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7.4

MICHIGAN STATE UNIVERSITY LIBRARIES

l Ill/"ll lllllllllllllMill/Illll

31293 01020 6161

ll

 

 

 

 

 

 

 

 

 

 

 

This is to certify that the

thesis entitled

Factors Influencing the Competitive Advantage of
Pseudomonas sp. Strain KC for Subsequent Remediation
of a Carbon Tetrachloride Impacted Aquifer

 

presented by

Wilfred Hans Knoll

has been accepted towards fulfillment '
of the requirements for

M.S. Environmental Engineering

degree in

 

 

555% Jet/K

Major professor

Date M—

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

 

LIBRARY
Michigan State
Unlversity

 

 

 

PLACE DI RETURN BOXto rammed“ Mountain your record.
To AVOID FINES Mum on or bdoro duo duo.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FACTORS INFLUENcrNG THE COMPETITIVE ADVANTAGE OF
PSEUDOMONAS sp. STRAIN KC FOR SUBSEQUENT REMEDIATION
OF A CARBON TETRACHLORIDE IMPACTED AQUIFER

By

Wilfred Hans Knoll

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

1994

ABSTRACT

FACTORS INFLUENCING THE COMPETITIVE ADVANTAGE OF
PSEUDOMONAS SP. STRAIN KC FOR SUBSEQUENT REMEDIATION
OF A CARBON TETRACHLORIDE IMPACTED AQUIFER

By

Wilfred Hans Knoll

TO be effective for biorernediation purposes, organisms that are added to a contaminated
environment must be able tO survive and compete with the indigenous organisms.
However, to minimize ecological disturbance, colonization should ideally be constrained.
These paradoxical Objectives can be satisfied by creating a temporary niche in which the
introduced organism can grow, persist, and express the necessary genes. When the niche
is no longer maintained, the organism dies out. A simple but powerful niche adjustment is
alkalinity addition. Modifying the pH Of a neutral or acidic environment to near 8.0
decreases the bioavailability of essential trace metals, such as iron, favoring those
organisms that possess efficient trace metal scavenging systems. Alkaline niche adjustment
with Pseudomonas sp. strain KC, a denitrifying aquifer organism originally isolated under
alkaline conditions, was evaluated. Bioaugmentation with strain KC Offers both pathway
and kinetic advantages for the removal Of carbon tetrachloride (CT), provided it can be
transported through porous media and can grow and compete with indigenous organisms.
Growth Of strain KC in batch cultures as measured by l-lmax and yield was more efficient
than that Of its competitors in Schoolcraft groundwater. In the stationary phase, strain KC
was highly persistent at pH 8.2, but died rapidly when carbon dioxide was added tO reduce
the pH Of the medium. These Observations indicate that alkaline niche adjustment can be
used to both facilitate and limit colonization Of strain KC, and to control expression Of its

biodegradative activity.

 

I humbly dedicate this thesis tO Jesus Christ, Lord and Savior Of my life,
without whom I am nothing, and tO my parents who raised me in His love.

iii

ACKNOWLEDGMENTS

It is my desire tO show appreciation and give due credit tO the people that made the
completion Of this research and thesis possible.

My wife Alisa has been a constant source Of encouragement throughout the two
years Of my graduate school experience. I thank her for challenging me to press forward
and yet still keeping me lighthearted.

I never would have produced the quality Of results or learned nearly as much if
Greg Tatara had not taken the time to teach me all I now know about experimental
techniques in microbiology. He deserves a lOt Of praise not only for his abilities and
dedication, but also for his patience and desire tO selflessly share his talents with those
around him.

Dr. Craig Criddle is the most enthusiastic and student oriented professor I have ever
met. His constant encouragement, guidance, and patience with my endless interruptions
and questions deserves my deepest appreciation.

A special thanks goes tO Dr. Michael Dybas and Blake Key. Dr. Dybas gave me
much advise and direction thanks tO his deep understanding and extensive knowledge in
microbiology. Blake taught me the intricacies Of ion chromatography which was crucial tO

my final result

iv

TABLE OF CONTENTS

Page

LIST OF TABLES .......................................................................... vii
LIST OF FIGURES ........................................................................ ix
LIST OF SYMBOLS ....................................................................... xi
CHAPTER 1 INTRODUCTION ............................................... l

ln-situ Remediation with Pseudomonas sp. Strain KC ...... 1

Theory Of Competition .......................................... 3
CHAPTER 2 MATERIALS AND METHODS ............................... 6
CHAPTER 3 EFFECTS OF INTI‘IAL pH MEDIUM ON GROWTH OF

CHAPTER 4

CHAPTER 5

STRAIN KC AND SCHOOLCRAFI‘ AQUIFER

ORGANISMS .................................................... 8
Materials and Methods ........................................... 8
Results and Discussion .......................................... 10

DEVELOPMENT OF SCHOOLCRAFT GROUNDWATER

PASTEURIZATION PROCESS ............................... 17
Materials and Methods ........................................... 17
Results and Discussion .......................................... l9

EFFECTS OFpH ON STRAIN KCANDTHE
INDIGENOUS ORGANISMS IN SCHOOLCRAFT

GROUNDWATER ............................................... 24
Materials and Methods ........................................... 24
Results and Discussion .......................................... 25

CHAPTER 6 — KINETIC PARAMETER CHARACTERIZATION OF
STRAIN KC AND THE INDIGENOUS ORGANISMS
IN SCHOOLCRAFT GROUNDWATER ..................... 28
Materials and Methods ........................................... 28
Results and Discussion .......................................... 30
CHAPTER 7 — ENGINEERING APPLICATION ............................. 47
CHAPTER 8 — CONCLUSIONS ................................................. 50
FUTURE WORK RECOMMENDATIONS .................. 51
LIST OF REFERENCES ................................................................... 52
APPENDD( A — ORIGINAL DATA AND CALCULATIONS FOR
PSEUDOMONAS SP. STRAIN KC KINETIC
PARAMETERS IN MEDIUM D ................................ 54
APPENDIX B — ORIGINAL DATA AND CALCULATIONS FOR KINETIC
PARAMETERS IN SCHOOLCRAFT GROUNDWATER. 57
APPENDIX C — ORIGINAL DATA USED FOR DETERMINING THE

HALF-VELOCITY COEFFICIENTS IN SCHOOLCRAFT
GROUNDWATER ............................................... 66

vi

Table 1.

Table 2.
Table 3.

Table 4.

Table 5.

Table A-1.

Table A-2.

Table B-1.

Table B-2.

Table B-3.

Table B-4.

Table B-5.

LIST OF TABLES

Effect Of initial medium pH on protein production for Pseudomonas
sp. strain KC and Schoolcraft aquifer microbial flora ................. 13

Kinetic parameters for Pseudomonas sp. strain KC in medium D... 14

Pseudomonas sp. strain KC and native aquifer consortium
growth/decay after 69 hours in Schoolcraft groundwater under

various conditions. Initial acetate, phosphate, and pH was 10 mM,
0.100 mM, and 8.2, respectively ........................................ 22

Kinetic parameters and yield coefficients for strain KC and
Schoolcraft aquifer flora in Schoolcraft groundwater adjusted
to an initial pH of 8.2 ...................................................... 42

Half-velocity coefficients and maximum specific rates Of substrate
utilization for Pseudomonas sp. strain KC and the indigenous
organisms in Schoolcraft groundwater .................................. 46

Original data used for determination Of um, b, and protein per
cell for P. KC in medium D .............................................. 54

Data used for calculations Of u and b for Pseudomonas sp.
strain KC .................................................................... 55

Data used for determining the maximum specific growth rate ilm
Of Schoolcraft organisms in Schoolcraft groundwater ................. 57

Data used for determining the maximum specific rate Of
substrate utilization (km) Of Schoolcraft organisms in Schoolcraft
groundwater ................................................................ 58

Data used for determining the yield Of Schoolcraft organisms in
Schoolcraft groundwater on nitrate reduced to gaseous
end products ................................................................ 59

Data used for determining the yield Of Schoolcraft organisms in
Schoolcraft groundwater on nitrate reduced tO nitrite .................. 60

Data used for determining the yield Of Schoolcraft organisms in

Schoolcraft groundwater on nitrite reduced to gaseous
end products ................................................................ 61

vii

Table B-6.

Table B-7.

Table B-8.

Table B-9.

Table B-lO.

Table C- 1 .

Table C-2.

Table C-3.

Table C-4.

Table C-S.

Table C-6.

Data used for determining the maximum specific growth rate Ilm
Of Pseudomonas sp. strain KC in Schoolcraft groundwater ......... 61

Data used for determining the maximum specific rate Of
substrate utilization (km) Of Pseudomonas sp. strain KC in

Schoolcraft groundwater .................................................. 62

Data used for determining the yield Of Pseudomonas sp. strain KC
in Schoolcraft groundwater on nitrate reduced tO gaseous
end products ................................................................ 63

Data used for determining the yield Of Pseudomonas sp. strain KC
in Schoolcraft groundwater on nitrate reduced to nitrite ............... 64

Data used for determining the yield Of Pseudomonas sp. strain KC
in Schoolcraft groundwater on nitrite reduced to gaseous
end products ................................................................ 65

Supporting data for the determination Of the maximum specific
rate Of nitrate utilization km Of Schoolcraft organisms in

Schoolcraft groundwater .................................................. 66

Supporting data for the determination Of the maximum specific rate
Of nitrite utilization km Of Schoolcraft organisms in Schoolcraft

groundwater ................................................................ 66

Calculated half-velocity coefficients Ks and supporting data for
Schoolcraft organisms in Schoolcraft groundwater .................... 66

Supporting data for the determination Of the maximum specific
rate Of nitrate utilization km Of Pseudomonas sp. strain KC in
Schoolcraft groundwater .................................................. 67

Supporting data for the determination Of the maximum specific rate
Of nitrite utilization km Of Pseudomonas sp. strain KC in
Schoolcraft groundwater .................................................. 67

Calculated half-velocity coefficients Ks and supporting data for
Pseudomonas sp. strain KC in Schoolcraft groundwater ............. 67

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

LIST OF FIGURES

Growth curve Of Pseudomonas sp. strain KC in Medium D.
Error bars represent one standard deviation Of three independently
grown cultures .............................................................. 11

Accumulation Of total protein Of Pseudomonas sp. strain KC in
medium D. Error bars represent one standard deviation Of three
independently grown cultures ............................................ 12

Effect Of various concentrations Of nitrite on Pseudomonas sp.
strain KC in medium D with specified nitrite and/or nitrate
concentrations .............................................................. 15

Comparison Of growth Of Schoolcraft aquifer consortium in
medium D in aerobic and anoxic conditions. (Time = 92 hours) ..... 16

Growth Of Pseudomonas sp. strain KC in sterile filtered Schoolcraft
groundwater with various initial values Of acetate. Initial phosphate
concentration and pH was 0.100 mM and 8.2, respectively .......... 20

Pasteurization effect on indigenous organisms by incubating
Schoolcraft groundwater at 65° C ........................................ 23

Effect Of various initial pH values on an existing population Of
indigenous aquifer organisms in Schoolcraft groundwater ........... 26

Persistence Of strain KC under nO-growth conditions in Schoolcraft
groundwater at pH 8.2. After 2 weeks Of incubation, the pH was
reduced tO 7.5 .............................................................. 27

Figure 9.1-9.3. Growth pattern, substrate utilization, and pH increase for

Figure 10.

