg/1; Media: Sand 2).
60
50
30
ui
o
'20
10
150
300 350
INFL. TCE CONC.(UG/L)
100
USD
500
Figure 3.13. Effect of Influent TCE Concentration on TCE
Biodegradation. (Water Flow: 6.1 ml/min; Air Flow
7.6 ml/mm; Influent Methane Concentration: 15 mg/1-
Media: Sand 2). '
29
-------
60
50
MO
I
§30
ui
20
10
01
100
'6° 18° 20° "° 240 260 280 300 320 3UO 360 360 400
INFL. TCE CONC.(UG/L)
Figure 3.14. Effect of Influent TCE Concentration on TCE Degradation
(Water Flow: 4.2 ml/min; Air Flow: 7.3 ml/rnin; Influent
Methane Concentration: 14 mg/1; Media: Sand 2).
60
50
UJ
o
I30
s
tr
M
30
10
200 300
INFL. TCE CONC.(UG/L)
1400
500
Figure 3.15. Effect of Influent TCE Concentration on TCE B.degradation
OVaterFlow: 4.1 ml/min; Air Flow: 7.5 ml/min; Influent
Methane Concentration: 13 mg/1 (upper curve)
3.4 mg/1 (lower curve); Media: Sand 3).
30
-------
60
50
10
'30
20
10
BO 100 120 140 160 180
INFL. TCE CONC.(UG/L)
200
220
210
Figure 3.16. Effect of Influent TCE Concentration on TCE Biodegradation.
(Water Flow: 4.1 ml/min; Air Flow: 7.6 ml/min; Influent
Methane Concentration: 13.4 mg/1; Media: Sand 3).
60
50
-i
o
§30
LJ
10
100
160 180
INFL. TCE CONC.(UG/L)
200
220
240
Figure 3.17. Effect of Influent TCE Concentration on TCE Biodegradation.
(Water Flow: 4.1 ml/min; Air Flow: 7.6 ml/min; Influent
Methane Concentration: 13.4 mg/1; Media: Sand 3).
31
-------
Table 3.7. Decrease of TCE Removal Resulting from Inhibition of Methanotrophs
Influent
Methane
Concentration
(tng/1)
3.8
1.0
3.0
0.70
2.7
0.7
6.7
1.4
Influent
TCE
Concentration
(Ug/1)
470
460
1070
1070
1500
1500
1400
1400
%
Removal
Methane
98.8
95.1
96.5
86.5
91.2
69.7
98.3
94.1
TCE
Removal
%±SD*
48.6 ± 3.8
40.9 ± 3.0
48 toO
41 toO
1.4 ±2.3
0.1 ± 0.8
9.5 ± 4.4
1.2 ± 1.7
*n^ 10
At TCE concentrations as high as 1000 ug/1 or greater, a significant impact was
observed for the columns with an inflow methane concentration of 0.7 mg/1, in this case there
was a 13 percent reduction. With regard to the removal of TCE, the system was far more
sensitive to inhibition as can be seen in Tables 3.7 and 3.8. For an influent TCE of 1070, ug/1 the
percent removal of TCE gradually decreased to zero over a period ranging from 12 to 24 days,
even though a relatively high percent removal of methane was being achieved. An increase of
TCE to 1460 ug/1 further reduced the percent removal of methane and a total inhibition of the
microbiota removing TCE occurred; however, as the influent methane concentration was
increased to 6.7 mg/1 for one column, the percent TCE removal increased to 9.5 which was still
low.
Previous studies have shown that the inhibition of methanotrophs occurred at lower
concentrations when pure methane was used as the sole carbon source (Janssen et al., 1987). The
authors reported that the addition of another substrate caused a relief from growth inhibition and
suggested that the toxicity is a result of the inhibition of some specific steps in the assimilation of
methane, rather than a general inhibitory effect. It is anticipated that a combination of substrates
will enhance the microbial capacity to resist inhibition at such low concentrations.
Inhibition of methane oxidation by higher concentrations of TCE was observed to be
a function of both microbial density and TCE concentration. Figure 3.18 shows the effect of the
influent TCE concentration on methane removal for a low microbial density (ie influent
methane concentration = 0.8 mg/]£ As the TCE concentration was increased the percenl removal
decreased in a parabolic trend (r = 0.998). At TCE concentration greater than 1000 ug/1 the
impact on the microbiota was significant, and TCE removals less than 80 percent of the
maximum observed were noted. A similar but less pronounced effect was noted when the
influent methane concentration was 3.2 mg/1 (Figure 3.19).
32
-------
100
70
300
600 9QO
INFL. TCE CONC.(UG/L)
1200
1500
Figure 3.18. Inhibition of Methanotrophs by TCE for an Influent Methane
Concentration of 0.8 mg/1.
100
90
600 900
INFL. TCE CONC.(UG/L)
1200
1500
Figure 3.19. Inhibition of Methanotrophs by TCE for an Influent Methane
Concentration of 3.2 mg/1.
33
-------
Table 3.8: Specific TCE Utilization Rate
Water Row
(tnl/min)
4.1
4.0
5.9
6.0
6.4
4.2
4.1
4.0
6.2
6.1
5.9
6.1
4.1
Air Flow
(ml/min)
7.6
7.7
6.8
6.6
6.4
7.3
7.5
7.7
6.0
5.7
6.5
6.2
7.6
Influent
Methane
Concentration
(mg/1)
13
13
13
14
13
3
3
4
3
4
24
9
1
Per Unit Mass of Methane Consumed for Sand 3
Influent
TCE
Concentration
(ug/1)
150
390
270
41
430
150
390
460
430
440
270
420
460
%
TCE
Removal
38
44
27
33
34
34
49
49
20
17
35
30
41
Ug TCE Consumed
permg
Methane Consumed
(Ug TCE/mg Methane)
2.3
8.5
4.6
8.5
9.6
10
30
30
25
24
3.5
14
102
The inhibitory effect was somewhat offset with the higher initial methane
concentration, but the inhibition effect was noticeable. When the inhibitory effect of TCE was
incorporated into the model (mentioned previously), the following equation resulted:
where
ln(Se/So) = K^l/So - 1/Se) - K^H"/ (1 + CTCE/KJ
CTCE = Influent concentration of TCE (u.g/1)
K = TCE inhibition constant (ng/1)
K, K= constants.
1 4.
Regression analysis was performed following the same process as described above with the
following results:
K =0.0284
K = 2.961
KI = 763.44
n = 0.31
r =0.9568
An effort was made to optimize the schedule of the application of methane to enhance TCE
removal. Four experimental conditions were considered: (1) Column operated as before; (2)
Two columns in series with more methane injected into the second column; (3) Multiport
injection of methane into one column; (4) Intermittent injection of methane (endogenous
respiration).
Five columns were operated simultaneously, one of them as a single regular column,
two of them in series, another one with multiport injection, and the last one as a control with no
methane injection. In a latter time period, four of the five columns were subjected to endogenous
34
-------
respiration by intermittently injecting methane (every other day) and monitoring the removal of
TCE and methane in the endogenous period.
