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THE-181$

STYI TYIELIBRAR

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3 1293 0088

 

 

 

 

This is to certify that the

thesis entitled

Variables Effecting Trichloroethylene
Transformation by Methanotrophs

presented by

Linda M. Clowater

has been accepted towards fulfillment
of the requirements for

Master of

 

degree in Wtal

Science Engineering,

a“; 5 éo/a/g

[Major professor

Datefi Zé: /7ZZ__.

0-7 639

MS U is an Affirmative Action/Equal Opportunity Institution

 

 

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PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.

DATE DUE DATE DUE DATE DUE ll

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU Is An Affinnetlve Action/Equal Opportunity Institution
chimera»!

VARIABLES EFFECTING TRICHLOROETHYLENE
TRANSFORMATION BY METHANOTROPHS

By

Linda M. Clowater

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Civil and Environmental Engineering

1992

ABSTRACT

VARIABLES AFFECTING TRICHLOROETHYLENE
TRANSFORMATION BY METHANOTROPHS

By

Linda M Clowater

This research focuses on environmental variables affecting co-metabolism of
trichloroethylene (TCE) by methanotroph enrichment MMl, derived from aquifer material
sampled at the Moffett Field Naval Air Base, Mountain View, California. Variables evaluated
include incubation pH, reducing power availability, and TCE concentration. In order to
quantify the effects of these variables, the kinetic models used to describe co-metabolism were
reviewed, and inter-relationships between the models were determined.

The effects of pH on transformation capacity, Tc, of the culture were evaluated at pH
values of 5.5, 6.8, 8.5 and 10. Transformation capacity decreased at pH 5.5 and 10.

Reducing power was evaluated by addition of forrnate. At low TCE concentrations,
formate addition had no effect on TCE transformation, but at high TCE concentrations, formate
addition increased the transformation capacity from 0.023 mg TCE/mg cells to 0.139 mg
TCE/mg cells. Formate addition at pH 5.5 increases the Tc from 0.013 mg TCE/mg cells to
0.033 mg TCE/mg cells, but had no effect at pH 10.

The loss of transformation capacity was also evaluated over a five day aeration period
in the absence of methane. Transformation capacity decayed at a rate of 0.0381 day'l.
Experiments with low levels of TCE gave a transformation capacity of 0.0346 mg TCE/mg
cells. This value is close to the values observed at higher concentrations ( ~ 0.04 mg TCE/mg
cells), but does not include the loss of Tc due to decay. If the low level TCE value is adjusted

for decay, the initial value for To would be 0.192 mg TCE/mg cells. This suggests that Tc may

actually increase at low TCE concentrations.

This thesis is dedicated to my family, whom I love.

ACKNOWLEDGMENTS

I would like to express my gratitude and appreciation to several people who provided
guidance and support for this project.

First I would like to thank my advisor Dr. Craig Criddle for his patience and guidance
in an area that was very new to me when I first started. His help was invaluable and greatly
appreciated. I would like to thank Dr. Susan Henry for her help and advise during and after
the time I studied under her at Stanford University. In addition, thanks to Jim Grant who put
in many frustrating hours dealing with methane utilization rates and Brad Kach and Chien-
Chun Shih for helping obtain some final data.

I must also express my appreciation to Dr. Robert Hickey and Michigan Biotechnology
Institute, the Center for Microbial Ecology, and the College of Engineering for their support.

Finally, I would like to thank my fiance Sean and my dog Kedzie. No matter what
kind of day I had or what kind of mood I was in, they were always glad to see me.

iv

TABLE OF CONTENTS

Page
LIST OF TABLES .................................................................................... vii
LIST OF FIGURES .................................................................................. viii
LIST OF SYMBOLS ................................................................................. x
CHAPTER 1 - INTRODUCTION ................................................................. 1
Background ......................................................................... l
Biotransformation of Chlorinated Organics ...................................... 2
Methanotrophs ....................................................................... 3
Aerobic Degradation of Trichloroethylene ....................................... 6
Modeling ............................................................................. 10
CHAPTER 2 - MATERIALS AND METHODS .................................................. 16
Mixed Culture Development ....................................................... 16
TCE Solution and Analysis ........................................................ 17
TCE Transformation Studies ...................................................... 18
Culture Density ..................................................................... 18
CHAPTER 3 - EFFECTS OF TCE CONCENTRATION ON
TRANSFORMATION CAPACITY .............................................. 20
Introduction .......................................................................... 20
Materials and Methods .............................................................. 20
Results ................................................................................ 21
Discussion ........................................................................... 28
CHAPTER 4 - EFFECTS OF pH AND FORMATE ADDITION .............................. 32
Introduction .......................................................................... 32

V

Materials and Methods .............................................................. 32

Results ................................................................................ 33
Discussion ........................................................................... 39
CHAPTER 5 - EFFECTS OF ENDOGENOUS DECAY ........................................ 40
Introduction .......................................................................... 40
Materials and Methods .............................................................. 40
Results ................................................................................ 41
Discussion ........................................................................... 44
CHAPTER 6 - ENGINEERING APPLICATION ................................................ 48
CHAPTER 7 - CONCLUSIONS ................................................................... 50
Future Work ......................................................................... 51
LIST OF REFERENCES ............................................................................ 52
APPENDIX A - Whittenbury Mineral Medium ................................................... 55
APPENDIX B - Methane Utilization ............................................................... 56
APPENDIX C - Mixed Culture MMl .............................................................. 59
APPENDIX D - Propagation of error .............................................................. 60
Sensitivity analysis .................................................................. 61

vi

Table 1.1
Table 1.2
Table 2.1
Table 3.1
Table 3.2
Table 3.3

Table 4.1
Table 4.2
Table 8.]

LIST OF TABLES

Summary of chemical and physical parameters .................................. 6

Kinetic expressions for cometabolism ............................................ 12
Methane utilization results .......................................................... 16
Evaluation of model 1 at five different TCE concentrations .................... 25
Evaluation of model 2 at five different TCE concentrations .................... 26

Evaluation of model 3 and 'measured' Tc with five different TCE

concentrations ....................................................................... 27
Summary of pH effects at low TCE concentration .............................. 35
Summary of pH effects at high TCE concentration ............................. 36
Methane utilization spreadsheet .................................................... 58

vii

Figure 1.1
Figure 1.2
Figure 1.3

Figure 1.4
Figure 3.1
Figure 3.2
Figure 3.3
Figure 3.4
Figure 3.5
Figure 3.6

Figure 4.1

Figure 4.2

Figure 4.3
Figure 4.4
Figure 4.5
Figure 4.6
Figure 4.7
Figure 5.1
Figure 5.2
Figure 5.3
Figure 5.4
Figure 5.5
Figure 5.6

LIST OF FIGURES

The metabolism of methane ........................................................ 4
Proposed mechanism for aerobic TCE degradation ............................. 9
Dependence of transformation capacity on substrate concentration for

Model 1 ............................................................................... 14
Assumptions associated with models 1, 2, and 3 ............................... 14
TCE degradation at high initial substrate concentration ........................ 21
TCE degradation at medium initial substrate concentration .................... 22
TCE degradation at low initial substrate concentration ......................... 22
Continuous respike experiment for low levels of TCE ......................... 23
Cumulative TCE degradation ...................................................... 23
Typical degradation curve for high-range TCE concentrations ................ 28

Effects of formate addition on TCE degradation at low TCE
concentrations ....................................................................... 34

The effect of formate on TCE degradation at at high TCE

concentrations ....................................................................... 34
Effects of pH on TCE transformation at low TCE concentrations ............ 35
Effects of pH on TCE transformation at high TCE concentration ............. 36
Formate effects at pH 5.5 .......................................................... 37
Formate effects at pH 6.8 .......................................................... 38
Formate effects at pH 10 ........................................................... 38
Aeration experiment for day 1 ..................................................... 41
Aeration experiment for day 2 ..................................................... 41
Aeration experiment for day 3 ..................................................... 42
Aeration experiment for day 4 ..................................................... 42
Aeration experiment for day 5 ..................................................... 43
Transformation capacity over time including endogenous decay .............. 44

viii

Figure 5.7

Figure 5.8
Figure 5.9
Figure C.1
Figure D.l

Figure D.2

Figure D.3

Figure D.4.

Figure D.5

Transformation capacity for the five day respike experiment (Chapter 3)
including endogenous decay effects compared to transformation capacity

corrected for endogenous decay effects of 0.381 day”l ...................... 45
Pr0posed explanation of TC vs. S based on above findings ................... 45
Pathways for potential loss of transformation activity ......................... 47
Scanning electron micrograph (SEM) of mixed culture MMl ................. 59

Sensitivity analysis for model 1b and 2b for the TCE concentration range
of 0015-0017 mg/L ............................................................... 61

Sensitivity analysis of models 1a and 2a at a TCE concentration range of
0.22-0.24 mg/L ..................................................................... 63

Sensitivity analysis for model 1a and 2a for the TCE concentration of
0.91-0.97 mg/L ..................................................................... 64

Sensitivity analysis for model 3 for the higher TCE concentration ranges of
10.2-12.5 mg/L and 5.5-5.7 mg/L ................................................ 65

Sensitivity analysis for model 3 at the lower initial TCE concentrations of
0.91-0.97 mg/L, 0.22-0.24 mg/L, and 0015-0017 mg/L .................... 66

LIST OF SYMBOLS

b first order decay coefficient (per day)

k maximum specific rate of substrate utilization (mg substrate/mg biomass-day)
Ks half saturation coefficient (mg substrate/L)

k' second order rate coefficient: K/Ks (Umg—day)

S non- growth substrate concentration (m g/ L)

t time

Tc transformation capacity (mg substrate/mg cells)

X active organism concentration (m g/L)

X0 initial active organism concentration (m g/L)

So initial non-growth substrate concentration

CHAPTER 1

INTRODUCTION

Background

Contaminated water is a problem facing many communities throughout the world. It is
estimated that 16 to 28% of randomly selected U.S. groundwater drinking supplies are
contaminated (Westrick et al., 1984). A major cause of groundwater contamination is
halogenated aliphatic compounds. These compounds are widely used in industry as solvents
for degreasing, paint stripping and dry cleaning. They enter the water supplies mainly through
accidental spills and improper disposal and storage. As use of these chemicals has grown, so
has the potential for groundwater contamination as these chemicals are mobile and will migrate
through soil into groundwater. Once present in a drinking water supply, they pose a threat to
public health since many of these chemicals are known or suspected carcinogens.

