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This is to certify that the

thesis entitled

Theoretical and Experimental

Competitiveness of Pseudomonas stutzeri KC

presented by

Mark Lee Sneathen

has been accepted towards fulfillment

of the requirements for

 

 

 

 

M. S. degree in Environmental Engineering
are? 7
Major professor
Date December , 1996
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TO AVOID FINES return on or baton date duo.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU loAn Affirmative Adlai/Emil Opportunity Inuituion
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THEORETICAL AND EXPERIMENTAL COMPETITIVENESS 0F
PSEUDOMONAS STUTZERI KC

BY

Mark Lee Sneathen

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

1996

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ABSTRACT

THEORETICAL AND EXPERIMENTAL COMPETITIVENESS OF
PSEUDOMONAS STUTZERI KC

By

Mark Lee Sneathen

The competitiveness of Pseudomonas stutzen’ KC with groundwater flora
indigenous to an aquifer impacted by carbon tetrachloride (CT) was evaluated
by measuring kinetic growth parameters, predicting competitive outcomes
based on those parameters, and testing the predictions in batch experiments.
The maximum specific growth rates of KC and the groundwater flora occurred at
pH 8.2 and pH 7.5. KC displaced the groundwater flora at pH 8.2 and became
the dominant organism when both populations were at similar densities initially.
At pH 7.5, if 150 KC colonies existed for every groundwater colony initially, K0
was able to remain the dominant species for at least nine days. Simulated
groundwater (SGW) medium proved to be the best starter medium of those
tested for promoting the dominance of KC. KC displaced the groundwater flora
in some regions of an aquifer when it was introduced as part of a bioremediation

project.

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ACKNOWLEDGMENTS

Thanks to following people who aided in the completion of this thesis:

Amy who reminds me of the important things in life.

My parents for bringing me as far as they could and having the wisdom to let
me make my own mistakes.

Todd and Shelly for the fun times when I allowed myself a break.

Dr. Criddle for having many, many good ideas, contagious enthusiasm, and an
unsurpassed desire to teach all of his students.

Dr. Dybas for his innumerable practical solutions, and for the wisdom of
designing complicated experiments in an uncomplicated manner.

Dr. Wiggert for stimulating and challenging classes and for information when l
was contemplating grad school.

Blake Key for interesting conversations about science and other important
topics.

George Baley for helping pay the rent.

Gre

cuis

Mik

Jim

Mlk

bre

Lin

Greg Tatara for help with day to day questions and information about Polish
cuisine.

Mike Witt for analytical assistance and travel anecdotes.

Jim Potter for hours spent counting smelly bacterial colonies.

Mike Josephs for assistance with experiments, media preparation and for not
breaking all of our glassware.

Linda Steinman for much needed bureaucratic assistance.

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TABLE OF CONTENTS

Page
LIST OF TABLES ............................................................................................ vii
LIST OF FIGURES .......................................................................................... xi
LIST OF SYMBOLS ........................................................................................ xvii
CHAPTER 1 — INTRODUCTION ............................................................. 1
CHAPTER 2 — MATERIALS AND METHODS ........................................... 8
CHAPTER 3 — MAXIMUM SPECIFIC GROWTH RATE ............................ 12
Materials and Methods ...................................................... 12
Results and Discussion ..................................................... 14
CHAPTER 4 — MIXED-STRAIN INCUBATIONS ....................................... 19
Materials and Methods ...................................................... 19
Results and Discussion ..................................................... 21
CHAPTER 5 -— HABITAT MODIFICATION ................................................ 51
Niche Adjustment .............................................................. 51
Pre-inoculation Disinfection ............................................... 52

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CH.

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Materials and Methods ...................................................... 52

Results and Discussion ..................................................... 53
CHAPTER 6 —— FIELD DATA ...................................................................... 56
Materials and Methods ...................................................... 56
Results and Discussion ..................................................... 59
CHAPTER 7 — ENGINEERING APPLICATIONS ...................................... 71
CHAPTER 8 -— CONCLUSIONS ................................................................ 74
FUTURE WORK RECOMMENDATIONS .......................... 75
LIST OF REFERENCES .................................................................................... 77

vi

Tabl

Tabl

Tabl

Tabl

Tabl

Tabl

Tabl

LIST OF TABLES

Table 1: Sm," calculation assuming b= 0.1 days’1 and K, = 11.97 mglL for

strain KC and b= 0.1 days'1 and Ks = 9.40 mg/L for the

groundwater flora. Values calculated using the geometric mean of

triplicate incubations ......................................................................... 16
Table 2: Pre-inoculation Liquid Phase Field Results, Before and After

Niche Adjustment ............................................................................. 61
Table 3: Plate Count Results from Schoolcraft Aquifer Solids Before

Inoculation with Pseudomonas stutzen' KC ...................................... 62
Table 4: Solid phase denitrifier assay results from Schoolcraft aquifer

solids before inoculation with strain KC. Geometric mean and

standard deviation of triplicate samples ........................................... 63

Table 5: Liquid phase plate counts from Schoolcraft Aquifer After

Inoculation (C.F.U. / mL) .................................................................. 65
Table A-1: Data used to calculate umax of PKC at pH 7.0 ........................... 81
Table A-2: Data used to calculate pm,,( of PKC at pH 7.5 ........................... 81

vii

Table
Table
Table
Table
Table
Table
Table
Table
Table
Table
Table

Table

Table

Table A-3: Data used to calculate umax of PKC at pH 8.0 ........................... 82
Table A-4: Data used to calculate um.x of PKC at pH 8.2 ........................... 82
Table A-5: Data used to calculate pm, of Groundwater Flora at pH 7.0.... 83
Table A-6: Data used to calculate pm“ of Groundwater Flora at pH 7.5 ..... 84
Table A-7: Data used to calculate pm, of Groundwater Flora at pH 8.0 ..... 84
Table A-8: Data used to calculate umx of Groundwater Flora at pH 8.5 ..... 85
Table B-1: Mixed-strain experiment, initial pH 7.5, KC starter medium:

SGW medium, initially 150:1 (PKC:Schoolcraft) ............................... 86
Table B-2: Mixed-strain experiment, initial pH 8.0, KG starter medium:

SGW medium, initially 18:1 (PKC:Schoolcraft) ................................. 87
Table B-3: Mixed-strain experiment, initial pH 8.2, KC starter medium:

SGW medium, initially 100:1 (PKC:Schoolcraft) ............................... 88
Table B-4: Mixed-strain experiment, initial pH 8.2, KC starter medium:

SGW medium, initially 160:1 (PKC:Schoolcraft) ............................... 89
Table B-5: Mixed-strain experiment, initial pH 8.2, KC starter medium:

SGW medium, initially 7.2:1 (PKC:Schoolcraft) ................................ 90
Table B-6: Mixed-strain experiment, initial pH 8.2, KC starter medium:

SGW medium, washed inoculum, initially 11:1 (PKC:Schoolcraft)... 91
Table B-7: Mixed-strain experiment, initial pH 8.2, KC starter medium:

SGW medium, washed inoculum, initially 15:1 (PKC:Schoolcraft)... 91

viii

 

 

 

 

Table I

Table

Table

Table

Table

Table

Table

Tabb

Tabl.

Tabl

Table B-8: Mixed-strain experiment, initial pH 8.2, KC starter medium:

Schoolcraft medium (acclimated culture), initially 32:1

(PKC:Schoolcraft) ............................................................................. 92
Table B-9: Mixed-strain experiment, initial pH 8.2, KC starter medium:

Schoolcraft medium (acclimated culture) , initially 308:1

(PKC:Schoolcraft) ............................................................................. 93
Table B-10: Mixed-strain experiment, initial pH 8.0, KC starter medium:

Medium D, washed lnoculum, initially 3:1 (PKC:Schoolcraft) ........... 94
Table B-11: Mixed-strain experiment, initial pH 8.0, KC starter medium:

Medium D, washed inoculum, initially 10:1 (PKC:Schoolcraft) ......... 94
Table B-12: Mixed-strain experiment, initial pH 8.0, KC starter medium:

Medium D, washed inoculum, initially 12:1 (PKC:Schoolcraft) ......... 95
Table B-13: Mixed-strain experiment, initial pH 7.5, KC starter medium:

SGW medium, initially 8.5:1 (PKC:Schoolcraft) ................................ 95
Table B-14: Mixed-strain experiment, initial pH 8.0, KC starter medium:

SGW medium, initially 9.2:1 (PKC:Schoolcraft) ................................ 96
Table B-15: Mixed-strain experiment, initial pH 8.2, KC starter medium:

SGW medium, initially 6.8:1 (PKC:Schoolcraft) ................................ 97
Table C-1: Percent CT remaining, Schoolcraft acclimated culture, pH 8.2. 98

Table C-2: Percent CT remaining, SGW, pH 8.2 ........................................ 98

ix

 

Te

Te

Ta

Table C-3: Percent CT remaining, SGW, pH 8.0 ........................................ 99
Table D-1: Groundwater disinfection with hydrogen peroxide (CFUImL).... 100

Table D-2: Groundwater disinfection with bleach (CF U I mL) ..................... 101

 

 

Figi

Figr

FigL

FigL

LIST OF FIGURES

Figure 1: pH dependence of pm”. Arithmetic mean plotted, error bars

represent plus/minus one standard deviation ................................... 15
Figure 2: Mixed-strain plate count results. Initial pH 8.0, 18:1 (strain KC:

groundwater flora), SGW medium as the starter medium.

Geometric mean plotted, error bars represent one standard

deviation ........................................................................................... 24
Figure 3: Mixed-strain plate count results. Initial pH 8.2, 160:1 (strain KC:

groundwater flora), SGW medium as the starter medium.

Geometric mean plotted, error bars represent one standard

deviation ........................................................................................... 25
Figure 4: Mixed-strain plate count results. Initial pH 8.2, 100:1 (strain KC:

groundwater flora), SGW medium as the starter medium.
Geometric mean plotted, error bars represent one standard

deviation ........................................................................................... 26

xi

 

 

Figure

Figure

Figure

Figure

Figure 5: Carbon tetrachloride transformation in the presence of

groundwater flora. Mixed-strain experiment, initial pH 8.0, 18:1

(strain KC: groundwater flora), SGW medium as the starter

medium. Geometric mean plotted, error bars represent standard

deviation ........................................................................................... 27
Figure 6: Carbon tetrachloride transformation in the presence of

groundwater flora. Mixed-strain experiment initial pH 8.2, 160:1

(strain KC: groundwater flora), SGW medium as the starter

medium. Geometric mean plotted , error bars represent one

standard deviation ............................................................................ 28
Figure 7: Mixed-strain plate count results. Initial pH 7.5, 150:1 (strain KC:

groundwater flora), SGW medium as the starter medium.

Geometric mean plotted, error bars represent one standard

deviation ........................................................................................... 30

Figure 8: Mixed-strain plate count results. Initial pH 8.2, 32:1 (strain KC:
groundwater flora), groundwater medium as the starter medium.
Geometric mean plotted, error bars represent one standard
deviation ........................................................................................... 32

Figure 9: Mixed-strain plate count results. Initial pH 8.2, 308:1 (strain KC:
groundwater flora), groundwater medium as the starter medium.
Geometric mean plotted, error bars represent plus one standard

deviation ........................................................................................... 33

xii

Figur

 

Figur

 

Flgu

Figu

Figure 10: Carbon tetrachloride transformation in the presence of
groundwater flora. Mixed-strain experiment initial pH 8.2, 308 and
32:1 (strain KC: groundwater flora), SGW medium as the starter
medium. Geometric mean plotted, error bars represent one
standard deviation ............................................................................ 34

Figure 11: Mixed-strain plate count results. Initial pH 8.2, 11:1 (strain KC:
groundwater flora), SGW medium as the starter medium, washed
inoculum. Geometric mean plotted, error bars represent one
standard deviation ............................................................................ 36

Figure 12: Mixed-strain plate count results. Initial pH 8.2, 15:1 (strain KC:
groundwater flora), SGW medium as the starter medium, washed
inoculum. Geometric mean plotted, error bars represent one
standard deviation ............................................................................ 37

Figure 13: Mixed-strain plate count results. Initial pH 8.0, 10:1 (strain KC:
groundwater flora), Medium D as the starter medium, washed
inoculum. Geometric mean plotted, error bars represent one

standard deviation ............................................................................ 38

xiii

 

 

Figure

Figure

Figu|

Flgu

Figure 14: Mixed-strain plate count results. Initial pH 8.0, 6:1 (strain KC:

groundwater flora), Medium D as the starter medium, washed

inoculum. Geometric mean plotted, error bars represent one

standard deviation ............................................................................ 39
Figure 15: Mixed-strain plate count results. Initial pH 8.0, 12:1 (strain KC:

groundwater flora), Medium D as the starter medium, washed

inoculum. Geometric mean plotted, error bars represent one

standard deviation ............................................................................ 40
Figure 16: Mixed-strain plate count results. Initial pH 8.2, 7.2:1 (strain KC:

groundwater flora), SGW medium as the starter medium.

Geometric mean plotted. error bars represent one standard

deviation ........................................................................................... 41
Figure 17: Mixed-strain plate count results. Initial pH 7.5, 8.5:1 (strain KC:

groundwater flora), SGW medium as the starter medium.

Geometric mean plotted, error bars represent one standard

deviation ........................................................................................... 45

xiv

 

 

Figure

Figure

Figun

Flgm

Figure 18: Ion analysis from a mixed-strain experiment. Initial pH 7.5,

8.5:1 (strain KC: groundwater flora), SGW medium as the starter

medium. Geometric mean plotted, error bars represent one

standard deviation ............................................................................ 46
Figure 19: Mixed-strain plate count results. Initial pH 8.0, 9.2:1 (strain KC:

groundwater flora), SGW medium as the starter medium.

Geometric mean plotted, error bars represent one standard

deviation ........................................................................................... 47
Figure 20: lon analysis from a mixed-strain experiment. Initial pH 8.0,

9.2:1 (strain KC: groundwater flora), SGW medium as the starter

medium. Geometric mean plotted, error bars represent one

standard deviation ............................................................................ 48
Figure 21: Mixed-strain plate count results. Initial pH 8.2, 6.821 (strain KC:

groundwater flora), SGW medium as the starter medium.

Geometric mean plotted, error bars represent one standard

deviation ........................................................................................... 49

Figur

Figu

 

Figu

 

Figure 22: Ion analysis from a mixed-strain experiment. Initial pH 8.2,

6.8:1 (strain KC: groundwater flora), SGW medium as the starter

medium. Geometric mean plotted, error bars represent one

standard deviation ............................................................................ 50
Figure 23: Plate count results from disinfection of Schoolcraft

groundwater with hydrogen peroxide. Geometric mean plotted,

error bars represent one standard deviation .................................... 54
Figure 24: Plate count results from disinfection of Schoolcraft

groundwater with bleach. Geometric mean is plotted, error bars

represent plus or minus one standard deviation ............................... 55

 

Sminv ‘

Um

xa?‘

Smim Jr R.

“max

LIST OF SYMBOLS

substrate concentration below which net growth does not occur
(mg/L)

maximum specific growth rate (days’1)

decay coefficient (days’l)

half-velocity coefficient (mg/L)

concentration of microorganisms (mg/L)

maximum specific rate of substrate utilization (days'l)

specific growth rate (days'1)

rate-limiting substrate concentration (mg/L)

time (days)

maximum organism yield (mg organisms/mg substrate)

 

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CHAPTER 1

INTRODUCTION

In-sltu Bloremediation with Pseudomonas stutzeri KC

In-situ bioremediation of groundwater offers the potential for remediation
of many contaminated sites. The two most prominent methods of 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 desired metabolic capabilities. For situations
where indigenous flora are not sufficiently numerous or lack the desired
metabolic capabilities, bioaugmentation may prove to be a cost effective clean-
up strategy. Many chlorinated solvents are biologically transformed via co-
metabolic pathways (Criddle et al. 1991). If an electron donor and acceptor, as
well as required growth factors, can be supplied to an organism that transforms
the compound(s) of interest co-metabolically, bioaugmentation of sites impacted
by this compound may be feasible.