For the columns in series, the effluent air and water flows from the first column were
injected to the top of the second column; however, methane was injected in both columns since
the effluent methane concentration from the first column approached zero. The total mass of
methane injected was twice as much as in one single regular column. In the columns with
multiport injection, methane was injected at three different ports spaced at the third points in the
columns. The total mass of methane was the same as the mass injected in one single column. A
constant influent TCE concentration of 420 ug/1 was injected until steady state was reached
(Table 3.9). Greater than 99 percent removal of methane was achieved for all the columns. The
TCE removal in the first column in series (34 percent) was similar to the removal observed in the
single regular columns (33 percent); this means that the former behaved as a single column. The
first of the columns in series was used for comparison purposes.
The multiport injection column showed a 10.5 percent increase in TCE removal
efficiency in comparison with a single column. Although the same mass of methane was
introduced to the multiport column as the single column, it was concluded that a better
distribution of the microorganisms significantly improved the removal of TCE.
The total TCE removal of the columns in series was 54 percent which is 20 percent
greater than the removal observed in the first column. From this result it can be concluded that
columns in series produced a beneficial improvement in TCE removal.
Next, the TCE concentration was reduced to 270 jig/1 (Table 3.9) until steady state
operations were reached. Greater than 99 percent removal of methane was achieved for all the
columns. The removal of TCE followed a pattern similar to the one described at 430 ug/1 but
the percent removed decreased in agreement with the general findings that as the influent TCE
decreases the percent removal decreases.
The multiport injection column removed more TCE than the single column
confirming the beneficial use of an even distribution of methanotrophs for the same mass of
methane. A 17 percent increase in TCE removal was observed in the columns in series with
regard to the single column. Removal was significantly greater in the columns in series. The
total removal was 44 percent.
Finally, the systems were subjected to endogenous respiration by feeding with
methane for 24 hours and then shutting off the methane source for 24 hours. Methane samples
were taken 1 hour after the methane source was reconnected to the columns The TCE
concentration was kept constant at 270 ug/1. The results, presented in Table 3.9, were taken one
hour after the methane source was reconnected to the columns.
The percent methane removal during the feeding phase was greater than 99 percent
for all the columns, this shows that the methane removals were relatively unaffected even with
alternate periods of methane addition. The TCE removal followed a pattern similar to the case
with continuous methane addition, but the percent removals were lower and more variable
indicating that intermittent methane addition did produce a detrimental effect on TCE removal '
Microorganisms growing on natural gas as a primary substrate would probably'be
more diverse in structure than microorganisms living on pure methane. Three experimental
35
-------
Influent
TCE
Cone.
Single column 410
First column in Series 430
Second column in series
(total removal)
Multiport Injection 420
Control Column 410
First Column in Series 270
Second Column in Series
(total removal)
Multiport Injection 270
Intermittent Methane Addition
First Column in Series 270
Second Column in Series 270
(total removal)
Multiport Injection 270
*n* 10
Table 3. 9. Removal
Influent
Water
Flow
(ml/m)
6.0
6.1
6.1
6.0
6.0
6.0
6.0
5.9
5.8
5.8
5.7
of TCE and Methane
Air Methane Methane
Flow Cone.
(ml/m) (mg/1)
6.6 14
6.4 13
6.5 13
6.6 14
6.1 0.0
6.8 13
7.0 14
6.7 14
6.7 13
7.0 14
6.6 14
Removal
%
99.8
99.8
99.8
99.6
-
99.8
99.8
99.7
99.8
99.8
99.6
TCE
Removal
% ± SD*
33 ± 1.9
34 ± 2.9
54 ± 4.3
44 ±5.3
1.6 ± 2.4
27 ± 4.3
44 ±4.6
34 ±4.1
16 ±9.1
31 ±5.0
22 ± 3.8
Table 3.10:
Summary of Results
Water Flow Air Row
Media (ml/min)
Sand 3 3.8
Sand 3 3.6
Sand 3 3.7
Sand 3 3.9
Berl Saddle 5.4
Berl Saddle 5.4
Berl Saddle 5.4
(ml/min)
6.7
6.7
6.4
7.5
6.9
6.6
7.0
of Bioreactors Fed with Natural Gas
Gas Flow
(ml/min)
0.3
0.2
0.1
0.1
0.2
0.1
0.2
Influent
TCE
Concentration
(|*g/l)
77
107
190
910
150
210
910
TCE Removal
(%)
38
51
22
55
57
54
38
36
-------
reactors were designed to assess the response of the methanotrophs exposed to natural gas to
degrade TCE. A summary of the results is shown in Table 3.10.
Removal of TCE with sand 3 ranged from 22 to 55 percent, with the highest
efficiency obtained for a maximum TCE concentration of 910 ^g/1. No inhibitory effects were
noticed at this high concentration. The percent removal of TCE in the column packed with berl
saddles ranged from 38 to 57 percent. It is assumed that some inhibition occurred at an influent
TCE concentration of 910 u.g/1 as the percent removal decreased in contrast with the general
observed pattern that the percent TCE removal increased as a function of the influent TCE
concentrations. The high percent removal achieved in the berl saddle reactor might have
occurred because the methanotrophs were more evenly distributed than in the coarse sand
reactor, allowing a longer contact between the biofilm and the TCE-laden water. Flow in the berl
saddle reactor was quiescent compared with that of the coarse sand packed reactor, resulting in
less TCE stripped.
It appears that similar percent TCE removals were attained by the bioreactor packed
with sand 3 and fed with natural gas than the bioreactor fed with pure methane. The relatively
high percent removal obtained for the low influent methane and TCE concentrations of the
former was never achieved for the bioreactors fed with pure methane. The inhibitory effect of
high concentrations of TCE was lower in the natural gas-bioreactors; concentration in the order
of 910 u.g/1 did not appear to repress the microbial activity as high removals of both methane and
TCE were attained. Greater diversity of microorganisms would probably be promoted using
natural gas as a primary substrate compared to pure methane. (Wilkinson et a/., 1974).
37
-------
SECTION 4
VAPOR PHASE TREATMENT OF TRICHLOROETHYLENE
AND 1,1,1-TRICHLOROETHANE
METHODS AND MATERIALS
The treatment of air streams contaminated with TCE and/or TCA is needed because
of soil venting for remediation of unsaturated subsurface materials and air stripping of ground
water pumped to the surface. Diverse geochemistries of ground waters may present problems to
biological-based systems for treating water; treating air would circumvent this complication.
Another potential advantage would be the dilution of high concentrations of TCE in water by the
10-to-l or 20-to-l air-to-water ratios of stripping towers.
Two bioreactors were constructed using borosilicate glass columns 60 cm long by 5
cm in diameter. A schematic of the bioreactors is shown in Figure 4.1. Celite biocatalyst carrier
R-635 by Mannville was used as the solid support for microbial growth in bioreactor A;
bioreactor B was filled with Celite biocatalyst 630 by Mannville. These diatomaceous-silica
supports vary in diameter, distributions of porosity, and mechanical integrity. The porous nature
of the supports allows for great surface areas per unit of volume and increases the chances of
biomass survival after periods of shock loadings. The R630 is a sphere with a mesh size of (3/5)
and a surface area of 1.3 m /g. No information on the R635 was provided by Mannville,
however, the R630 was a larger sphere.