Methods for remediation of contaminated groundwater include carbon adsorption, air
stripping, chemical oxidation and biological methods. The advantage of chemical and
biological processes over the others is that these processes provide partial or complete
destruction of contaminants. Adsorption and air stripping simply transfer contaminants to a
different phase, and may still require further treatment

One emerging technology for the treatment of contaminated water is bioremediation.
During the past decade, aerobic degradation of chlorinated aliphatics, such as trichloroethylene
(TCE), has been widely demonstrated using methanotrophic bacteria. Unlike anaerobic
degradation which can produce toxic by-products such as vinyl chloride, the products
produced aerobically are thought to be relatively harmless. To date, however, there have been

relatively few systematic investigations of environmental factors influencing the rate and extent

2

of methanotrophic transformations. An understanding of these factors is essential in the design

and operation of engineered systems and in the comparison of cultures from different sources.

Scom and Objectives

The overall goal of this research is to evaluate the role of environmental factors on the
rate and extent of degradation of TCE by methanotrophs. More specifically, this work
evaluates the following environmental factors:

1) TCE concentration
2) Reducing power (forrnate addition)
3) pH

4) Loss of transformation capacity

Biotra_n_sfonnation of chlorinated organics

 

Many organic compounds found in the environment are mineralized as a result of
biological activity. There are, however, some compounds which resist microbial degradation
or are only partially transformed. Reasons for this include environmental conditions that
prevent competent microbial populations from growing, such as lack of primary substrate or
nutrients.

For many years, it was held that many chlorinated aliphatics could only be degraded
anaerobically. In 1985, however, Wilson and Wilson demonstrated aerobic degradation of
TCE in soil enriched with natural gas. Since then, much research has focused on aerobic
biodegradation of chlorinated aliphatics.

Methanotrophic transformation of chlorinated aliphatics can be classified as ”co-

metabolic”. One definition of co-metabolism is ”transfonnation of a non-growth substrate in

3

the obligate presence of a growth substrate or another transformable compound" (Dalton and
Stirling, 1982). In a more general sense, however, co-metabolism can be defined as any
transformation of a non-growth substrate that depends upon the concurrent or previous
metabolism of a growth or energy substrate. This broader definition is more appropriate for
methanotrophs inasmuch as methanotrophic transformation of non-growth substrates, such as
TCE, can continue, although at decreasing rates, in the absence of a growth substrate (e. g.

methane) or an energy substrate (e. g. f orrnate).

Methanotrophs

Methylotrophs are bacteria that can oxidize methane and methanol for energy and
growth. Obli gate methylotrophs grow on reduced carbon compounds that contain one or more
carbon atoms but no carbon-carbon bonds. Facultative methylotrophs can grow on a variety of
other organic multi-carbon compounds (Anthony, 1982). Methylotrophic bacteria that are able
to grow on methane are called methanotrophs. .

Methanotrophs are classified as either Type I or Type 11 depending upon their internal
membrane structure. Type I methanotrophs have a membrane that is arranged in bundles of
vesicular discs, use the ribulose monophosphate cycle to assimilate carbon, have an incomplete
TCA cycle, and form cysts (Azotobacter—like) for resting stages. Type II methanotrophs have
their membranes arranged in pairs around the cell periphery, use the serine pathway to
assimilate carbon, have a complete TCA cycle, and have exospores or 'lipid' cysts (unique
structures) for resting stages (Higgins, 1979).

The metabolism of methane is shown in the Figure l. 1. In the conversion of methane
to methanol, several oxidation reactions occur. The initial oxidation is catalyzed by the
methane monooxygenase (mmo) enzyme system. Methanol dehydrogenase catalyzes the
oxidation of methanol to formaldehyde. Formaldehyde is either incorporated into biomass or

oxidized further for energy. Formaldehyde dehydrogenase catalyzes the oxidation of

4

formaldehyde to formic acid, and fonnate dehydrogenase catalyzes the final oxidation of formic

acid to carbon dioxide and water.

Methane Methanol Formaldehyde Formate
Monooxygenase Dehyckogenase Dehydogenase Dehyckogenase

02 ”2°

 

 

 

cu4%%) CI~§OH * > HCHO .779 HCOOH /\ > co2
NADH + H+ NAD+ NAD"’ NADH + H+
w
Anabolic
Pathways

Figure 1.1. The metabolism of methane. (Modified from Higgins, 1979;
Anthony, 1982; and Fox, 1990), * co-factors
required for these reactions vary.

The mmo system has been identified and characterized in three species of
methanotrophs: Methylococcus capsulatus (Bath), Methylosinus trichosporium OB3b, and
Methylobacterium sp strain CRL-26. The enzyme is a multicomponent system which catalyzes
the oxidation of methane to methanol. The mmo system has two known forms: a particulate
form associated with the cell membrane and a soluble or least soluble form (Higgins, 1979).
The dominant form of mmo depends upon growth conditions (Patel et al., 1982, Burrows et
al., 1984). The actual location of mmo in the cell is dependent upon copper availability and
biomass concentration. I

For Methylococcus capsulatus (Bath) and Methylosinus trichosporium 0B3 b, soluble
mmo dominates at low copper concentration; as the copper concentration increases, so does the

particulate to soluble ratio until the only form remaining is the particulate form. Dalton et al.,

S

(1984) showed similar variations if the copper concentration was held constant and the biomass
concentration varied.

The substrate specificities of the two forms of the enzyme also differ. Particulate mmo
efficiently oxidized n-alkanes and n—alkenes, but poorly oxidized alicyclics and aromatic
compounds (Stirling et al... 1979, Burrows, 1984). In in vitro studies, the soluble fraction of
Methylococcus capsulatus (Bath) catalyzes the hydroxylation of primary and secondary C-H
bonds, the formation of epoxides from internal and terminal alkenes, the hydroxylation of
aromatic compounds, the N -oxidation of pyridine, and the oxidation of CO to C02. (Colby et
al., 1977).

Using crude extracts from Methylosinus trichosporium OB3b and Methylobacterium
Sp. CRL-26, Stirling et al., (1979) and Patel et al., (1982) showed that the substrate specificity
and oxidation products were the same as those associated with Methylococcus capsulatus
(Bath). There is a wide but slightly more limited range of transformations that occur with
whole-cell suspensions as opposed to the cell-free enzyme. In studies conducted by Stirling
and Dalton (1979), resting-cell suspensions of Methylococcus capsulatus (Bath) oxidized the
following compounds: chloromethane, bromomethane, dimethyl ether, ethylene and
propylene. Using formeldehyde to regenerate reducing power, the following additional
compounds were oxidized: carbon monoxide, diethyl ether, ethane, propane, 1-butylene, cis-
2-butylene, and trans-2 butylene. Higgins et. al . (1979) showed that whole cell suspensions
of Methylosinus trichosporium OB3b had a broader specificity than Methylococcus capsulatus
(Bath) by oxidizing n-alkanes, n-alkenes, alicyclic and terpenoid hydrocarbons, aromatics,
alcohols, phenol, pyridine, and ammonia.

Mixed culture MMl (Appendix C, Figure C.1) used for this study was originally
enriched from aquifer solids from the Moffett Field Naval Base in California (Henry, 1990).
W1 is a stable consortium consisting of one methanotroph and three or four heterotrophs
containing predominantly Gram-negative pleomorphic coccobacilli and prosthecates as well as

some Gram-negative bacilli and cocci. The methanotroph was a pleomorphic coccobacillus that

6

contained the internal membrane structures characteristics of Type II methanotrophs (paired
membranes inside the periphery of the cell.) Extracts of mixed culture MMl were tested with
an antibody specific to the soluble MMO of M. trichosporium OB3b and they cross reacted
with it (T sien et al., 1990). This indicates that the methanotrophs in mixed culture MMl
expressed soluble MMO similar to that of Methylosinus trichosporium OB3b under the

specified growth conditions (Henry, 1991a).

Aerobic deggdation of trichloroethylene

Several investigators have examined the degradation of chlorinated aliphatics. Much of
this effort has focused on the chlorinated solvent trichloroethylene (TCE).
For a better understanding of this compound, some of the physical and chemical

properties associated with trichloroethylene are summarized in Table 1.1

 

 

Trichloroethylene
(TCE)
Molecular Weight 131.39 9 (a)
Melting Point -73°C(a)
Boiling Point 87°C (a)
Density (20°C) 1.464 g/mL (a)
Solubility (25°C) 1100 mg/L(b)
Henry's Constant 0.33 (c)
(dimensionless)
21°C

 

 

 

(a) = hand book of Physics and chemistry, 1989
(b) a Horvath (1982)
(c) - Gossett (1987)

Table 1.1 Chemical and physical properties of trichloroethylene.

7

Fogel et al.. (1986) showed that in a mixed culture, 1""C-labeled TCE was transformed
to C02, cell biomass, and nonvolatile or nonchlorinated compounds. Acetylene, a known
inhibitor of mmo activity, inhibited degradation indicating that methane-oxidizing bacteria
probably initiated TCE oxidation.

Henson et al.. (1989) examined the degradation of several chlorinated aliphatics
including TCE Mixed culture experiments were conducted in serum tubes to which a mixture
of halogenated hydrocarbons was added. When grown in the presence of methane, the culture
transformed TCE

Using a pure culture, strain 46-1, Little et al . (1988) demonstrated degradation of TCE
only when grown on methane or methanol. These studies were conducted using liquid cultures
in inverted serum bottles. Degradation stopped when methane was depleted and continued if
additional methane was added. These researchers concluded that TCE degradation is a
cometabolic process that provides little or no benefit to methanotrophs because strain 46-1
initiated the degradation of TCE but was unable to metabolize the intermediates. Preliminary
evidence indicated that glyoxylic acid and dichloroacetic acid were the breakdown products.
Finally, a mechanism of degradation was proposed. It was hypothesized that TCE is first
converted to its epoxide, which breaks down spontaneously, yielding dichloroacetic acid,
glyoxylic acid, f orrnate and carbon monoxide.

This proposed pathway seems consistent with the findings of Henschler (1979) who
evaluated TCE-epoxide reactivity in aqueous systems. Henschler found that the epoxide
decomposes to form formate, carbon monoxide, glyoxylic acid and dichloroacetic acid and that
the distribution of products was pH-dependent. As pH decreased, fewer l-carbon products
were observed.

Recently a new pathway was proposed for Methylosinus trichosporium OB3b
(Newman, 1991). Four different methanotrophs expressing soluble methane monooxygenase
produced 2,2,2-trichloroacetaldehyde (chloral hydrate). Chloral hydrate was biologically

transformed to trichloroethanol and trichloroacetic acid. Figure 1.2 is a proposed pathway

8

based on the findings of Henschler et al. (1979), Little et at. (1988), and Newman, (1991)
with some modifications.