Nutrient delivery presents considerable problems for in-situ
bioremediation. Because of the low solubility of oxygen in water, it is difficult to
deliver sufficient quantities of oxygen to aerobic organisms using gas delivery.
Delivering electron acceptors, such as nitrate, with much higher water

solubilities than oxygen should not be as difficult. An aqueous solution with high

1

2
nitrate concentrations can be pumped to the desired location. Denitrifying
organisms often grow nearly as fast as aerobes. The relative ease of nutrient
delivery combined with rapid growth rates may make denitrifiers better suited for
in-situ bioremediation applications than aerobes.

Carbon tetrachloride (CT) transformation by indigenous denitrifying
microorganisms typically leads to the production of chloroform (CF). In a field
scale experiment at Moffett Naval Air Station, for example, CF production
resulted when acetate was added to biostimulate denitrifying populations
(Semprini et al. 1992). Pseudomonas stutzen' KC is a denitrifying organism that
rapidly and completely co-metabolizes tetrachloromethane, commonly known as
carbon tetrachloride (CT), to carbon dioxide, formate and an unidentified non-
volatile product, without the production of chloroform (Criddle et al.1990, Lewis
et al. 1993, Dybas et al. 1995). To transform CT, an actively growing culture of
strain KC requires iron limiting conditions (Criddle et al. 1990, Tatara et al.
1993), trace concentrations of copper (Tatara et al. 1993) and an incubation
temperature of 4-23° C (transformation is inhibited above 25° C; growth is
inhibited above 30° C). Iron solubility (as ferric ions) in water is at a minimum in
the pH range of 8.0 to 8.2 (Stumm et al. 1981). Copper is required for CT
transformation (T atara et al. 1993), but inhibits growth at neutral pH (Criddle et

al. 1990) when it is more soluble than at alkaline pH. CT transformation by

3
strain KC is co-metabolic and believed to be linked to an iron-scavenging
system (Criddle et al. 1990, Tatara et al. 1993, Dybas et al. 1995). No similar
activity has yet been detected in other isolates or indigenous consortia (Criddle
et al. 1990, Lewis et al. 1993).

For bioaugmentation, transport of the exogenous organisms and
competition with the indigenous microbial community for limiting resources
present formidable challenges. This thesis focuses on predicting the most fit
competitor, verification of those predictions, methods and effects of habitat
modification for promoting the dominance of an exogenous organism,
specifically Pseudomonas stutzen' KC, and the results of a field-scale

bioaugmentation project.

Competition Theory
Microbial growth rate can be modeled using the original formulation by
Monod (1942), which was later modified by van Uden (1967) to include the rate

of decay:

 

- b (1)

u... = kam (2)

where u = §£ = specific growth rate (days “) (3)
and b = decay coefficient (days‘I)
km = maximum specific rate of substrate utilization (days‘l)

K, = half-velocity coefficient (mg/L)

um, = maximum specific growth rate (days’l)

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

X = concentration of microorganisms (mg/L)

Ym = maximum organism yield (mg organisms/mg

substrate)

A mechanistic parameter, 8min, describing the subsistence concentration of a
limiting resource can be derived from equation 1 by setting dX/dt = 0 (Rittman
and McCarty 1980):

DK
8 . = __£__..
u... _ b (4)

where Sm,n = substrate concentration where growth rate

equals death rate (mg/L).

Experimental field evidence has led to a proliferation of models for
interspecific competition for resources (eg. Stewart and Levin 1973, Lehman et
al. 1975, Petersen 1975, Taylor and Williams 1975, Leon and Tumpsen 1975,
Hsu et al. 1977, Hansen and Hubbell 1980, Tilman 1977,1980). If numerous
species are all limited by the same nutrient and they have similar decay rates, b,
and half-velocity coefficients, K,, for a limited resource, single nutrient
competition theory predicts that the species with the highest maximum specific
growth rate, pm”, will completely displace all other species at equilibrium in
continuous culture (Hsu et al. 1977, Tilman 1977, Hansen et al. 1980). The
Monod model describes the nutrient-limited growth of single species.
Parameters that are mathematically identical to Sm," such as J (Hansen and
Hubbell 1980) and R* (Tilman 1981) have been derived and extend to

competition between numerous species:

b. Ka
. = —— (5
“maxi ' bi )
where Sm"... = J, = Rf
Smin'hJi, R*, = substrate concentration where growth rate

equals death rate (mg/L) for species i,

6

b, specific death rate of species i,

Ksi

half-velocity coefficient of species i, and

um“ = maximum specific growth rate of species i.

According to equation 5, the dominant species will have the lowest Sm," value.
It follows from equation 5 that if competitors have equal death rates and half-
velocity coefficients, the competitor with the greatest maximum specific growth
rate will be the dominant species (Hsu et al. 1977, Tilman 1977, Hansen and
Hubbell 1980). Sm," is commonly used in engineering literature and it will be

used throughout this thesis to remain consistent with that notation.

EXPERIMENTAL OVERVIEW

This thesis addresses microbial competition issues relevant to
engineering the bioaugmentation of a carbon tetrachloride impacted aquifer.
Laboratory studies were designed to evaluate the competitiveness of
Pseudomonas stutzen' KC with a microbial groundwater community. The
maximum specific growth rates of Pseudomonas stutzen' KC and the
groundwater flora were determined over the pH range from 7.0 to 8.5. Single
nutrient competition theory was then used to predict whether strain KC or the

groundwater consortium will dominate at a given pH. Theoretical predictions

7
were tested with mixed-strain laboratory experiments. A mixed-strain
experiment in which strain KC was grown under similar conditions to a field
inoculum (Schoolcraft, Ml, Field Experiment) was also conducted to test the
outcome in the laboratory prior to field demonstration.

Disinfection could be used as a preemptive habitat modification step to
promote the dominance of Pseudomonas stutzen’ KC. Bleach and hydrogen
peroxide were evaluated as groundwater disinfectants.

Aquifer field samples have been collected and analyzed to show that
bioaugmentation with strain KC is a viable treatment alternative for carbon

tetrachloride impacted aquifers.

CHAPTER 2

MATERIALS AND METHODS

Organisms. Pseudomonas stutzen' KC (DSM deposit no. 7136. ATCC deposit
number 55595), derived originally from aquifer solids from Seal Beach, CA,
(Criddle et al. 1990) is routinely maintained in our laboratory on nutrient agar

plates.

Chemicals. All chemicals were American Chemical Society reagent grade
(Aldrich or Sigma Chemical Co.). All water used in reagent preparation was
deionized 18 MO resistance or greater. Tetrachloromethane (CT; 99 % pure)

was obtained from Aldrich Chemical Company, Milwaukee, Wisconsin.

Media. Simulated groundwater (SGW) medium (recipe provided by R. Skeen,
Battelle Pacific Northwest Laboratory) contained per liter of deionized water:
0.455 g of NaZSiO3 - 9HZO, 0.16 g NaCOa, 0.006 g of NaZSO4, 0.02 g of KOH,
0.118 g of MgCl2 - 6HZO, 0.0081 g of CaClz - 2HZO, 13.61 g of KHZPO4, 1.6 g of

NaOH, 1.6 g of NaNOa, 1.6 g of acetate and 1 mL of trace element solution. The

9

trace element solution contained per liter of deionized water: 0.021 g of LiClz,
0.08 g CuSO4 - 5H20, 0.106 g of ZnSO4 - 7HZO, 0.6 g of H3803, 0.123 g of
Al2(SO4)3 - 18H20, 0.11 g of MCI; - 6HZO, 0.109 g of CoSO4 - 7HZO, 0.06 g of
TiCl4, 0.03 g of KBr, 0.03 g of Kl, 0.629 g of MnClz - 4H20, 0.036 g of SnClz -
2H20, 0.3 g of FeSO4 - 7H20. The pH of SGW medium was adjusted to 8.2 with
NaOH pellets. The resulting medium was autoclaved at 121° C for 20 minutes.

Medium D contained per liter of deionized water: 2.0 g KHZPO4, 3.5 g of
KZHPO4, 1.0 g of (NH4)ZSO4, 0.5 g MgSO, - 7H20, 3.0 g of sodium acetate, 2.0 g
of sodium nitrate, 1 mL of 0.15 M Ca(MO3)3, and 1 mL of trace nutrient stock
TN2. Stock solution TN2 contained per liter of deionized water: 1.36 g of FeSO,
- 7HZO, 0.24 g of NazMoO4 . 2H20, 0.25 g of CuSO4 - 5H20, 0.58 of ZnSO4 -
7HZO, 0.29 g of Co(NO3)2 - 6H2P, 0.11 g of NiSO, - 6HZO, 35 mg of NazseO3,
62 mg of H3803, 0.12 g of NH4VO3, 1.01 g of MnSO4 - H20, and 1 mL of H2804
(concentrated). Typically, Medium D is adjusted to pH 8.2 using NaOH pellets
followed by 1 M NaOH stock solution. To sterilize Medium D, it is autoclaved for
20 minutes at 121 ° C. Cultures were grown at 20° C with 150 rpm shaking
under aerobic conditions (Lab-Line Orbital Shaker model 3590). Nutrient broth,
nutrient agar (Difco) and R2A (Difco) plates were prepared according to

manufacturer's instructions.

1 0
Groundwater. All groundwater used in experiments was collected from a CT
impacted aquifer in Schoolcraft, Ml. Groundwater was obtained by manually
collecting samples with a Teflon® bailer from a 2" steel well screened at 30 feet
below the water table. Groundwater samples were stored in pre-sterilized
sealed five gallon Nalgene® carboys or in Wheaton bottles equipped with

Teflon‘ID lined caps at 4°C.

Analytical methods. CT was assayed by removing samples from the
headspace gas above aqueous samples and injecting them into a gas
chromatograph, as described previously (Tatara et al. 1993). External
calibration standards were prepared by addition of a primary standard (7.8 ng of
CT per uL of methanol) to secondary aqueous standards having the same
gaszwater ratio, ionic strength, incubation temperature, and speed of shaking as
the assay samples. A four-point calibration curve was prepared over a
concentration range bracketing that of the assay samples. Measurements of pH
were made with an Orion model 720A pH meter. Nitrate, nitrite, and acetate
ions were assayed by ion chromatography (Dionex° model 2000i/SP ion
chromatograph with suppressed ion conductivity detection equipped with a
Dionex” lonpak AS4-A anion exchange column and utilizing a 1.8 mM

bicarbonate/1.7 mM carbonate mobile phase at 1 mUmin). Chromatograms

1 1
were recorded and data integrated using Turbochrom 4” software (Perkin Elmer
Corp.). External calibration curves which bracketed the concentrations of the
test samples were prepared by diluting primary ion standards into deionized
water with at least 18 MO resistance. Optical density measurements was

measured at 660 nm using a Shimadzu UV—160 spectrophotometer.

Bacterial enumeration. To determine the density of strain KC and aquifer flora
in competition experiments and aquifer field samples, spread plate counts

were used. Serial dilutions of extracted samples were performed in sterile
phosphate buffer (50 mM, pH 8.0). Phosphate buffer was prepared by
dissolving equimolar quantities of mono and dibasic sodium phosphate in 18
MO resistance deionized water. 1.8 mL quantities of phosphate buffer was then
dispensed into Kimax cultures tubes (which are subsequently capped and
autoclaved for 20 minutes at 121° C). A tenfold dilution can then be achieved by
adding 0.2 mL of sample to sterile Kimax tubes containing 1.8 mL of buffer and
mixing well. Diluted aliquots (100 pL) were spread on R2A plates. Total colony
forming units (C.F.U.) were obtained by counting colonies after 6 days of
incubation at 20° C. When grown on R2A plates, a “fried egg” colony

morphology characteristic of strain KC is observed.

CHAPTER 3

MAXIMUM SPECIFIC GROWTH RATE DETERMINATION

MATERIALS AND METHODS

Medium preparation. In order to evaluate the maximum specific growth rate of
strain KC and the groundwater flora, 500 mL Wheaton bottles, with TeflonO lined
septa and holes drilled in the bottle caps, containing 250 mL of groundwater
(pasteurized at 65° C for 8 hours for strain KC experiments), were amended with
30 mM acetate, 12 mM nitrate, 0.1 mM phosphate and 2 g/L of NaHCO;, (Knoll
1994). Pasteurization was used to kill indigenous flora because autoclaving
produced a precipitate that interfered with growth (Knoll 1994). Filter-sterilization
also had an adverse effect on organism growth (Knoll 1994). Gaseous C02 was
used to reduce pH for experiments below pH 8.2 and 1 N NaOH was added to

raise pH to 8.5.

Starter cultures. An individual strain KC colony was transferred from nutrient

agar and grown for 60 hours at 20° C with 150 rpm shaking under aerobic

12

13

conditions (Lab-Line Orbital Shaker model 3590) in 28 mL sterile serum tubes,
capped with crimp tops, that contained 10 mL of Medium D. After 60 hours,
strain KC starter cultures were centrifuged at 14,000 rpm for five minutes, re-
suspended in pasteurized, amended groundwater and centrifuged again at 8,000
rpm for 10 minutes. Starter cultures undergoing this procedure are considered
“washed”. Washing was done to minimize nutrient carryover from starter cultures

into experiments.

Removal of oxygen from headspace. Wheaton Bottles (500 mL with loosened
caps) were passed through a Coy anaerobic glove box interlock (Coy Laboratory
Products, Ann Arbor, Michigan) three times to remove oxygen from the
headspace. Thirty (30) milliliters of gas (90% nitrogen, 10% hydrogen) was
added to the bottles in the anaerobic glove box so that they were under positive
pressure and removing samples would not pull oxygen into the bottles. Strain KC
was added as a one percent inoculum (approximately 106 CFU/mL) from 60 hour

Medium D tubes.

Maximum specific growth rate determination. Microbial growth was followed

by optical density measurements at 660 nm. Optical density was measured on a

14
Shimadzu UV-160 spectrometer from 1.5 mL samples as described above
(Chapter 2). The maximum specific growth rate, um, was calculated for cells in
log phase growth using optical density measurements and the following
relationship: umax = [ ln (XJX,)] / (t, - t,), where X, and X. represent final and initial

optical densities, and t, and t. are the final and initial time.

RESULTS AND DISCUSSION

Batch incubations over the pH range of 7.0 to 8.5 were used to determine
maximum specific growth rate, pm”, as a function of pH. Maximum specific
growth rates for the groundwater consortium and strain KC were determined
independently and are compared in Figure 1. No trace metals were added and
incubations were nitrate limited. Because the aquifer water may lack some
required nutrients, the batch incubations could be limited by other resources.

Strain KC has a higher maximum specific growth rate than the
groundwater flora between approximately pH 8.0 and 8.5 (Figure 1). Net growth

must be occurring for maximum specific growth rate to be measured. Death rate,

15

b, must therefore be less than the maximum specific growth rate. Assuming a
death rate of 0.1 days‘1 and given the half-velocity coefficients, K,, previously
determined for Pseudomonas stutzen’ KC and the groundwater community (Knoll
1994), strain KC has a lower Sm,n than the groundwater flora between pH 8.0 and
8.5 (Table 1). It is predicted that KC will outcompete the groundwater flora over
this range in continuous culture. Death rate and half-velocity coefficients are
functions of pH. To calculate Sm,n more accurately, b and K, should be measured

as a function of pH over the experimental pH range.

 

 
  
 

 

 

 

5 r r
A 4 __
F L Aquifer Flora
g 3 - ‘ Strain KC
to =
'U /
v E
x -
“I 2
§

1 _.

l.
06 . - .

Figure 1: pH dependence of um. Arithmetic mean plotted, error bars represent
plus/minus one standard deviation.