Figure l 1.

indigenous aquifer organisms in Schoolcraft groundwater adjusted
tO an initial pH Of 8.2 (Replicates #1-3) ................................. 32

Biomass accumulation Of indigenous organisms in Schoolcraft
groundwater measured as dry weight .................................... 35

Growth Of indigenous organisms in Schoolcraft groundwater
measured by bacterial plate counts ....................................... 36

Figure 12.1-12.3. Growth pattern, substrate utilization, and pH increase for

Pseudomonas sp. strain KC in Schoolcraft groundwater adjusted
tO an initial pH Of 8.2 (Replicates #1-3) ................................. 37

Figure 13.
Figure 14.
Figure 15.

Figure 16.

Biomass accumulation Of Pseudomonas sp. strain KC in
Schoolcraft groundwater measured as dry weight ..................... 40

Growth Of Pseudomonas sp. strain KC in Schoolcraft groundwater
measured by bacterial plate counts ....................................... 41

Nitrate utilization by indigenous aquifer organisms in Schoolcraft
groundwater adjusted tO an initial pH of 8.2 ............................ 43

Nitrate utilization by Pseudomonas sp. strain KC in Schoolcraft
groundwater adjusted to an initial pH Of 8.2 ............................ 44

LIST OF SYMBOLS

b decay coefficient, day‘l

Ks half-velocity coefficient, mg/L

Ilm maximum specific growth rate, day‘1

S rate-limiting substrate concentration, mg/L

t, T time, days

X concentration Of microorganisms (or protein), mg/L

SO the initial nitrate concentration, mg/L

8 time dependent nitrate concentration, mg/L

km the maximum specific rate Of substrate utilization, hour"1

5min. R*, J substrate concentration at which growth and decay are equal, mg/L

CHAPTER 1

INTRODUCTION

In-situ Remediation with Pseudomonas sp. Strain KC

In-situ bioremediation Offers the potential for remediation Of many contaminated sites.
The two most important Options for in-situ bioremediation are biostimulation, the addition
Of growth-limiting factors to increase the concentration Of indigenous organisms, and
bioaugmentation, the introduction Of organisms with unique catabolic capabilities. Both
approaches have advantages and disadvantages. For bioaugmentation, transport Of the
added microorganisms and competition between the added microorganisms and indigenous
populations present formidable challenges. A possible means Of sunnounting the challenge
Of competition is to create a temporary niche that favors growth and maintenance Of the
introduced organism. The niche adjustment evaluated in this work was addition Of

alkalinity tO favor growth and maintenance Of Pseudomonas sp. strain KC.

Carbon tetrachloride (CT) transformation by indigenous denitrifying microorganisms
typically leads to the production Of chloroform (CF). In a field-scale experiment at the
MOffett Naval Air Station, for example, CF production resulted from acetate addition to
biostimulate indigenous denitrifying populations (Semprini et al., 1992). Pseudomonas

sp. strain KC is a denitrifying microorganism that rapidly transforms CT tO C02, forrnate,
and an unidentified non-volatile product, without the production Of CF (Criddle et al.,
1990, Lewis et al., 1993, Dybas et al., 1994 a). The requirements for CT transformation
by strain KC are: (1) adequate concentrations Of nitrate and electron donor, (2) iron-limiting

conditions, (3) trace levels Of copper, and (4) an incubation temperature Of 4-23°C

2
(transformation is inhibited above 25°C; growth is inhibited above 30°C). Iron-limited

conditions can be achieved by adjusting the pH to near 8.0 (Tatara et al., 1993) or by
adding iron chelators (Lewis et al., 1993). Copper is required for CT transformation

(T atara et al., 1993), but inhibits growth at neutral pH (Criddle et al., 1990). The
transformation is cometabolic, and it is linked to mechanisms for trace metal scavenging
(Criddle et al., 1990, Tatara et al., 1993). NO similar activity has yet been detected in other
isolates or in indigenous enrichment communities (Criddle et al., 1990, Lewis et al.,

1993). Thus, use Of strain KC for bioaugmentation may Offer certain pathway and kinetic
advantages, provided it can compete with indigenous organisms and can express the genes

needed for CT degradation in environments that contain appreciable levels Of iron.

The value Of pH adjustment in creating a niche favorable for growth Of strain KC is
associated with changes in speciation Of iron and copper at high pH. Iron solubility (as
ferric ions) is lowest in the pH 8.0-8.2 range (Stumm et al., 1981). Addition Of base
results in the precipitation Of ferric salts from solution. Consequently, base addition favors
strain KC and Other organisms capable Of scavenging iron in low iron environments.
However, upon adjustment Of pH tO near neutral conditions, say, for example, after mixing
with carbonated waters, then trace copper becomes inhibitory tO growth. These
considerations suggest that pH control may be used tO both enable and confine

colonization Of strain KC.

In this thesis, the effectiveness Of niche adjustment and the potential for successful
bioaugmentation with Pseudomonas sp. strain KC is evaluated in groundwater samples.
Evidence is provided demonstrating that alkaline niche adjustment favors growth Of strain

KC giving it a competitive advantage over the indigenous flora.

3
Kinetic characterization Of both Pseudomonas KC and the native flora found in the

Schoolcraft aquifer under alkaline conditions is crucial for determining the outcome Of
experiments in which Pseudomonas KC is in direct competition with the aquifer
consortium. Kinetic parameters Obtained will be used in modeling efforts for field scale
studies. With this data it will be possible tO predict the approximate yield Of biomass
expected from certain substrate concentrations facilitating estimation Of the degree Of carbon

tetrachloride degradation.

Theory of Competition

There has been much debate in literature over the topic Of predicting the qualitative
outcomes Of mixed-growth competition using kinetic parameters Obtained on organisms
grown alone (Hansen et al., 1980, Tilman, 1981). When microbial strains compete for the
same limiting nutrient in continuous culture, resource-based competition theory predicts
that only one strain will survive and all others will die out (Hansen et al., 1980).
Experimental results have shown that two species were Observed to coexist only if either
(1) each was limited by a different resource and met the theoretical criteria for coexistence
or (2) the species were limited by the same resource and did not differ significantly in their
resource requirements (Tilman, 1981). Taylor and Williams (1975) concluded that tO
sustain a mixed population Of a number Of species in a chemostat-type continuous-flow
system it is necessary that there are at least as many growth-limiting substrates as there are

different species.

The experiments reported herein attempt to create single-nutrient competition for nitrate.
The superior competitor as predicted by resource-based competition theory will be the

organism with the lowest resource requirement, as measured by R* (T ilman, 1981). This

4
resource requirement is also termed subsistence or "break-even" concentration of the

limiting resource, defined by the J parameter in other literature (Hansen et al., 1980).
Predictions using the J parameter have been confirmed in the case Of auxotrophic bacterial
strains competing for limiting tr'yptophan (Hansen et al., 1980). Rittrnan and McCarty
(1980) have also referred to this resource requirement parameter labeling it 5min. Although
all three parameters mentioned above are named differently, each are defined as the
substrate concentration at which growth and decay are equal (Hansen et al., 1980, Tilman,
1981, Rittman et al., 1980). The derivation of this parameter (hereafter referred to as 5min)
begins with Monod's (1942) formulation of microbial growth which was later modified by

van Uden (1967) to consider organism decay as well:

_5_
I“: 1““ (S+Ks) ' b
also. u = (dX/dt)/X = specific growth rate, day-1

where:

b = decay coefficient, day‘l

K5 = half-velocity coefficient, mg/L

Ilm = maximum specific growth rate, day”1

S = rate-limiting substrate concentration, mg/L
t = time, days

X = concentration of microorganisms, mg/L

This crucial parameter (3min) is found from the differential form of the equation for u by
letting dX/dt = O (Rittman et al., 1980):

When the substrate concentration is equal to or below 3min, there is no net growth of

microorganisms (Criddle et al., 1991).

It is readily obvious that such a parameter is a powerful tool for predicting outcomes of
single-nutrient competition between organisms when kinetic parameters have been obtained
for the organisms grown alone. The following predictions can be made for competition
between two types of organisms: (i) if two strains have equal Ilm'S and b's, the strain with
the lower Ks wins; (ii) if two strains have identical Ks's and b's, the strain with the higher
Ilm wins; and (iii) if two strains have different Ks's and thus, but in spite of this still have

identical Smin's, then the species or strains will coexist indefinitely (Hansen et al., 1980).

In this thesis, kinetic parameters were determined for Pseudomonas sp. strain KC and the
indigenous microorganisms in Schoolcraft groundwater. Using these kinetic parameter
values and resource-based competition theory, it is possible to predict the winner of direct
competition between Pseudomonas sp. strain KC and the indigenous microorganisms in

Schoolcraft groundwater.

CHAPTER 2

MATERIALS AND METHODS

Organisms. Pseudomonas sp. strain KC (DSM deposit no. 7136, ATCC deposit
number 55595), derived originally from aquifer solids from Seal Beach, CA (Criddle et al.,

1990), was routinely maintained on nutrient agar plates.

Chemicals. All chemicals used were ACS reagent grade (Aldrich or Sigma Chemical

Co.). All water used in reagent preparation was deionized 18 Mohm resistance or greater.

Media. Medium D contained per liter of deionized water: 2.0 g Of KH2P04, 3.5 g of
K2HP04, 1.0 g of (NH4)2S04, 0.5 g MgSO4 - 7H20, 3.0 g of sodium acetate, 2.0 g of
sodium nitrate, 1 m1 of 0.15 M Ca(N03)2, and 1 ml of trace nutrient stock TN2. Stock
solution TN2 contained per liter of deionized water: 1.36 g of FeSO4 - 7H20, 0.24 g of
Na2MoO4 - 2H20, 0.25 g of CuS04 - 51-120, 0.58 g of ZnSO4 - 7H20, 0.29 g of
Co(NO3)2 - 61120, 0.11 g of NiSO4 - 61120, 35 mg of Na2Se03, 62 mg of H3303,
0.12 g of NH4VO3, 1.01 g of MnSO4 - H20, and 1 m1 of H2804 (concentrated).
Typically, medium D is adjusted to pH 8.2 using KOH pellets followed by 3 M KOH stock
solution. Medium D was prepared and dispensed in 28 mL serum tubes, 120 ml serum
bottles, or 500 ml Wheaton bottles. Nutrient broth and nutrient agar (Difco) plates were
prepared according to manufacturer's instructions. Cultures were grown at 20°C with 150

rpm shaking under aerobic or denitrifying (N2 head space) conditions.

Groundwater. Groundwater from a CT-contaminated aquifer in Schoolcraft, MI, was
used in all batch. Groundwater samples were obtained manually by withdrawing

groundwater from a 2" steel well screened at 30 feet below the water table with a Teflon®

7
bailer. Groundwater samples were stored in pre-sterilized sealed N a] gene® carboys or in

Wheaton bottles equipped with Teflon® lined caps at 4°C.

Analytical methods. Nitrate, nitrite, and acetate ions were assayed by ion
chromatography (Dionex model 2000i/SP ion chromatograph with suppressed
conductivity detection equipped with a Dionex® Ionpak AS4-A anion exchange column
and utilizing a 1.8 mM bicarbonate/1.7 mM carbonate mobile phase at 1 ml/min).
Chromatograrns were recorded and data integrated using Turbochrom® 3 software (Perkin
Ehner Corp.) External standard calibration curves which bracketed the concentrations of
the test samples were prepared by diluting primary ion standards into deionized water
having at least 18 Mohm resistance. Measurements of pH were made with an Orion model
720A pH meter. Protein was determined by the modified Lowry method, with bovine
serum albumin as the standard (Markwell et al., 1981 ). Optical density was measured at
660 nm using a Shirnadzu UV-160 spectrophotometer.