Once TCE degradation and its degradation pathway were well established, it became
possible to test a number of environmental variables in an attempt to enhance transformation
rates and capacity. Most efforts to enhance the transformation have focused on the availability
of reducing power. As previously described, the mmo enzyme system requires a source of
reducing power to carry out the transformation of TCE. This reducing power is provided by
electron carriers in the cell, such as NAD(P)H (Figure 1.1). When a growth substrate, such as
methane, or energy substrate such as formate, is metabolized, reducing power is regenerated.
Oxidation of any one of the metabolites of methane (methanol, formaldehyde or f ormate) can
regenerate reductant (Figure 1.1). When mmo oxidizes a non-growth substrate, such as TCE,
reducing power is not regenerated, and the oxidation will eventually stop. Providing
methanotrophs with methanol, f ormeldehyde or f onnate will not alleviate the need for methane,
but will support continued TCE oxidation. Few methanotrophs grow well on methanol, none
grow well on formaldehyde, and fonnate is not a growth substrate (Anthony, 1982).

Even when methane is provided, reducing power can still be depleted. Using a pure
culture, Henry (1991b) showed that carbon monoxide competitively inhibited methane
oxidation until forrnate was added as an exogenous electron donor. She also showed that
formate addition to mixed culture MMl did not enhance degradation rates at low TCE
concentrations and assumed this was because MMl methanotrophs possess lipid storage
granules which can serve as an alternate source of electrons. Henry (1991a) also tested the
effect of f orrnate addition on the rates of TCE transformation during methane starvation. At a
TCE concentration of 30 -60 [lg/L, fonnate addition did not increase rates for mixed culture
MMl. However, the rates were enhanced for the the first ten hours of methane starvation
when pure culture MM2 was incubated with 2 mM fonnate. When the culture was incubated
without fonnate and the fonnate was added simultaneously with the TCE, transformation rates

remained significantly enhanced throughout 62 hours of testing.

 

Cl Cl
C— C/
TCE / \
Cl H
a?
m“ \\\
\Fg‘L—o-
Y
CI
F§+ \ /CI
/ C
OH 3 I
\Fe+_o\ c
/ \

 

 

 

 

 

 

 

 

 

 

 

 

Cl 0
\ /
m—c—c/
/ \
Cl H
Chloral
Cl
Cl l
\ /OH C l— C — CH CH
m—c—q\ I Z
/ \
Cl 0 m.
2,2,2-Trrchloro-
Trichloroacetate ethanol

Heterotrophic
Organisms

 

 

 

 

Dichloroacetate

 

 

 

C0z + cells I

 

 

 

 

 

TCE Epoxide
CI H
\/ o\/
c—
Cl/
_C<CI o\\ /
c—c’
I I \
0H
Glyoxylate

   
 

Heterotrophic
Organisms

Figure 1.2. Proposed mechanism for aerobic TCE degradation
(After Little et al., 1988; Henschler et al, 1979; and Newman, 1990)

10

Alvarez-Cohen and McCarty (1991a) introduced the concept of transformation capacity,
as the amount of non-growth substrate (TCE in this case) degraded per unit biomass. This
parameter serves as a useful index making possible the comparison of different co-metabolizing
cultures. Alvarez-Cohen (1991b) tested the effect of forrnate on transformation capacity using
a TCE concentration of approximately 20 mg/L. With the addition of 20 mM of formate,
transformation capacity of the culture increased from 0.036 to 0.073 mg TCE/mg cells. The
transformation rates also increased from 0.6 to 2.1 mg TCE/mg of cells per day. Alvarez-
Cohen (1991b) also tested the effect of aeration on a culture's ability to transform TCE.
Freshly harvested cells were spiked with TCE and compared to cells that had been aerated for
24 hours before TCE addition. The freshly harvested cells had an initial linearized
disappearance rate of 0.5 mg TCEng cell-day compared to the aerated cells which had a rate of
0.03 mg TCE/mg cell-day.

The effects of TCE concentration were evaluated by both Henry (1987) and Alvarez-
Cohen (1991b). Henry showed that increased TCE concentration resulted in increased
transformation rates. When the TCE concentration was increased from 0.1mg/L to 1.0 mg/L,
there was approximately an order of magnitude increase in the TCE transformation rate.

Alvarez-Cohen showed that fluctuations in TCE concentration had no effect on transformation

capacity.

Modeling

Michaelis—Menton or Monod expressions are commonly used to describe cometabolic
degradation. One limitation of these models is that they don't account for the loss of
transformation activity in the absence of growth substrate or the loss of transformation activity
due to other factors such as product toxicity. Consequently, parameters estimated by these
models may be in error. Models are needed that account for terms that include loss of biomass

or enzyme activity caused by consumption of reducing power, endogenous decay, product

11

toxicity and suicide inactivation. Recently, a few models that can account for loss of
transformation activity have emerged, but to date, no effort has been made to identify the inter-
relationships among these models or to critique their basic assumptions. In this section, three
models are described together with their underlying assumptions.

All of the models proposed to date can be derived from the following three equations:

 

—-d—S- kSX (1.1)
dt K,+S

92S.--bx (1.2)
(It

dS

_-T 1.3
dx 5 ( )

where:
S = non- growth substrate concentration (mg/L)
X = active organism concentration (mg/L))
k = maximum specific rate of substrate utilization
(mg substrate/m g biomass-day)
Ks = half saturation coefficient (mg substrate/l)
k'= second order rate coefficient = k/Ks (L/mg-day)
b = first order decay coefficient (per day)
Tc = transformation capacity (mg substrate/ mg cells)
t = time

Table 1.1 summarizes different ways in which these equations can be combined. Here,
80: initial substrate concentration (mg/L), and X0: initial concentration of active organisms
(mg/L). Model 1 is obtained by combining equations 1.1 and 1.2. Model 2 is obtained by
combining equations 1.1 and 1.3. Model 3 is obtained by combining equations 1.2 and 1.3.
Also shown are simplifications for S°<< Ks (equations 1b & 2b) and S0>> Ks (equations 1c
& 2c).

In models 1 and 3, transformation activity is lost with time as a result of the decay of

cometabolizing biomass. This is modeled using a simple first-order exponential decay term, as

12

proposed for enzymes by Bailey and Ollis (1986) and for whole cells by Galli and McMarty

 

 

 

 

 

 

 

 

 

 

 

(1989).
Table 1-2. Kinetic expressions for cometabolism
Differential equations for .
model substrate utilization rate integrated form ref.
dS kSX dX
and - -bX
‘cfi Ks+ 8 Th" 3 40¢ ,., 14.
1a 8 -13- kSX° 6., Kslr(§)+S-S°—T(l-e ) 32
dt Ks+ S
dS
-—-k'SXand§-—bX 0
lb dt dt (k'X (e_u-1)) 9
b
S<<KS so '%I' k'SX"e"" S=S’e
—%f- kx anci%- -bx s =S°~TCX’(1-e"" )
1c
ds 0 -u _ k
S>>Ks so dt kX e whereTc-B—
dS kSX dS
dt-Ks+S and-‘fi- TC tz—l {—KS—)ln— SX +TCI X
k S’lTC-X° FS° _F 1
23‘ kS(X°—1(S’- 3)) 1
so -15- TC where F: X°—— (S’ -S)
dt Ig+s Tc
dS dS
2b “at - kSX andfi -T° S=S°_F§::_ where F'=X°.S"/Tc
xo_ge-th
S<<Ksso:?X'-k'S(-—T1C-(S° 3)) TC
-9; .. kX andsd _ (ix-TC
2c dtdS S-S°-TCX°(l-e"‘)
S>>Ks SO-E -(kX°—-:( 60- 8))
a(--bX and-d—S- -Tc
dt dx 0 _u
3 d5 s=s -TCX°(1-e ) 31
so-d-t --bTCX°e'°‘

 

 

 

 

 

 

In model 2, the loss of transformation activity is directly proportional to the amount of
non-growth substrate transformed. A key concept in models 2 and 3 is the idea of a

”transformation capacity" To for the cometabolizing cells. As stated earlier, Alvarez-Cohen and

13

McCarty (1991a) defined Tc as the mass of non-growth substrate transformed per unit of
biomass, as expressed mathematically by equation 1.3. In both the second and third models, it
is assumed that transformation capacity is independent of concentration and is constant. In the
first model, however, transformation capacity, while not explicitly defined, can be derived by
dividing equation 1.1 by equation 1.2. In this case, Tc is not constant, but depends upon the

concentration of the non-growth substrate (equation 1.4).

Tc = i (1.4)
b(K,+S)

Equation 1.4 has the same form as the Monod expression for substrate utilization, as illustrated
in Figure 1.3. For high substrate concentration (S>>Ks), transformation capacity Tc becomes
constant, Tc = k/b, and model 1 converges with models 2 and 3 (compare 1c, 2c, and 3c).
When Tc is close to Ks, the transformation capacity is half its maximum value. At low
concentrations (S<<Ks), model la predicts a linear increase in transformation capacity with
substrate concentration.

In addition, models 1 and 2 assume a constant maximum substrate utilization rate over
all substrate concentrations, and models 1 and 3 assume that the biomass decay rate is
independent of substrate concentration and is also constant. A summary of all the assumptions
behind the models is shown in Figure 1.4.

Saez and Rittmann ( 1991) used model 3 and classified their experiments as a "success"
or ”failure" depending upon the rate and extent of transformation. For a ”success” test, the
target compound was degraded steadily and completely; for a failure test, only minor amounts

of the compound were transformed. The substrate to biomass ratio correlated with the success

or failure of an experiment. During failure tests, Tc decreased with increasing time. This was

14

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

k/ b
T C Tc/Z ~
KS
S
Figure 1.3. Dependence of transformation capacity on substrate
concentration for model 1.
MODEL ASSUMPTIONS
Tc k b
S 5
Model 1
T C k
5
Model 2
T c
b
S 8
Model 3

Figure 1.4. Assumptions associated with models 1, 2, and 3.

15

attributed to increased inhibition as the S:X ratio increased. In other words, the biomass was
dying off faster than the compound was transformed. If Tc was greater than the S°:X° ratio,

the test was classified as a success. If Tc was less than the S°:XO ratio, the test failed.

CHAPTER 2

MATERIALS AND METHODS

Mixed culture development.

Mixed culture MM], a methanotrophic enrichment obtained from aquifer material at the
Moffett Field test site, was used for all experiments in this study (courtesy of S. Henry,
Stanford University). This culture was grown in Whittenbury Mineral media (Appendix A). A
l-liter culture was grown at room temperature in a continuously stirred 4»liter bottle with
approximately 30% methane in air supplied at 68 mL/min. Growth curves were monitored
and as stationary phase approached, approximately 10 mL of culture was transferred to 1 liter
of Whittenbury Media and incubated as previously described. Cells were harvested in mid-log
growth for all testing.

In order to ensure that mid-log growth stage was the optimal stage of growth to utilize,
preliminary work was done involving methane utilization rates for different stages of the
culture's growth. The materials and methods for this procedure is described in Appendix B,

with a summary of the results shown in Table 2.1.