16

TABLE 1: Sm... as a Function of pH for the Groundwater Flora and Strain KC

 

 

 

 

smln (mg/L)
pH Groundwater Flora1 Pseudomonas
stutzeri Kc2
7.0 1.08 2.4
7.5 0.23 0.60
8.0 0.60 0.47
8.2 1.32 0.40
8.5 2.35 2.86

 

 

 

 

1: Assumes nitrate limiting conditions, b = 0.1 d '1, and
K, = 9.40 mg NO'3/ L.
2: Assumes nitrate limiting conditions, b = 0.1 days", and

K, =11.97 mg NO'3/ L.

Nutrient concentrations decrease with time in actively metabolizing
microbial batch incubations. Because there is no flow out of batch incubations,
metabolites accumulate with time unless they decay chemically or biologically.
Although single-nutrient competition theory was developed for continuous culture
applications, its utility for batch cultures has not been tested and was therefore

evaluated experimentally in growth rate and mixed-strain experiments.

17

Resource-based competition theory was developed for species, not
consortia. Growth parameters of the groundwater consortium, as determined by
the experiments detailed above, can be attributed to the kinetics of the fastest
growing organism(s) in the community. Because death rate is assumed constant,
as long as the half-velocity coefficient determined for the community corresponds
to the same member of the community for which a maximum specific growth rate
was determined, the Sm,n calculated for the community will actually be for an
individual species. Both strain KC’s and the groundwater consortium's Sm,"
would then be representative of individual species. Experimental conditions
are designed to optimize growth of Pseudomonas stutzen’ KC. These conditions
promote the growth of species that will be most competitive with strain KC.
Comparing the Sm,n parameter of strain KC with that of the groundwater
consortium is in effect a comparison between strain KC and the species in the
consortium that is most competitive with strain KC. Single nutrient competition
theory is intended to facilitate interspecies comparisons and may be a valid
predictive tool in this case.

Single nutrient competition theory however was derived for continuous
culture. In continuous culture, once steady-state is reached, nutrient and cell
concentrations do not vary temporally and if the vessel is well-mixed, are

constant spatially as well. Nutrient and cell densities are time dependent in batch

1 8
culture. Due to the physical and therefore mathematical differences between
batch and continuous cultures, single nutrient competition theory probably has

limited applicability to batch cultures.

CHAPTER 4

MIXED-STRAIN INCUBATIONS

MATERIALS AND METHODS

To determine if single nutrient competition theory can be applied to batch
experiments and to determine the conditions where strain KC is able to displace
the groundwater flora, 10 mL of amended groundwater was put in 30 mL serum
tubes. The groundwater was amended with 60 mg of sodium acetate, 13.6 mg
of KHZPO4, and 250 mg of NaHCO;, per liter. The pH was increased by 1 N
NaOH addition and decreased by 1 N HCI addition. After the groundwater was
added to the serum tubes they were passed through the Coy glove box interlock
chamber to remove oxygen from the headspace (Chapter 3). The tubes were
then capped in the Coy glove box (Chapter 3) with Teflon coated septa under
anaerobic conditions (90% nitrogen, 10% hydrogen). Strain KC starter cultures,
which were grown as described below, were used to inoculate the anaerobic
serum tubes. CT concentration, strain KC and groundwater flora densities were

recorded with time.

19

20
Starter cultures. An individual KC colony was transferred from nutrient agar
and grown for 48 hours at 20° C with 150 rpm shaking under aerobic conditions
(Lab-Line Orbital Shaker model 3590) in 10 mL sterile serum tubes with crimp
tops that contained 10 mL of Medium D (pH 8.2). After 24 hours, a 100 mL
Erlenmeyer flask containing 50 mL of sterile SGW medium (pH 8.2, aerobic)
was inoculated with 1 mL of the Medium D grown KC culture and placed on a
stir plate. Flasks were loosely covered with aluminum foil to prevent
contamination and to allow oxygen into the headspace. Strain KC was grown for
48 hours in SGW medium and used to inoculate anaerobic serum tubes at the

desired density.

Bacterial enumeration. Pseudomonas stutzen’ KC and the groundwater flora
were enumerated with plate counts on R2A agar. Samples were diluted
(Chapter 2) and a range of dilutions was selected for plating that would produce
between thirty and three hundred colonies per plate. R2A was chosen because
higher plating efficiencies were achieved with it than with nutrient agar or aquifer
water agar (aquifer water amended with 10% nutrient broth and noble agar). As
mentioned above strain KC has a distinctive “fried egg” morphology. To
determine if strain KC could be reliably differentiated from the groundwater flora

by morphology. twenty-five (25) suspected KC colonies and twenty-five (25)

21
suspected non-KC colonies were sampled and placed in medium D vials
containing CT. Vials were incubated for 6 days at 20° C. All twenty-five (25) KC
colonies degraded CT to non-detectable levels on a gas chromatograph (CT
assays performed as described in Chapter 2), whereas the non-KC colonies

had not significantly reduced the concentration of CT in the headspace.

RESULTS AND DISCUSSION

Pseudomonas stutzen' KC and the groundwater flora have previously
been incubated separately in competition experiments. In those experiments,
kinetic parameters were measured and used to predict competitive outcomes.
To experimentally evaluate the application of single nutrient competition theory
to batch cultures, mixed-strain batch incubations (strain KC and the
groundwater flora incubated in the same vessel) were prepared. Mixed-strain
batch incubations were also used to estimate the inoculum density required for
domination of the groundwater community by strain KC. The effectiveness of
two starter culture growth media were also compared. Mixed-strain results will
help to determine if the maximum specific growth rate of a community is a

quantitative measure of its competitiveness in batch experiments.

22

Competitive interactions between species have been studied by additive
design, in which the density of one species is held constant while the other is
varied, or by substitutive design, in which the density of two species is varied
while total density is held constant (Firbank et al. 1990, Harper 1977, Snaydon
1991, Wilson et al. 1994). The substitutive design or replacement series,
introduced by de Wit (1960), has the advantage that the effects of proportion
and density are not confounded. When strain KC is introduced into an aquifer,
the background flora are present at a fairly constant natural density. In order to
closely model field conditions and to avoid the complexity of maintaining a
constant total density, additive experiments were conducted with constant
groundwater flora density and varying densities of strain KC.

Mixed-strain experiments test the predictive capabilities of single nutrient
competition theory. As discussed in Chapter 3, nutrient and cell concentrations
vary with time in batch incubations, but are constant in continuous culture once
steady-state has been achieved. This mathematical inconsistency is one of the
reasons that competition theory predictions may not be validated in batch
culture. Also, if more than one nutrient is limiting the growth of the competing
organisms or complex ecological relationships develop, theoretical predictions

may not be verified in mixed-strain incubations.

23

Mixed-strain experiments will help determine if single-nutrient competition
theory can be applied to batch incubations. As nutrients are consumed in batch
incubations, the community structure will change. Initially, when nutrient
concentrations are high, an r strategist (a fast growing organism that prevails
when nutrients are not severely limiting) may be dominant and as the nutrients
are consumed a K strategist (an organism that reproduces more slowly than an
r strategist, but prevails under resource limiting conditions) may displace it. Both
types of strategists could also coexist, but proliferate under different conditions.

Initial um experiments indicated that the minimum Sm," for
Pseudomonas stutzen’ KC (under the specified experimental conditions) occurs
at pH 8.2 (Table 1). Strain KC is predicted to outcompete the groundwater flora
at this pH because it has a lower 8min. Mixed-strain batch incubations near pH
8.2 were used to test this prediction. Support for single nutrient competition
theory was obtained at pH 8.0 and 8.2 when strain KC starter cultures were
grown in SGW medium. At pH 8.0 (Figure 2) and pH 8.2 (Figures 3 and 4),
strain KC replaced the groundwater flora as the dominant population and readily
transformed CT (Figures 5 and 6). Additional experiments, over a longer time
. frame, might help to answer the question of whether single nutrient competition

theory can be used to reliably predict batch incubation results.

C.F.U. I mL

24

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1.(E+08 E
5 I.
1.0‘5-1-07E I'LL n r: F
F Stra’nKC *
10905: Emma
10305.;
1m P l ,1 l
0 2.5 46 70 117 168 204
Tine(hous)

Figure 2: Mixed-strain plate count results. Initial pH 8.0, 18:1 (strain
KC: groundwater flora), SGW medium as the starter medium.
Geometric mean plotted, error bars represent one standard

deviation.

C.F.UJmL

25

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1.0E+08
H I I- H

1.0E+07 E

E:
1.0E+06 4?

E
1-°E+°5 “5 DStrain KC

[ .Groundwater Flora

q
1.0E+04 {a
1.0E+03 I . 4. i i i i A.

0 48 72 96 144 168

Time (hours)

Figure 3: Mixed-strain plate count results. Initial pH 8.2, 160:1 (strain
KC: groundwater flora), SGW medium as the starter medium.

Geometric mean plotted, error bars represent one standard deviation.

C.F.U. I mL

26

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1.0E+09 E
: -&
1.0E+08
1.0E+07 E
g .L
1.0E+06
1.0E+05 E; nStrain KC
-_ Groundwater Flora
1.0E+04
1.oe+03 3— 1 . .
0 22 96 120 144

Time (hours)

Figure 4: Mixed-strain plate count results. Initial pH 8.2, 100:1 (strain KC:
groundwater flora), SGW medium as the starter medium. Geometric

mean plotted, error bars represent one standard deviation.

°/o CT Remaining

27

 

 

   
  

 

 

 

 

 

 

 

 

 

100 -_
80
60 a
40 --
.Uiinoculabd Control
20 .- uStrain KC, Initially 13:1
0 [ t i s
0 21

Time (hours)

Figure 5: Carbon tetrachloride transformation in the presence of
groundwater flora. Mixed-strain experiment, initial pH 8.0, 18:1 (strain
KC: groundwater flora), SGW medium as the starter medium.

Geometric mean plotted, error bars represent standard deviation.

28

 

 

 
 

 
 

 

  

 

 

 

100
g 80
.E
E 60 » _
& .Uninoculated Control
5 40 . uStrain_KC: Initially EL
3
20
0

 

 

 

 

 

0.8 3.6 5.1 120.0
Time (hours)

Figure 6: Carbon tetrachloride transformation in the presence of
groundwater flora. Mixed-strain experiment initial pH 8.2, 160:1
(strain KC: groundwater flora), SGW medium as the starter
medium. Geometric mean plotted, error bars represent one

standard deviation.

To verify that the groundwater consortium would be the dominant
population when it has a lower Sm.n than KC, a mixed-strain experiment was
conducted at pH 7.5 with SGW medium as the starter medium (Figure 7). Strain
KC maintained higher cell densities than the groundwater consortium
throughout the experiment. Strain KC concentration however did not increase

above the its initial density. Groundwater flora concentrations increased more

29
than one order of magnitude (from 105 to 106) between 26 and 168 hours after
inoculation. These results suggest that single nutrient competition theory may
have limited utility for predicting the outcome of batch experiments. In batch
experiments, initial conditions such as inoculum size have a strong influence on
the outcome. The groundwater flora would probably displace strain KC in
continuous culture at pH 7.5, as predicted. The groundwater flora would also
most likely displace strain KC in a batch experiment (at pH 7.5) that was
repeatedly spiked with microorganisms (strain KC and groundwater flora) and
nutrients. As the spiking frequency increases, batch experiments look more and
more like continuous cultures. In this batch experiment however, strain KC and

the groundwater organisms coexisted.

30

 

 

 

      
 

 

 

 

 

 

 

1.0E+08 E
E .—
1.0E+07 1: :—
_l :
E .
3' 1.0E+06 HE
l: ..
U - usuéin—kc
1.0E+05 “E .Groundwater Flora
:
1.0E+04

 

 

 

 

   

 

0 25.5 74 95 168 217
Tlme (hours)

Figure 7: Mixed-strain plate count results. Initial pH 7.5, 150:1 (strain
KC: groundwater flora), SGW medium as the starter medium.

Geometric mean plotted, error bars represent one standard deviation.

Mixed-strain competition experiments under denitrifying conditions were
also used to compare prospective starter culture media. Prior to inoculating the
aquifer, strain KC must be grown to the cell density required for competitive
domination. Pasteurized aquifer water amended with acetate, nitrate, and

phosphate, as described above, was tested as a prospective starter-culture

31
medium. Cells pre-adapted to an environment similar to one in which they will
be introduced should experience a minimal lag phase after inoculation (Brock
and Madigan 1991). When pre—grown in amended groundwater, acclimated
strain KC cells did not displace the groundwater species in mixed-strain
experiments at pH 8.2 (Figures 8 and 9) and did not generate a population with
high levels of CT transformation activity (Figure 10). Carbon tetrachloride losses
in incubations that did not contain KC (Figure 10) could be due to abiotic losses
(leaking septa) or biological transformations by the groundwater flora. SGW
medium proved to be a superior starter medium because strain KC was able to
displace the groundwater flora in mixed-strain experiments at pH 8.2 when the
initial strain KC to groundwater flora ratio was approximately equal (Figures 3

and 4).

32

 

1.0E+08
1.05007 E
1.0....

n Stra'n

1.0E-I-04 J
_ .GromdwaterFlora
1.0303 , _. , _,

0 19 42 66 88 109 170
Time(hours)

IIIIIT]

    

C.F.U. I mL

T TTTTTTI

 

 

 

 

 

Figure 8: Mixed-strain plate count results. Initial pH 8.2, 32:1 (strain KC:
groundwater flora), groundwater medium as the starter medium.

Geometric mean plotted, error bars represent one standard deviation.

Presumably, SGW medium provides nutrients in early growth stages that
make strain KC more competitive. Cell growth rates depend on the growth
medium. Rapidly growing cells produce more proteins and copies of genes than
cells growing slowly. Increases in cell protein density and quantity of gene

copies increase competitiveness (Bailey and Ollis 1986). Because it is growing

33

much more rapidly, an inoculum in log phase growth should be more

competitive than one in the stationary phase.

C.F.U. I mL

 

    

 

 

 

 

 

 

 

 

 

1.0E+08
1.0E+07

: 1:1
1.0E+06 E

E
1.0305 I
1-051'04 as a trainK

r
1.0903 ll .-

 

0 19 42 66 88 109 170
Time (hours)

Figure 9: Mixed-strain plate count results. Initial pH 8.2, 308:1 (strain
KC: groundwater flora), groundwater medium as the starter medium.
Geometric mean plotted, error bars represent plus one standard

deviation.

 

 

 

 

 

 

120 — — — — ———— —— f
3
.5 100 .
12’ 25 .1: — » _\ l':‘.g.;‘.‘iniiiaiiy 308:1"
g a 80 _- ‘ -..,. o -lnitially 32:1
c .

3 'E
g .3 60 —l—
.n E
'ii 2
0 4o --
H
C
8
3 20 a»
D.

0 at —»+-—- +—-----—~ a - 1—--~-—+ l i

 

 

0 24 48 72 96 120 144 168 192
Time (hours)

Figure 10: Carbon tetrachloride transformation in the presence of
groundwater flora. Mixed-strain experiment initial pH 8.2, 308 and
32:1 (strain KC: groundwater flora), SGW medium as the starter
medium. Geometric mean plotted, error bars represent one

standard deviation.

Although inoculum growth rate was not quantified, it probably varies in
each medium and could contribute to differences in inoculum competitiveness.

All starter cultures regardless of growth medium were incubated for 48 hours

35

prior to inoculation. Starter cultures from different starter media could therefore
be in different growth phases at inoculation. The composition of SGW medium
is known. A chemical analysis of the groundwater is recommended so that the
two media can be compared and the critical component(s) determined.