CHAPTER 3

EFFECTS OF INITIAL pH MEDIUM ON GROWTH
OF STRAIN KC AND SCHOOLCRAFT AQUIFER ORGANISMS

Materials and Methods

Growth Curve and kinetic parameters of strain KC in medium D. Strain KC
starter cultures were prepared by transferring cells from nutrient agar plates to previously
autoclaved 28 ml test tubes containing nutrient broth using aseptic technique. After 24
hours, previously autoclaved test tubes containing medium D were inoculated with a 1%
inoculum of the nutrient broth grown culture. One hundred milliliter aliquots of medium D
at pH 8.2 under anaerobic conditions (nitrogen head space) were inoculated with a 48 hour
1% medium D inoculum. Growth was followed using serial dilutions and nutrient agar
plate counts. Each colony forming unit was scored after 6 days of incubation at 20° C. The
geometric mean and the standard deviation for the geometric mean was used to statistically
analyze the data. Total protein accumulation was monitored using the modified Lowry
protein assay (Markwell et al., 1981).

The maximum specific growth rate 14m was obtained for log growth phase cells using plate
counts and the relationship: 11m = [ln(Xf/Xi)] / (tf-ti), where Xf and Xi represent the
geometric mean of the final and initial plate counts, respectively, and if and ti are the final
and initial time, respectively. The decay rate was obtained in similar fashion using the
relationship: b = - [ln(Xf/Xi)] / (tf-ti). Yield on nitrate was determined by dividing the

maximum protein concentration by the initial concentration of nitrate. This estimate is based

9
on the assumption that all the nitrate was converted to gaseous end products. The maximum

specific rate of nitrate utilization km was estimated by dividing the yield on nitrate by the
maximum specific growth rate (um/Y N). The fraction of electrons diverted for energy fe
and the fraction of electrons used for synthesis fs were calculated using balanced theoretical
stoichiometric relationships between nitrate and cells (Criddle et al., 1991). It was assumed
that the dry weight of each cell was equal to exactly twice the measured protein. Protein per
strain KC cell is shown in Table 2 and dry weight per strain KC cell can be found in Table
4. These values lend credibility to the above assumption. The ratio of acetate to nitrate was
based on balanced stoichiometric relationships using the estimated fe and f5 values. Yield
on acetate was an estimate based on the mathematical relationship: Ym = (fs)max (h/g),
where Ym represents the maximum yield, (fs)max is the maximum fraction of electrons
diverted to synthesis, g is the electron equivalent mass of the electron donor (acetate), and h
represents the electron equivalent mass of biomass (4.04 when nitrate is the nitrogen
source) (Criddle et al., 1991). See Appendix A for raw data and calculations supporting the

above determinations.

Utilization of nitrite as terminal electron acceptor by strain KC. Medium D
was prepared as indicated in Chapter 2 of this thesis with the exception of the sodium
nitrate addition. Concentrated nitrite and nitrate stocks were used to obtain the desired
concentration of each. Strain KC inocula were prepared as above then centrifuged, washed,
and resuspended in medium D containing no nitrate. Protein was analyzed using the

modified Lowry protein assay.

Effects of pH on growth. The dependence of growth of Pseudomonas sp. strain KC
and the Schoolcraft aquifer native microbial flora as a function of initial medium pH was
determined by preparing medium D at various initial pH levels (modified by addition of

nitric acid or NaOH). Ten milliliter aliquots of medium D at pH 7—10 were inoculated

10
with an aerobic 24 hour 1% inoculum of medium D grown strain KC or aquifer microbial

flora and sealed under anaerobic conditions. Growth was assayed for protein levels using
the modified Lowry protein assay after 92 hours of growth. The aquifer microbial flora
originated from an inoculum of Schoolcraft groundwater into nutrient broth which then
served as an inoculum source for the medium D starter culture. Aerobic samples of
Schoolcraft flora at pH 8.2 were prepared as above with the exception of being sealed

under anaerobic conditions.

Results and Discussion

Effect of alkaline niche adjustment on growth and persistence of indigenous
aquifer organisms and strain KC. The growth curve of Pseudomonas sp. strain
KC in Medium D with an initial pH of 8.2 is illustrated in Figure 1. Total accumulated
protein, shown in Figure 2, did not hydrolyze as rapidly as cell number decreased.

Because protein did not hydrolyze before the fourth day, protein measurements taken up to
4 days accurately represent maximum accumulated protein. This result allowed for adequate
accumulated protein comparisons of strain KC and Schoolcraft organisms even after strain
KC had passed through the decay phase. As shown in Table 1, initial medium pH had a
significant effect on the concentration of protein produced in medium D. The buffer
capacity of the system will also effect this result but to a lesser degree. The effects of
medium pH on growth of strain KC and Schoolcraft aquifer native microbial flora are

shown for comparison.

ll

CFU/ml

 

 

 

 

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
Time (days)

Figure 1. Growth curve of Pseudomonas sp. strain KC in Medium D. Error bars
represent one standard deviation of three independently grown cultures.

12

 

 

1000
100 , . ___
E
a
5' 10
.s
‘2’
a:
r _
.1! ' r ' I ' U ' I
o 1 2 3 4

Time (days)

Figure 2. Accumulation of total protein of Pseudomonas sp. strain KC in Medium D. Error
bars represent one standard deviation of three independently grown cultures.

1 3
Table 1. Effect of initial medium pH on protein production for Pseudomonas

sp. strain KC and Schoolcraft aquifer microbial flora.1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Final Culture pH2 Protein concentration
Initial (3g Imr)2
Culture pH Strain KC Schoolcraft Strain KC Schoolcraft
consortium consortium
7.03 7.06 : 0.01 7.17 i 0.03 32.3: 0.5 19.7: 3.3
7.39 7.40 : 0.01 7.64 :1: 0.08 69.5: 1.9 35.2: 4.8
7.74 9.17 :1: 0.01 7.76 :1: 0.04 134: 2 11.0: 1.3
8.12 9.42 :1: 0.03 8.19 : 0.10 126: 11 14.4: 3.8
8.33 9.36 :1: 0.01 8.36 :1: 0.04 125: 4 12.99: 6.12
8.62 9.38 : 0.04 8.53 : 0.01 110: 9 7.4: 0.2
9.04 9.07 :1: 0.29 8.99 : 0.02 71.4: 14.3 6.2: 0.2
9.93 9.93 : 0.03 9.91 : 0.05 4.2: 0.8 5.7: 1.6

 

1- grown 92 hours in medium D.

2° average : one standard deviation for triplicate cultures.

Highest protein concentrations for strain KC were obtained in the moderately alkaline range

(pH 8-9), indicating that its growth is Optimal under these conditions. Schoolcraft

microbial flora showed a pH Optimum for growth in the neutral range (pH 7-7.4).

Reduced growth of strain KC at pH levels less than 8.0 are likely related to speciation

changes that increase the toxicity of copper (Criddle et al., 1990). Changes in the speciation

and solubility of iron and cobalt may also have affected protein production (Criddle et al.,

1990, Tatara et al., 1993). Denitrification by strain KC caused the pH to increase to 9.2 to

9.4, at which point no additional growth was observed.

 

14
Kinetic parameters for strain KC Obtained from the results shown in Figures 1 and 2 are

presented in Table 2. Assumptions underlying the calculations are complete nitrate
conversion to gaseous end products and balanced stoichiometric equations. The amount of
protein per strain KC cell was obtained by using plate counts and protein data throughout
the first 54 hours. This limited data was used in order to avoid inclusion of dead cell

protein in the calculations.

The rapid decay of strain KC shown in Figure 1 and Table 2 may have been the result of
nitrite toxicity. Presumably, strain KC reduces nitrate to nitrite throughout its utilization of
acetate. If the 23.5 mM nitrate in medium D is converted to nitrite before nitrite utilization
begins, the nitrite concentrations may reach as high as 23.5 mM nitrite. Figure 3 displays
possible toxic effects of 35 mM nitrite which may potentially be exhibited at 23.5 mM. As a
control, strain KC was subjected to relatively low nitrate and nitrite concentrations in
Schoolcraft aquifer water supplemented with 10 mM acetate. The growth under these

conditions is shown in Figure 3.

Table 2. Kinetic parameters for Pseudomonas sp. strain KC in medium D.

, max
deca coefficient
YN, Yield
A.

Ratio Of mmol Acetate/mmol
max

N

 

15

 

 

 

 

 

 

100 _-
I
35mM Nitrite
553, 101’
'2. —-~
E 645uM Nitrate ‘ 3
430uM Nitrite
70 mM Nitrite
1 A L n 1 A l n Lg l A l l A l n 1 J l n l L I
0 10 20 30 40 50 60 70 80 90 100 110 120
Time (hours)

Figure 3. Effect of various concentrations of nitrite on Pseudomonas sp. strain KC in
medium D with specified nitrite and/or nitrate concentrations.

Because CT is cometabolized by strain KC only under anoxic conditions, it became a
concern as to whether or not the Schoolcraft aquifer is anoxic. One method Of addressing
this issue is to determine whether the indigenous organisms prefer aerobic or anaerobic
conditions. If the resulting growth is much greater under aerobic conditions it is possible
that the percentage of denitrifying bacteria is low. This would be an indication that anoxic
conditions are not the norm for this aquifer. A comparison of aerobic and anaerobic growth
for Schoolcraft aquifer organisms is shown in Figure 4. It is possible that the greater yield
under aerobic conditions is due to the greater amount of energy obtained from oxygen
versus nitrate as the terminal electron acceptor. A related explanation is that the Schoolcraft

organisms have a higher growth rate when using oxygen. Enumeration of denitrifying

l6
bacteria in the Schoolcraft aquifer is recommended as an area of future study. This will

serve as an aid in narrowing down the relevant factors influencing the competitiveness of

strain KC.

Proteinmg/ml)

 

 

Conditions

Figure 4. Comparison of growth of Schoolcraft aquifer consortium in medium D
in aerobic and anoxic conditions. (Time = 92 hours).

CHAPTER 4

DEVELOPMENT OF SCHOOLCRAF T GROUNDWATER
PASTEURIZATION PROCESS

Materials and Methods

Growth of strain KC in Schoolcraft groundwater with varying
concentrations of acetate. Schoolcraft groundwater was adjusted to pH 8.2 by
addition of NaOH. It was previously shown by M. Dybas (personal communication) that a
final concentration of 0.18 mM NaOH produced a final pH of 8.2 in Schoolcraft
groundwater. One hundred microliters of a 100 mM phosphate buffer was added to each
100 ml sample to produce a 0.100 mM phosphate concentration to serve as a phosphorus
source. An appropriate amount of a concentrated acetate stock solution was added to yield
the desired acetate concentrations. The resulting solution was sterilized by filtration through
a 0.22 um pore vacuum filtration unit pressurized to approximately 15 psi. Each sample
was transferred to a sterile 250 ml Erlenmeyer flask and passed through the anaerobic
vacuum chamber for three full cycles to ensure anaerobic conditions. Once in the anaerobic
hood, 10 ml aliquots were aseptically transferred to 4 sterile test tubes and sealed with
sterilized heavy black rubber septa. Common crimp tops were used to seal the test tubes.
Strain KC inocula consisted of a 48 hour 1% aerobic medium D inocula that had been
washed and resuspended in Schoolcraft groundwater with nutrients added that had been
sterilized by filtration. Starter cultures were centrifuged at 14,000 rpm for 5 minutes,
resuspended and centrifuged again at 8,000 rpm for 10 minutes. This was to eliminate trace

carryover of nutrients from the starter cultures into the growth tubes.