 

 

 

Table 2.1. Methane Utilization Results
Elapsed Dry Wt.
Time Conc. dM/dt/VL
(h r) (mg/ L) (mg/L-day)
24.5 40 -1 15.4
43 140 -1 1 7
48.5 240 -349.2
73.5 490 -574.6
90 760 -97S.3
96 820 -810.6
145.5 1590 -308.67

 

 

 

 

 

16

17

From the data, it is evident that as the culture approaches mid-log phase (dry weight
between 500-800 mg/L), the specific rates of methane utilization (dM/dt/VL) increased
indicating greater activity within the culture. Based on these results, additional experiments
were conducted using cells with high activity (in mid-log growth phase) for the TCE

degradation studues.

TCE solutions and analysis.

Water saturated with TCE was used as the spike solution in all experiments. The TCE
saturated solution was prepared by adding 3 mL pure TCE (99%+ Aldrich Chemical Company,
Milwaukee, MI.) to a 120-mL vial with Teflon®-lined rubber septum containing 117 mL
water. The vial was vigorously shaken and stored in a refrigerator until needed. One hour
before use, it was shaken again and allowed to settle. A Hamilton microliter syringe was
injected through the Teflon® septum and the appropriate amount of TCE saturated water was
withdrawn and injected directly into batch bottles containing the culture.

TCE concentrations were determined using headspace analysis. Headspace samples of
0.2 mL were removed from the test bottles with a Precision gas-tight syringe and analyzed on a
Perkin Elmer 8500 Gas Chromatograph (GC). For low TCE concentrations (less than 0.5
mg/l), samples were analyzed on a GC equiped with an electron capture detector and a stainless
steel packed column with nitrogen as the carrier. For high TCE concentrations (1-10 mg/L),
samples were analyzed on a GC equipped with a flame ionization detector and a squalane
packed column with helium as the carrier. The oven-temperature program was set at an
isothermal temperature of 90°C for both the FID and ECD.

Standards for calibration were prepared by adding approximately 25 mg of TCE to 25
mL of methanol for a concentration of 1 mg TCFJmL MeOH. The vial was sealed with a

Teflon®-lined rubber septum and aluminum crimp-top cap. From this stock solution, known

18

amounts of TCE were transferred to bottles containing mineral media, shaken vigorously on a
rotary shaker and sampled three minutes later. A calibration curve was produced for each data
set with at least five concentration levels bracketing the concentrations expected in the samples.
The TCE concentration in the samples was obtained using the calibration curve, a
dimensionless Henry's constant of 0.33 for a temperature of 21°C (Gossett, 1987), and known

liquid and gas volumes. Controls without cells were spiked and analyzed in all experiments.

TCE Transformation Studies

TCE transformation studies were performed using 250-mL glass bottles sealed with
Teflon Mininert® valves. These bottles were inoculated with 100 mL of a mixture of mineral
media and culture. The mass of culture added varied from 10 mg to 16 mg per bottle
depending on the desired rate .of transformation. TCE saturated water was injected through the
Mininert valves into the bottles, shaken vigorously for 20 seconds and placed upside-down on
a rotary shaker (250 rpm). The bottles were shaken at 100 rpm and 350 rpm with no increase
in TCE utilization, indicating that the system was not mass transfer limited. Doubling the
biomass concentration resulted in a doubling of the transformation rates, also indicating that the
system was not mass transfer limited under the experimental conditions used. After TCE
addition, 0.2 mL gas samples were withdrawn periodically using a l-mL Precision gas tight

syringe and injected on to a GC for analysis.

Culture Densig

Cell biomass was determined on a dry weight basis using 0.2 pm filters (Gelman
Sciences Inc., Ann Arbor, MI.). The filters were prepared by first soaking them in mineral
media for 10 minutes, rinsing on a vacuum filter with deionized water, drying overnight in 3

103°C oven, and cooling in a desiccator until needed. The filters were weighed, and once a

19

known amount of culture was filtered through them, they were rinsed, dried, cooled and
reweighed. Optical density was also determined by measuring culture transmittance at 540 nm

on a Shimadzu UV-l60 spectrophotometer.

CHAPTER 3

EFFECTS OF TCE CONCENTRATION ON
TRANSFORMATION CAPACITY

Introduction

As discussed in Chapter 1, it is now clear that the both cell-free mmo enzyme and the
whole-cell suspensions of methanotrophs can oxidize many different compounds, including
trichloroethylene. From an engineering prospective, the key to the degradation of these
compounds is manipulating the environmental conditions of the culture to maximize the rate
and capacity of degradation. This chapter focuses on the effects of TCE concentration on
transformation capacity.

According to Alvarez-Cohen and McCarty (1991a), transformation capacity (T c) can be
obtained experimentally by determining the maximum amount of TCE degraded by a specific
amount of biomass. In this thesis, this method is referred to as the 'measured' method of
obtaining Tc. This method can only be applied to data sets in which the TCE concentration
does not go to zero because the microorganisms may not have reached their capacity to degrade
TCE. A respike may need to be performed until the degradation levels off at a point which is

IIOt ZCI'O.

Materials and Methods

All materials and methods used for this section are described in Chapter 2. For the
batch studies, 30 mL of cells were harvested, giving a mass of cells per bottle of 15 - 17 mg,
and placed in 250-mL bottles containing 70 mL mineral media.

20

21

Beanie

The models of Table 1.1 were applied to triplicate samples run at five different
concentrations: 0.016 - 0.018 mg/L, 0.22 - 0.26 mg/L, .91 - .97 mg/L, 5.5 - 5.7 mg/L, and 10

- 12.5 mg/L A graph of this data is shown in Figures 3.1 through 3.3.
12

 

   

-—-EI-— TCE = 5.5-5.7 mg/L

-+—- TCE = 10.2-12.5 mg/L
10 a

TC! conc.(ngtl.)

 

 

o ' l I I 1

 

0.0 0.1 0.2 0.3 0.4
Days

Figure 3.1. TCE degradation at high initial
substrate concentrations.

Figure 3.1 shows data from two of the higher initial substrate concentration ranges.
The data points are an average of triplicate samples with the error bars representing the
respective standard deviation. Figure 3.2 shows data in the middle range of initial substrate
concentrations, and Figure 3.3 shows data from the low initial substrate concentration. In

addition, a respike experiment was done at the low TCE concentration to determine

22

transformation capacity. The first six hours of this experiment are shown in Figure 3.4.

Cumulative TCE degraded over the five days for the same set of data is shown in Fr gure 3.5.

 

1.0 _

- —ra— TCE=.91-.97 mo/L
1 —0— TCE= .224-.25 mg/L

 

 

 

 

 

. , .
0.00 0.01 0.02 0.03 0.04
Days

Figure 3.2. TCE degradation at medium initial substrate concentrations.

 

0.02

—El— TCE = .016-.018 mg/L

TCE seminal.)

 

 

 

 

0.00 WT T v I v I u
0.00 0.01 0.02 0.03 0.04
Days

Figure 3.3. TCE degradation at low initial substrate concentrations.

TCE Cornell-git)

23

0.2 1-
I
0.18 --
0.16 'l'
0.14 -' '
0.12 J. 1 \
0.1 ‘| .\ .
0.08 - . 1
0.06 --- \_ \
0.04 -~ \_
\ '1 '
0.02 .. .- -,,r g, \_ ‘..
0 J—LII.-__';_r__'i'uu I'I' "-.I.--.-+-__'.!I_.i
0 0.1 0.2 0.3 0.4

I”.

 

l
. l
\ \.\_

 

 

 

 

 

Figure 3.4. Continuous respike experiment for low levels of TCE
(experiment continued for five days and required 22 spikes:
only the first six spikes are shown here.)

 

 

3 0.6 ""

9

z 0.5 q- /./._—————""I

I ./-

8 0.4 -- l/

3 ... r’

I- E 0.3 .. I!

5’- ‘” .I"

or- _ I

g 0.2 ' .4-

E 0.1 T:

a 3-

° 0 9' : . i . : .
0 1 2 3 4 5 6

Days

Figure 3.5 Cumulative TCE degradation

24

Figure 3.5 shows the degradation of TCE to be leveling off after about three days. The
cumulative TCE degraded after five days was 0.53 mg. Dividing this mass by the cell mass of
15 mg gives an apparent transformation capacity of 0.036 mg TCE/mg cells.

These data can be compared with To obtained for the high concentration data set. For
the high concentration data, the total TCE degraded was 0.89 mg TCE. Dividing this mass by
the amount of cells in the bottle (16.2 mg) gives a Tc of 0.055 mg TCE/mg cells.

Finally, Tables 3.1 through 3.3 provide a summary of all the current models used to
describe cometabolism for a wide range of TCE concentrations (covering three orders of
magnitude). Nonlinear regression analysis was performed using Systat 5.1 application
software (Systat, Inc.) Table 3.1 gives best fit parameter estimates for model 1 at five different
initial concentrations. A key assumption for use of model 1 is that the decay term b is constant
and therefore independent of substrate concentration. Accordingly, b was estimated from the
high concentration data set and model 1c and used as a constant in evaluating the mid—range and
low concentration data sets with models 1a and lb. Model 1 also assumes that the maximum
rate of substrate utilization, k, is independent of substrate concentration. Consequently, the
value for k obtained using the high concentration data set was used to evaluate the mid-range
data set in estimating Ks.

Table 3.2 gives best fit parameter estimates for model 2 at all concentrations. A key
assumption for model 2 is that the transformation capacity and the maximum rate of substrate
utilization remain constant and are independent of substrate concentration. Consequently, Tc
and k were estimated from the high concentration data and used as a constant in evaluating
model parameters at lower concentrations.

Table 3.3 gives the best fit parameter estimates for model 3 with varying initial
concentrations. For comparison, the 'measured' values of Tc are also provided. For low
concentrations, the measured transformation capacity could only be determined by continuous
long-term respiking. In addition, model 3 was used to estimate the initial TCE concentration,

80, and consistently underestimated the transformation capacity by choosing a lower S0 and

25

 

 

 

Model 1 - Fitting
Parameters
Datasetand
Model used k' k Ks b R42
Model 1b
S°-.015-.017 mg/L 1.4:.23 N/A N/A 9.03 (a) 9981.001
Model la
S°=.22-.25 mg/L N/A 0.422 (b) .257i.013 9.03 (a) .98zt.007
Model 1a
S°=.91-.97 mg/L N/A 0.422 (b) .184zt.034 9.03 (a) 981.002
Model 1c
S°=5.5-5.7 mg/L N/A 0.38:.01 N/A 9.03 (a) .97:I:.005
Model 1c
S°-10.Z-12.5 mg/L N/A 04221.06 N/A 9.031.63 .94:I:.01

 

 

 

 

 

 

(a)- used average b from high concentration data to predict other parameters
(b)-used average K from high concentration data to predict other parameters

N/A- model did not estimate these parameters

Table 3.1 Evaluation of model 1 at five different TCE concentrations.