Stressing an inoculum could decrease its colonization efficiency.
Washing cells may cause them to lyse. When strain KC was centrifuged and
washed prior to inoculation (to minimize the amount of starter medium added to
experiments at inoculation), strain KC was not able to displace the groundwater
flora regardless of the starter medium or inoculum size (Figures 11, 12, 13, 14
and 15). This suggests that the stress on the inoculum due to washing
significantly reduced the competitive fitness of strain KC. Strain KC starter
cultures used in maximum specific growth rate determination experiments
(Chapter 3) were always washed prior to inoculation. Thus the umx data may
have been affected by the washing. Strain KC could actually have higher
maximum specific growth rates (and lower Sm...) than were measured
experimentally. To determine the maximum specific growth rate of the
groundwater organisms, nutrients were added to groundwater and optical
density was measured. Because strain KC cultures were more stressed than
the groundwater flora, strain KC may be an even better competitor with the

groundwater flora than single nutrient theory predictions suggest.

36

 

1.0E+08

1.0307

T ‘TTTTT

1.0E+06

         
    

C.F.U. I mL

1.0E+05 -

TITTTT T

. [a Strain KC
_ I Groundwater Flora
1.0E+04 - -

nrr

 

 

 

 

 

 

1.0303 I

   

0 22 51 72
Time (hours)

Figure 11: Mixed-strain plate count results. Initial pH 8.2, 11:1 (strain KC:
groundwater flora), SGW medium as the starter medium, washed inoculum.

Geometric mean plotted, error bars represent one standard deviation.

C.F.U. I mL

37

 

1.0E+08
1.0307
1.0E+06 .;

1.0E+05 -

ITTTTTT T I TITT

u Strain KC

1.0E+04

 

 

 

 

 

1.0E+03 ——

 

Time (hours)

Figure 12: Mixed-strain plate count results. Initial pH 8.2, 15:1
(strain KC: groundwater flora), SGW medium as the starter medium,
washed inoculum. Geometric mean plotted, error bars represent

one standard deviation.

C.F.U. I mL

38

 

1.0E+08

T

1.0E+07 ~

 

 

[j SIra'n KC

1.0E+06a .9 I I Flora

T

 

1.0E+05 l

1.0E+04 —

T

 

 

 

 

1.0E+03 l

 

 

 

Time (hours)

Figure 13: Mixed-strain plate count results. Initial pH 8.0, 10:1 (strain
KC: groundwater flora), Medium D as the starter medium, washed
inoculum. Geometric mean plotted, error bars represent one standard

deviation.

C.F.U. I mL

39

 

‘L0E+08—E

 

1.05.07 [JStrain KC - _, —
‘ .Groundwater Flora

 

 

in r rrrrr

1.0E+06 -

1.0E+05 -

ITTTTTTT T TTTTI

iOE+04-E

 

 

 

 

 

 

 

 

 

iOE+03_L_

 

0 22 51
Time (hours)

Figure 14: Mixed-strain plate count results. Initial pH 8.0, 6:1 (strain KC:
groundwater flora), Medium D as the starter medium, washed inoculum.

Geometric mean plotted, error bars represent one standard deviation.

40

 

 

 

 

 

 

 

 

 

 

 

1.0E+08
1.0E+07
.I
E 1.0E+06 +
11.: 1.0E+05 -_ DStrain KC
0 .Groundwater Flora
1.0E+04 _-
1.0303 ,_..._ i I E
0 72 96

Time (hours)

Figure 15: Mixed-strain plate count results. Initial pH 8.0, 12:1 (strain
KC: groundwater flora), Medium D as the starter medium, washed
inoculum. Geometric mean plotted, error bars represent one standard

deviation.

Although inoculum density was varied in the mixed-strain experiments,
the minimum dose of strain KC required to displace the groundwater flora has
not been determined. A 0.1% inoculum (105 C.F.U. / mL) yields a ratio of
approximately 7.221 (strain KC:groundwater Flora) (Figure 16). Strain KC was
able to displace the aquifer flora under these conditions, but the smallest

density difference (for unwashed SGW medium cultures) between the two

41

competitors was observed. A 0.1% inoculum (105 C.F.U. / mL) is probably close

to the minimum required dose when native flora are present at concentrations

less than 105 C.F.U. / mL.

C.F.U./mL

 

 

 

 

 

 

 

 

 

1.0E+08

1.0E+07

1.05%

1.0E+05

1.0E+04

105403 ‘luStr’air’ikc '1

1.0302 ; I 'Wfaflfl .

0 22 45 68 92 120

Time (hours)

Figure 16: Mixed-strain plate count results. Initial pH 8.2, 7.2:1
(strain KC: groundwater flora), SGW medium as the starter
medium. Geometric mean plotted, error bars represent one

standard deviation.

 

42

To better understand nutrient consumption in the mixed-strain
incubations, cell densities and nutrient concentrations were measured at time
points throughout experiments with initial pHs of 7.5, 8.0, and 8.2. To test the
competitive fitness of the KC inoculum used in the field demonstration, a colony It
collected during field inoculation was used as the starter culture for these

experiments and grown according to the field inoculum procedure. Plate count

 

results are presented in Figures 17, 19, and 21; ion chromatography data in l
Figures 18, 20, 22.

Most of the nitrate and nearly equimolar concentrations of acetate were
consumed within twenty-four hours after inoculation. After nitrate was
consumed, KC density did not increase (Figures 17,19, and 21). No nitrite
concentrations above detection limits were measured in the experiment at pH
7.5 (Figure 18). In the incubations at pH 8.0 and pH 8.2, as nitrate was utilized,
low nitrite concentrations were measured (Figures 20 and 22). No nitrite
concentrations above detection limits were recorded in any of the incubations
after 48 hours. KC remained at greater densities than the groundwater flora in
all three experiments (Figures 17, 19, and 21). KC also transformed CT to non-

detectable levels during the initial twenty-four hours after inoculation.

43

Significant quantities of acetate were not metabolized after all the
available nitrate was consumed. It is possible that the groundwater flora does
not contain very many denitrifiers and may not be directly competitive with KC.
In earlier mixed-strain experiments, groundwater flora densities increased after
a four or five day lag period. The previously observed increase may not have
occurred in these experiments because of their reduced duration. The long lag
period for the groundwater flora could be explained if the consortia has
significant numbers of fennentative organisms. Strain KC may outcompete other
denitrifiers in the groundwater consortia and at later time points the fermentative
groundwater organisms begin to grow. An experiment with increased nitrate
concentrations and a longer duration could provide more information about the
relationship between KC and the groundwater flora.

In previous experiments where KC was grown in pure culture, it was
shown that nitrite concentrations increased as nitrate was used and then nitrite
concentrations gradually decreased (Knoll 1994). In those same experiments,
strain KC decayed rapidly after growth stopped. Knoll observed a decay rate of
8.92 clays'1 for strain KC growing as a pure culture in Medium D. The maximum
decay rates calculated, from the data presented in Figures 17, 19, and 21, at
pHs 7.5, 8.0, and 8.2 are 1.3 days", 1.7 days", and 1.6 days'1 respectively. The

lower nitrite concentrations and smaller decay rates in mixed culture versus

44

pure culture may be related. A plausible explanation is that the groundwater
flora are utilizing the nitrite produced by KC. By keeping the nitrite concentration
low, the groundwater flora protect KC from nitrite toxicity and help to stabilize
the KC population.

Samples were saved from the final mixed-strain experiments for some
type of genetic analysis. Genetic assays could provide valuable information
about the types of organisms present and the changes in community structure

occurring after nutrient addition and inoculation.

C.F.U. I mL

45

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

1.00E+08
1.00E+07 ._
1.00E+06 -_
1.00E+O5 ..
flStrain KC
.Groundwater Flora
1.00E+O4 -_ I I
1.00E+O3 -..... I, ,
24 48 96 140

 

Tim (hours)

Figure 17: Mixed-strain plate count results. Initial pH 7.5, 8.521 (strain
KC: groundwater flora), SGW medium as the starter medium. Geometric

mean plotted, error bars represent one standard deviation.

m, nltnh I. nitri- (ppm)

46

 

 

 

 

 

 

 

 

 

 

130.0 9.0
ii
140.0 [ .. 30
120.0 .. 7.0
'l
100.015 .. 6-0
\ \ . AI A
\ \‘ /
30.0 "it: ....................... i ~~~~~~~~~~~~~ ./.. ._ 5.0 g
0 \' ........ u ........ / """" I!
\ i’ /
00.0 \I- - ,__~ ”T7 ’r ..4.0
40.0 .....Aoatato . 3.0
+Nltmt0
- Nitrite
20.0 . “I «~10
I a... Phosphate
0.0 t f: s e = t 4. 1.0
40 00 so 100 120 140
-200 0.0
Time(hours)

Figure 18: Ion analysis from a mixed-strain experiment. Initial pH
7.5, 8.5:1 (strain KC: groundwater flora), SGW medium as the
starter medium. Geometric mean plotted, error bars represent one

standard deviation.

C.F.U. I mL

47

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

           

1.00E+08
flStrain KC
100E+07 fl ‘ .Groundwater Flora r
1.00E+06 +
1.00E+05 «_
1.00E+04 T E»
1.00E‘I'03 s—
0 48 96 140
Time (hours)

Figure 19: Mixed-strain plate count results. Initial pH 8.0, 9.221 (strain
KC: groundwater flora), SGW medium as the starter medium.

Geometric mean plotted, error bars represent one standard deviation.

48

 

 

 

 

 

 

 

 

 

100.0 7'0
II. J
140.0 .1
/ 0.0
120.0 .. ‘ , /
‘. / .. 5.0
1000 ' /
u - _, ‘L’TL- # -_ vi 7H _ —
'__ ~’—’-’ fi' --------------------------------------------- A
m! """ 1“” g
E
00.0 .. .. 3.0
. . 9 - -Aceiat—°
40.0
-—o— Nitrate .. 2.0
.W - .Nitrite
20.0 .. x
_._ Phosphate
\ —~—————~ .. 1.0
0.0 1 ¢ \1: t i = l #
40 60 00 100 120 140
-200 «— 0'0
Tlme (hours)

Figure 20: Ion analysis from a mixed-strain experiment. Initial pH
8.0, 9.2:1 (strain KC: groundwater flora), SGW medium as the
starter medium. Geometric mean plotted, error bars represent one

standard deviation.

C.F.U. I mL

49

1%

 

1.(IE+O7 -l_

1% --

 

 

1.00305 ..

 

1.(IE+04 ..

 

 

 

 

 

 

 

1.(XE+03 -_

     

24 48 %

Figure 21: Mixed-strain plate count results. Initial pH 8.2, 6.8:1 (strain
KC: groundwater flora), SGW medium as the starter medium.

Geometric mean plotted, error bars represent one standard deviation.

mumsnmm)

50

 

 

 

 

 

 

 

100.0 7.0
0,
140.0
(0.0
120.0 . )1
/ a: 5.0
............................ °/1E40
\ \ ‘1’ 3.0 g
.....Aoetate .. 2.0
+8“!th
__,, - Nitrite
N... Phosphate .. 1.0
‘r —I_TT_ +
00 00 100 120 140
-20.0 0.0

 

Time (hours)

Figure 22: Ion analysis from a mixed-strain experiment. Initial pH 8.2,
6.8:1 (strain KC: groundwater flora), SGW medium as the starter
medium. Geometric mean plotted, error bars represent one standard

deviation.

 

CHAPTER 5

HABITAT MODIFICATION

NICHE ADJUSTMENT

To enhance the competitiveness of exogenous organisms, a temporary
niche can be created in aquifers selected for bioaugmentation. Colonization of
this niche by the introduced organism would facilitate remediation. Initial
maximum specific growth experiments revealed that the optimum pH for growth
of strain KC is 8.2 (Figure 1). Schoolcraft groundwater (Schoolcraft, Ml, Field
Project) has a natural pH of 7.5 which, as expected, is the optimum pH for
groundwater flora growth (Figure 1). Alkalinity addition was used to raise the
groundwater pH to 8.2. Subsequently, the aquifer was inoculated with strain KC.
The favorable niche created by alkalinity addition may have helped establish the
Pseudomonas stutzen‘ KC population in the aquifer.

If strain KC outcompetes the indigenous flora and transforms CT at a pH
less than 8.2, less niche adjustment will be required and costs will be reduced.
Ideally, the treatment zone will have a pH near 8.2. Near the boundary of the

treatment zone, pH will begin to decrease and as the distance from the

51

52

treatment zone increases, groundwater pH will return to the background value
(pH 7.5). If strain KC can displace the groundwater flora in regions where the
pH is less than 8.2, a larger than anticipated treatment zone will be created.

Mixed-strain experiments were used to evaluate the competitiveness of
strain KC at pH 8.0. Strain KC has a lower Sm,n than the groundwater
consortium at pH 8.0 and the mixed-strain experiment verified its predicted
dominance (Figure 2). The treatment zone should therefore extend to regions

where the pH is less than 8.2.

PRE-INOCULATION DISINFECTION

MATERIALS AND METHODS

To determine the effects of disinfection on the groundwater flora, 50 mL
of groundwater was put in 125 mL Erlenmeyer flasks. Bleach (HOCI) and
hydrogen peroxide (H202) were added to the flasks at the desired
concentrations. Flasks were incubated at 20° C on a rotary shaker at 150 rpm

(Lab—Line Orbital Shaker model 3590) and samples were plated on R2A agar.

 

53
Organism density was determined according to the enumerative procedure in

Chapter 2.

RESULTS AND DISCUSSION

Aquifer solids at the field site (Schoolcraft, Ml) have been colonized by
indigenous organisms (Chapter 6). In a model aquifer experiment, strain KC
was no longer detected in the liquid phase 4 weeks after inoculation, but was
found on the solid phase (Zhu 1994). Pumping bleach or ozone into the aquifer
prior to inoculation may remove many of the indigenous flora from the solids
and create available surface sites for colonization by Pseudomonas stutzeri KC.

Disinfection with hydrogen peroxide and bleach was tested in liquid
phase batch experiments. Hydrogen peroxide proved to be an excellent
disinfectant (Figure 23), but it is not commonly used for well disinfection. Bleach
is also an effective disinfectant and it is commonly used as a well disinfectant.

Bleach was most effective at 100 ppm (Figure 24).

Groundwater Flora

(C.F.UJmL)

54

 

  
   

 

 

 

 
 

 

 

 

1.0E+m

1.0E+05 --

1.0E+O4 -_

1.0903 __ ,
.le Hydrogen Peroxide
.0.1% Wdrogen Peroxide

1.0E+02 (31.5 % hydrogen Perow'de
[33% I-ydrogen Peroxide

1.0901 - I , ,

 

 

 

0 30 60 120 180
Contact'l’ime(ninutes)

Figure 23: Plate count results from disinfection of Schoolcraft
groundwater with hydrogen peroxide. Geometric mean plotted,

error bars represent one standard deviation.

Groundwater Flor

(C.F.UJmL)

1 .OE+08

55

 

1.0E+O7 - —

1.0E+06 ~

r

 

1.0E+05 w

1.0E+04 —

  

 

1 .OE+03

 

+I\b Bleach
-..o—. 2 Bleach
"0-- 100 pmeleach

 

 

 

 

 

 

 

 

 

 

0 10
Contact Time (minutes)

Figure 24: Plate count results from disinfection of Schoolcraft
groundwater with bleach. Geometric mean is plotted, error bars

represent plus or minus one standard deviation.

CHAPTER 6

FIELD DATA

MATERIALS AND METHODS

Extraction buffer. As previously reported by Warren et. al 1992, extraction
buffer can be used to remove microorganisms from solid media. To prepare
1000 mL of 20 mmol phosphate buffer solution add 400 mL of 50 mmol
phosphate buffer (Chapter 2) to 600 mL of DI water. Add KOH to adjust to pH
8.0, if necessary. Add the following chemicals to a 1 L Wheaton bottle: 1000
mL of 20 mmol phosphate buffer solution, 1000 uL of 400 mg/L Tween, 380 mg
of Ethylene bis N,N,N’,N’ tetraacetic acid, and 70 mg of yeast extract.
Autoclave the solution in the Wheaton bottle with the lid on loosely at 250° F at

17 psi.