17

18
Effect of groundwater filtration on strain KC growth. The experimental

procedure used was identical to the above with the exception of filtration. Half the samples
were filtered before addition of phosphate, N aOH, and acetate; the other half were filtered

after the addition of nutrients.

Effect of various environments on strain KC and the Schoolcraft

organisms. Several different environments were prepared in order to determine the
reason for minimal growth of strain KC in Schoolcraft groundwater. Aerobic conditions
were established for the first set of samples by following the procedure used to test growth
at various acetate concentrations. Samples were sterilized by filtration before addition of
nutrients and sealed under aerobic conditions. Excess nitrate was added to a second set of
samples in the event that the nitrate present in Schoolcraft water (about 55 ppm) was
insufficient to support adequate growth. The nitrate added stoichiometrically balanced the
10 mM acetate concentration. Samples were sealed in the anaerobic hood to produce
denitrifying conditions. A third set was autoclaved previous to inoculation with strain KC
to determine if trace nutrients were locked in the precipitate that formed from the
autoclaving process. A fourth set consisted of Schoolcraft groundwater adjusted to pH 8.2
with NaOH and supplemented with phosphate and acetate. N 0 strain KC cells were added.
Samples in the fifth set were prepared in identical fashion as those in the experiment testing
the varying acetate concentrations except that the nutrients were added after sterilization by
filtration. The final or sixth set consisted of Schoolcraft groundwater with all nutrient
additions and pH adjustment with a 2% washed strain KC inoculation. No sterilization

technique was used on sample set six.

Pasteurization of Schoolcraft groundwater using 65° C incubation.

Schoolcraft groundwater was dispensed in triplicate sterile serum bottles (20 ml aliquots)

19
then incubated at 20° C and 65° C. Serial dilution and nutrient agar plate counts were used

to determine the effectiveness of 65° C pasteurization.

Results and Discussion

There are many experimental methods which can be used to measure the kinetic parameters
of bacteria. Before any of these can be successfully implemented, certain basic parameters
must first be established. The minimum concentration of the electron donor (acetate)
required to achieve maximum growth was the first parameter to be determined. Obviously,
as acetate concentration is increased, growth should increase as long as adequate electron
acceptor and trace nutrient concentrations are available. This trend will continue until the
acetate concentration is no longer limiting growth. If acetate is supplied at concentrations
that stoichiometrically balance the bacterial demand for nitrate, then no additional growth
will occur as acetate concentrations are increased further. This upper limit for acetate was
evaluated to determine an appropriate concentration to be used in future experiments aimed
at Obtaining kinetic parameters for strain KC. In an initial experiment, acetate was added at
different concentrations to Schoolcraft groundwater and growth was monitored by Optical
density. The results shown in Figure 5 indicate that growth of strain KC in Schoolcraft
groundwater is insufficient for measurement by optical density, regardless of acetate
concentration. Note the optical density scale on the vertical axis. Typically, adequate

growth measurements are an order of magnitude greater than those shown here.

20

 

 

0.03 -
0.02 -

35 i \ _ émMAm

5 - A 1 \

3" 0.01 - _ / ‘ \ V110“

. . ,- ,- ' I", \

g “'A ’\ I \v ‘ 'A/ 1111M

5' 090' 11¢ iii/{13¢ V \'

g ‘ \il 0,2.3mM
-o.or - r
.OOOZOU‘UZBUULO“.UenoUJusJoUl-llao ..... 1 £0 ..... 1:0...

Time (hours)

Figure 5. Growth of Pseudomonas sp. strain KC in Schoolcraft groundwater sterilized by
filtration with various initial values of acetate. Initial phosphate concentration and pH was
0.100 mM and 8.2. respectively.

The results in Figure 5 were obtained by addition of sodium hydroxide, phosphate, and
acetate to Schoolcraft groundwater before sterile filtration (no additional nitrate was
provided) and inoculation under anaerobic conditions. The lack of growth of strain KC
under these conditions was unexpected and puzzling. It was hypothesized that the cause for
this may have been related to the sterile filtration process preceding inoculation of strain
KC. Trace nutrients, such as iron, may have adhered to the filter membrane and caused
growth limiting conditions. To test this hypothesis, growth in samples that were filtered
before nutrient addition was compared to growth results in samples that had been sterilized
by filtration after nutrient addition. Protein measurements over a period of six days showed
that filtration before or after nutrient addition had no apparent effect on growth of strain
KC. Samples from both sets again showed very little growth.

21

A possible explanation for the lack of growth of strain KC was inadequate electron acceptor
concentration. Oxygen and nitrate can both be utilized by strain KC as the terminal electron
acceptor. Accordingly, the next experiment tested growth of strain KC under aerobic and
excess nitrate conditions in Schoolcraft groundwater that had been sterilized by filtration
before addition of nutrients. As a control, strain KC was also subjected to identical
conditions as the previous experiment in which nutrients were added after sterilization by
filtration. Autoclaved Schoolcraft groundwater containing phosphate and acetate at pH 8.2

was also used as the medium for strain KC. Results are provided in Table 3.

Autoclaving caused precipitate to form that may have converted trace nutrients to
inaccessible forms. The decrease in CFU/ml of strain KC in the samples sterilized by
filtration and autoclavin g indicates that these processes have some adverse effects on the
growth of strain KC in Schoolcraft groundwater. Results for sample number 4 show a
significant increase in indigenous organisms in Schoolcraft groundwater adjusted to pH 8.2
with nutrient addition. Strain KC was not inoculated into sample 4 and the Schoolcraft
groundwater was neither filter sterilized nor autoclaved. Sample number 6 consisted of
identical conditions as sample 4 except that strain KC was inoculated in sample 6. Strain
KC and Schoolcraft organisms were differentiated by colony morphology in sample 6
which may have been a source of some inaccuracy. Be that as it may, the results of samples
4 and 6 show significant growth of both strain KC and the Schoolcraft organisms. These
results give further credibility to the hypothesis that filter sterilization and autoclaving cause
adverse effects on bacterial growth in Schoolcraft groundwater. Anorher interesting
conclusion which can be made from the results of samples 4 and 6 is that Schoolcraft
organisms show significant growth at pH 8.2. Previous to this experiment it was believed
that alkaline conditions were inhibitbry to indigenous organisms in Schoolcraft

groundwater. This issue will be addressed again in the results of later experiments.

22

Table 3. Pseudomonas sp. strain KC and native aquifer consortium growth/decay after 69
hours in Schoolcraft groundwater under various conditions. Initial acetate, phosphate, and
pH was 10 mM, 0.100 mM, and 8.2, respectively.

 

 

Amendruents Marion Method Initial population Final population
(104 CFU/ml) (104 CFU/ml)

 

 

 

1. Ox en Filtration 4.10 :1: 1.40 1.04 :1: 9.20

”FER. . cess Filtration 2.11 :1: 3.66 0.64 i 3.50
nitrate

. None Autoclaving 142.72 :1: 53.66 T19 .+_ 4.48

 

4. None Non-Sterile (Schoolcraft 8.88 :1: 2.34 779.17 :1: 101.29

or anisms only)

 

 

5. None F' nation 9.65 :1: 4.59 9.93 i 3.2; ,
6a. None Non-Sterile (P. KC, 3.11 i 1.55 58. :1: 1.
identified by colony
morphology)

 

6b. None Non-Sterile(Schoolcraft 8.24:8.88 537m :1 . ‘
organisms, identified by
colony morphology)

 

 

 

 

 

 

Because of the results discussed above, it became evident that a sterilization method other
than filter sterilization or autoclaving would be needed in order to eliminate indigenous flora
from Schoolcraft groundwater. An experiment was designed to determine whether the
organisms in the Schoolcraft groundwater were all psycrophilic or mesophilic bacteria. If
so, then incubation at higher temperatures without the excessive pressure and temperature
of an autoclave may provide adequate removal of indigenous flora. This pasteurization
process was tested, and the results are shown in Figure 6. Incubation of Schoolcraft
groundwater at 65° C for 8 hours removed all indigenous organisms that were capable of
forming colonies on nutrient agar plates. This process was used for all the following

experiments to determine growth of strain KC in Schoolcraft groundwater.

23

 

_9

100000

     

20° C Incubation

- - .....l -

65° C Incubation

CFU/ml
§

'8'

OH
O
- ----..J A ---...A

 

 

J
.1
1
.1
r
4
i
.1

0 2 4 6 8 10 12 14 16 18 20 22
Time (days)

Figure 6. Pasteurization effect on indigenous organisms by Schoolcraft
groundwater at 65° C.

CHAPTER 5

EFFECTS OF pH ON STRAIN KC AND THE INDIGENOUS
ORGANISMS IN SCHOOLCRAFT GROUNDWATER

Materials and Methods

Effect of various pH values on indigenous organisms in Schoolcraft
groundwater. Schoolcraft groundwater was adjusted to the desired pH values using a
sterile 100 mM Na2CO3 stock solution. This served to adjust the pH and as a carbonate
buffer which is the natrnal buffering system of the Schoolcraft aquifer. Amounts to be
added were determined by trial and error on test samples. Colony forming units were

measured using serial dilutions and nutrient agar plate counts.

Persistence of strain KC in groundwater. To evaluate long-term survival of strain
KC in Schoolcraft groundwater, strain KC ( 2x106-1x107 cells/ml) was added to
pasteurized Schoolcraft groundwater that had been adjusted to a pH of 8.2 using sterile 100
mM sodium carbonate solution and sterile CO2 gas. Pasteurized groundwater was used to
allow reliable enumeration of strain KC by plate counts. The water contained no added
carbon source or electron acceptor. Samples were removed at one to forn' day intervals and
assayed for strain KC using the serial dilution and bacterial plate count procedure described
above. After 14 days, the pH was adjusted to pH 7.5 by addition of carbon dioxide, and

levels of Pseudomonas sp. strain KC were monitored for an additional 7 days.

24

25
Results and Discussion

As previously discussed, alkaline pH values were suspected to be inhibitory to indigenous
organisms in Schoolcraft groundwater. This was thought to be a primary factor in the
competitive advantage of strain KC exhibited in previous model aquifer column studies.
Preliminary experiments (samples 4 and 6 of Table 3), however, indicated growth of
indigenous organisms in Schoolcraft groundwater at pH 8.2. To confirm this result,
Schoolcraft groundwater was adjusted to various pH values using a sterile Na2CO3 stock
solution, and the effect on the indigenous organisms was monitored using plate counts.
Results shown in Figure 7 exhibit little or no effect of various initial pH values on an
existing population of indigenous Schoolcraft organisms. Alkaline niche adjustment does
not reduce the concentration of indigenous organisms which will be in direct competition
with strain KC. The slight increase in organism concentration may have been due to cryptic

growth or to growth on carbon already present in the groundwater since none was added.

The persistence and containment of strain KC after release into an alkaline niche is of
interest for application purposes. Figure 8 illustrates long-term persistence of strain KC
under no-growth conditions (no electron acceptor or donor) and demonstrates a pH
dependence. A stable population of stationary phase strain KC (103-105 CFU/ml) rapidly
decayed upon a shift of pH from 8.2 to 7.5, resulting in a drop of three orders Of
magnitude in the population levels Of strain KC in 7 days. This experiment simulated what
would happen after substrate and/or base is no longer used to maintain the alkalinity of a
niche. Without substrate addition, denitrification will no longer occur and the increase in
pH caused by denitrification will also cease. If base is not added, carbonated groundwater
will eventually titrate the alkalinity. As shown in Figure 8, once the pH was decreased to
7.5, the concentration Of strain KC decreased dramatically. This decrease may have been

caused by the toxicity of copper to strain KC at near neutral pH values (Criddle et al.,

26
1990). This must not be taken to conclude that all strain KC cells will eventually die.