 

26

 

 

 

Model 2 - Fitting
Parameters
Datasetand
Model used k' k Ks Tc RAZ
Model 26
S°-.015-.017 mg/L 1.331.24 N/A N/A .047 (a) 9971.001
Model 2a
S°=.22-.25 mg/L N/A 0.422 (b) 0.29:.02 .047 (a) .97i.008
Model 2a
5°=1.1-1.2 mg/L N/A 0.422 (b) 0.21:.04 .047 (a) .98zt.001
Model 2c
S°=5.5-5.7 mg/L N/A 04061.03 N/A .047 (a) .97i.006
Model 2c
S°-10.2-12.5 mg/L N/A 0.422:l:.06 N/A 04710.0 .94:I:.01

 

 

 

 

 

 

(a)- used average Tc from high concentration data to predict other parameters
(b)- used average b from high concentration data to predict other parameters

N/A- model did not estimate these parameters

Table 3.2 Evaluation of model 2 at five different TCE concentrations.

 

27

 

 

 

 

 

Model 3 — Fitting
Parameters
Measured
Dataset b Tc R42 Tc
S°=.015-.017 mg/L 0.083101 0.047(a) 0.731.046 N/M
S°-.22-.25 mg/L 1.21.15 0.047(a) 0.631.016 N/M
5°=.91-.97 mg/L 5.1141637 0.047(a) 0.911.018 N/M
S°=5.5-5.7 mg/L 8.691.64 0.047(a) 0.971.006 0.0431003
S°-10.2-12.5 mg/L 9.0311635 0.047100 0.944101 0.055100
Respike data

S°-.12-.18 mg/L N/D N/D N/D 0.035
S°- .1S-.23 mg/L 0.034

 

 

 

N/Ms not measured
N/D- not determined

Table 3.3 Evaluation of model 3 and 'measured' Tc with five TCE concentrations.

 

28

leveling off higher than the data as shown in Figure 3.6

‘ --. model 3 best fit

Has: TCE (mg)
0 o
in on

.o .o
N -hu
1 i

 

 

0 0.2 0.4 0. 6
Days
Figure 3.6 Typical degradation curve for hi gh-range TCE concentration. The

measures mass of TCE degraded is compared with the fit for model 3 (note that
model 3 is the same as models 1c and 2c.)

It should be noted that due to the inaccuracy of a time zero measurement due to
nonequilibiium between the headspace and liquid phase of a volatile compound at the initial
injection of TCE, computing the 'mcasured' transformation capacity using the initial data point
for each set of data would be inaccurate. Instead, the initial data point was obtained by
averaging the controls for the experiment, which were injected with the same initial amount of

TCE, and subtracting the remaining TCE once the degradation leveled off.

Discussion

There are some conceptual differences in the models of Table 1 which should help in

their selection. In model 1, the loss of activity is not directly linked to transformation,

whereas models 2 and 3 assume a direct linkage. In model 3, transformation rate is

29

independent of substrate concentration and depends only upon the rate of decay of
transformation activity. This decay term and k were estimated at high TCE concentration and
used at lower concentrations to predict the necessary parameters. These parameters were then
used to compare the consistency of the model at all concentrations. From Table 3.1, dividing
the k by the K3 predicted from model 1a at the medium TCE concentration of 0.91-0.97 mg/L,
and using propagation of errors (Appendix D), gives a k' of 2.3 1.097 L/mg-day. This value
is almost twice as large as the value predicted by model lb at the low TCE concentration of 1.4
Umg-day. However, using the same procedure at the TCE concentration range of 0.22-0.24

mg/L gives a k‘ of 1.631. 15. This reveals some inconsistencies in model 1 over the range of

data tested. As for transformation capacity, model 1 predicts increasing Tc with increasing
TCE concentration. Since the apparent Tc at low concentrations was fairly close to Tc at high
concentrations (shown using the respike data), model 1 does not appear to be appropriate for
this data set.

In model 2, To is assumed to be independent of TCE concentration. For the evaluation
of model 2, Tc and k were estimated at the high TCE concentration and used in the models for

lower TCE concentration to predict the necessary parameters. From Table 3.2, dividing k by
the Ks as predicted with model 2a, and using propagation of errors, gives a k' of 2.05 1096

Umg-day (at the TCE concentration of 0.91-0.97 mg/L) which is again much larger than the
1.33 Umg-day value predicted by model lb at the lower concentration. However, the k' for
the TCE concentration of 0.22-0.24 mg/L was 1.461. 12. Again this indicates problems with
the consistency of model 2 over the concentration range tested.

The assumption behind model 3 is that both Tc and b are independent of TCE
concentration. Using model 3, Tc was held constant and the decay term allowed to vary.
From Table 3.3, the assumption that the decay term is constant does not seem to hold true for

the data set. The term b varies from 9.031 d‘1 at the high TCE concentration to .083 d‘1 at the

low TCE concentration with poor correlation coefficients at the lower end.

30

To evaluate the uniqueness of these non-linear parameter estimates and how important
each one is over the concentration range tested, a sensitivity analysis was performed (Beck,
1977). The detailed analysis is performed in Appendix D, but in summary, most of the models
gave unique parameters except for the low concentration data using model 3, and the low TCE
concentration range of 0.22-0.24 mg/L using models 1a and 23. This suggests that models 1a
and 2a should report k' (k/Ks) rather than k and Ks at the concentration in question, and for
model 3, another approach to modeling should be used at the lower concentration range,
possibly the one used by Saez and Rittmann (1991) discussed below. However, since models
1 and 2 converge to model 3 at high TCE concentrations, and model 3 appears to be valid in
this range, it will be used to model data obtained at TCE concentrations at or above 2 mg/L.

There are certain limitations of the models which should be considered. For model 2,
the solution is discontinuous when F = 0. Real solutions can only be obtained when F +
S/Tch > 0 which simplifies to XoTc > (So-S), indicating that the extent of transformation can
not exceed the transfonnation capacity (Alvarez-Cohen, 1991a).

It should be noted that Saez and Rittmann (1991) used a different approach for
parameter estimation with model 3. In this model, biomass is assumed to decay in a first order

manner given by:
x = x e'“ (3.1)

Saez and Rittmann (1991) linearized equation 3.1 and used biomass measurements to

determine X0 and b. From there, equation 3.2 was linearized using the estimated values for

X0 and b to determine Tc.

5 = s, — Texan - e'“) (3. 2)

31

For this project, the time rate of change in biomass was not obtained, so data involving
substrate concentration vs. time was fit to equation 6 using non-linear parameter estimation to

solve for To and b.

CHAPTER 4

EFFECTS OF pH AND FORMATE ADDITION

Introduction

Several investigators have shown that environmental conditions play a key role in the
degradation of TCE (Henry, 1991, Alvarez-Cohen, 1991b). These environmental factors
enhance or reduce transformation rates or capacity. This chapter explores two environmental
factors: the addition of f ormate, which provides additional reducing power for mmo oxidation
of TCE, and pH effects. These factors may be helpful in the optimal operation and design of
engineered systems and in the comparison of methanotroph enrichments from different
sources.

Initially, the effects of formate addition at low TCE concentrations were investigated.

Subsequently, high TCE concentrations were investigated at various pH values.

Materials and Methods

Culture preparation was as described in Chapter 2. For the formate experiments at
neutral pH, 10 to 15 mL of mid-log phase cells were harvested and placed in a flask. Two
different levels of sodium fonnate (Aldrich Chemical Co., Deerfield, 111.), 2 mM and 20 mM,
were added by weighing out the necessary amount of f ormate, adding it to the cell suspension,
and diluting up to 100 mL with mineral media. This solution was then transfered into a 250
mL bottle and sealed with a Mininert®valve. Depending on the experiment, various amounts
of TCE were added from the TCE saturated stock solution to the bottles. The bottles were I
hen shaken vigorously for 15 seconds, sampled, placed on a rotary shaker, and periodically

sampled thereafter.
3 2

33

Forthe pH experiments, the culture was grown at a neutral pH of 6.8, harvested and
incubated in 250—mL bottles buffered at a desired pH. The liquid volume in the 250-mL bottles
contained 50 mL of a universal buffer (Perin and Dempsey, 1979) with the remaining 50 mL
consisting of a mixture of 42 mL of mineral media and 12 mL of culture to obtain final pH

values between 5.5 and 10.

Results

Formate effects at low TCE concentrations were examined initially. Two different
levels of formate were used: 2 and 20 mM. Samples without formate were also tested. The
initial aqueous phase TCE concentration was approximately 0.19 mg/L. At these TCE
concentrations, fonnate had no effect on the degradation rates. (Figure 4.1).

The next logical step was to determine whether fonnate addition had any effect at high
TCE concentrations. For experiments run at high TCE concentrations (4.5 mg/L), 20 mM
f ormate was used to ensure that transformation would be visible if fonnate made a difference.

With the addition of 20 mM formate, k increased from 0.145 to 2.4 mg TCE/mg
biomass-day. In addition, the transformation capacity increased from 0.043 mg TCE/mg cells
to 0.082 mg TCE/mg cells. This data was fit using model 3 and the results are shown in
Figure 4.2. It should be noted that because the TCE concentration decreased to zero (and no
additional TCE was added), the transformation capacity is underestimated.

Degradation parameters for pH of 5.5, 6.8, 8.3, and 10 were determined.
Transformation rates decreased in the more basic region (near pH = 10). The acidic region of
pH = 5.5 had slightly slower rates, and the neutral range between pH = 6.8 to 8.5 seemed to
contain the fastest rates as shown in Figure 4.3. Table 4.1 provides a summary of the
parameters obtained from this data when evaluated with model 1b. Due to the difficulty of
obtaining consistent initial TCE concentrations at this level of TCE, data from triplicate samples

were averaged.

0.2
0.18
0.16
0.14
0.12

0.1
0.08
0.06

TCE Dancing”)

0.04

0.02

 

34

 

—D— No Formate

+ 2mM Formate

—"— 20mM Formate

 

 

 

 

 

0.005 0.01 0.015 0.02 0.025 0.03
Time (days)

Figure 4.1. Effects of f orrnate addition on TCE Degradation at low TCE concentrations

TCE Cowling!"

(initial TCE concentration 2 0.18 mg/L)

 

 

 

 

 

5 T
4 5 :-
‘1.
4
3 5 }.\_\
' '\. Tc .. .043, b = 3.384
3 » \
2.5 ._ !\ No formate
2 -- fi'fi—
1.5 --l “2.4 k-.14S
1 .. " Tc = .082, b = 29.371
0-5 i °. 20 mM formate
o 4 ° °=4¢ c : : 4 : a
0 0.2 0.4 0.6 0.8 1 1.2

Days

Figure 4.2. Effect of fonnate on TCE degradation at a high TCE concentrations.