Plate counts. To determine the density of strain KC and aquifer flora in
groundwater and solids samples, spread plate counts were used. Groundwater
samples were diluted (Chapter 2) and a range of dilutions was selected for

plating that would produce between thirty and three hundred colonies per plate.

56

57
To determine bacterial density in solid samples, approximately 0.1 gram of soil
was placed in sterile 1.5 mL microcentrifuge tubes. To extract organisms from
the soil into the liquid phase, 1 mL of extraction buffer was added to the tubes
which were then sealed and shaken for approximately two minutes (at high
speed on a vortexer). Extraction buffer was then diluted in the same manner as
groundwater samples. Soil from which samples had been extracted was dried
so that the solid phase plate count data could be normalized to colony forming
units per gram of dry soil. Serial dilutions of samples were performed in sterile
phosphate buffer (50 mM, pH 8.0). Diluted aliquots (100 pL) were spread on
R2A plates. Total colony forming units were counted after 6 days of incubation

at 20° C.

Denitrifier quantification procedure. The most reliable and convenient
method to measure denitrifier populations is to measure the denitrification
process in most probable number (MPN) tubes by screening for nitrate and
nitrite disappearance as the presumptive test (T iedje 1994). The following
description summarizes the method previously published by Tiedje (1994).
Culture medium is prepared by adding 8.0 g of nutrient broth and 0.5 g of
potassium nitrate to one liter of ultra-pure deionized water. Culture medium is

added in 10 mL aliquots to 16 mm by 125 mm Hungate tubes (have butyl rubber

58
septa in screw caps, Bellco Glass Inc., \fineland, NJ), inverted Durham tubes
are also added and sealed Hungate tubes are autoclaved for 20 minutes at
121° C. For the nitrate and nitrite presumptive test, 0.2 g of diphenylamine
[(C5H5)2NH] should be dissolved in 100 mL of concentrated sulfuric acid. The
reagent bottle containing the diphenylamine solution should be wrapped in foil
and stored in a refrigerator. To extract organisms from the liquid to solid phase,
approximately 0.1 gram of soil is placed in sterile 1.5 mL microcentrifuge tubes.

Extraction buffer, 1 mL, is added to the tubes which are then sealed and shaken

 

for approximately two minutes (at high speed on a vortexer). Serial dilutions of
the extraction solution are performed in autoclaved Hungate tubes (three per
dilution), final dilutions of 10'3 to 10‘8 should suffice. Tubes are incubated at 12°
C for fourteen days. At that time, 0.1 to 1.0 mL of medium is withdrawn by
syringe and tested for nitrate and nitrite by adding drop-wise up to six drops of
the diphenylamine reagent. A blue color indicates the presence of nitrate or
nitrite; a colorless response is considered presumptive evidence of
denitrification. The denitrifier population can be estimated using the MPN
procedure outlined below. For confirmation of the presence of denitrifiers,

visually inspect the inverted (Durham) tubes for bubble formation.

 

59
Most probable number determination. The most probable number (MPN)
method permits estimation of population density without an actual count of
single cells or colonies (Alexander 1982). On the basis of probability theory, it is
possible to combine the results from successive serial dilutions in such a way
that a single value is obtained for the most probable number of microorganisms. r-
As previously described by Halvorson and Ziegler (1933), an equation can be

solved for its one unknown variable which is the most probable number of

' V2

 

organisms.

If: ......-

RESULTS AND DISCUSSION

The fieId-scale aspect of this project involves collection of field data and
comparison with laboratory data. A grid of monitoring wells has been placed at a
field site (Schoolcraft, Ml, Field Experiment) to allow sampling throughout the
bioaugmentation zone. Prior to inoculation of the aquifer with strain KC,
groundwater was collected from the sampling wells and soil cores were
removed from the aquifer strata to provide baseline natural flora data. At the
time of inoculation and throughout the treatment process, groundwater samples

were collected so that strain KC and aquifer flora colonies could be quantified. A

60
statistically significant number of the samples were analyzed with a polymerase
chain reaction (PCR) gene probe as a confirmatory assay and to verify the
reliability of the morphology differentiation technique. Use of mixed-strain liquid
batch incubations as a predictive tool for field applications will also be
discussed.

An initial microbial site characterization should be conducted to evaluate
the effects, of introducing Pseudomonas stutzen' KC into a carbon tetrachloride
impacted aquifer (Schoolcraft, Ml, Field Experiment), on the indigenous flora.
Microbial habitat in an aquifer consists of two phases: liquid and solid. To
sufficiently quantify the density of the indigenous microbial population,
enumerative procedures should be conducted on both of these phases.

Total platable colonies and denitrifler density were identified as important
microbial indicators. Plate counts have proven to be a simple and reliable
method for enumerating both strain KC and the aquifer flora. Liquid phase plate
count data from before niche adjustment and after niche adjustment prior to
inoculation are shown in Table 2. Baseline platable colony density of
groundwater flora was approximately 105. The data from after niche adjustment
show higher platable colony densities than prior to niche adjustment. Niche
adjustment may have increased indigenous groundwater flora concentrations,

but only one of the sample locations (3.6) was assayed during both sampling

 

61
events. A larger data set is needed to draw conclusions about the effects of
niche adjustment on the indigenous population density. Baseline solid phase
plate counts were between 105 and 107 (Table 3). As expected, solid phase
densities are greater than liquid phase densities, but only by about one order of
magnitude. Plate count results underestimate the actual population density B—

because only platable organisms are quantified.

Table 2: Pre-inoculation Liquid Phase Field Results, Before and After

 

Niche Adjustment

Sample: MSU-GW—MLS-3-6-Baseline (pre-niche adjustment, all depths)

Geometric Mean
Well (C.F.U./mL)
5.45E+05
2.96E+05
2.01 E+05
6.71 E+05
5.38E+05
6.50E+05

 

\lODU’I-hOON

Sample: MSU-GW-MLS-2,3-14 (post-niche adjustment)

 

Cell Depth Geometric Mean
Numbfl Well (feet) Mimi.)
2.8.14 2 8 1.63E+06
3.6.14 3 6 1.26E+06

3.8.14 3 8 9.79E+05

62

Table 3: Plate Count Results from Schoolcraft Aquifer Solids Before

Inoculation with Pseudomonas stutzerf KC

Geometric Mean of Standard Deviation of

Groundwater flora Groundwater flora
Depth (C.F.U. I gram (C.F.U. I gram

Boring # Core # (feet) of dry soil) of dry soil)
MSU-SO-MLS-3 2 60.5 5.44E+06 3.45E+06
MSU-SO-MLS-3 2 61.5 2.53E+07 1 .01E+07
MSU-SO-MLS-3 2 62.5 6.32E+06 2.10E+06
MSU-SO-MLS-3 2 64.0 5.49E+05 1 .92E+05
MSU-SO-MLS-3 2 65.5 3.81 E+06 1.27E+06
MSU-SO-MLS-6 1 61.5 1.29E+07 2.93E+06
MSU-SO-INW-1 2 61.5 3.50E+06 3.34E+05
MSU-SO-INW—1 2 62.5 2.33E+06 5.95E+05
MSU-SO-lNW—1 2 63.5 4.79E+06 1.54E+06

Pseudomonas stutzen' KC is a denitrifying organism (Criddle et al. 1990).
Of the many types of microorganisms in the indigenous population, denitrifiers
could prove to be the most competitive with strain KC for resources. Baseline
denitrifier Most Probable Number (MPN) assays were conducted on soil
samples to quantify this segment of the indigenous community. Results of MPN
assays are shown in Table 4. Approximately 103 to 105 denitrifiers per gram of

soil existed on the aquifer solids prior to niche adjustment. Solid phase assays

(both plate counts and MPN tubes) may underestimate the denitrifler population

 

63
due to less than one hundred percent extraction of the organisms from the solid

to liquid phase.

Table 4: Most Probable Number (MPN) Denitrifier Assay Results from
Schoolcraft Aquifer Solids Before Inoculation with Pseudomonas stutzen'
strain KC

Geometric Mean of Standard Deviation of

 

Groundwater flora Groundwater flora
Depth (denitrifiers I gram (denitrifiers / gram
Boring # Core # (feet) of dry soil) of dry soil)

MSU-SO-MLS-3 2 60.5 3.22E+04 3.13E+04
MSU-SO-MLS-3 2 61.5 1.87E+05 4.01 E+05
MSU-SO-MLS-3 2 62.5 7.66E+03 1.19E+04
MSU-SO-MLS-3 2 64.0 3.85E+04 4.1 1 E+04
MSU-SO-MLS-3 2 65.5 1 .22E+05 1 .08E+05
MSU-SO-MLS-6 1 61.5 6.25E+05 5.16E+05
MSU-SO-lNW-1 2 61 .5 5.07E+05 1 .96E+06
MSU-SO-lNW-I 2 62.5 8.32E+04 1.24E+04
MSU-SO—lNW-1 2 63.5 6.84E+05 3.36E+06

Plate counts have proven to be a relatively simple and reliable method

for enumerating both Pseudomonas stutzen‘ KC and the aquifer flora. Results

from plate count assays on liquid phase samples collected at the field site

(Schoolcraft, MI, Field Experiment) are shown in Table 5. Liquid phase

64

concentrations of strain KC varied greatly, both spatially and temporally. Some
test wells (#2 and #3) consistently showed approximately 103 to 10‘ colony
forming units of strain KC per milliliter. Other wells (#4, #6, and #7) showed little
evidence of strain KC. Groundwater flora densities remained between 104 and
105 colony forming units of groundwater flora per millimeter during the period of ""
nutrient addition to the aquifer.

In wells (#2 and #3) where strain KC was present in sufficient quantities

to allow successful enumeration in the first sampling event after inoculation

 

(#15), it appears that between sampling events 16 and 17, liquid phase KC
densities decreased by approximately two orders of magnitude. It was
previously shown in a model aquifer study that four weeks after inoculation,
strain KC was no longer detected in the liquid phase, but was detectable on the
solid phase (Zhu 1994). The decreased density of strain KC in the liquid phase
could therefore be due to preferential partitioning to the solid phase. Analysis of
aquifer solids at the completion of the field study will help to confirm whether or

not strain KC migrated to the solid phase.

 

65

Table 5: Liquid phase plate counts from Schoolcraft Aquifer After
Inoculation (C.F.U. I mL)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MLS Event #15 Event #16 Event #17
Sampling
Port PKC Aquifer PKC Aquifer PKC Aquifer
Flora Flora Flora
2.1 0 @ 10'3 1.88E+05 57651-06 6.80E+05 0 @ 10“ 3.50E+05
2.2 0@10‘ 3.30E+05 4.00E+04 5.20E+05
2.3 0 @ 10" 1.51E+06 2.56E+06 2.00E+05 3.00E+04 3.80E+05
2.4 0@10" 4.70E+05 1.00E+04 4.60E+05
2.5 6.70E+05 4.00E+04 3.08E+06 6.00E+05 3.00E+04 3.00E+05
2.6 4.70E+05 5.00E+04 3.00E+04 3.90E+05
2.7 1.40E+06 0@10'5 4.BOE+06 7.00E+05 1.00E+04 6.70E+05
2.8 4.60E+05 1.20E+05 3.00E+04 1.21E+06
MLS Event #1 8 Event #19 Event #20
Sampling
Port PKC Aquifer PKC Aquifer PKC Aquifer
Flora Flora Flora
2.1 3.00E+03 6.80E+05 1.00E+03 2.00E+05 2.00E+03 5.00E+05
2.2 4.00E+03 7.90E+05 1.10E+04 4.00E+05 1.50E+04 1.01E+06
2.3 7.00E+03 8.30E+05 1.00E+03 4.30E+05 2.00E+03 3.60E+05
2.4 6.00E+03 7.30E+05 6.00E+03 5.00E+05 3.00E+03 8.80E+05
2.5 4.00E+03 5.60E+05 0@10'3 3.00E+05 1.00E+03 3.80E+05
2.6 1.00E+04 4.00E+05 3.00E+03 3.50E+05 4.00E+03 4.80E+05
2.7 6.00E+03 7.60E+05 5.00E+03 7.20E+05 4.00E+03 3.80E+05
2.8 4.00E+03 2.24E+06 4.00E+03 9.80E+05 7.00E+03 7.00E+05

 

 

 

 

 

 

 

 

Em

 

 

66

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 5 (cont’d)
MLS Event #15 Event #16 Event #17
Sampling
Port PKC Aquifer PKC Aquifer PKC Aquifer
Flora Flora Flora
3.1 0 @ 10'r 1.80E+05 4.76E+06 7.60E+05 0 Q 10" 5.60E+05
3.2 0 @ 10'3 1.58E+05 0 @ 10" 6.20E+05
3.3 0 @10‘3 4.80E+05 3.32E+06 4.40E+05 1.00E+04 7.20E+05
3.4 0 @ 10" 1.55E+05 0 @ 10“ 1.16E+06
3.5 2.27E+06 1.50E+05 1.72E+06 4.10E+05 0 @10“ 3.20E+05
3.6 5.00E+05 9.00E+04 0 @ 10'5 2.80E+O5
3.7 2.60E+05 9.00E+04 2.88E+06 1.60E+05 2.00E+04 3.80E+05
3.8 1.80E+05 1.10E+05 0 @ 10" 1.40E+06
MLS Event #1 8 Event #19 Event #20
Sampling
Port PKC Aquifer PKC Aquifer PKC Aquifer
Flora Flora Flora
3.1 0@10"5 1.48E+05 1.00E+03 1.48E+05 1.00E+03 1.18E+06
3.2 3.00E+03 1.48E+05 2.00E+03 4.00E+05 0@10’3 5.60E+05
3.3 9.00E+03 5.60E+05 2.00E+03 6.00E+05 3.00E+03 7.70E+05
3.4 1.00E+03 1.36E+06 0 @ 10'3 2.32E+05 1.20E+04 2.34E+06
3.5 1.70E+04 1.12E+06 0@10’3 8.00E+05 1.00E+04 1.82E+06
3.6 6.40E+04 1.56E+06 o @10“ 8.40E+05 2.70E+04 1.82E+06
3.7 2.00E+03 9.60E+05 0 @ 10¢ 9.20E+05 1.80E+04 2.31 E+06
3.8 0@10'5 2.00E+06 3.00E+04 2.00E+06 1.00E+04 5.10E+06

 

 

 

 

 

 

 

 

 

 

 

 

67

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 5 (cont’d)
MLS Event #15 Event #16 Event #17
Sampling

Port PKC Aquifer PKC Aquifer PKC Aquifer
Flora Flora Flora

4.1 0 @ 10‘3 8.50E+04 1.60E+04 2.56E+05

4.2 0 @ 10'3 1.24E+05 5.00E+03 8.60E+04

4.3 0 @ 10‘3 8.90E+04 8.00E+03 1.72E+05 0 @ 10'3 1.12E+05

4.4 0 @ 10'3 7.90E+04 0 @ 107‘F 6.40E+04

4.5 0 @ 10'3 7.60E+04 0 @ 10" 4.50E+05 0 @ 10‘r 9.60E+04

4.6 1.00E+03 9.60E+04 0 @ 10“ 1.51E+05

4.7 0 @ 10'3 1.63E+05 0 @ 10" 1.60E+06 0 @ 10’3 1.60E+05

4.8 0 Cg} 10‘3 1.29E+05 0 @ 10" 1.53E+06

MLS Event #18 Event #19 Event #20

Sampling

Port PKC Aquifer PKC Aquifer PKC Aquifer
Flora Flora Flora

4.1 0 @ 10'3 1.20E+04 0 @ 10" 2.36E+05 0 @ 10" 4.90E+05

4.2 o @ 10'T 9.20904 0 @ 10" 1.28E+05 o @ 10" 5.90905

4.3 0 @ 10'3 8.60E+O4 0 @ 10'3 3.24E+05 0 Q 10’3 6.50E+05

4.4 0 @ 10'3 9.20E+04 0 @ 10'3 1.36E+05

4.5 0 @ 10’3 1.20E+05 0 @ 10'3 2.68E+05 0 @ 10'3 5.70E+05

4.6 0 @ 10"r 1.16E+05 0 @ 10‘3 2.76E+05 0 @ 10* 3.80E+05

4.7 0 @ 10’3 5.20E+04 0 @ 10'3 4.30E+05

4.8 0 @ 10'3 2.28E+05 0 @ 10" 6.40E+04 0 Q 10’3 2.14E+05

 