The headspaces of the columns were plumbed to receive vapors of TCE and TCA, a
mixture of butane (technical grade, 95.0 percent minimum purity in the liquid phase; Linde
Specialty Gases) and air, and a nutrient media. The nutrient media solution contained 100 mg/1
NH Cl, 100 mg/1 K PO , 20 mg/1 MgSo , 1 mg/1 FeSO , and 1 mg/1 CaCl .
A slurry of Rollin muck soil previously adapted to n-butane was the inoculum for the
biofilm (Kampbell et al, 1987). Ten grams of the soil was added to 250 ml of the nutrient media
and circulated throughout the columns until substantial removals of butane were observed in the
effluent gas port.
Four pairs of butane sampling ports and air sampling ports are located along each
column (Figure 4.1). The butane-enriched air was pulled throughout the column by a vacuum
line placed in the last air sampling port. An overflow line returns the effluent media to the
nutrient solution reservoir to provide recirculation of that solution to the columns.
Influent and effluent butane samples were collected by gas-tight syringe at the
appropriate ports (H,J,L,N). Air was pumped into a neat solution of TCE and TCA (D) by a
peristaltic pump (C) to saturate the air going to the column with TCE and TCA vapors. The
peristaltic pump also pulled a slight vacuum which mixed the air and butane as well as metering
the flow of butane from a cylinder (A). Influent concentrations of TCE and TCA were
determined by inserting a Tenax TA trap in the first air port (I) located in the headspace of the
column and placing the vacuum line (0) onto the end of the trap with an air flow of 13 ml/min.
38
-------
Figured. Schemauc of Bioreactors Treating Air Streams. A. Butane (primary
substrate); B. Nutrient media reservoir; C. Peristaltic pump-
D. Chemical reservoir; E. Influent air; F. Influent butane- '
Port
c '-- ynge;
I.K.M.O. Sample port by trap; P. Effluent water sampling port
39
-------
The air retention time of the column containing the R630 carrier was 18 minutes
while that of the column containing the R635 carrier was 20 minutes. Additional air samples
could he taken at the remaining ports located along the column (K,M,O). The initial
concentration were approximately 90 \ig TCE/1 air, 200 u.g TCA/1 air, and 8 mg butane/1 air. The
chemicals used in the bioreactor study were high purity TCE and TCA obtained from Aldrich
Chemical Co., Milwaukee, WI. All purities were at least 97 percent.
The effluent nutrient solution was transferred to a serum bottle then purged onto a
Tenax TA trap. The traps from both air and solution samples were analyzed by EPA Method 624
(1982), modified for GC/FID. Each trap was desorbed at 225°C for 5 minutes onto a Megabore
DB 624 column. The column was held at 30 °C for 5 minutes then programmed at 8°C/minute
to 175°C and held for 15 minutes; detection was by flame ionization. Influent and effluent
butane samples were analyzed by the method of Mindrup (1978).
Biomass estimates were determined by acridine-orange direct counts.
Approximately 300 milliliters of the effluent waters were examined for protozoa, with both
ciliates and amoeba observed . The amoeba were identified as Arcella vulgaris; the ciliates were
identified as the genus Tetrahymena.
RESULTS AND DISCUSSION
Reactors A and B were exposed to butane approximately 2.5 weeks before the
addition of TCE (90 ug/1 air) and TCA (200 ug/1 air) on day 18. Removals of TCE, TCA, and
butane were 82 percent, 93 percent, and 65 percent, respectively, on day 19 in bioreactor A
(Tables 4.1 and 4.2). TCE removals did not remain constant over the next seven days, with a
maximum removal of 94 percent observed on day 25. Removals of TCA gradually decreased to
73 percent during this same time interval.
In bioreactor B, respective removals of TCE, TCA, and butane were 74 percent, 76
percent, and 59 percent on day 19 (Tables 4.3 and 4.4). Decreases in removal efficiencies of
TCE were seen after day 20, with no removal of TCE observed on day 28; the removals of TCA
gradually decreased to 28 percent on day 28.
To test for the ability of the organisms to metabolize greater quantities of chemical,
the concentrations were increased to approximately 770 jig TCE/1 air and 990 ug TCA/1 air on
day 28. The ability of bioreactor A to remove TCE and TCA was dramatically reduced with
maximum removals on day 32 of 24 percent for TCE and on day 31 of 16 percent for TCA.
Interestingly, the removals of TCE and TCA in bioreactor B remained similar to those at the
lower concentrations, with maximum removals of TCE and TCA of 25 percent and 21 percent,
respectively, on day 30.
Removals of butane were seen throughout the length of the columns (Tables 4.5 and
4.6), indicating that the Celite biocatalyst carriers allowed microbial growth throughout the entire
length of the bioreactor. The larger biomass should result in increased removals of TCE and
TCA.
40
-------
Time
(Days)
1
9
15
19
21
22
25
29
31
32
Time
(Days)
1
9
15
19
21
22
25
29
31
32
Table 4.1
Butane
Quantity
Applied
(mg/L air)
7.6
8.3
7.6
8.5
6.7
7.2
6.2
10.5
7.6
7.5
Table 4.2.
Butane
Quantity
Applied
(mg/L air)
7.6
8.3
7.6
8.5
6.7
7.2
6.2
10.5
7.6
7.5
. Removal of TCE from Bioreactor Packed with R635
—
Air
98.4
61.9
34.2
35.1
60.2
55.9
67.9
59.7
84.7
75.8
— ^^— ^— •^—•.^
Removal
Aii
98.4
61.9
34.2
35.1
60.2
55.9
67.9
59.7
84.7
75.8
Ajr
10.9
9.2
36.2
4.8
56.7
68.4
60.5
TCE
Water
. _
7.5
11.7
15.3
1.3
32.6
14.3
15.8
~~~^ — — «
............
Total
—
18.4
20.9
51.5
6.1
89.3
82.7
76.3
of TCA from Bioreactor Packed with R635
AJI
— - _
1.0
10.6
23.7
23.9
87.3
79.4
87.5
TCA
Water
— — — — __
5.8
7.6
9.4
2.9
11.2
6.3
6.2
— ^ ___
. — — ..._.._ _.
Total
_
6.8
18.3
33.1
26.8
98.4
85.7
93.7
41
-------
Time
(Days)
1
3
5
15
19
22
25
26
28
30
Table 4.3.
Butane
Quantity
Applied
(mg/L air)
9.3
9.7
8.9
8.7
10.7
8.8
7.0
7.6
8.6
7.7
Removal of TCE from Bioreactor Packed with R630
Air
98.9
77.9
62.2
49.3
41.2
60.5
73.3
66.0
87.5
83.4
An
14.4
40.2
66.3
40.1
90.3
59.1
TCE
Water
11.5
8.1
11.8
11.9
21.9
16.2
Tofaj
— »B*~ _
25.9
48.3
78.1
52.0
112.2
75.3
Time
(Days)
1
3
5
15
19
22
25
26
28
30
Table 4.4.