The Tc reported was obtained by parameter estimation.

35

0.18
0.16
0.14

 

 

 

A
AT, 0.12
E
V 0.1
2'
is 0.08
1'3 0.06
i- \ \ \

0.04 -- B~\‘.‘ 0“

fig ‘0“ pH = 5.5
0.02 "- \i‘ég ~.o__
o :fixn.‘ - pt. 3 6.8 & 8.3
0 0.05 0.1 0.15
Days

Figure 4.3. Effects of pH on TCE transformation at low TCE concentrations.
(initial TCE concentration = 0.12-0.17 mg/L)

 

 

 

 

Table 4.1
Summary of pH Effects at Low TCE Concentration
Start pH End pH R2 k' b
5.6 5.5 1 1.061 20.47
6.82 6.78 0.995 1.225 9.682
832 8.2 1 1.357 8.605
9.98 9.86 0.999 0.698 35.64

 

 

 

 

 

 

The effects of pH were also evaluated at a higher TCE concentration of 5.0 - 5.5 mg/L.
These results were modeled with model 3 and are shown in Figure 4.4 with a summary of the

kinetic parameters listed in Table 4.2. Incubation pH had a dramatic effect on transformation

TCE conc.( lull)

 

36

 

 

T
3:
5 is?“ 9 e
s‘,‘ ‘~'.:;j_'_;:'""°' -------------- ' --------- . pH---- 10
4 4" ‘0 ~-—_~.~~ —————— ~I _____ . pH 55
4 =
3 “- \*§g‘\l
s“.. ‘ g
~._.~-_ ‘ pH 8.5
2 1- -~-~'“'-.""~----
O pH=6.8
0 0.2 0.4 0.6 0.8 1 1.2 1.4
days

Figure 4.4. Effects of pH on TCE transformation at high TCE concentrations.

(initial TCE concentration = 4.5 - 5.5 mg/L)

Table 4.2

 

Summary of pH Effects at High TCE concentrations

 

 

 

 

 

 

 

 

(Triplicate Samples)

PH (begin) P140114) R2 TC b k
5.47 5.41 0.86 0.0197 3.385 0.067
5.48 5.5 0.869 0.0212 2.969 0.063
5.48 5.41 0.917 0.0212 4.304 0.0912
6.86 6.86 0.976 0.0515 2.943 0.151
6.95 6.92 0.97 0.053 2.772 0. 147
6.98 6.91 0.969 0.053 2.415 0.128
8.45 8.53 0.963 0.038 7.286 0.277

8.4 8.45 0.96 0.039 8.825 0.309
8.42 8.44 0.966 0.036 9.053 0.326
10.11 10.19 0.914 0.017 30.827 0.524
10.05 10.14 0.918 0.017 40.504 0.688
10.02 10.1 0.905 0.015 47371 0.71

 

 

37

capacity. The effects of incubation pH were further evaluated by providing 20 mM fonnate
with a high levels of TCE (5 mg/L). The results for pH 5.5, 6.8 and 10 are shown in Figures
4.5, 4.6, and 4.7 respectively. Again, all data were evaluated with model 3. From Figure 5,
at pH = 5, the maximum specific rate of substrate utilization without formate was .0896 mg
TCE/mg cells-day. While formate was provided this value increased to 0.392 mg TCE/mg
cells-day. The transformation capacity almost tripled from 0.019 mg TCE/mg cells to 0.049
mg TCE/mg cells.

In one sample, illustrated in Figure 4.6, TCE was respiked at neutral pH in order to
provide a better estimate for Tc. Formate addition increased transformation capacity from .034
mg TCE/mg cells to .139 mg TCE/mg cells. In the more basic region (pH = 10) fonnate

addition did not increase TCE transformation rates or capacity (Figure 4.7).

 

 

4.5 q
4 1., .
D! 3., To = .0.019 mg TCE/mg cels
3.5 -- \ ""'--.,.___‘._ noformate
:- 3 n'\ """"" ' ------------------- -I--------.----
3 i" ”x, k= .0896
,5, 2 5 .. l3\
2 i- ‘0‘0 D
8 Z ‘ “ ....... D. ........... intimate.
3 1 5 -- D D
" 1 .. Tc = .0049 mg TCE/mg cells
k- .392
0.5 «-
0 : : J. : : : :
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Days

Figure 4.5. Formate effects at pH = 5.

TCE cone. (mall)

TCE tie-clung")

I" I"
-IU'IN01

.0
001

s» .4-
(11450101

I"
vice

._s
dU'IN

.0
OUT

 

  
   

 

'measured' Tc =- .139 mg TCE/mg cells

38

Tc - .034 mg TCE/mg cells
no formate

--
----§-‘-‘-~
--

 

 

 

 

 

 

20 mM fonnate
0.4 0.6 0.8 1 1.2
Days
Figure 4.6. Formate effects at pH = 6.8.
Tc = .015
, 20 mM formate
9‘ Q «a -------- .3 --------------- ecu-3n
.. No fonnate
.. Tc a .014
..
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Days

Figure 4.7. Formate effects at pH = 10.

39

W

The results indicate that formate addition is helpful only under certain conditions. At
low TCE concentrations, there appears to be enough reducing power available to degrade TCE.
However, if respike experiments were performed at these low TCE levels with formate, the
transformation capacity would probably increase. Additional TCE would require additional
reducing power, which the formate could provide.

Formate addition at high TCE concentrations is definitely advantageous in the neutral
pH range and also in the more acidic region of pH = 5.5 , but it does not enhance degradation
at the more basic region of pH = 10.

Table 2 indicates that there is an increasing k with increasing pH. This could indicate
an enzyme which increases activity at high pH but also looses activity at an accelerated rate at
higher pH.

Based on the high TCE concentration data, transformation capacity appears to be a
more accurate measure of capabilities of a culture than either the decay coefficient b or the
maximum rate of substrate utilization k. At pH = 10, the k value is very high, but if the decay

coefficient is also large, the overall efficiency of Tc is low.

CHAPTER 5

EFFECTS OF ENDOGENOUS DECAY

Introduction

As mentioned in Chapter 3, it has been suggested that current models to describing
cometabolism of TCE should possibly by modified to distinguish between the loss in
transformation activity caused by TCE toxicity and the loss cause by endogenous decay. In
order to do this, however, it is necessary to understand the mechanism or mechanisms behind
the loss of activity.

To facilitate part of this objective, an experiment was designed to study endogenous
decay to see how this parameter would effect the transformation capacity of a culture over a

certain period of time.
Materials and Methods

The culture was prepared as described in Chapter 2. For these studies, 30 mL of
culture containing 15 mg cells and 70 mL of mineral media was transferred to five 250-mL
bottles. Transformation capacity at time zero was measured by spiking one of the five bottles
with 5.0 mg/L TCE and monitoring the disappearance over time. In order to measure the
decline in transformation capacity over a 4 day period, the remaining bottles were incubated
without TCE addition for 1, 2, 3, or 4 days prior to the measurement of transformation
capacity. Once spiked with TCE, each bottle was measured for a certain period of time until

the degradation ceased and the transformation capacity leveled off.

40

Results

41

The results from each day is shown in Figures 5.1 through 5.5. All data was evaluated

using model 3 unless otherwise stated. As shown, transformation capacity decreased from .04

mg TCFng cells in day 1 to 0.0081 mg TCE-7mg cells in day 5.

TCE Conc.(mgIL)

TCE Conc.(lnglL)

 

 

 

5 I‘
It'-
4 Ilia}.
'--,.. Tc =0.041
3 \ I
{ b = 2.31
2 \\
a 4 - g

l ‘L‘i
0 . 1 : :

0 0.5 1 1.5

Days

Figure 5.1. Aeration experiment for day l.
5 -r
4 '1; Tc = 0.027

' "-_ b = 3.08
3 .- '-\.

\

2 -- \. -"""—-—a
1 .-
0 : 1 1

0 1 2 3

Days

Figure 5.2. Aeration experiment from day 2.

TCE Colic. (mail)

TCE Conc.( llltjll.)

42

 

 

5
T
4 1'"; Tc = 0.02
"‘k .__ b = 1 84
3 -- 1 _____ .___ -
I ~- ——— I-
2 ..
1 ..
0 1
0 l 2

Days

Figure 5.3. Aeration experiment for day 3.

 

 

5 .._
\l.. -
\
4db “'~‘LI ————— '
3" Tc=0.014
21- b.- 1.18
1..
0 : .
0 1 2
Days

Figure 5.4. Aeration experiment for day 4.

43

 

 

 

5
A I ."""\. .
Fl. 4 " I—IN .
5 3 -- model does notapply
E J, Measured Tc = .0081
. 2
1.1
8 1 --
.-
0 1 1 1 .1
0 0.5 1 1.5 2
Days

Figure 5.5. Aeration experiment for day 5.

As shown in Figure 5.5, model 3 was unable to predict a transformation capacity for
the data because there was very little degradation, so the 'measured' transformation capacity

was used for comparison.
When the individual transformation capacities for each day of aeration are plotted
against time, there is an obvious trend which was modeled with equation 5.1 listed below.

Tc - 13(max) 6‘” (5.1)

Figure 5.6 shows a graph of the data and the modeled equation. Using equation 5.1,
the data gives a decay term of 0.381 day‘1 and a TCO of 0.04 mg TCE/mg cells. It is necessary
to keep in mind that the transformation capacity values reported for each day are a result of two
separate processes: endogenous decay and, due to the high TCE concentration, toxicity. In
order to compare this with low concentration data, an assumption was made that endogenous

decay (aeration only) does not effect susceptibility to toxicity. In other words, the shape of the

44

 

 

0.045
A 0.04 :.\
0 \
fg' 0.035 1- \\
. ons« ‘\\1
5 0.025 .. \\\ b =O.381
U \\‘
g 0.02 '4' \{‘\
2 0.015 .. “‘\_|‘\
V 0.01 "' ~\—~~
0 I
'- o.oos ..
o : : : a
0 1 2 3 4
Days

Figure 5.6. Transformation capacity lost over time due to aeration.

curve would remain the same whether at high or low TCE concentration, hence all the

parameters estimated from this curve would be the same.

Discussion

If equation 5.1 is applied to the low concentration respike data shown in Figure 3.5, it
is possible to calculate a TCO that takes into account endogenous decay. By using the above
decay value for this culture of 0.381 day'l, and using the individual spiked quantities of TCE
at their respective times, equation 5.1 gives a TCO of 0.067 mg TCE/mg cells. Comparing this
to the measured Tc of 0.035 mg TCE/mg cells (Chapter 3) which does not account for
endogenous decay, it is evident that the Tc is drastically reduced by endogenous decay at low
TCE concentrations (Figure 5.7).