 

 

 

 

 

 

 

 

 

 

68

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 5 (cont’d)
MLS Event #15 Event #16 Event #17
Sampling
Port PKC Aquifer PKC Aquifer PKC Aquifer
Flora Flora Flora
5.1 0 @ 10" 2.40E+05 1000 1.38E+05 0 @ 10" 1.07E+05
5.2 0 @ 10'“ 1.36E+05 0 @ 10" 6.10E+06
5.3 0 @ 10" 1.40E+05 0 @ 10'3 1.98E+05 0 @ 10‘5 6.80E+06
5.4 0 @ 10‘r 1.92905 0 @ 10" 1.31906
5.5 0 @ 10" 8.10E+04 0 @ 10'3 1.61E+05 0 @ 10‘ 2.80E+05
5.6 0 @ 10“ 9.10904 0 @ 10" 1.52906
5.7 0 @ 10“ 1.17E+05 0 @ 10'3 1.59E+05 0 @ 10‘ 2.80E+05
5.8 0 @ 10'“ 7.20E+04 0 Q 10" 3.80E+05
MLS Event #18 Event #19 Event #20
Sampling
Port PKC Aquifer PKC Aquifer PKC Aquifer
Flora Flora Flora
5.1 0 @ 10‘ 8.70E+05 3.00E+03 4.70E+04 0 @ 10‘3 2.39E+05
5.2 0 @ 10" 5.10E+05 4.00E+03 1.70E+04 0 @ 10'3 2.96E+05
5.3 0 @ 10“ 1.02E+06 7.00E+03 1.40E+04 0 @ 10"r 1.54E+05
5.4 0 @ 10“ 8.20E+05 6.00E+03 2.30E+04 0 @ 10"r 4.96E+05
5.5 0 @ 10‘ 2.64E+06 4.00E+03 2.00E+04 0 @ 10“r 1.88E+05
5.6 o @ 10“ 3.16906 1.00904 3.20905 0 @ 10" 4.20904
5.7 0 @ 10“ 1.04E+06 6.00E+03 2.50E+05 0 @ 10" 2.26E+05
5.8 0 @ 10J 1.32E+06 4.00E+03 1.50E+04 0 @ 10'3 3.08E+05

 

 

 

 

 

 

 

 

 

 

 

69

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 5 (cont’d)
MLS Event #15 Event #16 Event #17
Sampling
Port PKC Aquifer PKC Aquifer PKC Aquifer
Flora Flora Flora
6.1 0 @ 10’3 6.80E+04 0 @ 10’3 1.83905 0 @ 10“ 3.20E+05
6.2 0 @ 10'3 1.02E+05 0 @ 10“ 3.80E+05
6.3 0 @ 10'3 1.68E+05 0 @ 10'3 1.52E+05 0 @ 10" 3.00E+05
6.4 0 @ 10" 7.20904 0 @ 10‘ 3.50905
6.5 0 @ 10'3 4.10E+04 0 @ 10'3 1.07E+05 0 @ 10" 2.23E+06
6.6 0 @ 10'3 2.70E+04 0 @ 10‘ 2.48E+06
6.7 0 @ 10'3 8.20E+04 1000 1.55E+05 0 @ 10"
6.8 0 @ 10'3 2.70E+04 0 @ 10" 3.64E+06
MLS Event #18 Event #19 Event #20
Sampling
Port PKC Aquifer PKC Aquifer PKC Aquifer
Flora Flora Flora
6.1 2.00E+04 2.08E+06 0 @ 10"“ 2.64E+05 0 @ 10" 7.70E+04
6.2 0 @ 10‘ 3.48E+06 0 @ 10'3 1.16E+05 0 @ 10'3 4.60E+04
6.3 1.00E+04 6.50E+05 0 @ 10'3 3.40E+05 0 @ 10'3 6.70E+04
6.4 0 @ 10‘ 4.50E+08 0 @ 10'3 2.12E+05 0 @ 10’3 4.10E+04
6.5 0 @ 10" 2.80E+07 0 @ 10’3 2.00E+05 0 @ 10'3 1.88E+05
6.6 2.00E+04 1 .44E+06 0 @ 10’3 1.47E+05
6.7 0 @ 10" 9.60E+05 0 @ 10’3 3.00E+05 0 @ 10“ 1.02E+05
6.8 0 @ 10‘ 1.4BE+06 0 @ 10’3 1.64E+05 0 @ 10'3 5.40E+04

 

 

 

 

 

 

 

 

 

 

70

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 5 (cont’d)
MLS Event #15 Event #16 Event #17
Sampling
Port PKC Aquifer PKC Aquifer PKC Aquifer
Flora Flora Flora
7.1 0 @ 10'3 3.50E+05 0 @ 10'3 2.60E+05 0 @ 10" 4.90E+06
7.2 0 @ 10'3 3.00E+05 0 @ 10‘5 3.00E+06
7.3 0 @ 10" 4.10E+05 0 @ 10‘3 1.99E+06 0 @ 10'5 5.30E+06
7.4 0 @ 10‘3 2.06905 0 @ 10‘5 3.50906
7.5 0 @ 10'3 3.20E+05 0 @ 10" 1.41E+06 0 @ 10’” 4.60E+06
7.6 o @ 10‘3 2.90905 8.20E+06
7.7 0 @ 10'3 9.50E+05 0 @ 10'3 1.80E+06 8.80E+06
7.8 0 @ 10'3 1.18E+06 1.00E+05 7.60E+06
MLS Event #18 Event #19 Event #20
Sampling
Port PKC Aquifer PKC Aquifer PKC Aquifer
Flora Flora Flora
7.1 1.00E+05 4.40E+06 0 @ 10'3 3.20E+04 0 @ 10'2 1.20E+04
7.2 2.00E+05 3.90E+06 0 @ 10'3 7.00E+03 0 @ 10'3 9.20E+04
7.3 6.00E+04 1.60E+05 0 @ 10" 5.70E+04 0 @ 10'3 8.60E+04
7.4 2.00E+04 5.10E+06 0 @ 10'3 1.40E+04 3.00E+04 3.88E+06
7.5 0 @ 10‘ 3.90E+05 0 @ 10‘ 3.30E+05 0 @ 10'3 5.00E+05
7.6 0 @ 10“ 7.00E+05 0 @ 10'3 1.92E+05 0 @ 10" 4.20E+05
7.7 0 @ 10“ 4.50E+05 0 @ 10'3 6.10E+05 6.00E+04 3.40E+05
7.8 0 @ 10“ 1.52E+06 0 @ 10'3 7.70E+05 0Q 10'3 2.06E+06

 

 

 

 

 

 

 

 

 

 

 

CHAPTER 7

ENGINEERING APPLICATION

Bioaugmentation is emerging as a promising technology for groundwater
applications. If an introduced species cannot coexist with or displace an
indigenous population, bioaugmentation may not be a plausible option. Addition

of alkalinity can be used to create a favorable niche for colonization by an

 

exogenous population. Strain KC multiplies rapidly at pH 8.2, but not at pH 7.5.
For successful remediation with KC, it is therefore imperative to maintain pH
within the remediation zone near 8.2. When remediation activities are complete,
niche adjustment provides another advantage. If alkalinity is no longer added,
the pH of the remediation zone should return to its natural level. Wlth the return
of natural conditions, indigenous flora should displace the exogenous
population.

If pH adjustment is not sufficient to provide the exogenous population
with a competitive advantage, pre-inoculation disinfection could be used to
reduce the indigenous flora concentration. After disinfection, exogenous
organisms would have a better chance of successfully colonizing an aquifer.
Further experimental investigations are necessary if in-situ bioremediation is to

become a viable alternative for remediating chemically impacted sites, but niche

71

 

72
adjustment and pre-inoculation disinfection should provide competitive
advantages to exogenous populations.
Although single nutrient competition theory was derived for continuous

culture experiments, it was used to predict the outcome of batch experiments

 

described in earlier chapters. Some of the batch experiment results at pH 7.5, '—
where the groundwater flora was predicted to outcompete KC, seemed to

contradict single nutrient theory predictions. At pH 7.5, although KC did not

appear to growing, it maintained a higher population density than the L

groundwater flora. In continuous culture however, KC would probably be
displaced by the groundwater flora at pH 7.5. Although single nutrient theory
predictions do not strictly apply to batch incubations, they do indicate which
species would dominate as the incubations became less like batch experiments
and more like continuous cultures.

One of the concerns that arose in the planning stages of the associated
bioaugmentation project was the problem of conducting laboratory experiments
using only the liquid phase when in reality the introduced organism would have
to compete for nutrients with organisms present in both the liquid and solid
phases. Even though KC is able to dominate the organisms suspended in the
liquid phase, will KC be able to compete with the organisms attached to the

solid phase? As it turned out, KC was able to persist in the aquifer over the time

 

73
frame of the bioaugmentation project. It was previously demonstrated (in the
laboratory) that KC preferentially attaches to solids. Liquid phase KC
concentrations in the field decreased in one sampling location two to three
weeks after inoculation. This liquid phase decrease could be attributed to solid
phase attachment. If so KC was able to colonize the solid phase as well.
Analysis of solids removed from the aquifer should indicate whether this is an
accurate conclusion. The subsistence of strain KC in the aquifer suggests that,
at least in some cases, the observed dominance of an organism in the liquid
phase demonstrates that it can persist when a biologically colonized solid phase

is present.

CHAPTER 8

CONCLUSIONS

1) Pseudomonas stutzen’ KC is most competitive with the groundwater flora at
pH 8.2. Strain KC’s highest pmax and lowest S,,,,,, values occur at pH 8.2. Strain

KC achieved the greatest density in mixed-strain experiments at pH 8.2.

2) Washing a starter culture probably stresses it, causing a decrease in its
competitiveness. Washing the KC starter cultures used in the um
determination experiments may have affected their outcome. Strain KC may

actually have a higher maximum specific growth rate than is reported here.

3) Single nutrient competition theory is not a viable tool for predicting the
outcome of all batch competition experiments. For example, ifthe inoculum
density is high enough, strain KC can maintain cell densities comparable to the

groundwater flora even when strain KC's Sm,n is greater.

4) Strain KC does not experience rapid decay after growth has ceased in
mixed-strain experiments. In batch, pure culture experiments, it was previously
shown that the density of strain KC decreased rapidly after the cessation of
growth . This suggests that a factor present in the mixed-strain experiments
helps stabilize the strain KC p0pulation. This stabilizing factor could be the
groundwater flora. Utilization of nitrite by the indigenous flora may protect KC

from nitrite toxicity and as a resultt stabilize the KC population.

74

75

5) The minimum inoculum size of KC required to dominate the groundwater flora
is approximately 105 CFU/mL when the groundwater flora concentration is less
than 105 CFUImL.

6) Of the starter media tested, SGW medium proved to be the best for
promoting the proliferation of KC in mixed-strain experiments. KC inocula grown
in SGW medium may be in log phase growth at inoculation and may have
access to all the nutrients it requires. KC inocula grown in the other media may

not have these two distinct advantages.

7) 100 ppm of bleach proved to be an effective groundwater disinfectant. Pre-
inoculation disinfection could be used to create niches in situations where the

indigenous population is too dense to overcome by competition.

FUTURE WORK RECOMMENDATIONS

1) Another mixed-strain competition experiment in groundwater amended with
nitrate would be useful. Ion and cell concentrations as well as CT transformation
capacity could be measured. This experiment would be similar to the
experiment where ion concentrations were assayed, but adding nitrate would
provide additional information about the relationship between strain KC and the

groundwater flora.

2) Significant differences in the competitive fitness of strain KC are observed

when it is grown in SGW versus it being acclimated to amended groundwater.

 

 

76

Identifying the chemical differences between the two growth media should

increase the understanding of strain KC growth requirements.

3) Samples from the mixed strain experiment in which ion concentrations were
quantified were collected and frozen for genetic analysis. Genetic assays could
provide information about the initial community structure and changes in the

community during experiments.

4) Chemostat studies involving strain KC and the groundwater flora could be

used to demonstrate the applicability of single nutrient competition theory to

 

interactions of groundwater bacteria. Maximum specific growth rates for strain
KC and the groundwater community could also be measured more accurately.
Maximum specificdecay rates as a function of pH could be recorded. If strain
KC was not the dominant species under certain conditions, samples could be
collected and the remaining species could be evaluated. Many interesting
experiments (in addition to those mentioned here) could be conducted in a

chemostat.

 

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Mjgmbialfiopulatjons, ASA-SSSA. Madison, Wlsconsin. 815-820.

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Publishing Company, Inc. Redwood City, CA

. Bailey. J.E.. D.F. Ollis. 1986. Bimbennicalfinflneedmflndamflals.
McGraw-Hill, Inc.: New York, New York.

. Brock, TD. and MT. Madigan. 1991. W, Prentice
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. Criddle, CS. LA. Alvarez, and P. L. McCarty. 1991. J. Bear and MY.

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

. de Vlfit, CT 1960. On competition. Versl. Landbouw. Onderz. 66: 1-82.
. Dybas, M.J., G.M. Tatara, C.S. Criddle. 1995. Localization and
characterization of the carbon tetrachloride transformation activity of

Pseudomonas stutzen' KC. Appl. Environ. Microbiol. 61: 758-762.

. Firbank, L..,G A. R. Watkinson.1990. On the effects of competition. from
monocultures to mixtures, p. 165-192. InJ. B. Grace and D. Tilman (eds.,)

Wm. Academic Press, San Diego, Calif.

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10. Halvorson, H.O., N.R. Ziegler. 1933. Application of statistics to problems in
bacteriology: I. A means of determining bacterial populations by the dilution
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11.Hansen S.R., S.P. Hubbell. 1980. Single-nutrient microbial competition:
qualitative agreement between experimental and theoretically forecast
outcomes. Science. 207:1491-1493. "

12. Hsu, S.B., S.P. Hubbell, P. Waltman. 1977. A mathematical theory for
single-nutrient competition in continuous cultures of microorganisms. Society
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32:366-383.

 

13.Jensen, H.L. 1963. Carbon nutrition of some microorganisms decomposing .
halogen-substituted aliphatic acids. Acta Agr. Scand. 13: 404-412.

14. Harper, J.L. 1977. The population biology of plants. Academic Press,
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15. Keshtacher-Liebson, E., Y. Hadar, Y. Chen. 1995. Oligotrophic bacteria
enhance algal growth under iron-deficient conditions. Appl. Environ. Mirobiol.
61: 2439-2441.

16. Knoll, W.H. 1994. MS. Thesis. Michigan State University. East Lansing,
Mich.

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

18. Lawrence, A. W., 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.

19. Lehman, J.T., D.B. Botkin, and GE. Likens. 1975. The assumptions and
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Limnology and Oceanography. 20: 343-364.

79

20. Leon, J.A., D.B. Tumpsen. 1975. Competitions between two species for two
complementary or substitutable resources. Journal of Theoretical Biology.
50: 185-201.

21 . Monod, J. 1942. Recherches sur la croissance des bacteriennes. Hermann
and Cie (eds.) Rue de la Sorbonne, Paris.

22. Novick, A., L. Szilard. 1950. Description of a chemostat. Science. 112: 715-
716.

23. Petersen, R. 1975. The paradox of the plankton: an equilibrium hypothesis.
American Naturalist. 109: 35-49.

24. Rittmann, B.E., P.L. McCarty. 1980. Model of steady-state biofilm kinetics.
Biotechnolgy and Bioengineering. 22: 2243-2357.