Replicate 2 shows persistence of a significant concentration of strain KC cells well after pH
adjustment. It is highly likely that mutations may allow strain KC to survive at pH 7.5 and
continue to persist in the aquifer (Lensld, 1992). This should not be a serious concern as
the pathogenicin and toxicity of strain KC to plant and animal life has been tested and no

adverse effects were reported (M. Dybas, personal communication).

 

 

 

 

 

 

 

 

 

 

 

10 1
l
1 pH 7.7 ..-I
10"; pH 8.0 .. -
1
1051
S ‘ pH 9.0
U
1041 pH 8.2
i
l
1031
1 Jr
1
102 T T 1 T— V T ' I ' l " l ' U "' 1
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
Time (days)

Figure 7. Effect of various initial pH values on an existing population of
indigenous aquifer organisms in Schoolcraft groundwater.

27

 

   

 

 

 

10°
107
105 § pH adjusted from 8.2 to7.5
105 \
E
E 10
U
103 .
ReplrcateZ
10
Replicate]
1
10 Replicate3
10° A
T'I‘I'lfHTT‘l‘j I ITI'ITI'I
o 2 4 6 81012141618202224262830

Time (days)

Figure 8. Persistence of strain KC under nO-growth conditions in Schoolcraft groundwater
at pH 8.2. After 2 weeks of incubation, the pH was reduced to 7.5.

CHAPTER 6

KINETIC PARAMETER CHARACTERIZATION OF STRAIN KC AND
THE INDIGENOUS ORGANISMS IN SCHOOLCRAFT GROUNDWATER

Materials and Methods

Kinetics of growth of strain KC and indigenous groundwater organisms in
niche adjusted groundwater. In order to evaluate the kinetics of growth of strain KC
and the indigenous Schoolcraft groundwater organisms and their specific rate of utilization
of acetate, nitrate, and nitrite, 500 mL Wheaton bottles containing pasteurized Schoolcraft
groundwater (not pasteurized for the Schoolcraft organism samples) were supplemented
with 30 mM acetate, 12 mM nitrate, 0.1 mM phosphate, and 10 g Nal-ICO3/L to give an
initial pH of 8.2. The groundwater was previously pasteurized by heating at 65°C for 8
hours. Pasteurization was used to remove indigenous flora because autoclaving produced a
precipitate that interfered with organism growth and filter-sterilization had a similar adverse
effect on organism growth (Chapter 4). Strain KC was added as a 1% inoculum from an
aerobic culture grown for 72 hours in medium D and washed in pasteurized Schoolcraft
aquifer water. Growth of both strain KC and Schoolcraft flora was followed by optical
density measurements, plate counts, and dry weight. Acetate, nitrate, and nitrite utilization

were followed by ion chromatography.

The maximum specific growth rate 11m was obtained for log growth phase cells using
optical density measurements at a wavelength of 660 nm and the relationship: 11m =

[1n(Xf/Xi)] / (tf-ti), where Xf and Xi represent the final and initial Optical density,

28

29
respectively, and tf and ti are the final and initial time, respectively. The observed yield on

nitrate and nitrite for both cultures was calculated using dry weight measurements Obtained
by filtering a known volume through a 0.2 um pore filter membrane (Gelman Sciences)
and washing the collected cells with phosphate buffer adjusted to pH 5. The filter
membranes were then dried overnight at 104° C and weighed. The filters were dried in this
fashion and weighed before the cells were filtered allowing for an accurate measure to be
obtained. Ion chromatography was used to measure the nitrate and nitrite consumption. The
dry weight biomass concentration of each sample was corrected for the slight precipitate
present in uninoculated, pasteurized groundwater. The observed yield was calculated by
dividing the corrected dry weight biomass concentration increase by the total nitrate or
nitrite consumed over the identical time period. Actual substrate data at specific time points
were used but (by weight measurements were obtained from the plot Of dry weight versus
time. This was necessary due to the fact that substrate and dry weight data time points were
taken at different times throughout each day. The maximum specific rate of nitrate and
nitrite utilization km was estimated by dividing the instantaneous rates of nitrate or nitrite
consumption by the dry weight biomass concentration at that time. See Appendix B for raw

data and supporting calculations.

To determine the half-velocity coefficient Ks cells were grown as in the determination of
um, Y, and km but were harvested anaerobically during log phase. Cells were centrifuged
and washed with Schoolcraft groundwater supplemented with 10 mM acetate and 0.1 mM
phosphate. The final cultures contained approximately twice the biomass concentration of
the original cultures to ensure rapid nitrate utilization. Schoolcraft groundwater was
supplemented with additional nitrate to give a final nitrate concentration of approximately
1.3 mM. Samples were taken about every half hour, frozen, and later analyzed for nitrate

and nitrite using ion chromatography.

30
Results and Discussion

As shown in Figures 9.1 through 14, both strain KC and the Schoolcraft groundwater
flora exhibited similar biphasic growth patterns at an initial pH of 8.2: in each case, nitrate
was first converted to nitrite, and the nitrite was subsequently converted, presumably to
NO, N20, and N2 although these gaseous products were not quantified A longer lag
phase occurred with the Schoolcraft flora, but this is probably due to differences in the
number Of the organisms initially present (105/mL for Schoolcraft flora vs. 106/mL for
strain KC). The increase in pH exhibited for KC and for the Schoolcraft organisms is
expected due to denitrification. This effect is dependent on the buffering capacity of the
system. Schoolcraft groundwater was amended with 10 grams sodium bicarbonate per
liter, but the buffering capacity of the carbonate system is at a minimum around pH 8.
Highly buffered groundwater may prevent this increase in pH. This increase in pH may be
advantageous for field scale niche adjustment. With KC producing alkaline conditions as a
by product of growth, no supplemental base may be needed to produce alkaline conditions.
Note the higher pH values obtained in the KC samples coinciding with the higher values
for optical density and dry weight.

Results of kinetic characterization under alkaline conditions reveal a competitive advantage
for Pseudomonas sp. strain KC over the indigenous Schoolcraft organisms. As shown by
the Optical density, plate count, and dry weight data in Figures 9.1 through 14 and by the
growth parameters in Table 4, a critical difference between strain KC and Schoolcraft flora
was in the observed yield: for the first phase of growth (nitrate to nitrite), the observed
yield for strain KC was four times the Observed yield for the Schoolcraft flora. This
translated into a fourfold higher maximum specific growth rate for strain KC. Because Of

its high yield, strain KC also has a higher value for fs, the fraction of electrons diverted for

cell synthesis. Snain KC is able to utilize over half the available energy for cell synthesis.

31
Supporting data for the parameters in Table 4 are located in Appendix B. A possible

explanation for this yield difference is the competitiveness of strain KC under iron limiting
conditions. Dybas et al. (1994 a) have shown that strain KC produces a wide spectrum Of

iron-binding activities at pH 8.2.

In this thesis, kinetic parameters were determined for Pseudomonas sp. strain KC and the
indigenous microorganisms in Schoolcraft groundwater. As previously mentioned, l-lrn for
P. KC was four times larger than the 11m for the indigenous organisms. The Ks's for the
two organism types were virtually indistinguishable. Decay rates were not obtained and
thus 3min values could not be accurately calculated. However, in light of the large I’m for
P. KC, a relative value for 3min significantly lower than that for the indigenous flora can
be predicted Assuming relatively equal decay rates, resource-based competition theory
predicts that if the strains also have equal Ks's, the strain with the higher 11m outcompetes
all other species (Hansen et al., 1980). Classical competition theory asserts that two-
species outcomes are independent of the intrinsic rates Of increase of the two species, a
claim that the more mechanistic resource-based approach shows to be incorrect (Hansen et
al., 1980). Pseudomonas sp. strain KC will outcompete the indigenous microorganisms in
Schoolcraft groundwater under alkaline conditions on the grounds of resource-based

competition theory and the assumptions above.

32

9.2
9.0
8.8
8.6
8.4

pH

 

1'6 18

 

r 0.30

    

U)
C
1-

" 0.25

A

Di

Acetate -- 0.20

u—o
M

 

Concentration (mM)

 

 

 

0 2 4 6 8 10 12 14 l6 18
Time (days)

Figure 9.1. Growth pattern, substrate utilization, and pH increase for indigenous aquifer
organisms in Schoolcraft groundwater adjusted to an initial pH of 8.2. (Replicate #1)

Optical Density (660 nm)

33

 

 

 

 

 

I- 0.20

>- 0.15

r-O.‘IO

Concentration (mM)
Optical Density (660 nm)

1o . . Nitrite

‘ Optical Density t

5 q ‘ - 0.05
‘ ’ A k

0 ... .... ‘1‘

 

 

- ,..-. - -o.oo
o 2 4 6 a 10 12 14 16 13

Time (days)

Figure 9.2. Growth pattern, substrate utilization, and pH increase for indigenous aquifer
organisms in Schoolcraft groundwater adjusted to an initial pH of 8.2. (Replicate #2)

pH

 

9.2
9.0
8.8
8.6
8.4

 

   

Nitrate

 

i6
30;
1
i
25.1 A
cetatt
E :
5 20;
.5 :
g 15.‘
g 1
U

 

" 0.30

" 0.25

. 0.20

" 0.15

"' 0.10

' 0.05

 

 

0 2 4 6 8 10 12 14 16
Time (days)

 

0.00

pH

Optical Density (660 nm)

Figure 9.3. Growth pattern, substrate utilization, and pH increase for indigenous aquifer
organisms in Schoolcraft groundwater adjusted to an initial pH of 8.2. (Replicate #3)

35

550-
500‘
450‘

350‘

Dry Weight (ms/L)

 

 

 

Figure 10. Biomass accumulation of indigenous organisms in Schoolcraft groundwater
measured as dry weight.

36

109

CFU/ml

10‘

 

105

 

10‘ ' - r
0 2 4 6 8 10 12 14 16 18
Time (days)

' I ' T fi U ' U V I ' 1 V U f 1

Figure l 1. Growth of indigenous organisms in Schoolcraft groundwater measured by
bacterial plate counts.

37

 

 

 

 

 

 

9.2 ~9.2
9.0 _ _ — ~9.0
8.8 L33
I . :1:
9 8.6 -8.6 a
8.4 L8.4
8.2 7 .
0 2 4 6 8 10 12 14 16 1882
Time
35 (daYS) -o.35
30 . -o.3o
1 Optical Density
25.: r025 A
E a ' §
7:? 20: Acetate mm 3’.
O ‘ ....
t: 151 Nitrate -O.15 g
i? 1 a
8 ' .—
10. -o.10 3'
5% -o.os
0 4 - 0.00
0 2 4 6 8 10 12 l4 l6 18
Time (days)

Figure 12.1. Growth pattern, substrate utilization, and pH increase for Pseudomonas sp.

strain KC in Schoolcraft groundwater adjusted to an initial pH of 8.2.