Based on the above findings, it is likely that Tc is a function of the substrate

concentration S, and that it's value increases at low S, if endogenous decay can be accounted

 

45

for (Figure 5.8).

0.07 -

Tc - 003/
./

0.06 -
0.05 -
0.04 d
0.03 d
a 0.02 ~

I

'\

I
I

 
    

To -= 0.035

Cummultivc Tc
TCEIIng cells)
I
\

(In

0.01 -
o d. :

 

cil-
d-

 

Days

Figure 5.7. Transformation capacity for the five day respike experiment (Chapter 3)
including endogenous decay effects (Tc = 0.035) compared to transformation capacity

corrected for endogenous decay effects of 0.381 day’1 ("PC = 0.067).

 

Tc

 

 

3

Figure 5.8. Proposed explanation of Tc vs. S based on above findings.
(drawing of 'measured' Tc data for various TCE concentrations)

46

This finding indicates that the assumption behind model 2 that To does not change with
time may appear to be valid only because endogenous decay plays such a large role at low TCE
concentration. At low TCE concentrations, rates of TCE transformation are low, permitting
more extensive endogenous decay during the transformation.

As indicated by Figure 5.8, the transformation capacity at low substrate concentration is
quite large. This raises the question that if endogenous decay could be overcome, is there
some threshold concentration of TCE below which damage to the cells does not occur? The
idea of a threshold has long been debated by toxicologists but could be one possible approach
to low level TCE modeling.

It is obvious that the loss of transformation activity of the cells with time is a key issue.
Specific questions need to be answered as to how this activity is lost. Figure 5.9 gives
suggestions as to how this may happen. Fundamental research and models are needed that will
differentiate between all these potential activity sinks.

The models currently in use assume that the decay term indicates dead cells. This
particular issue is quite important because if the cells are dead, it requires a certain amount of
time to regrow them. However, if the cells are not dead, but exhausted and out of reducing

power, then the time needed to rejuvenate them would be less than it takes to regrow them.

 

47

TCE oxidation depletes Death rate - due to
reducing power TCE toxicity

 
 
 
 

      

I’ll
\\\\\\\
IIIIIII’I

Endogenous decay

 

Figure 5.9. Pathways for potential loss of transformation activity.

CHAPTER 6

ENGINEERING APPLICATION

As discussed in Chapter 1, some researchers found formate addition to be beneficial to
TCE transformation. In studies conducted, f orrnate addition at low TCE concentration had no
effect on degradation rates, but at high TCE concentration, the addition of formate provided
readily available reducing power to enhance degradation.

I-Iigh concentrations of TCE depleted the reducing power of cells as evidenced by the
effect of fonnate addition. To prevent this, it is necessary to reduce the amount of TCE
transformed per cell per unit time. This could be accomplished by reducing the amount of TCE
that individual cells were exposed to. One way of doing this would be to increase cell density.
It has been shown that increasing the cell density increased TCE transformation capacity of a
mixed culture (Alvarez-Cohen, 1991b). Reducing liquid TCE concentrations could be
accomplished by diluting process wastes with larger volume reactors or by removing TCE
using absorptive materials. The addition of activated carbon reduced the concentration in
solution and increased transformation capacity on a suspended culture of M. trichosporium
OBBb (Oldenhuis et al., 1991).

The pH of a system has also been shown to effect the transformation of TCE. A shif t
from pH 7 to 7.5 in a continuous recycled expanded bed reactor decreased TCE transformation
by 85% (Phelps, et al.,1990). The pH also effects mineral nutrient availability (Stumm and
Morgan, 1981) and the formation of transformation intermediates which may have some
influence on TCE oxidation toxicity. The TCE epoxide rearranges in water to form formate,
carbon monoxide, dichloroacetic acid and glyoxylic acid (Henschler et al., 1979). The ratio of
products formed depends upon the pH of the system, with CO and fonnate predominating at
neutral regions and becoming more dominant at basic regions (Henschler et al., 1979). It is

interesting to note that CO has been shown to inhibit TCE degradation (Henry, 1991b). This
48

49

could explain the considerably reduced transformation capacity of a culture at high pH as
opposed to neutral pH. In addition, some enzymes can be denatured at this high pH (pH =
10).

In order to provide optimal pH for a treatment system, the pH in the transformation
reactor should be maintained in the neutral range of pH 6.8 to pH 8.5 for mixed culture MMI.
Additional studies are needed to deterrrrine whether this conclusion can be generalized to other
methanotroph cultures. If a waste tends to vary in pH, as is typical for industrial wastes, a pH
control system would appear to be warranted.

Alvarez-Cohen showed that transformation capacity does not change with TCE
concentration (model 2a). Chapter 5 indicates that this may appear to be true only because at
low TCE concentration, the transformation rates are slow and endogenous decay reduces the
transformation capacity of a culture. If it is true that To increases at low TCE concentration,
then an operating system that exposed cells to lower TCE concentrations, such as a completely
mixed reactor, may be most effective if the negative effects of endogenous decay can be
reduced. Endogenous decay might be decreased by periodically feeding the cells methane,
reducing the mean cells residence time, or alternating the level of oxygen exposure.

If the problem associated with differentiating cells that are dead and cells that are out of
reducing power could be solved, this would have a major impact on processes that involve
sequencing reactors. The question of how long to recharge cells to restore transformation
capacity would is dependent on whether or not the cells are dead or exhausted.

Environmental conditions clearly play a role in the efficiency of methanotrophic
degradation of TCE Reducing power availability , mineral nutrient availability as a function of
pH, endogenous decay and TCE concentration all play a role. All these factors must be taken
in to account during the design and implementation of treatment systems. By optimizing these
factors, the use of methanotrophs for the treatment of TCE and other chlorinated solvents

would appear to have a promising future.

 

 

CHAPTER 7

CONCLUSIONS

Based on research described in this thesis, the following conclusions can be made for

mixed culture MMl:

Formate addition increases transformation capacity at higher TCE concentrations (4.5-5
mg/L). At low TCE concentrations (224—.25 mg/I.) , formate addition had no apparent
effect on the TCE degradation rate.

Basic and acidic incubations reduce transformation capacity of a culture.

The transformation capacity of a decaying culture decreases with time at a rate of 0.38

d-l.

The transformation capacity of a culture at low TCE concentrations is artificially low

because TCE transformation rates are slow compared to endogenous decay.

The assumptions underlying models 2 and 3 that transformation capacity does not
change with TCE concentration appears to be incorrect. Model 2 might appear valid
only because endogenous decay reduces transformation capacity, and the decrease is

most significant at low TCE concentrations when TCE degradation rates are slowest.

Model 1 does not describe TCE transformation for the full range of TCE

concentrations. Transformation eapacity appears to increase rather than decrease with

50

 

51

decreasing substrate concentration if the transformation capacity is corrected for

endogenous decay.

FUTURE WORK

Determine if increasing the reducing power with formate addition to a low TCE

concentration respike experiment would increase the transformation eapacity.

Determine if increasing the biomass concentration at all TCE concentrations increases

the transformation capacity.

Determine the effect of DO concentration (or the partial pressure of oxygen) on

endogenous decay.

Determine the effects of pH on cell growth.

Detemrine the relationship between methane utilization activity and TCE transformation

activity.

Finally, a model is needed or experiments designed to distinguish between the cell

death rate and the cell inactivation rate, and what processes are responsible for each.

 

10.

ll.

12.

13.

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36.

37.

38.

54

Patel, R. N., C. T. Hou, A. I. Laskin, and A. Felix. 1982. Microbial oxidation of
hydrocarbons: properties of a soluble methane monooxygenase from a facultative
methane-utilizing organism, Methylobacterium sp. strain CRL-26. A221. Environ.
Microbiol. fl: 1 130-1 137.

Perin, D. D., and B. Dempsey, 1979. Buffers for 2H and Metal [on Control.
Chapman and Hall Laboratory Manuals in Physical Chemistry and Biochemistry.

Phelps, T. J., J. J. Niedzielski, R. M. Schrarn, S. E. Herbes, and D. C. White. 1990.
Biodegradation of trichloroethylene in continuous-recycle expanded-bed reactors.
Appl. Environ. Microbiol. 54:604-606.

Saez, P. B. and Rittmann, 1991. Biodegradation kinetics of 4-chlorophenol, an
inhibitory co-metabolite. Research Journal of the Water Pollution Control Federation
Q (6), 838-847.

Schmidt, S. K., S. Simkins, and M. Alexander, 1985. Models for the kinetics of
biodegradation of organic compounds not supporting growth. Appl. Environ.
Microbiol. $323831.

Stirling, D. I., J. Colby, and H. Dalton. 1979. A comparison of the substrate and
electron-donor specificities of the methane monooxygenases from three strains of
methane-oxidizing bacteria. Biochem. J. mz361-364.

Stumm, W., and J. J. Morgan. 1981. Aquatic chemistry. An introduction
emphasizing chemical equilibria in natural waters, 2nd ed. John Wiley and Sons, New
York.

 

Topping, J., Errors of Observation and Their Treatment The Institute of Physics,
London, 1955.

Tsien, H. C., B. J. Bratina, K. Tsuji, and R. S. Hanson. 1990. Use of
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soil. Appl. Environ. Microbiol. 31:331-331.

APPENDIX A

APPENDIX A

Whittenbury Mineral Medium

The recipe for NMS(nitrate mineral salts medium - whttenbury medium was as follows:
per Liter

1.0 g Mg304. 7H20
1.0 g KNO3
0.2 g CaClz . 2H20
3.8 x 10-3 g FeEDTA
5 x 104 g Na2M004 . 21120
1 mL trace element stock (below)

use deionized water and after autoclaving, add 10 mL per liter of
autoclaved phosphate stock (below).
Trace element stock for NMS
per liter
500 mg FeSO4 . 7H20
400 mg ZnSO4 . 7H20
20 mg MnClz . 4H20
50 mg CoClz . 6H20
10 mg NiC12 . 6H20
15 mg H3BO3 (boric acid)
250 mg EDTA

use 1 mL per liter

Phosphate Stock for NMS
per liter

26 g KH2P04
33 g Nazi-IPO4

use 10 ml per liter (autoclaved)

55

 

 

APPENDIX B

 

 

APPENDIX B

Methane Utilization

Materials and Methods

The culture was grown as describe in Chapter 2. During a growth cycle, the culture
was harvested at beginning, middle and stationary phases of growth.