Butane
Quantity
Applied
(mg/L air)
9.3
9.7
8.9
8.7
10.7
8.8
7.0
7.6
8.6
7.7
Removal of TCA
Aii
98.9
77.9
62.2
49.3
41.2
60.5
73.3
66.0
87.5
83.4
from Bioreactor Packed with R630
Aii
18.8
44.4
70.0
61.4
65.3
73.1
TCA
Water
% Influent
5.4
7.2
5.8
6.3
6.4
6.2
Total
24.2
51 6
7S8
67.7
71.7
79.3
42
-------
Day
1
3
4
8
12
14
16
18
19
25
27
29
30
*see Figure 4.1
**not determined
Port 1 (H>*
mg/1
7.6
8.8
7.9
8.3
8.2
7.6
9.2
8.5
7.1
7.3
10.5
7.6
7.0
Table 4.5. Gas Removals
Port 2 (J)*
mg/1
nd**
nd
nd
6.6
5.1
5.2
4.1
6.5
6.0
6.4
9.3
7.3
6.2
In Bioreactor A With R635
Port 3 (L)*
mg/1
nd
nd
nd
6.0
2.0
3.6
3.9
4.5
2.8
5.6
8.7
6.8
5.9
Port 4 (N)*
mg/1
7.4
7.9
7.2
5.2
1.0
3.7
2.9
3.0
1.6
4.6
6.4
6.4
5.4
Day
1
3
4
8
12
14
16
18
19
25
27
29
30
*see Figure 4.1
**not determined
Port 1 (H)*
mg/1
9.3
9.7
8.9
9.7
9.1
8.7
6.2
10.7
8.6
7.6
8.6
7.8
12.0
Table 4.6. Gas Removals
Port 2 (J)*
mg/1
nd**
nd
nd
8.3
6.9
7.4
5.6
8.4
7.4
6.9
7.7
7.3
12.0
In Bioreactor B With R630.
Port 3 (L)*
mg/1
nd
nd
nd
7.6
4.5
5.9
4.8
6.3
6.1
5.9
7.6
7.0
10.4
Port 4 (N)*
mg/1
9.2
7.5
5.6
6.3
2.2
43
"T»«7
3.8
4.4
A \
*T.l
5.0
7.3
6.5
9.2
43
-------
No products of biotransformation were determined in this study. However CO and
cel1ula[4conStituents have been observed as the final products of biotransformntion in studies
using C-labeled TCE (Wilson and Wilson, 1985; Fogel et af., 1986) or CCL (Strand and
Shippert, 1986) with methane as the primary substrate. The probable imermedia?e products of
the oxulation of low molecular weight alkanes such as methane, propane, or butane are alcohols
and ketones, which are easily metabolized in oxygenated systems (Haber ct al 1983- Atlas
1981). Epoxides are probably formed during the oxidation of chlorinated ethylenes with rapid
conversion to biodegradable hydrolysis products. Recent work has shown that oxidation of TCA
yields 2,2,2-trichloroethanol as a chlorinated intermediate (Oldenhuis et al 1989)
In this study, the pH of the effluent media solution changed from 6.5 to 5 0 during
the study interval. It is possible that sufficient quantities of butyric acid might have been formed
from the oxidation of butane (van Ginkel et al., 1987) with the resultant change of pH This
change in pH would probably decrease the efficiency of the removal process The reactive
nature of the epoxide produced by the oxidation of TCE would also decrease the efficiency of
removal by potentially destroying biomass. The effect of these intennediate compounds on
biofilms in engineered systems and the effluent waters to be disposed should not be ignored
The biotransformation of TCA seen in this study has also been seen in saturated soil
columns containing poorly sorted silty sand and gravel from a semi-confined alluvial aquifer
with propane as the primary substrate (Wilson et al., 1987). However, other studies have not
observed the burfogical removal of TCA when using an unsaturated soil column containing
Lincoln fine sand with natural gas as the primary substrate (Henson et al., 1988) or in fixed-film
bioreactors usmg propane as the primary substrate (Wilson and White, 1986). The three soils
differ greatly in texture, organic content, and clay content, but the effect of these differences on
the distribution of organisms capable of cometabolically degrading TCA is not known
A bacterial count of 1.8 x 10 showed approximately 6400 protozoans in an acridine-
orange direct count smear on a microscopic slide. Since ciliates and amoebas are free-living
protozoans, they can be found in every type of fresh and salt water, soils, or other mois!
environment including fixed-film bioreactors. Bioreactors can hold many fa tors m^g
the proliferation and distnbution of ciliates and amoebas. These factors are the temperature
hght chemical composition, acidity, kinds of food and the amount, and the adaptabili^ole
individual protozoan to various changes of the habitats and environment. The ProtoZ«
probably ennched along with bacteria from the Rollin muck soil used as an JcuTum for Z
bioreactor The proliferation of a butane-utilizing bacterial consortia would provTde 1 Iple
. . , ~»viv«. ilic reauction of biomass
protozoan predation would provide no benefit if TCE 'H '
cell phase of growth. Only if an actively growing b^ atl°n WCre aSS°°iated with the resting
TCE would protozoan predation seem beneficial. ""^ ^ ^"^ ^ cometabolism of
44
-------
SECTION 5
VAPOR PHASE TREATMENT OF ALKYLBENZENES
METHODS AND PROCEDURES
The laboratory methods and procedures developed and used for the soil bioreactor
and microcosm studies are presented. A description of the experimental apparatus and
experimental set-up are explained along with a review of procedures for sample gathering and
analysis.
The chemicals used in the vapor phase treatment project were Baker
spectraphotometric grade benzene, Baker reagent grade toluene, Eastman industrial grade
ethylbenzene, and Aldrich 97 percent purity o-xylene. Chemical purity was confirmed with
analysis by gas chromatography.
Soil columns were constructed of 7.6 cm internal diameter beaded process pipe. The
column length varied from 86.4 cm to 96.5 cm. The columns were filled with soil by lightly
tamping the soil into the column as well water was pumped up through the bottom of the column.
The well water was obtained from a local source near Ada, Oklahoma. This technique allowed
settling of the soil without physically packing and destroying the natural soil aggregates. Once
filled, the water was pumped out of the column; and an air stream was introduced into the bottom
of the column to establish a flow up through the column.
A hydrocarbon vapor stream was generated by purging air through vials containing
the desired hydrocarbon and then gathering the individual vapors into one stream to be directed
into the bottom of the soil column. Figure 5.1 shows a schematic of the soil column apparatus.
Air was pumped through the vials with a Technicon Autoanalyzer pump and the rate was
controlled by using different sized pump tubing. Tubing was replaced once a week. All lines
were made of Technicon pump tubing or teflon tubing.