(Replicate #1)

38

 

f 9.2
P 9.0
L 8.8
l 8.6
' 8.4

 

 

10 1'2

 

35 (dayS)

.AAIAAA-IA‘AA

8

Nitrate

H
U!
‘..|

Concentration (mM)

10:

 

 

 

0 2 4 6 8 10 12
Time
(dayS)

1'4 16

14

Optical Density

Acetate

16

18 8.2

l- 0.30
- 0.25
' 0.20
i. 0.15

" 0.10

.. 0.05

 

r 0.00
18

pH

Optical Density (660 nm)

Figure 12.2. Growth pattern, substrate utilization, and pH increase for Pseudomonas sp.
strain KC in Schoolcraft groundwater adjusted to an initial pH of 8.2. (Replicate #2)

39

 

 

 

Optical Density (660 nm)

 

 

 

9.2
9.0
8.8 I
8.6 9-
8.4
18 8.2
35 (daYS) no.3o
30 l
~025
25.‘ . . ’
: Optlcal Denslt) P020
3 .
5 20: .
'8 1 ' 015
a . Nitrate Acetate '
§ 15. b
8 1
i-O.10
U 10..
' L
5.: -o.os
0‘ ._ 4 . . . _o_oo
0 2 4 6 8 10 12 14 16 18

Time (days)

Figure 12.3. Growth pattern, substrate utilization, and pH increase for Pseudomonas sp.
strain KC in Schoolcraft groundwater adjusted to an initial pH of 8.2. (Replicate #3)

550‘
500‘
450‘

350‘

DIV Weight (mam)
E

Replicate #2

Replicate #1

 

 

 

Tune (claw)

Figure 13. Biomass accumulation of Pseudomonas sp. strain KC in Schoolcraft

groundwater measured as dry weight.

41

 

 

 

10 91 .l
: fl" ""~ ‘ ‘
I
. VI . .
. laboratory temp. Replleate #3
disturbance ,
10 8. Replicate #1
i ..‘ an»
E 1 Replicate #2
lo 71
1
10 6 ' I f I f I ' I I I fl I ' I ' I ' I ' I f T V I ‘ I ' I '
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

Time (days)

Figure 14. Growth Of Pseudomonas sp. strain KC in Schoolcraft groundwater measured
by bacterial plate counts.

42

Table 4. Kinetic parameters and yield coefficients for strain KC and Schoolcraft aquifer
flora in Schoolcraft groundwater adjusted to an initial pH of 8.2.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

W
Kinetic Parameter KCl Floral

Nitrate to nitrite
[2,)“, max. specific growth rate during N03' 6311 6:1

nversion to NO; (cl-1)

bserved yield (mg cell dry weight per mg Ofli 0.04 0.05:1: 0.01
NO3'converted to NOz')
Observed yield (109 CFU per mg NO3' 0.64 :t 0.19 0.033: 0.02—
converted to NQ')
km, Maximum specific rate of nitrate removal TH: 1.8 W 1. :1: .
(mg NO3' per mg cell dry weight per day)
'R—atio Acetate to N03' 00,13qu 1.01 :1: 0.03 1.05 :t 0.01
Nitrite to gaseous end products
um, max. specific growth rate during N02' 0133: 6.65 6.3721: 6.63
conversion to gaseous end products (d'l)
Observed yield (mg cell dry weight per mg 0.46:1: 0.18 0.18:1: 0.07—
NOz'leduced)
NOz'reduced)
km, Maximum specific rate of nitrite removal 0.59: 0%— 3371 0.30
(mg NOz'per mg cell dry weight per day)
Nitrate to gaseous end products
Overall Observed yield (mg ceH dry weight 0.40: 0.04 0121—0703-—
per mg N031 ,
vaet-all observed yield (109 CFU per 1.01 :t 631 5.53 i 5153
mg N03')
f5, fraction of electrons diverted for synthesis 0.61 :1: 0.03 0m—
fc, fraction of electrons used for energy 0.39 i 0.03 0.3’2 i 0.03 I
23: weight per cell 007 pgper CFU) 4.08 i 1.52 3.09 :l: 1.10

 

 

 

 

 

1 Average :1: one standard deviation for three independently grown cultures at 21.1°C.

43
The half-velocity coefficient Ks is another microbial kinetic parameter effecting competition

between microbial groups. It is numerically equal to the nutrient concentration at which the
specific growth rate is half of its maximum value (Brock er al., 1991). The approximate
values can be estimated by identifying the points at which the plots in Figures 15 and 16
change from first order to zero order. As shown in theses figures, the nitrate half-velocity
coefficients for strain KC and the indigenous Schoolcraft organisms are essentially

indistinguishable.

1.5
1.4
1.3

1.2
1.1 .. Nitrite
1.0- /

0.9 1 ‘
0.8 1
0.7 r
0.6 1
0.5 1
0.4 1
0.3
0.2

0. 1
0.0

Nitrate

Concentration (mM)

 

Model

“A 4A” ~u .

012345678 9101112131415161718192021
TimeChours)

 

Figure 15. Nitrate and nitrite utilization by indigenous aquifer organisms in Schoolcraft
groundwater adjusted to an initial pH of 8.2.

15
1.4
1.3 _
1.21 ‘ y” .

1.1 1 Nitrite
1.01
0.9 -
0.8 1
0.7 -

0...; it“

Coneentntim (mM)

05 1
0.4 1

0.3 1 X . .
0.2 1
0.1 1

0.0 T11 I'
5

 

 

 

T
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Timealours)

Figure 16. Nitrate and nitrite utilization by Pseudomonas sp. strain KC in Schoolcraft
groundwater adjusted to an initial pH of 8.2.

Calculated values for the half-velocity coefficients (Ks) for strain KC and the Schoolcraft
organisms can be found in Table 5. These values were calculated using the computer
modeling software package Systat®. This model computes the best fit value for Ks using
time and nitrate depletion data. The maximum specific rates Of substrate utilization km
shown in Table 5 are comparable to approximately half the value of those shown in Table
4. This is under the assumption that prOtein represents half the dry weight of each cell. See
Appendix C for raw data and calculations supporting the parameters in Table 5. The model
plots in Figures 15 and 16 were developed by using the average Ks, km, protein, and

nitrate data for triplicate samples in the relationship below:

T = (Ks*ln(So/S)/(km*X) + (SO-S)/(km*X)

45
where:

Ks = the half-velocity coefficient

So = the initial nitrate concentration

S = time dependent nitrate concentration

km = the maximum specific rate of substrate utilization (mg NO3-lmg dry weight *hour)

X = average protein concentration

The above equation is based on the theory that substrate utilization generally follows a
similar kinetic model as that for bacterial growth (Lawrence et al., 1970). It has the same
form as the well-known Michaelis—Menton expression for enzymatic degradation, but it is
an empirical relationship based on observed patterns of substrate consumption by whole
cells, and its coefficients may or may not be related to the activity of a specific enzyme. As
shown in Figures 15 and 16, the model does not fit the actual data perfectly. This may be
caused by conditions in which a nutrient or nutrients other than nitrate are limiting growth.
The initial lag period of the data could be the result of cell acclimation to a new environment

(Brock er al., 1991).

As discussed in the introduction of this thesis and the beginning of this chapter, it is
apparent that strain KC has the competitive advantage on the grounds of resource-based
competition theory. Assuming approximately equivalent decay rates for strain KC and the

Schoolcraft organisms, strain KC has a much lower 5min and hence would be expected to

outcompete the indigenous organisms at alkaline pH levels (>8).

Table 5. Half-velocity coefficients and maximum specific rates of substrate utilization for
Pseudomonas sp. strain KC and the indigenous organisms in Schoolcraft groundwater.

 

 

 

 

 

 

 

 

 

 

W
Kinetic Parameter KCl Floral

Nitrate to nitrite

Maximum specific rate of nitrate removal, km
L(mg NO3' per mg protein per day) 19.51 :1: 2.41 21.68 i 0.49

velocity coefficient, Ks
(mg NO3'IL) 11.97 :1: 1.34 9.40 d: 0.32
itrite to gaseous end products
Maximum specific rate of nitrite removal, 1cm
(mg NOz‘ per mg protein per day) 0.77 :1: 0.58 5.60 :1: 0.22

 

1 Average :1: one standard deviation for three independently grown cultures at 21.1°C.

CHAPTER 7

ENGINEERING APPLICATION

There has been much discussion in the literature concerning the introduction of genetically
engineered microorganisms into the environment. Pseudomonas sp. strain KC is not a
genetically engineered microorganism, but many of the concerns surrounding its
introduction are addressed in such reports. The introduction of novel organisms into a new
environment is termed bioaugmentation. This process has much untapped potential for

remediation of many environmental contaminants in a variety of treatment schemes.

Many important scientific issues must be considered in evaluating the potential ecological
consequences of the planned introduction of organisms into the environment. These include
survival and reproduction of the introduced organisms, interactions with other organisms in
the environment, and effects of the introduced organisms on ecosystem function (Tiedje et
al., 1989). When considering the environmental application of some microorganism, one
of the most important series of questions to ask concerns the opportunity for persistence of
the population after it has been introduced into the target environment (Lenski, 1992). Is it
desirable for the introduced population to be self-sustainin g? Or is it better if the introduced
population performs its intended function and then dies out, being reintroduced only as
need arises? The answer will depend, of course, on a comparison of the magnitude of the
additional benefits that may derive from prolonged persistence with the possible costs, if
any, that might arise from potential adverse effects caused by persistence (Lenski, 1992).
Once this comparison has been made, it is appropriate to ask: What efforts, if any, have

been made to enhance or limit the persistence

47

48
of the introduced population, as so desired? Results contained herein have shown that

persistence and relawd containment of strain KC can be controlled to a certain degree by
alkaline niche adjustment. Alkaline conditions have been shown to support a stable
population of strain KC which rapidly decreased upon lowering the pH to 7.5. Copper
toxicity is the suswa cause for the rapid decay of strain KC under near neutral
conditions (Criddle et al., 1990). However, this effect is not immune to mutations within

strain KC which may allow it to persist.

Another necessary question to ask is: What empirical data are there concerning the
indefinite persistence of the introduced microbial population in the target enviromnent
(Lenski, 1992)? Figure 8 provides evidence for the ability to contain strain KC through pH
adjustment, but there is also evidence for the lack of ability to completely eliminate its
persistence. It is quite likely that strain KC will not completely die out but will continue to
persist. Lewis and Crawford (1993) observed persistence of strain KC for one year in
aquifer materials stored at 4°C. This may be the result of genotype differences between
generations. A key element in determining the likelihood of persistence of any introduced
organism is its fitness in the new environment (Lenski, 1992). In most cases, a target
environment will be supporting an indigenous population that is closely related to the
organism proposed for introduction. Interactions between these two organism types is
likely to be quite significant for the fate of the introduction; even slight differences in kinetic
parameters or the ability to withstand various conditions may affect the opportunity for
persistence of the introduced population. It becomes clear that the fimess of an introduced
organism relative to a closely related indigenous population is likely to be especially useful
in predicting the fate of an introduced population (Lenski, 1992). This thesis has addressed
the fitness of Pseudomonas sp. strain KC in comparison to the indigenous organisms in
Schoolcraft groundwater. Inherently the Schoolcraft groundwater contains organisms that

are somewhat related to strain KC, but it is possible that the most competitive organisms

49
are fixed to soil particles in the Schoolcraft aquifer and not present in the groundwater that

was used for the experiments in this thesis .

A textbook definition of fimess is 'The average contribution of one allele or genotype to the
next generation or to succeeding generations, compared with that of other alleles or
genotypes' (Lenski, 1993). The Darwinian fimess of an organisms therefore refers to its
capacity for survival and reproduction, which depends on its environmental circumstances
as well as on its genotype (Lenski, 1993). Fitness of an organism is dependent upon
environmental conditions. Therefore, fitness is best regarded as a relative property, not an
absolute one (Lenski, 1992). The results contained in this thesis have shown that under
alkaline conditions, strain KC has the competitive advantage over the indigenous organisms
(as a group) in Schoolcraft groundwater. This is not to infer that strain KC is the best

competitor under all environmental conditions.