25.Semprini, L., 6.0. Hopkins, P.L. McCarty, P.V. Roberts. 1992. ln-situ
transformation of carbon tetrachloride and other halogenated compounds
resulting from biostimulation under anoxic conditions. Env. Sci. Tech. 26:
2454-2461.

26. Snaydon, R.W. 1991. Replacement or additive designs for competition
studies? J. Appl. Ecol. 28: 930-946.

27.Stewart, F .M., BR. Levin. 1973. Partitioning of resources and the outcome
of interspecific competition: a model and some general considerations.
American Naturalist. 107: 171-198.

28.Stumm, W., J.J. Morgan. 1981. W, 2nd ed.; John Wlley &
Sons: New York, 248.

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

30.Taylor, P. P. Wllliams. 1975. Theoretical studies on the coexistence of
competing species under continuous flow conditions. Canadian Journal of
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31 .Tiedje, J.M. 1994. Denitflflers, ASA-SSSA. Madison, Wisconsin. 815-820.

 

80

32.Tilman, D. 1977. Resource competition between planktonic algae: an
experimental and theoretical approach. Ecology. 58:338-348.

33.Tilman, D. 1980. Resources: a graphical-mechanistic approach to
competition and predation. American Naturalist. 116: 362-393.

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

35.van Uden, N. 1967. Transport-limited growth in the chemostat and its
competitive inhibition; a theoretical treatment. Archiv fur Mikrobiologie. 58:
145-1 54.

36.Vogel, T.M., C.S. Criddle, P.L. McCarty. 1987. Transformations of
halogenated aliphatic compounds. Env. Sci. Tech. 21: 722-736.

37.Warren, T.M., V. Wllliams, M. Fletcher. 1992. Influence of Solid Surface,
Adhesive Ability, and lnoculum Size on Bacterial Colonization in Mlcrocosm
Studies. Appl. Environ. Microbiol. 58: 2954-2959.

38.V\filson, M., S.E. Lindow. 1994. Ecological similarity and Coexistence of
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39.Zhu, D. 1994. MS. Thesis. Michigan State University. East Lansing,
Michigan.

APPENDICES

APPENDIX A

MAXIMUM SPECIFIC GROWTH RATE RESULTS

Table A-1: Data used to calculate um of PKC at pH 7.0

 

 

 

 

 

 

 

 

 

PKC, pH 7.0
Tlme Replicate #1 Replicate #2 Replicate #3
(hours) 0.0 u (days") 0.0 u (days") 0.0 u (days")
0 0.001 3.5176 0.002 2.5670 0 —--
17.5 0.013 0.6621 0.013 0.6621 0.013 0.5281
49 0.031 -0.0593 0.031 0.2909 0.026 0.3604
76 0.029 0.1591 0.043 -0.2348 0.039 0.0500
100 0.034 0.0730 0.034 0.1661 0.041 0.0887
169.5 0.042 0.0221 0.055 -0.0176 0.053 0.0176
220 0.044 0.1396 0.053 -0.0396 0.055 0.0180
267.5 0.058 0.049 0.057

 

Table A-2: Data used to calculate pm, of PKC at pH 7.5

 

 

 

 

 

 

 

 

 

PKC, pH 7.5
Time Replicate #1 Replicate #2 Replicate #3
(hours) 0.0 u(days'1) on u (days‘) 0.0 u (days‘)
0 -0.006 --- -0.008 --- 0.005 2.296
15 0.018 4.349 0.014 6.613 0.021 0.372
18 0.031 0.443 0.032 0.000 0.022 2.090
23 0.034 1.776 0.032 0.224 0.034 2.097
29.5 0.055 0.988 0.034 2.466 0.060 1.250
39.5 0.083 0.353 0.095 -1.589 0.101 -1.538
45 0.090 -2.603 0.066 2.301 0.071 2.660
I 48 0.065 0.588 0.088 -0.377 0.099 0.091
63.5 0.095 0.083 0.069 0.361 0.105 -0.352
72.5 0.098 0.079 0.092

 

81

 

 

 

Table A-3: Data used to calculate pm of PKC at pH 8.0

 

 

PKC, pH 8.0
Time Replicate #1 Replicate #2 Replicate #3
(hours) 0.0 u (days‘) 0.0 u(days'1) 0.0 u (days‘)
0 0.001 2.639 0.001 2.996 0.002 2.251
24 0.014 0.932 0.020 0.234 0.019 1.121
29 0.017 0.373 0.021 0.500 0.024 0.393
84 0.040 0.231 0.066 0.049 0.059 -0.086
98.5 0.046 0.093 0.068 0.032 0.056 0.566
120 0.050 0.396 0.070 0.210 0.093 0.134
150 0.082 -0.110 0.091 0.053 0.110 0.055
169.5 0.075 0.156 0.095 0.103 0.115 0.104
190.5 0.086 0.104 0.126

 

 

 

 

 

 

 

 

Table A-4: Data used to calculate pm, of PKC at pH 8.2

 

 

 

PKC, pH 8.2
Time Replicate #1 Replicate #2 Replicate #3
(hours) 0.0 u (days‘) on u (days1 ) 0.0 u (days‘)
0 0 --- 0.001 3.078 0.001 2.877
20 0.009 0.120 0.013 -0.300 0.011 0.498
41 0.010 0.662 0.010 0.392 0.017 0.172
77 0.027 0.018 0.022

 

 

 

 

 

 

 

 

83

Table A-5: Data used to calculate pm of Groundwater Flora at pH 7.0

 

Groundwater Flora. pH 7.0

 

 

 

 

 

 

 

 

 

Time Replicate #1 Time Replicate #2A Replicate #3A
(hours) 0.0. uldays"l (hours) 0.0. uldays") 0.0. utdays")
0.0 0.001 0.853 3.0 -0.002 —— -0.001 ...-

19.5 0.002 ....- 22.5 0.002 -2.218 0.001 ....

27.0 0.000 ...- 30.0 0.001 3.197 0.000 -—-

45.0 0.003 0.739 48.0 0.011 0.739 0.006 1.564
67.5 0.006 0.619 70.5 0.022 0.314 0.026 0.201

103.0 0.015 0.463 106.0 0.035 0.121 0.035 0.000
129.5 0.025 -0.292 132.5 0.040 0.000 0.035 0.127
140.0 0.022 0.118 143.0 0.040 0.000 0.037 -0.107
166.0 0.025 0.251 190.0 0.040 -0.105 0.030 0.236
211.0 0.040 0.362 214.0 0.036 0.133 0.038 -0.055
234.5 0.057 -0.029 237.5 0.041 -0.011 0.036 0.012
264.5 0.055 -0.079 290.5 0.040 0.062 0.037 0.380
287.5 0.051 -0.550 309.5 0.042 0.024 0.050

306.5 0.033 0.141 333.5 0.043

330.5 0.038

 

Note A: Data recorded at different time scale 3.0, 22.5, 30, 48,
70.5, 106, 132.5, 143, 190, 214, 237.5, 290.5, 309.5, 333.5.

 

Table A-6: Data used to calculate pm of Groundwater Flora at pH 7.5

 

 

Groundwater Flora, pH 7.5

 

Time Replicate #1 Replicate #3 Time Replicate 1192A
(hours) O.D. u (days") 0.0. u (days'1l (hours) 0.0. u ldays")
0.0 0.000 --- 0.000 --- 6.0 0.007 ---
17.0 0.004 1.024 0.000 --- 19.0 -0.002 0.000
26.5 0.006 --- 0.002 2.931 22.0 -0.002 ---
43.0 0.000 --- 0.015 -3.353 27.0 0.003 ---
47.5 0.013 0.209 0.008 0.630 33.5 -0.001 ---
56.0 0.014 3.718 0.010 4.015 43.5 0.013 4.322
65.5 0.061 -3.467 0.049 2.331 49.0 0.035 -1.785
68.3 0.041 0.944 0.064 0.513 52.0 0.028 1.073
77.5 0.059 0.070 0.078 -0.027 67.5 0.056 1.353
89.0 0.061 0.266 0.077 0.025 76.5 0.093 0.100
113.5 0.080 -0.459 0.079 -0.551 121.0 0.112 0.064
122.0 0.068 -O.288 0.065 -0.185 137.5 0.117 -0.063

146.0 0.051 0.054 157.5 0.111

 

 

 

 

 

 

 

 

Note A: Data recorded at different time scale: 6,17,26.5,43,47.5,56
hours.

Table A-7: Data used to calculate pm of Groundwater Flora at pH 8.0

 

Groundwater Flora, pH 8.0

 

 

 

Time Replicate #1A Replicate #2 Replicate #3
(hours) 0.0. u ldays“) 0.0. u ldays") 0.0. u ldays“)
0 -0.002 0.000 0.001 ....- 0 ~-
19 -0.002 -2.559 0 —— 0.004 ~-
32 -0.001 ....- 0.001 2.944 0 ~-
56 0.013 -0.729 0.019 1.787 0.021 1.652
63 0.011 1.538 0.032 0.137 0.034 0.105

88.5 0.036 0.037 0.038

 

 

 

 

 

 

 

Note A: Data recorded at different time scale: 0, 17,
26.5, 43, 47, 56 hours.

 

85

Table A-8: Data used to calculate pm, of Groundwater Flora at pH 8.5

 

Groundwater Flora, pH 8.5

 

 

Time Replicate #1 Time Replicate #2A Replicate #3A
(hours) 0.0. u (days'1) (hours) 0.0. u Idays") 0.0. u (days’1)
3.0 0.001 0.853 0.0 0.001 0.000 -0.001 ——
22.5 0.002 --- 19.0 0.001 1.344 0.001 1.040
30.0 0.000 --- 51.0 0.006 1.677 0.004 1.180
48.0 0.004 0.666 67.5 0.019 0.255 0.009 0.556
106.0 0.020 0.241 116.5 0.032 0.212 0.028 0.141
143.0 0.029 -O.214 144.5 0.041 0.000 0.033 0.028
169.0 0.023 0.225 170.0 0.041 -0.014 0.034 0.063
190.0 0.028 -0.074 212.5 0.040 0.038
214.0 0.026 -0.082

 

 

 

 

 

 

 

 

Note A: Data recorded at different time scale: 0, 19, 51, 67.5, 116.5,
144.5, 170, 212.5 hours.

 

APPENDIX B

RESULTS FROM MIXED STRAIN COMPETITION EXPERIMENTS

Table B-1: Mixed-strain experiment, initial pH 7.5, KC starter medium: SGW
medium, initially 150:1 (PKC:Schoolcraft)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Initially 150:1 (PKC:Schoolcraft) 148.89
Time PKC Geo Sthev Ground- Geo Sthev
Mean water Mean

(hrs) (CFU/mL) (CFU/mL)
0 1.11E+07 1.25E+05 Rep. 1
0 1.07E+07 1.79E+07 2.25E+07 1.11E+05 1.20E+05 8.09E+03 Rep.2
0 4.82E+07 1.25E+05 Rep. 3
25.5 1.22E+07 1.00E+05 Rep.1
25.5 1.40E+07 1.21E+07 1.75E+06 2.00E+05 1.26E+05 5.84E+04 Rep. 2
25.5 1.05E+07 1.00E+05 Rep. 3
74 2.50E+07 5.00E+05 Rep. 1
74 2.30E+07 2.31E+07 1.85E+06 1.20E+06 8.14E+05 3.57E+05 Rep.2
74 2.13E+07 9.00E+05 Rep. 3
95 1.35E+07 1.90E+06 Rep. 1
95 2.33E+07 1.4OE+07 7.58E+06 1.10E+06 1.40E+06 4.19E+05 Rep. 2
95 8.70E+06 1.30E+06 Rep. 3
168 1.06E+07 1.36E+07 Rep. 1
168 5.60E+06 7.70E+06 2.53E+06 9.00E+05 3.40E+06 7.43E+06 Rep. 2
168 7.70E+06 3.20E+06 Rep. 3
217 Rep. 1
217 2.34E+06 3.32E+06 1.69E+06 7.50E+05 1.28E+06 1.06E+06 Rep.2
217 4.70E+06 2.20E+06 Rep. 3

 

86

 

87

Table B-2: Mixed-strain experiment, initial pH 8.0, KC starter medium: SGW
medium, initially 18:1 (PKC:Schoolcraft)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Initially 18:1 (PKC:Schoolcraft) 17.92
Time PKC Geo Sthev Schoolcraft Geo Sthev
Mean Mean
(hours) (CFU/mL) (CFU/mL)
0 1.40E06 1.44E05 Rep.1
0 2.30E06 2.74E06 2.77E06 1.39E05 1.53E05 2.18E04 Rep.2
0 6.40E06 1.79E05 Rep.3
22.5 4.10E06 Rep.1
22.5 7.70E06 6.74E06 2.89E06 1.00E00 1.22E00 Rep.2
22.5 9.70E06 Rep.3
46 9.10E06 1.00E00 Rep.1
46 1.17E07 1.11E07 1.99E06 1.00E00 1.00E00 0.00E00 Rep.2
46 1.30E07 1.00E00 Rep.3
70 1.51E07 1.00E05 Rep.1
70 1.84E07 1.61E07 1.94E06 2.00E05 2.15E05 2.17E05 Rep.2
70 1.50E07 5.00E05 Rep.3
117 1.38E07 Rep.1
117 1.17E07 1.67E07 9.60E06 2.00E05 7.75E05 2.30E06 Rep.2
117 2.90E07 3.00E06 Rep.3
168 7.10E06 1.00E05 Rep.1
168 7.40E06 8.15E06 1.77E06 1.00E05 1.26E05 5.84E04 Rep.2
168 1.03E07 2.00E05 Rep.3
204 5.40E06 5.00E05 Rep.1
204 5.60E06 6.78E06 2.80E06 1.00E05 2.15E05 2.17E05 Rep.2
204 1.03E07 2.00E05 Rep.3

 

 

 

 

 

 

 

88

Table B-3: Mixed-strain experiment, initial pH 8.2, KC starter medium: SGW
medium, initially 100:1 (PKC:Schoolcraft)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Initially 100:1 (PKC:Schoolcraft) 96.14
Time PKC Geo Sthev Ground- Geo Sthev
Mean water Mean
(hours) (CFUImL) (CFU/mL)
0 4.00E06 3.00E04 Rep. 1
0 1.00E06 2.52E061.83E06 3.00E04 2.62E04 5.80E03 Rep.2
0 4.00E06 2.00E04 Rep. 3
22 2.00E08 1.00E00 Rep. 1
22 3.00E08 2.36E08 5.31E07 1.00E00 1.00E00 0.00E00 Rep. 2
22 2.20E08 1.00E00 Rep. 3
96 1.00E07 1.00E00 Rep. 1
96 1.40E07 1.25E07 2.32E06 1.00E00 1.00E00 0.00E00 Rep.2
96 1.40E07 1.00E00 Rep. 3
120 1.70E07 1.00E06 Rep. 1
120 3.20E07 2.90E071.43E07 2.00E06 1.41E06 7.17E05 Rep.2
120 4.50E07 Rep. 3
144 1.65E07 2.90E06 Rep. 1
144 1.12E07 1.44E07 3.02E06 2.90E06 2.33E-09 Rep.2
144 1.63E07 Rep. 3

 

 

 

 

 

 

 

 

 

89

Table B-4: Mixed-strain experiment, initial pH 8.2, KC starter medium: SGW
medium, initially 160:1 (PKC:Schoolcraft)

Initially 160:1 (PKC:Schoolcraft)