A solvent vapor generator was constructed to produce a vapor stream which could be
injected into crimp-top serum bottles. The solvent vapor generator consisted of a glass manifold
which distributed an air supply through several flowmeters. The flowmeters then adjusted the
individual air streams which were bubbled through vials containing the individual aromatic
hydrocarbons. The streams were gathered into a single stream which could be injected into the
soil microcosm. Figure 5.2 illustrates the solvent vapor generator which was located in an
exhaust hood.
Sample analysis was performed using a Varian 3700 gas chromatograph. The Varian
3700 was equipped with a flame ionization detector and a 0.32 cm by 182.9 cm stainless steel
packed column containing 5 percent SP - 1200 / 1.75 percent Bentone 34 on 100/120
Supelcoport. Nitrogen
-------
Figure 5.1. Soil Column Apparatus.
Needle
. Valve
Laboratory
Air Supply \
Puraino
Vials
Cydohexanone
Figure 5.2. Solvent Vapor Generator Apparatus.
46
-------
The hydrocarbon vapor stream was sampled at the inlet and the outlet of the soil
column. Samples were collected onto Tenax traps. Each trap consisted of a 0.64 cm by 10 cm
stainless steel tube which contained five cm of 60/80 mesh Tenax held in place with glass wool
plugs. Hydrocarbon removal was determined by comparison of the inlet and outlet
concentrations. Flow rates were measured prior to each sampling period. Upon completion of
the soil column study, the columns were taken apart and sections of soil were stored in separate
sealed containers.
Moisture determinations were conducted on the acclimated soil from the soil
columns. Soil microcosms were then prepared to determine the hydrocarbon removal rate. A
predetermined amount of acclimated soil was added to crimp top serum bottles; 50 and 160 ml
serum bottles were used for the microcosm studies. The amount of soil added to a bottle was
dependent upon the moisture content and estimated porosity of the soil. A headspace to open
pore space ratio of 60:1 was used so that hours in a microcosm would correspond to minutes in a
soil bed. Each microcosm was injected with hydrocarbon vapors using the solvent vapor
generator. The injection was accomplished by holding a silicon-Teflon septa over the mouth of
the serum bottle and injecting vapors through the septa. An outlet line connected to a soap film
bubble meter allowed monitoring of the injection volume. After injection of a predetermined
volume of vapors, an aluminum faced Teflon disk was inserted between the bottle top and the
punctured septa. Both septa and disk were then crimped onto the serum bottle. This sealed the
serum bottle with an unbroken aluminum seal. Neglecting to insert the additional disk between
the bottle top and the punctured septa would result in loss of the hydrocarbons within 24 hours.
Sampling of a microcosm was accomplished by connecting a Tenax trap to the
microcosm with a short section of teflon tubing. Ten ml of tap water spiked with an internal
standard were then injected into the bottle. The slurry was purged for 9 minutes (for 50 ml
bottles) or 15 minutes (for 160 ml bottles) with nitrogen at a flow rate of 40 ml/min. The bottle
was shaken on a bottle shaker during the purging period. The sample was desorbed onto a
Varian 3700 gas chromatograph with aTekmar LSC-2 for analysis. The microcosm studies were
conducted in triplicate with samples of acclimated soil and controls of unacclimated soil. Soil
column and microcosms were conducted in a constant temperature box maintained at 12° C.
RESULTS AND DISCUSSION
The experimental data and results from this research is presented in two sections:
soil column data and microcosm data. The soil column experiments examined the variables of
column preparation, inlet concentration, inlet flow rate, and soil type. Microcosm experiments
examined the effect of different soil moistures on hydrocarbon removal rates.
The rate of biodegradation should be dependent on one of two factors: (1) transport
of the carbon source to the soil microbe; or (2) utilization of the carbon source by the microbe.
Soil microbes exist within water films and pore space waters of the soil matrix. The carbon
source must move from the vapor (gas) phase across the gas/liquid interface and through the
liquid to the microbe. Diffusion is the primary driving force and diffusion is dependent upon the
concentration gradient. Transport, or availability, will probably be the limiting step under
conditions of lower concentrations because the concentration gradient (driving force) will be low.
47
-------
Moving the carbon source to the microbe would take longer than its utilization by the microbe.
High vapor phase concentrations will produce a large driving force for transport, thus utilization
by soil microbes will probably be the limiting step.
Soil columns need to be consistently packed to minimize variations in loading which
may influence interpretation of the removal efficiency of the soil column. Duplicate columns of
Rubicon sand were used to examine packing variability. Table 5.1 presents a summary of the
removals obtained for the individual compounds in each column. Comparison of the two
columns reveals a good correlation at both hydrocarbon loading rates. Duplication between the
columns was adequate for experimentation purposes for each of the compounds. Removal
differences were due to the slight difference in the initial inlet concentrations. It can be
concluded that the method of column packing is reproducible and minimizes packing variation.
Table 5.2 contains a summary of the removals of each compound for the three soils.
In general, when the inlet concentrations were increased, the relative percent of removal
decreased only very slightly or remained the same which indicates the overall removal process
follows first-order kinetics. When the inlet concentrations were decreased from a higher level,
the relative percent removal increased due to the initially inflated biomass to hydrocarbon ratio.
The greatest change was for benzene. The high level of removal was only temporary, however.
As the biomass to hydrocarbon ratio adjusted back to the initial value, the percent removals were
approaching their initial values. Percent removals which are independent of loading rate are
characteristic of first-order removal kinetics.
Residence time within a soil column is determined by the flow rate of the inlet
stream. Reducing the flow rate in half will theoretically double the residence time but, at the
same time, will cut the loading rate in half. The biomass within the soil column will 'in turn
adjust to the new loading rate and, as shown in the previous section, would produce the same
percent removal if the residence time were equal and first-order removal kinetics were
controlling the removal process. Since the residence time increases with reductions in the flow
rate, increases in percent removal may be expected from the soil columns. The increases in
removal will follow first-order removal kinetics until removal becomes transport limited.
Removals then become a combination of the rate of the removal reaction and the rate of
transport. During periods of low loading rates the concentration gradient between the bulk fluid
and the reaction site (which drives the transport process) will be small and transport would be
reduced. As the retention times are increased within a given column, the outlet concentration
would asymptotically approach a minimum concentration which is limited by the transport of the
reactant to the reaction site. As the outlet concentration approaches the minimum concentration
allowed by the transport limitations, increases in retention time would produce diminishing
increases in percent removals.