The concept of niche adjustment has broad implications for bioaugmentation efforts, where
competition with native microorganisms is a major hurdle. Addition of alkalinity is a
simple procedure that may be effective at certain sites. Of course, many other niche
adjustment strategies can be envisioned. The optimal choice of strategies will depend upon
the physiology of the organism to be introduced, the nature of the indigenous organisms,

and prevailing environmental conditions at a targeted site.

CHAPTER 8

CONCLUSIONS

Pseudomonas sp. strain KC has a higher yield than Schoolcraft aquifer organisms

at all pH values in medium D.

Alkaline pH values are optimal for growth of strain KC in medium D.

Niche adjustment does not reduce the concentration of indigenous organisms.

Strain KC will not persist at significant concentrations in a post niche-adjusted

environment.

Higher maximum specific growth rate and yield are the primary reasons for the

competitive advantage of strain KC under moderately alkaline conditions.

The half-velocity coefficient (Ks) for nitrate is insignificant in the assessment of

strain KC's growth advantage.

Using resource-based competition theory, a lower estimated 3min is evidence that

strain KC would outcompete the indigenous organisms in Schoolcraft groundwater.

The competitiveness of strain KC and its ease of transport through columns packed with

Ottawa sand and Schoolcraft aquifer material (Mayotte et al., 1994) indicate that field-scale

50

5 1
studies are jusu'fied. It may be possible to establish and maintain a (Tr-degrading zone or

biofence by colonizing a pH-adjusted region in front of a migrating CI' plume. Considering
the diverse environments that strain KC was able to colonize and remediate (Dybas et al.,
1994 b), it can be concluded that alkali niche-adjustment is a useful means of maintaining a

competitive population of strain KC under non-sterile operating conditions.

FUTURE WORK RECOMMENDATIONS

1. Complete lo'netic growth parameter characterization of strain KC and the indigenous

organisms in Schoolcraft groundwater at pH 7.5.
2. Determination of the kinetic growth parameters of strain KC and the indigenous
organisms in Schoolcraft groundwater with nitrite as the sole initial electron

acceptor.

3. Determination of the minimum P. KC dose needed for adequate colonization in the

Schoolcraft aquifer

4. Enumeration of denitrifying bacteria in the Schoolcraft aquifer.

LIST OF REFERENCES

10.

11.

LIST OF REFERENCES

Brock, TD., and MT Madigan. 1991. WWW, Prentice Hall:
Englewood Cliffs, New Jersey.

Criddle,C. S., L. A. Alvarez, andP. L. McCarty. 1991. J. Bear andM. Y.
Corapcioglu (eds 1W Kluwcr Academic
Publishers. The Netherlands, 639-691.

Criddle, C.S., J .T. DeWitt, D. Grbric-Galié, and PL. McCarty. 1990.
Transformation of carbon tetrachloride by Pseudomonas sp. strain KC under
denitrification conditions. Appl. Environ. Microbiol. 56:3240-3246.

Dybas, M. J ., G. M. Tatara, and CS. Criddle. 1994(a). Localization and
characterization of the carbon tetrachloride transformation activity of Pseudomonas
sp. strain KC. Submitted for publication in Applied and Environmental
Microbiology.

Dybas, M. J., G. M. Tatara, W. H. Knoll, and CS. Criddle. 1994(b). Alkaline
niche adjustment to enable colonization and remediation by Pseudomonas sp. strain
KC. Submitted for publication in Environ. Sci. Technol.

Hansen, S. R., and S. P. Hubbel, 1980. Single-nutrient microbial competition:
qualitative agreement between experimental and theoretically forecast
outcomes. Science, 207: 1491 - 1493.

Lawrence, A. Wm, and P. L. McCarty, 1970. Unified basis for biological
treatment design and Operation, Jour. Sanitary Engineering Division, Amer. Soc. of
Civil Engineers, 96/SA3, 757-778.

Lenski, R. E., 1992. M. A. Levin, R. J. Seidler, andM. Rogul, (eds). Relative
fitness: its estimation and its significance for environmental application of

microorganisms WWW.
McGraw-Hill, Inc. New York, Chapter 9, pg. 183-198.

Lenski, R. E., 1993. Evaluating the fate of genetically modified microorganisms in
the environment: Are they inherently less fit? Experientia, 49(3): 187-276.

Lewis, T. A., and R. L. Crawford. 1993. Physiological factors affecting carbon
tetrachloride dehalogenation by the denitrifying bacterium Pseudomonas sp. strain
KC. Appl. Environ. Microbiol., 59: 1635-1641.

Markwell, M.A., S. M. Haas, N.E. Tolbert, and LL. Bieber. 1981. Protein

determination in membrane lipoprotein samples: manual and automated procedures.
Methods Enzymol., 72:296-301.

52

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

53

Mayotte, T. J., M.J. Dybas, and CS. Criddle. 1994. Bench-scale evaluation of
bioaugmentation to remediate carbon tetrachloride-contaminated aquifer materials.
Submitted for publication.

Monod, J., 1942. Recherches sur la croissance des cultures bacteriennes, Hermann
and Cie, Editors, Rue de la Sorbonne, Paris.

Rittrnan, B. E., and P. L. McCarty, 1980. Model of steady-state-biofilrn kinetics.
Biotechnology and Bioengineering, 22:2343-2357.

Semprini, L., G. D. Hopkins, P.L. McCarty, and RV. Roberts. 1992. In-situ
transformation of carbon tetrachloride and other halogenated compounds resulting
from biostimulation under anoxic conditions. Environ. Sci. T echnol., 26:2454-
2461.

Stumm, W. and J.J. Morgan. 1981. W, 2nd ed.; John Wiley &
Sons: New York, p. 248.

Tatara, G.M., M.J. Dybas, and CS. Criddle. 1993. Effects of medium and trace
metals on kinetics of carbon tetrachloride transformation by Pseudomonas sp. strain
KC. Appl. Environ. Microbiol., 59:2126-2131.

Taylor, P. A., and P. J. Lab. Williams, 1975. Theoretical studies on the
coexistence of competing species under continuous-flow conditions. Can. J.
Microbial. 21:91-98.

Tiedje, J. M., R. K. Colwell, Y. L. Grossman, R. E. Hodson, R. E. Lenski, R.
N. Mack, and P. J. Regal, 1989. The planned introduction of genetically
engineered organisms: ecological considerations and recommendations. Ecology,
70(2):298-315.

Tilman, D. 1981. Tests of resource competition theory using four species of Lake
Michigan algae. Ecology, 62(3):802-815.

van Uden, N., 1967. Transport-limited growth in the chemostat and its competitive
inhibition; a theoretical treatment. Archiv fur Mikrobiologie, 58:145-154.

APPENDIX A

APPENDIX A

ORIGINAL DATA AND CALCULATIONS FOR PSEUDOMONAS SP.
STRAIN KC KINETIC PARAMETERS IN MEDIUM D.

Table A-1. Original data used for determination of um, b, and protein per cell for P. KC in
medium D.

verage . .
(hr) Protein /ml Protein/ml .KC Cells/ml .KC Cells Prot./Cell

 

was not a

Average ttg Protein/Viable P.KC Cell = 1.72 X 10-7 i 1.01 X 10-7
The correlation for the above relationship is shown by R2 = 0.996

Time points 21 - 54 hours used to ensure that cells were in log growth phase and to avoid
including dead cell protein in calculations.

54

55
Table A-2. Data used for calculations of 11m and b for Pseudomonas sp. strain KC.

 

um (days-1) = 10.73
Overall b (days-1) = 8.92

Time range for calculation of overall b = 60 to 78 hours.

Balanced stoichiometric equations (shown below) using f3 and fc were used for the

theoretical determination of the ratio of acetate to nitrate for cells (Criddle et al., 1991).

Rd: 1/8 CH3coo- + 1/4 H20 = 1/4 coz + 7/8 11+ +e-
stc: f5 * [5/28 C02 + 1/28 NO3' + 29/28 H+ + e' = 1/28 C5H702N + 11/28 H20]
fcRa: fc * [1/5 N03: + 6/5 11+ + e' = 1/10 N2 + 3/5 H20]

where:

= half reaction for the oxidation of an electron donor normalized by the moles of
electrons removed from the donor.

Ra = half reaction for the reduction of an electron acceptor used for energy normalized by
the moles of electrons added to the acceptor.

RC = half reaction for the reduction of an electron acceptor used for synthesis normalized
by the moles of electrons added to the acceptor, and f3 + fe =1.

56
Using the balanwd equations above and yield data, the following relationship can be made:

Yield = W219 :-
[fe (1/5) +fs (1/28)] 62 g NO3'lmole e’

 

Using the relationship f¢ = l-fs, the values for fc and fs were calculated It was assumed
that all the nitrate was converted to nitrogen gas and that the dry weight of each cell was

equal to twice the measured protein. The maximum overall protein measurements were

used in the above calculations. The assumed formula for cells was C5H702N which has

not been proven to for Pseudomonas sp. strain KC.

APPENDIX B

APPENDIX B

ORIGINAL DATA AND CALCULATIONS FOR KINETIC PARAMETERS
IN SCHOOLCRAFT GROUNDWATER.

Table B-1. Data used for determining the maximum specific growth rate 11m of Schoolcraft
organisms in Schoolcraft groundwater.

 

 

 

 

 

 

 

 

 

11m 011 N03‘ =(lnX2/X1)/(t2-t1) 11m 011 N02’ =(1nX2/X1)/(t2-t1)
(da s-1>
rrne -1 - — ' - -
(dags)
. - 0.76 7.021- 0.69
3.604 8.042
T5712. - 1.04 . 1- 0.74
5.875 7.500
3.604- 062' "77521- 0.59
4.125 7.50
Average 0.81 Average 0.67
Standard 0.21 Standard 0.08
Dev. Dev.

 

 

 

 

 

 

 

 

 

 

57

58

Table B-2. Data uwd for determining the maximum specific rate of substrate utilization
(km) of Schoolcraft organisms in Schoolcraft groundwater.

8:1mMaxNO3‘ Util. -1 Max N02 Util.

03 (mM 02- (mM) I' - ,-
- dam?» mine-mi
50379

7. 11L7l- 0.— -I.o°6 I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

    
 

 

 

 

 

7. 02
with; 0221a;
m /m cells*d) m /m cells*d)
34 E 11.69 24 .51
me I 03 (mM) I' ate (mg N03 ““68 I02' (W) is (mg N02
(days) *d) *d)
7....85y-) ‘.I5- I'- '0. 0
4.13 7. 02 1.031
Dry tan-2 Dry ltm-f
Weight (mg NO3- Weight (mg N03-
(mg/L) /m cells*d) (mg/L) /mg cells*d)
—'_2'7'_E'—21 .51 50] 3721
{-s 3 Max N03 Util. 1-3 Max NOz’Util.
Time I (mM ) I’ are (m N - imc I 02' (mM)
a...) a...)
‘4.1-..rt -i.7 -.11. t .47.1 - 4.4 -
5.88 0.284

 

 

Dry km-3
Weight (mg NO3'
(mg/L) /mg cells*d)

Dry kin-3
(mg/L) /mg cells*d)

 

 

 

 

 

 

 

 

28 10 80] 48 3. 48

: choolcr t ' Max. pec' c Nltrate i choolcraft ora Max. pec 1c Nltnte .
t111tion,krn ”tilization, km ,
1 ..’

 

 

 

 

 

59

Table B-3. Data used for determining the yield of Schoolcraft organisms in Schoolcraft
groundwater on nitrate reduced to gaseous end products .