A 250-mL bottle containing 50 mL of mineral media and 50 mL of culture was sealed
with a Mininert® cap. The mass of the culture added to each bottle varied considerable
depending on the stage of growth. Using a 251 f t3 cylinder of 99.5% pure methane, 5 mL of
methane was drawn out and injected into the bottle using a 30-mL syringe equiped with a 22
gage 1.5 inch sterile needle. The bottle was then shaken for 25 seconds, after which a 0.7 mL
headspace sample was taken with a 1.0 mL Precision-Lok gas syringe and injected into a HP
5890 II gas chromatograph equiped with a thermal conductivity detector (TCD). This was
repeated until 7% of the methane was oxidized

A calibration curve was generated for the data using 99% pure methane gas from a 14
liter cylinder of Scotty Analyzed Gases. The calibration curve was made up of at least five
points which bracketed the data. Anywhere from 10 yL to 100 [4L injections were made using
a 100 yL Precision-Lok syringe.

In order to determine the amount of methane that was being injected into the GC for the
calibration curve, a spreadsheet was devised which converted the volume of methane present

into the number of moles injected based on the ideal gas law:

PV = n RT
Where:

P = pressure (101.326 kPa)
56

 

 

 

57

V = volume (varied)

R = gas constant (8.314 kJ.kmol.K)

T = temperature (298 K)

n = number of moles
The number of moles of methane was then converted into a mass of methane by dividing by the
molecular weight. Using this calibration curve, the headspace data for the sample in the 250

mL bottle was converted into a mass of methane present To determine the concentration of

methane in the bottle, the mass was divided by the injection volume which was 0.7 mL(CG).
The concentration of the methane present in the liquid (CL) was determined using Henry's

constant of 27.2 at 21°C for mass transfer equilibrium of methane ( Mackay, 1981):

H =CG

° El
Therefore, the total mass of methane present at any time can be calculated as:
M( t) = CGVG + CL Vr.

A linear regression of the total mass of methane (M(t)) versus elapsed time was run to obtain

the methane utilization rate, dM/dt. A sample output of the spreadsheet is shown in Table B-1.

 

 

S 8
Table 8.1

METHANE UTIUZATION SPREADSHEET

Methane Utilization Rate for 08-29-91, Culture Monitoring (at 8:00 am)

16 a molecular mass of methane in g/mole

 

CAUBRATION CURVE
Volume (mL) mass (mg) area
0.01 0.006544 7580
0.02 0.013088 32157
0.03 0.019632 47975
0.04 0.026176 91952
0.05 0.03272 112141
SAMPLE ANALYSIS

k a maximum rate of substrate (methane) consumption in days-1
M(t) - time total mass of methane present at time t

100 -= volume of the aqueous solution in mL

150 - volume of the headspace in mL

0.7 - volume of the samples tested in mL

27.2 a Henry's Constant for methane

760 :- cell concentration in mg/L (from dry weight)

 

W
time (min) area mass (mg) conc. (mg/L) cone. (mg/L) M(t)
0 70097 0.0263 37.6094 1.3827 5.7797
5.8 63218 0.0241 34.4254 1.2656 5.2904
10.8 59882 0.023 32.8813 1.2089 5.0531

Methane Utilization = -975.3 mg/Ld

 

 

APPENDIX C

APPENDIX C

MIXED CULTURE MMl

   

Figure C.1. Scanning electron micrograph (SEM) of mixed culture MMl.
(Courtesy of S. Henry)

59

 

 

APPENDIX D

 

 

APPENDIX D
PROPAGATION OF ERROR

Originally taken from J. Topping, Error of Observation and Their Treatment;

If the function is a division :

R:—
C

Then the absolute error of the calculated result is given by the difference of the
individual relative absolute errors, i.e.
I a r)
- —-— R
e“ \A C
where:
a = the absolute error associated with A.

Y = the absolute error associated with C.

60

 

61

SENSITIVITY ANALYSIS

The sensitivity equations were derived by taking the equations associated with each
model and taking the first derivative of the dependent variable with respect to the parameter of
interest (e.g. dS/dk'). This was then multiplied by their respective parameter to yield
consistent units. Since the dependent variable is S, those models that did not have a direct
solution for S were solved using implicit differentiation. If there was a lack of proportionality
or no linear relationship between the curves, then the experimental conditions gave unique
parameter estimates over the concentration range tested.

Figure D.1 evaluates models 1b and 2b for the TCE concentration range of 0.015-
0.017 mg/L. The lack of proportionality between the three curves indicate that these are unique
solutions under the conditions of the experiment. It should be noted that in Figure D.l (A),
that the equation for k' is multiplied by 10. This indicates that for the indicated substrate
concentration, model 1b is relatively insensitive to changes in k'. In addition, Figure D.1 (B)
indicates that model 2b is extremely insensitive to changes in Tc since it was multiplied by
1000.

Figure D.2 shows a high proportionality between R and Ks for both models 1a and 2a
at the TCE concentration range of 0.22-0.24 m g/L, suggesting that unique estimates for k and
Ks may not have been obtained. In this case, it would be preferable to lump the two
parameters k and Ks into a single parameter k' (=k/Ks) as in model 1b and 2b.

Figure D.3, evaluates models la and 2a for the TCE concentration of 0.91-0.97 mg/L.
The figure indicates that both models yield unique solutions under the conditions applied

during the experiment.

 

 

 

 

 

 

 

0. 0050.010. 01509020. 0250. 030. 035
Days

62
g A
.— ul.
3' °~°°2 -So dS/dSo
g 0. 0015»
'2 0-001“ k'dS/dk
8 10de/db
3 0.0005»
g E
g 0. 0050. 010. 0150. 020. 0250. 030. 035
Days
.A. B
3 0 002-
E” ' SodS/dSo
3 0.0015" 'k' dS/dk
5’5 0.001-1 -1000 TcdS/ch
0)
C
:3 0.0005»
L
a
O
E
O
L
O
D.

Figure D.l Sensitivity analysis for model 1b and 2b for the TCE concentration range
of 0.015-0.017 mg/L (mass ~0 0024 mg TCE). (A) Model 1b, k'=l.4, b=9.031, So=.0024,
Xo=15. (B) Model 2b, k'=1.33, Tc=.0467, So=.0024, Xo=15.

63

0.030
0.0250
0.02"

       
 

-So dS/dSo

Y

0.0151

0.01.? /
f/ -l(s dS/sz

k dS/dk

0.0051

 

 
 
  

 

Parameter Sensitivity (mg TCE)

 

07305 0.01 0.015 0.02 0.025 0.03

 

   

 

quCE
§ 0.03” 3
g 0.025.. -SodS/d$o
g 0.021-
.2
E 0.015»
8 0.01«-/
:3
’2' 0.0051- -Ks dS/sz
E
O
O.

0.005 0.01 0.015 0.02 0.025 0. 03
mgTCE

Figure D.2 Sensitivity analysis of models 1a and 2a at a TCE concentration of 0.22-
0.24 mg/L (mass~.033 mg TCE. (A) Model la,So=.032, Xo=16.2, k=.4215, b=9.031,
Ks=.257. (B) Model 2a, So=.032, Xo=16.2, k=.4215, Tc=.0467, Ks=.288.

64

0.141-
0.12-

0.11
0.0%
0.051
0.041-
0.02-

-So dS/dS .

 

 

 

Parameter Sensitivitu (mg TCE)

l 0.02 0.304 0.060.138 0.71 0.12 .01
mgTCE

   
 

0.141 8
0.121-
0.1“
0.08”

0.051
0.041 .
0.021

-So dS/dSo

 

 

Parameter Sensitivity (mg TCE)

 

0.02 0.04 0.05 0.08 0T1 0.312 0.1

mg TCE

Figure D.3. Sensitivity analysis for model la and 2a for the TCE concentration of
0.91-0.97 mg/L (mass = .146 mg TCE). (A) Model 1a, So=.l46, Xo=15.6, b=9.031,
Ks=.184, k=.4215. (B) Model 2a, So=. 146, Xo=15.6, Tc=.0467, Ks=.206, k=.4215.

65

 

 

 

 

 

 

{I} A —

{-2 21 -3TcdS/ch

U

5 SodS/dSo

3' 1.51

E

E 1..

0

(O

L

.e

g 115“ -3de/db

Q

L

§ : : : 4 :
0.1 0.2 0.3 0.4 0.5

Days

A SodS/dSo

:6: 0.81- _

E B as

V -c /ch

3 0.61»

E

E 041

8

L

g 0.2.. -de/db

E

E

O

O. ; 4

 

 

0.1 0'2 0'3 0'4 05
Days

Figure D.4. Sensitivity analysis for model 3 for the higher TCE concentration ranges
of 10.2-12.5 mg/L (mass~1.56 mg TCE) and 5.5-5.7 mg/L (mass~.83 mg TCE). (A) Model
3, So=l.56, Tc=.0467, b=9.031, Xo=l6.2. (B) Model 3, So=.83,b=8.69,
Tc=.0467,Xo=16.2.

From Fr gure D4 which evaluates model 3 at the higher initial TCE concentration range
of 10.2-12.5 mg/L and 5.5-5.7 mg/L, it is also evident that the lack of pr0portionality indicates
unique solutions under these experimental conditions. However, comparing this to Figure
D5, which evaluates model 3 at the lower TCE concentrations of 0.91 mg/L down to 0.015
mg/L, shows a marked difference. In Figure D.5, there is a direct proportionality between the

curves at these lower TCE concentrations. This indicates that unique estimates of both b and

Tc may not have been obtained.

 

66

0.15- C /

0. 125» SodS/dSo -Tc dS/dT

0.1- -b dS/db
0. 075»

0. 025?

 

4

0.01 0.702 0.703 0.04 0.05

 

Days
0. 041- -
D Tc dS/%dS/db
o. 03.. SodS/dSo '
0. 02 -
0. 01‘

 

0:01 0.02 0.03 0.04 0:05

Parameter Senaitivitu (m0 TCE) Parameter Sensitivity {mg TCE)

 

 

 

Days
‘3 E
{1’ 0.0025- /
O
5 0.002-5°d5/d5°
3
E 0.0015-
§ 0.001.. -Tc dS/ch
‘E 0‘. 0005- 4" dS/db
C)
:5 - 5 e e s
g 0.01 0.02 0.03 0.04 0.05
o
0.

Days

Figure D.5. Sensitivity analysis for model 3 at the lower initial TCE concentrations of
0.91-0.97 mg/L (mass~.142 mg TCE), 0.22-0.24 mg/L (mass ~.034 mg TCE), and 015-017
mg/L (mass ~ .0024 mg TCE). (C) 80:. 142, Xo=15.6, Tc=.0467, b=5.l 14. (D) So=.034,
Xo=15.6, Tc=.0467, b=l.l87. (E) So=0.0024, Xo=15.6, Tc=.0467, b=.083.

 

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