Table 5.3 provides a summary of the compound removals for the varying flow rate
experiment. In general, decreased flow rates resulted in increased percent removals In the
Durant loam soil column, decreasing the flow rate from 40 ml/min to 20 ml/min increased
removals which indicated the removal process was controlled by first-order removal kinetics
Further reduction in the flow rate in the Durant loam soil column resulted in diminished increases
in the removals which indicated that transport was becoming limiting in the removal process
48
-------
Table 5.1. Hydrocarbon Removal In Duplicate Soil Columns
Rubicon Sand Soil Columns
Soil Type
Column F
Column G
Column F
Column O
Compound
Benzene
Toluene
Ethylbenzene
0-Xylene
Benzene
Toluene
Ethylbenzene
0-Xylene
Benzene
Toluene
Ethylbenzene
0-Xylene
Benzene
Toluene
Ethylbenzene
0-Xylene
Avg Inlet
ug/ml
3.1
1.1
0.9
1.0
3.1
1.5
1.1
1.1
20.3
4.5
1.3
0.9
20.5
4.7
1.2
0.9
Percent
Remaining*
66.4 ± 4.9
64.8 ± 3.0
53.2 ± 4.4
64.8 ± 5.6
67.5 ± 5.1
57.6 ± 4.8
58.1 ± 2.4
62.4 ± 3.4
70.2 ± 4.2
68.7 ± 3.3
61.2 ±5.0
68.2 ± 4.0
68.8 ± 3.9
66.9 ± 4.3
62.2 ±5.1
66.2 ± 6.0
* mean ± sample SD, n = 19
40 ml/min average inlet flow rate
Flow rate reductions in the Dougherty sand soil columns also produced increases in
the percent removals. Observed percent removal was possibly limited by transport effects. A
reduction in the inlet flow rate may not produce an increase in the percent removal which follows
purely first-order removal kinetics with no concentration minimum. An alternative to decreasing
the inlet flow rate (and reducing the biomass) would be to keep the flow rate constant and simply
double the length of the column. This would allow greater percent removals up to the point that
transport becomes the limiting step in the removal process. Further experiments need to be
conducted to determine the minimum concentrations caused by transport limitations. Further
work is needed to examine grain size distribution, organic carbon content, and other factors
which may reduce the minimum concentration.
Table 5.4 presents a summary of the removals of each compound obtained by the
three soils examined under similar operating conditions. Table 5.5 lists the final moisture
contents of the soil columns. The columns were constructed by settling the soil with water to
minimize destruction of the natural soil aggregates. Initial moisture contents were probably
higher than the moisture contents determined at the end of the column experiments. The amount
of drying which took place in the columns is unknown but each column still had adequate soil
49
-------
Table 5.2. Hydrocarbon Removal At Varying Inlet Concentrations
Average flow rates and concentrations
Soil Type Flow Rate
ml/min
Rubicon Sand 40
Column F
Rubicon Sand 40
Column G
Dougherty Sand 10
Durant Loam 10
jfc M«ann 4- QP) n "> ft
Compound
Benzene
Toluene
Ethylbenzene
0-Xylene
Benzene
Toluene
Ethylbenzene
O-Xylene
Benzene
Toluene
Ethylbenzene
O-Xylene
Benzene
Toluene
Ethylbenzene
0-Xylene
•
Inlet Cone
ug/ml
3.1
20.3
1.1
4.5
0.9
1.3
1.0
0.9
3.1
20.5
1.5
4.7
1.1
1.2
1.1
0.9
35.2
4.1
3.4
9.5
2.9
0.9
3.9
4.0
1.8
2.6
4.9
2.4
35.6
3.9
3.9
10.3
3.8
1.6
3.6
3.7
1.6
2.6
4.5
2.2
Percent
Remaining*
66.4 ± 4.9
70.2 ± 4.2
64.8 ± 3.0
68.7 ± 3.3
53.2 ± 4.4
61.2 ± 5.0
64.8 ± 5.6
68.2 ± 4.0
67.5 ± 5.1
68.8 ± 3.9
57.6 ± 4.8
66.9 ± 4.3
58.1 ± 2.4
62.2 ± 5.1
62.4 ± 3.4
66.2 ± 6.0
69.1 ± 2.4
4.2 ± 1.0
16.0 ± 8.5
76.7 ± 2.2
73.3 ± 4.7
69.1 ± 2.9
72.2 ± 3.1
72.8 ± 10.2
61.7 ± 8.3
82.2 ± 3.4
64.5 ± 23.2
desorption
69.9 ± 1.8
8.5 ± 5.9
26.0 ± 5.8
76.4 ± 1.9
65.0 ± 9.4
69.5 ± 3.3
73.1 ± 3.2
73.5 ± 8.0
74.8 ± 4.9
77.2 ± 3.6
74.8 ±11.4
84.6 ± 4.1
50
-------
Table 5.3. Hydrocarbon Removal Al Varying Flow Rates
Average flow rates and concentrations
Soil Type Flow Rate Compound
ml/min
Durant Loam 40 Benzene
Toluene
Ethylbenzene
0-Xylene
20 Benzene
Toluene
Ethylbenzene
0-Xylene
10 Benzene
Toluene
Ethylbenzene
0-Xylene
Dougherty Sand 40 Benzene
Toluene
Ethylbenzene
0-Xylene
20 Benzene
Toluene
Ethylbenzene
0-Xylene
10 Benzene
Toluene
Ethylbenzene
0-Xylene
Inlet Cone
ug/ml
31.2
8.7
3.4
2.7
33.0
8.9
3.3
2.5
35.6
10.3
3.6
2.6
33.1
9.6
3.5
2.4
31.9
8.7
3.6
2.5
35.2
9.5
3.9
2.6
Percent
Remaining
85.4 ± 3.2
86.2 ± 1.9
85.3 ± 1.2
86.4 ± 1.8
71.3 ±2.4
79.4 ± 2.1
74.3 ± 2.7
72.7 ± 2.6
69.9 ± 1.8
76.4 ± 1.9
73.113.2
77.2 ± 3.6
76.2 ± 3.8
90.8 ± 0.9
82.0 ±5.1
92.0 ± 3.0
75.4 ± 2.7
79.4 ± 2.5
72.0 ± 2.6
79.7 ± 2.2
69.1 ±2.4
76.7 ± 2.2
72.2 ±3.1
82.2 ± 3.4
moisture after three to four months of operation. Inhibition due to excessive moisture contents
was not directly addressed and was not observed in the experiments performed in this study.
Table 5.6 lists the individual characteristics of each soil examined in this study. The main
physical differences between the soils are the grain size distributions and the permeabilities.
51
-------
Additional differences between the soil columns were the length of the columns (50
to 75 cm) and the types of microbial populations present in each soil. The Rubicon sand
provided the greatest removal for each component tested of the three soils examined. The
Rubicon sand had the greatest moisture content throughout the soil column. The columns of
Durant loam and Dougherty sand each had upper sections of lower moisture contents. Later
microcosm experiments showed the upper sections had reduced microbial activities. The
residence times of the components within the active microbial zones in the Durant loam and
Dougherty sand soil columns were less than the residence time within the Rubicon sand column.
Direct comparison of the removals between the columns is not possible due to the differences but
relative removals of the hydrocarbons can be addressed.