1e . Data( rtratetogaseousenc p nucts)

 

 

 

 

 

 

 

 

 

 

- usrn est o we1_.htv ue) ove . rhest
NO3' I' . 1e . late ieH
Consumed 918m cunts
(mM) mg/L) mg cells/mg CFU/ml) 10"9 CFU/
' 03') , gNO3‘)
I 12.003I 93| O.12I1.50E+08] 0.20
- (usin' iar est - wei tvalue) overall hi_hest) I
N03' ID . 1e’r ' te reld
Consumed 518m ' unts
(mM) mg/L) mg cells/mg CFU/ml) 109 CFU/
I 03’) , gN03‘)
I 11.931I 68] 0.09I2.20F.+08 0.30
- (usin larest c wei ht value) overall hihest) I
1003- II Yield 'late reld
Consumed eight ‘ unts
(mM) mg/L) mg cells/mg CFU/ml) 109 CFU/
I 03') g NO3')

 

 

[ 11.938[ 108V 0.15I2.00E+08| ’ 0.27

60

Table B-4. Data used for determining the yield of Schoolcraft organisms in Schoolcraft
groundwater on nitrate reduced to nitrite.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

re ata (mtrate to nitrite)
- = 28)
NO3' Dry Yield
Consumed Welght
(mM) (mg/L) (mg cells/mg
N03' )
0.04 3 DOE-+07] 0.0
- = days) i
NO3' Dry Yield
Consumed W918i“
(mM) (mg/L) (mg cellS/mg
NO3' )
11.931 3 0.05 4 OOE+06 0.01
Piate Yield
Counts
(CFU/ml) (10"9 CFU/mg
N03“ )
ZOOE+O7 00

 

 

 

 

 

 

61

Table B-5. Data used fa determining the yield of Schoolcraft organisms in Schoolcraft
groundwater on nitrite reduced to gaseous end products.

ie ata 1tnte to gaseous end p cts)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

- =.-.1 s) 5.-.£1ays)
NOz' Dry Yleld 1002- Plate Yidld
Consumed W913” Consumed Counts
(mM) (mg/L) (mg cells/mg (mM) (CFU/m1) (1009 CFU/mg NOz')
N02')
6.791 63] 0.20] 5.5| 12015378 0.47
(5W -1 .71 days)
N02“ Plate Yield
Consumed Counts
(mg cells/mg (mM) (CFU/ml) (1009 CFU/mg N02“)
‘ 7.161 32] 0.101 77701 2.16E+O8 ' 0.61
To. - 0.7Ldays)
NOT—- Plate Y'ield
Consumed Counts

(mM) (CFU/m1) (1049 CFU/mg N02')
0.24 4.422 1.80E+08 0.88

 

 

 

 

 

 

 

 

 

 

Table B-6. Data used for determining the maximum specific growth rate (11m)
of Pseudomonas sp. strain KC in Schoolcraft groundwater.

' “In on NC)3. =(lnX2/X1)/(t2-t1) ttm on NOZ‘ =(lnX2/X1)/(t2-t1) i
If- “131)” ___ _,_ __ _____ ___ ___ __ ____)____.___ __ __ 1

    
 

_____ _ . ..__.

 

 

 

 

 

 

 

 

- - - rrne - - -

(da 5) (da 3)

l. 75- 3.21 5.021- 0.30 0.25

2.375 5.826

1.938- 376'. "Ti. 8 9.875- 0.13'
2.250 11.125

Average 3.1 1 Average 0.23

Standard 0.69 Standard 0.09

Dev. Dev.

 

 

 

 

 

 

 

 

 

 

62

Table B-7. Data used for determining the maximum specific rate of substrate utilization
(km) of Pseudomonas sp. strain KC in Schoolcraft groundwater.

 

 

 

 

 

 

 

 

 

 

 

 

    
    
   

 

 

 

 

 

 

 

 

 

P. KC -1 Max NO3‘ Utll :. -1M N- Util. f 1
”me I ()3- (mM) I' re rrne I 07011114) ,. te
(days) mg NO3‘/L*d) (days) mg N02’/L*d)
.OI - '.i - I 150.5. - 6.5 4- I 313.54
1.94 7.157 4.46 2.717
Dry km
Weight (mg N03- Weight (mg NOz'
(mg/L) oclls*d) (mg/L) g cells*d)

129] 1 1.68 I 390] 0.80]
l_ UIII- ___7 777_"77'7__7 I: ” _7._ [fl
rrne I 03‘ (mM) 1’ te (mg NO3‘ me I 02' (mM) 1' ate (mg N02'
7.38 - 5.595 - i 2T67

2.25 3.061
Dry ltm-z Dry ltm-2 ’
Weight (mg N03“ Weight (mg NO3‘
(m ) lmg cells*d) (mg/L) /m g cells*d)
114] 10.60] | 310] 0.68]

 

 

 

 
  

 

 

 
   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ate (mg N03' rrne 02 (mM) te (mg N02'
(days) *d)
| 76.8 1
. Dry.

Welght (mg N03" WClghI (mg N02'
(mg/L) ling cells*d) (mg/L) [mg cells*d)

64 14.06 254 0.30]
Pseudomonas sp. strarn K Max. seudomonas sp. straln K Max. pecr c
S cific Nitrate Utilization, km itrite Utilization, km
Average 1 .11] Average] 6.53]

 

 

 

[Standard I 1.77 Standard 0.26
Dev. Dev.

 

 

63

Table B-8. Data used for determining the yield of Pseudomonas sp. strain KC in
Schoolcraft groundwater on nitrate reduced to gaseous end products .

1e ata(N1trate to gaseous en products

 

 

 

 

 

 

 

 

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

P. - = .17 days)
NO3' Dry. Yield
Consumed W918”
(mM) (mg/L) (mg cells/mg
NO3')
11.716] 259 0.36.5.70E+08] 0.78]
P. - (T= . days) overall hi hest) [
NO3' Dry Yield Plate Yleld
Consumed Weight Counts
(mM) (mg/L) (mg cells/mg (CFU/ml) (10"9 /
NO3' ) mg NO3’ )
11.624] 299 0.41 9.9013408] 1.37]
. - (T=1 .1 s) [
N03" Dry. field
Consumed W918”
(mM) (mg/L) (mg cells/mg /
NO3-) mg N03" )
11.492] 309 0.43 6.50E+08] 0.91]

 

 

 

64

Table B-9. Data used for determining the yield of Pseudomonas sp. strain KC in
Schoolcraft groundwater on nitrate reduced to nitrite.

_l

     

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

'. ”' - verage (T: . .
between 2.38 and 2.94
days)
NO3' Dry 7176111
Consumed Weight
(mM) (mg/L) (mg cells/mg (CFU/ml) (10"9 CFU/mg
NO3’)
. 1 1 0.26 0.
P. K - (Average
between 2.38 and 2.94
days) _
1003- Dry Yiekl
Consumed Weight
(mM) (mg/L) (mg cells/mg (CFU/ml) (10"9 CFU/mg
NO3') N03’)
116271—129 0.1 5.20E+08 0'72
P. KC -3 (Average (12.46)
between 2.38 and 2.94
'i't'ield Plate Yield
Counts
(mg cells/mg (CFU/ml) (10"9 CFU/mg
NO3') NO3')
0720 2.98E+O8 0.421

 

 

 

 

 

 

 

65

Table B-10. Data used for determining the yield of Pseudomonas sp. strain KC in
Schoolcraft groundwater on nitrite reduced to gaseous end products.

1e ata (N itrite to gaseous end p cts

 

 

 

 

 

 

 

 

 

 

 

 

  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

. - .13- .1 days r
1002- Dry Yield 1002- Plate Yield
Consumed Weight Consumed Counts
(mM) (mg/L) (mg cells/mg (mM) (CFU/ml) (10A9 CFU/mg N02')
N02“)
11.050] 150] 0.30] 11.010| 3.42E+0 0.68
P. K - ( .1 - . da s) (3.13-6.13 days)
NOz' Dry_ Yield NOz' Plate Y'ield
Consumed Weight Consumed Counts
(mM) (mg/L) (mg cells/mg (mM) (CFU/ml) (1009 CFU/mg NOz‘)
N02")
10.943 220] 0.44 6.500] 6.6OE+08 2721
(W1 - .96 gays) _
NO2- Plate Yield
Consumed Weight Consumed Counts
(mM) (mg/L) (mg cells/mg (mM) (CFU/ml) (mag CFU/mg NOz')
N02)
9.000] 270 . 0E5 5.600] 435E+08 1.65

 

 

 

 

The relationship below and the method outlined in Appendix B was used to calculate fc and
f5. Yield on nitrate reduced to gaseous end products represented the yield for these

calculations.

 

Yield = _fsllafinllacellumle e-
[fc (1/5) +fs (1/28)] 62 g NO3-lmole e-

APPENDIX C

APPENDIX C

ORIGINAL DATA USED FOR DETERMINING THE HALF-VELOCITY
COEFFICIENTS IN SCHOOLCRAFT GROUNDWATER.

Table C-l. Supporting data for the determination of the maximum specific rate of nitrate
utilization km of Schoolcraft organisms in Schoolcraft groundwater.

W—NO3" “flag—c
consumed Progt/Leir; (mg NO3'lmg
- tein*d)
1'3

    

 

.
3 7. 5 0. 784 11.79 21.99
- . . 1 . 1.11

 

Table C-2. Supporting data for the determination of the maximum specific rate of nitrite
utilization km of Schoolcraft organisms in Schoolcraft groundwater.

Sample Trrne NOZ' Average km
rotein*d) J

l . 5.6
l S2 l8.75- 20. 7'75“ 0.776 1T3O 5.37 |
- 9. 5- . 5 .879 I 13.91 I 5.81 |

Table 03. Calculated half-velocity coefficients Ks and supporting data for
Schoolcraft organisms in Schoolcraft groundwater.

      
     

   

 

 

 

Ks (mg/L) ' .05

Ikm(hr" 1)I 0.914 I 05916 I 0.88 | 0.90 | 0.02 |
Protein I 12.09 I 11.79 I 12.34 I 12.07 I 0.2? I
(mg/L)

 

 

 

66

67

The above values for km and protein were used to calculate the half-velocity coefficients Ks
for Schoolcraft organisms. The average Ks was then used in developing the model plot
shown in Figure 15.

Table C-4. Supporting data for the determination of the maximum specific rate of nitrate
utilization km of Pseudomonas sp. strain KC in Schoolcraft groundwater.

 

 

 

 

Sample Time NO3‘ Average km
(hours) consumed Protein (mg NQ3-/mg
__r (mM) (mg/L) protein*d)
P.KC 1 1.5-5.5 0.940 18.25 19.16
P.KC 2 1.5-6 0.962 18.41 17.28
P.KC 3 1.5-6 1.01 15.12 22.08

 

 

 

 

 

 

 

Table C-5. Supporting data for the determination of the maximum specific rate of nitrite
utilization km of Pseudomonas sp. strain KC in Schoolcraft groundwater.

km 1
(mg N02'/mg l
rotein*d)

Average

N02- .
Protem
m

consumed

; Time
~ (hours)
1

P.K 1

 

 

 

 

 

 

 

 

Table C-6. Calculated half-velocity coefficients Ks and supporting data for Pseudomonas
sp. strain KC in Schoolcraft groundwater.

 

 

 

 

Parameter P.K 1 P.KC 2 P.KC Average Standard

Dev.
Ks (mg/L) 13.52 11.13 11.26 11.9% 1.34
[km(hr’\-l) 0.80 0.72 0.92 0.81 0.10 |
Protein 18.25 18.41 15.12 17.26 1.85 I
(mg/L)

 

 

 

 

 

 

The above values for km and protein were used to calculate the half-velocity coefficients Ks
for Pseudomonas sp. strain KC. The average K3 was then used in developing the model
plot shown in Figure 16.

"111111111111111111115