Table 5.4. Hydrocaibon Removal By Different Soils
Averaged inlet concentrations
Soil Type Compound
Rubicon Sand (F) Benzene
(G)
Dougherty Sand
Durant Loam
Rubicon Sand (F) Toluene
(G)
Dougherty Sand
Durant Loam
Rubicon Sand (F) Ethylbenzene
(G)
Dougherty Sand
Durant Loam
Rubicon Sand (F) 0-Xylene
(G)
Dougherty Sand
Durant Loam
Inlet Concentration
Ug
20.3
20.5
33.1
31.2
4.5
4.7
9.6
8.7
1.8
1.2
3.5
3.4
0.9
0.9
2.4
2.7
Percent
Remaining*
70.2 ± 4.2
68.8 ± 3.9
76.2 ± 3.8
85.4 ± 3.2
68.7 ± 3.3
66.9 ± 4.3
90.8 ± 0.9
86.2 ± 1.9
61.2 ±5.0
62.2 ± 5.1
82.0 ±5.1
85.3 ± 1.2
68.2 ± 4.0
66.2 ± 6.0
92.0 ± 3.0
86.4 ± 1.8
* mean ± SD, n a 6
40 ml/min flow rate
Rubicon sand soil columns removed nearly equal percentages of each hydrocarbon.
Equal utilization of the hydrocarbons possibly reveal a diverse microbial community within the
Rubicon sand. The columns removed 25.6-35.1 percent of the input benzene, 28.0-37.4 percent
of the input toluene, 32.9-43.8 percent of the input ethylbenzene, and 27.8-39.8 percent of the
input
-------
Soil Type
Rubicon Sand
Column F
Column G
Durant Loam
Dougherty Sand
Table 5.5.
lower 15 cm
middle 25 cm
upper 25 cm
lower 15 cm
middle 25 cm
upper 25 cm
lower 28 cm
upper 25 cm
lower 48 cm
upper 25 cm
Soil Column Moisture Contents
Percent Moisture
Content (Wet Basis)
17.7
11.2
9.4
13.4
9.5
10.0
19.6
11.4
10.4
6.8
Percent Of
Saturation
saturated
63
53
76
54
56
88
51
46
30
Dougherty sand showed a distinct preference for benzene (20-27 percent removal)
and virtually ignored o-xylene (5-11 percent removal). The Dougherty sand soil column had the
lowest percent water saturation values of the soil columns studied. In this case the low water
content would eliminate the possibility of transport limitations causing the low o-xylene
utilization. Possibly the types of microorganisms present in the Dougherty sand soil simply
could not utilize the doubly methylated o-xylene.
Table 5.6. Soil Characteristics
Soil Type
Dougherty
Sand*
Moist Bulk
Density^
(gm/cm" )
Permeability
(in/hr)
0.63 - 2
Soil
Reaction
PH
5.6 - 6 .5
% Organic
Matter no.4
4.7mm
0.79 inn
^Passing Sieve
no. 10 no.40
2.0 mm 0.42 mm
inn
no.200
0.074 mm
Durant Loam*
Rubicon Sand**
1.35 -1.55
68-80 13-30
<0.6 5.6 -6.5 0.75 100 100 95-100 55-85
6-20 4.5-6.0 .5-1.0 95-100 75-100 35-70 0-15
* (USDA^CS, 1973)
** (USDA/SCS. 1986)
53
-------
Similarly, the Dougherty sand soil column does not effectively remove toluene (8.3-
10.1 percent removal). Again, possibly the presence of the methyl group inhibits the
microorganisms from utilizing the hydrocarbon. Alternatively, the Dougherty sand soil column
did show a potential for ethylbenzene utilization (12.9-23.1 percent removal). The types of
microbes present in the Dougherty sand seem to make a distinction between the ethyl substituted
ethylbenzene and the methyl substituted toluene and o-xylene. Alternatively, the low relative
moisture content of the Dougherty sand soil column may cause the inhibition of the microbes
from utilizing the methyl substituted hydrocarbons. Additional studies of Dougherty sand soil at
other moisture contents would have to be performed to determine the possible cause of the lack
of methyl substituted hydrocarbon utilization.
Durant loam soil also showed a balanced removal of the four hydrocarbons. The
Durant loam soil column removed 11.4-17.8 percent of the benzene, 11.9-15.7 percent of the
toluene, 13.6-15.9 percent of the ethylbenzene, and 11.8-15.4 percent of the o-xylene introduced
into the column. The relative moisture content of the column was 51-88 percent of the saturation
value. This moisture content was close to the optimum range of 50-70 percent of saturation
capacity suggested by Dibble and Bartha (1979). Moistures within this optimum range possibly
allow microbial development which utilize the available hydrocarbon sources equally.
The rate of change of hydrocarbon concentration was monitored with soil
microcosms. Figures 5.3 and 5.4 are examples of experimental data expressed as percent
remaining versus time. Rate constants were determined for each contaminant of interest for each
of the soils. The rate of removal was assumed to be pseudo first-order or zero-order. First-order
rate constants were determined using the integral method for determining the reaction rate (Hill,
1977). First-order reaction rates should produce straight line graphs of In(concentration) versus
time. The slope of the resulting straight line is the rate constant of the removal reaction. Zero-
order reactions are straight line relationships when data are plotted as reaction time versus
concentration. The slope of the line is then the reaction rate constant.
A least squares line was fit to the removal data for zero-order and first-order relations
and the accompanying r-squared values were calculated. R-squared is the statistic that implies
how well a model fits a set of data. R-squared equal to one implies a perfect fit. The r-squared
values were used to determine whether the microcosm data displayed kinetics of zero- or first-
order. Table 5.7 lists the rate constants determined for the removal of each component. Least
squared fits which had nearly equal r-squared values for both zero-order and first-order list the
rate constants for both. Rate constants are listed as negative values to clarify that the reactions
are removal reactions.
Most of the removal reactions did not display a clear distinction between first-order
removals or zero-order removals. The r-squared values for the zero-order and first-order
relations were nearly equal in most cases. Table 5.8 lists the microbial densities determined for
each acclimated soil at the various moisture contents. In all but one case, greater microbial
densities accompanied higher moisture contents. In two out of three cases, greater moisture
contents were associated with faster removal rates. Figures 5.5 and 5.6 graphically illustrate the
effect of moisture content on removal rates. The Durant loam produced the largest benzene
54
-------
% Remaining
lOOt
90-
80-
70-
60-
50-
40-
30-
20-
10-
0-
>-
-8 I o
* Q
* * fl
*
* o
*
*
*
*
1 1 1 1 1 1 1 1
O
o
o
*
*
-1 i_l
10 15 20 25 30 35 40 45 50
Time (hours)
Moisture Content 13.4%
o - Control
* - Sample
Figure 5.3. Toluene Removal in Rubicon Sand Microcosm.
lOOt
90-
80-
70-
60-
% Remaining 50-
40-
30-
20-
10-
0-
h 8 «
- 1 8 . ,
i
$
*
1 1 1 1 1 1 fc-H MM 1
8
i — i — i — i * i
5 10 15 20 25 30 35 40 45 50 55 60 657075
Time (hours)
Moisture Content
o - Control
* - Sample
19.6%
Figure 5.4. Benzene Removal in Durant Loam Microcosm.
55
-------
Table 5.7. Removal Rate Constants For Hydrocarbons In Soil Microcosms
Soil Type Moisture gms Soil per Compound
Content Microcosm
(wet basis)
Rubicon Sand
13.4 % 27.3 Benzene
Toluene
Ethylbenzene
0-Xylene
9.5 % 12.5 Benzene
Toluene
Ethylbenzene
0-Xylene
10.0 % 13.3 Benzene
Toluene
Ethylbenzene
0-Xylene
Dougherty Sand
10.4 % 6.2 Benzene
Toluene
Ethylbenzene
O-Xylene
6.8 % 5.9 Benzene
Toluene
Ethylbenzene
O-Xylene
Durant Loam
19.6% 21.7 Benzene
Toluene
Ethylbenzene
0-Xylene
11.4% 6.9 Benzene
Toluene
Ethylbenzene
0-Xylene
Initial Reaction Constants
Cone. Zero First
(ug/ml) (ug/min) (min )
3.46
3.32
3.59
3.95
3.61
3.46
3.53
3.87
3.36
3.43
3.52
3.81
2.48
2.65
1.74
3.13
2.64
2.55
1.71
2.62
3.33
3.12
1.94
3.02
2.53
2.48
1.50
2.31
-2.966
-3.039
-0.860
-1.187
-3.075
-0.869
-1.177
-2.803
-1.866
-2.849
-1.362
-0.794
-1.474
-0.750
-1.135
-0.444
-4.626
-2.062
-0.660
-0.705
-3.076
-1.616
-0.655
-0.714
-0.039
-0.121
-0.032
-0.005
-0.007
-0.032
-0.013
-0.005
-0.007
-0.026
-0.013
-0.102
-0.014
-0.025
-0.006
-0.006
-0.003
-0.045
-0.012
-0.007
-0.012
-0.007
56
-------
removal rate constant. Rubicon sand had the largest ethylbenzene and o-xylene removal rate
constants.
Rubicon sand had the largest toluene removal rate constant at the higher moisture
content. At lower moistures, Durant loam had the largest removal rate constant for toluene.
Ethylbenzene and o-xylene removal seemed to be the most first-order related of the contaminants
tested. Shorter sampling times may be required to produce better first-order correlations for the
other components.
Table 5.8. Microbial Densities
Acridine Orange Fluorescent Direct Bacteria Counts
Soil Type
Rubicon Sand (G)
Durant Loam
Percent Moisture
(wet basis)
13.4
9.5
10.0
19.6
Microbial Density*
(Cells per g soil)
18.0(10)®
22.2(10)?
26.6(10)
9.1(10)*
.
Unacclimated soil 0.4(10)
Dougherty Sand 10.4 32.8(10)8
6-8 17.4(10)!*
Unacclimated soil 186(10)
* Acridine-orange Direct Counts follow the Poisson Distribution. The standard deviation of the microbial density is
equal to the square root.
In general, the Rubicon sand exhibited a preference for ethylbenzene followed by
toluene, benzene, and o-xylene when tested at the higher moisture content. The microbes present
in the Rubicon sand evidently contained microbial strains which preferentially utilized the ethyl
and methyl substituted benzenes over benzene itself. Higher moisture contents possibly restrict
the transport of o-xylene causing the removal rate constant for o-xylene to be slightly less than
for benzene. At the lower moisture content, a larger surface area would be available for
adsorption. And in the case of lower moisture contents, the resulting removal rate constant for o-
xylene is greater than either of the removal rate constants for benzene or toluene. At the lower
moisture content the order of preference was ethylbenzene, o-xylene, toluene, and benzene. The
order of preference corresponds well with the results obtained from the soil column studies.
Ethylbenzene was removed the most in the soil column, followed by nearly equal removals of
toluene and o-xylene, with benzene being removed the least.
Dougherty sand removed benzene the fastest of the four components tested
Ethylbenzene removal was second to benzene and preceded toluene and o-xylene removal. The
lesser removals of o-xylene probably reflect increased resistance to biodegradation due to
additional methyl substitution of o-xylene. A considerable reduction in microbial activity was
57
-------
6.8% Moisture
% Remaining
70-
60-
50-
40-
30-
20-
10-
0-
- -- e--^ /
* ^ o
* x-- " ^ - °
--.. * -» ^
*•. ***
"^ **^
'•--. " ^ ^ o
\ ° ^^-0
10.4% Moisture ^ "'••-,
Hours
Lines Generated Using Removal Rate Constants
o - 0.8% Moisture
* - 10.4% Moisture
Figure 5.5. Moisture Effect on Benzene Removal, Dougherty Sand Microcosm.
% Remaining 50--
10--19.6% Moisture
11.4% Moisture
5 10 " 20 25 ao 35 40 4550
Hours
* - 19.6% Moisture
Figure 5.6. Moisture Effec, on Benzene Removal, Duran, Loam Microcosm.
58
-------
observed between the 10.4 percent (46 percent of saturation capacity)and 6.8 percent (30 percent
of saturation capacity) moisture contents as illustrated in Figure 5.5. The 10.4 percent moisture
content is close to the optimum moisture range of 50-70 percent of saturation which has been
reported by Dibble and Bartha (1979).
Removal rate constants developed from Durant loam were greatest for benzene
followed by toluene, 0-xylene, and ethylbenzene. The removal constants of ethylbenzene and o-
xylene were almost equal. Nearly equal solubilities which influenced the transport of these
components along with the presence of suitable utilizing microorganisms probably contributed to
the nearly equal removal rates. Moisture content was an important parameter of hydrocarbon
removal as illustrated in Figure 5.6. Removal inhibition from excess moisture was not observed
for the limited number of moisture contents evaluated. Additional studies need to be performed
to determine optimum.soil moistures.
Direct comparison of the experimental reaction rates with previously reported values
is not possible because of the different types of experimental setups, but some generalizations
may be drawn. Thomas (1987) reported benzene removals for soil-water systems approximately
1000 times less than the benzene removals obtained in this study (Thomas:0.0004-0.0024 /min
versus 0.9-4.0 /min). Slower transport through water than air and limited availability of oxygen
in aqueous systems compared to air systems may contribute to this difference. Swindell (1988)
has reported removal rates for toluene removal in aqueous systems which are within an order of
magnitude to the values obtained in this study (Swindoll:0.00083 min compared to 0.006-0.045
min ). The values obtained in the aqueous system are less than those obtained in the air system.
The rates of aromatic hydrocarbon removal in the bioreactors were equivalent to
rates of biological removal of aliphatic hydrocarbons published for other soils. From Table 5.7
the zero-order rate for benzene degradation in the Rubicon sand at 13.4% moisture is equivalent
to 0.109 ^ig/g soil/min. The rate of removal of propane and butanes in a pilot-scale bioreactor at
Racine, Wisconsin, was 0.113 ug/g soil/min (Kampbell et al., 1987). Hoeks (1972) showed that
soils from The Netherlands could degrade methane at a zero-order rate of 0.083 mg/g soil/min
after a suitable period of acclimation.
59
-------
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