158.30

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Time PKC GeoMean Sthev Ground GeoMean Sthev
water
(hrs) (CFUImL) (CFUImL)
O 4.00E+06 3.00E+04 Rep.1
0 5.10E+06 4.15E+06 8.21E+05 3.00E+04 2.62E+04 5.80E+03 Rep.2
0 3.50E+06 2.00E+04 Rep.3
48 5.20E+07 6.00E+05 Rep.1
48 4.10E+07 4.86E+07 7.01E+06 6.00E+05 4.76E+05 1.76E+05 Rep.2
48 5.40E+07 3.00E+05 Rep.3
72 2.70E+07 0 @ 104-6 Rep.1
72 6.30E+07 4.51E+07 1.91E+07 0 @10"-6 1.00E+00 1.00E+00 Rep.2
72 5.40E+07 0 @ 104-6 Rep.3
96 5.10E+07 2.00E+06 Rep.1
96 4.50E+07 4.86E+07 3.22E+06 1.00E+06 1.26E+06 5.84E+05 Rep.2
96 5.00E+07 1.00E+06 Rep.3
144 4.50E+07 0 @ 104-6 Rep.1
144 4.30E+07 4.53E+07 2.52E+06 1.00E+06 1.00E+06 6.59E—10 Rep.2
144 4.80E+07 1.00E+06 Rep.3
168 1.60E+07 1.00E+06 Rep.1
168 2.10E+07 1.82E+07 2.52E+06 0@10"-6 1.00E+06 1.00E+00 Rep.2
168 1.80E+07 0 @ 104-6 Rep.3

 

 

90

Table B-5: Mixed-strain experiment, initial pH 8.2, KG starter medium: SGW

medium, initially 7.2:1 (PKC:Schoolcraft)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Initially 7.2:1 (PKC:Schoolcraft) 7.18
Time PKC GeoMean Sthev Ground- GeoMean Sthev
water

(hrs) (CFUImL) (CFUImL)

0 2.60E+05 9.00E+04 Rep.1
0 2.99E+05 3.12E+05 6.69E+04 1.30E+04 4.34E+04 4.36E+04 Rep.2
0 3.90E+05 7.00E+04 Rep.3
22 1.62E+07 2.00E+05 Rep.1
22 2.25E+07 1.89E+07 3.19E+06 1.41E+05 7.17E+04 Rep.2
22 1 .86E+07 1.00E+05 Rep.3
45 4.70E+07 1.20E+07 Rep.1
45 5.50E+07 4.95E+07 4.62E+06 2.00E+06 4.58E+06 5.57E+06 Rep.2
45 4.70E+07 4.00E+06 Rep.3
68 7.30E+07 2.00E+06 Rep.1
68 6.40E+07 6.62E+07 5.86E+06 4.00E+06 3.63E+06 2.05E+06 Rep.2
68 6.20E+07 6.00E+06 Rep.3
92 6.20E+07 2.90E+06 Rep.1
92 5.70E+07 6.06E+07 3.22E+06 3.00E+06 2.97E+06 5.77E+04 Rep.2
92 6.30E+07 3.00E+06 Rep.3
120 5.30E+07 5.00E+06 Rep.1
120 6.80E+07 5.93E+07 7.65E+06 1.00E+06 2.71E+06 2.22E+06 Rep.2
120 5.80E+07 4.00E+06 Rep.3

 

 

 

 

 

 

 

 

 

 

91

Table B-6: Mixed-strain experiment, initial pH 8.2, KC starter medium: SGW
medium, washed inoculum, initially 11:1 (PKC:Schoolcraft)

Initially 1 1 :1 (PKC:Schoolcraft)

11.18689

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Time PKC GeoMean Sthev Ground- GeoMean Sthev
water

(hrs) (CFUImL) (CFUImL)

0 2.00E+05 3.00E+04 Rep.1
O 4.20E+05 2.93E+05 1.11E+05 3.00E+04 2.62E+04 5.80E+03 Rep.2
0 3.00E+05 2.00E+04 Rep.3
22 5.00E+03 3.60E+05 Rep.1
22 1.00E+03 2.24E+03 2.66E+03 6.00E+05 3.46E+05 2.11E+05 Rep.2
22 1.91 E+05 Rep.3
51 3.10E+06 Rep.1
51 4.00E+05 4.00E+05 1.00E+00 4.00E+06 3.51E+06 4.52E+05 Rep.2
51 3.50E+06 Rep.3
72 1.00E+00 7.90E+06 Rep.1
72 1.00E+00 1.00E+00 1.00E+00 2.65E+07 9.66E+06 1.26E+07 Rep.2
72 1 .00E+00 4.30E+06 Rep.3

 

 

 

 

 

 

 

Table B-7: Mixed-strain experiment, initial pH 8.2, KC starter medium: SGW
medium, washed inoculum, initially 15:1 (PKC:Schoolcraft)

Initially 15:1(PKC:Schoolcraft)

15.04

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Time PKC GeoMean Sthev Groundwater GeoMean Sthev

(hours) (CFUImL) (CFUImL)
0 3.60E+06 7.00E+05 Rep.1
0 4.40E+06 3.98E+06 5.66E+05 1.00E+05 2.65E+05 4.65E+05 Rep.2
22 4.50E+05 2.60E+05 Rep.1
22 3.30E+05 3.85E+05 8.51E+04 1.50E+05 1.97E+05 7.85E+04 Rep.2
51 4.00E+05 3.10E+06 Rep.1
51 1.00E+05 2.00E+05 2.24E+05 3.50E+06 3.29E+06 2.83E+05 Rep.2
72 3.00E+05 7.90E+06 Rep.1
72 1.10E+06 5.74E+05 5.93E+05 8.10E+06 8.00E+06 1.41E+05 Rep.2

 

 

 

 

 

 

92

Table B-8: Mixed-strain experiment, initial pH 8.2, KC starter medium:

Schoolcraft medium (acclimated culture) , initially 32:1 (PKC:Schoolcraft)

Initially 32:1 (PKC:Schoolcraft)

32.1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Time KC GeoMean Sthev Ground- GeoMean Sthev
water

(hrs) (CFUImL) (CFUImL)

0 2.88E+05 3.00E+03 Rep.1
O 2.80E+05 3.10E+05 4.99E+04 3.00E+04 9.65E+03 1.51E+04 Rep.2
0 3.70E+05 1 .00E+04 Rep.3
19 4.00E+03 4.20E+04 Rep.1
19 3.46E+03 7.09E+02 7.00E+04 5.31E+04 1.44E+04 Rep.2
19 3.00E+03 5.10E+04 Rep.3
42 1.00E+00 5.40E+05 Rep.1
42 1.00E+00 1.00E+00 1.00E+00 4.20E+05 4.90E+05 6.44E+04 Rep.2
42 1.00E+00 5.20E+05 Rep.3
66 3.90E+06 Rep.1
66 2.40E+04 1.55E+04 1.01E+04 1.41E+06 2.31E+06 1.29E+06 Rep.2
66 1.00E+04 2.25E+06 Rep.3
88 7.70E+06 Rep.1
88 5.00E+04 3.16E+04 2.17E+04 7.20E+06 7.22E+06 4.51E+05 Rep.2
88 2.00E+04 6.80E+06 Rep.3
109 1.00E+00 5.40E+06 Rep.1
109 1.00E+00 1.00E+00 1.00E+00 1.06E+07 8.82E+06 3.53E+06 Rep.2
109 1.00E+00 1.20E+07 Rep.3
170 1.00E+00 1.97E+07 Rep.1
170 1.00E+00 1.00E+00 1.00E+00 1.58E+07 1.46E+07 4.87E+06 Rep.2
170 1.00E+00 1.01E+07 Rep.3

 

 

 

 

 

 

 

 

 

93

Table B-9: Mixed-strain experiment, initial pH 8.2, KC starter medium:
Schoolcraft medium (acclimated culture) , initially 308:1 (PKC:Schoolcraft)

Initially 308:1(PKC:Schoolcraft)

308

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Time PKC GeoMean Sthev Ground- GeoMean Sthev
water
(hour (CFUImL) (CFUImL)
8)
0 2.70E+06 1.00E+04 Rep.1
0 3.60E+06 3.08E+06 4.59E+05 1.00E+04 1.00E+04 1.11E-11 Rep.2
0 3.00E+06 1.00E+04 Rep.3
19 1.00E+05 ' Rep.1
19 1.00E+05 1.00E+00 1.00E+05 1.00E+05 1.00E+00 Rep.2
19 Rep.3
42 1.00E+00 4.00E+05 Rep.1
42 1.00E+00 1.00E+00 1.00E+00 2.00E+05 2.60E+05 1.11E+05 Rep.2
42 1.00E+00 2.20E+05 Rep.3
66 1.70E+06 Rep.1
66 8.00E+04 1.00E+00 3.80E+06 3.38E+06 2.22E+06 Rep.2
66 8.00E+04 6.00E+06 Rep.3
88 1.20E+05 5.00E+06 Rep.1
88 8.00E+04 7.83E+04 3.57E+04 6.70E+06 6.80E+06 2.24E+06 Rep.2
88 5.00E+04 9.40E+06 Rep.3
109 1.09E+07 Rep.1
109 2.20E+05 2.20E+05 1.00E+00 5.80E+06 8.29E+06 2.60E+06 Rep.2
109 9.00E+06 Rep.3
170 1.43E+07 Rep.1
170 3.00E+05 1.73E+05 1.60E+05 6.20E+06 7.47E+06 5.29E+06 Rep.2
, 170 1.00E+05 4.70E+06 Rep.3

 

 

 

94

Table B-10: Mixed-strain experiment, initial pH 8.0, KC starter medium:
Medium D, washed lnoculum, initially 3:1 (PKC:Schoolcraft)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Initially 3:1 (PKCquuifer Flora) 3.05
Time PKC GeoMean Sthev Ground- GeoMea Sthev
water n

(hrs) (CFUImL) (CFUImL)
0 1.60E+06 3.10E+05 Rep.1
0 6.00E+05 1.42E+06 1.26E+06 7.00E+05 4.66E+05 2.81E+05 Rep.2
0 3.00E+06 Rep.3
22 1.20E+05 1.80E+05 Rep.1
22 4.00E+04 8.32E+04 4.78E+04 3.20E+05 2.70E+05 8.81E+04 Rep.2
22 1.20E+05 3.40E+05 Rep.3
51 7.00E+04 7.00E+06 Rep.1
51 1.00E+04 3.66E+04 3.83E+04 4.20E+06 4.55E+06 1.99E+06 Rep.2
51 7.00E+04 3.20E+06 Rep.3
72 1.00E+05 1.04E+07 Rep.1
72 1.00E+04 3.16E+04 5.07E+04 7.60E+06 7.14E+06 2.94E+06 Rep.2
72 4.60E+06 Rep.3

Table B-11: Mixed-strain experiment, initial pH 8.0, KC starter medium:

Medium D, washed inoculum, initially 10:1 (PKC:Schoolcraft)

Initially 10:1 (PKC:Schoolcraft) 9.6

Time PKC GeoMean Sthev Ground- GeoMean Sthev

water

(hrs) (CFUImL) (CFUImL)
0 Rep.1
0 4.00E+05 9.59E+05 1.34E+06 1.00E+05 1.00E+00 Rep.2
0 2.30E+06 1.00E+05 Rep.3
72 4.50E+06 Rep.1
72 1.00E+00 1.00E+00 6.70E+06 4.63E+06 1.74E+06 Rep.2
72 3.30E+06 Rep.3
96 1.33E+07 Rep.1
96 1.00E+00 1.00E+00 7.10E+06 1.16E+07 4.86E+06 Rep.2
96 1 .65E+07 Rep.3

 

 

 

 

 

 

 

 

 

 

 

95

Table B-12: Mixed-strain experiment, initial pH 8.0, KC starter medium:

Medium D, washed inoculum, initially 12:1 (PKC:Schoolcraft)

 

 

 

 

 

 

 

Initially 12:1 (PKC:Groundwater) 12
Time PKC GeoMean Sthev Ground- GeoMean Sthev
water
(hrs) (CFUImL) (CFUImL)
0 1.00E+04 Rep.1
0 2.00E+04 1.20E+05 1.00E+05 1.00E+04 1.00E+04 1.58E-11 Rep.2
0 1.20E+05 1.00E+04 Rep.3
72 2.37E+06 Rep.1
72 3.00E+04 1.00E+00 2.48E+06 2.88E+06 6.48E+05 Rep.2
72 3.00E+04 2.88E+06 Rep.3

 

 

 

 

 

 

 

 

 

 

Table B-13: Mixed-strain experiment, initial pH 7.5, KC starter medium: SGW
medium, initially 8.5:1 (PKC:Schoolcraft)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(CFUImL)
Time Sample Acetate Nitrate Phos. Nitrite PKC Aquifer
(lire)

0 75-1 144.1 65.1 8.3 0.0 4.10E+07 3.00E+06

0 75-2 149.0 68.2 5.1 0.0 3.80E+07 4.00E+06

0 75-3 148.6 70.0 5.0 0.0 4.30E+07 9.00E+06

0 Avg 147.2 67.8 6.1 0.0 G.Mean 4.06E+07 4.76E+06

0 Sthv 2.721 2.48 1.877 0.0 Sthev 2.52E+06 3.29E+06
24 75-1 78.3 0.0 4.0 0.0 1.56E+07 1.60E+06
24 75-2 82.4 0.0 6.4 0.0 1.44E+07 8.00E+05
24 Avg 80.4 0.0 5.2 0.0 G.Mean 1.50E+07 1.13E+06
24 Sthv 2.899 0.0 1.697 0.0 Sthev 8.49E+05 5.74E+05
48 75-3 77.9 0.0 4.1 0.0 4.00E+07 2.00E+06
48 75-4 76.1 0.0 4.0 0.0 2.52E+07 2.00E+06
48 Avg 77.0 0.0 4.1 0.0 G.Mean 3.17E+07 2.00E+06
48 Sthv 1.273 0.0 0.071 0.0 Sthev 1.05E+07 9.88E-10
96 75-5 80.2 0.0 4.4 0.0 9.60E+06 0.00E+00
96 75-6 88.0 0.0 4.2 0.0 1.80E+07 1.20E+06
96 Avg 84.1 0.0 4.3 0.0 G.Mean 1.31E+07 1.20E+06
96 Sthv 5.515 0.0 0.141 0.0 Sthev 6.01E+06 2.33E-10
140 75-7 76.4 0.0 5.0 0.0 0.00E+00 0.00E+00
140 75-8 78.2 0.0 6.4 0.0 1.20E+06 0.00E+00
140 ES 77.3 0.0 5.7 0.0 G.Mean 1.20E+06 0.00E+00
140 Sthv 1.273 0.0 0.990 0.0 Sthev 1.20E+06 0.00E+00

 

 

 

 

 

 

 

 

 

 

 

 

96

Table B-14: Mixed-strain experiment, initial pH 8.0, KC starter medium: SGW
medium, initially 9.2:1 (PKC:Schoolcraft)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(CFUImL)
Time Sample Acetate Nitrate Phos. Nitrite PKC Aquifer
(We)

0 80-1 152.3 66.7 3.8 0.0 3.40E+07 2.00E+06

0 80-2 149.9 66.2 4.4 0.0 4.10E+07 5.00E+06

0 80-3 150.7 65.9 4.0 0.0 2.80E+07 5.00E+06

0 Avg 151.0 66.3 4.1 0.0 G.Mean 3.39E+07 3.68E+06

0 Sthv 1.222 0.404 0.306 0.0 Sthv 6.53E+06 1.77E+06
24 80-1 104.1 10.2 2.36E+07 8.00E+05
24 80-2 102.9 13.8 1.40E+07 4.00E+05
24 Avg 103.5 12.0 G.Mean 1.82E+07 5.66E+05
24 Sthv 0.849 2.546 Sthv 6.85E+06 2.87E+05
48 80-3 80.2 0.0 4.9 0.0 1.84E+07 2.00E+06
48 80-4 88.0 0.0 4.0 0.0 2.90E+07 2.00E+06
48 Avg 84.1 0.0 4.5 0.0 G.Mean 2.31E+07 2.00E+06
48 Sthv 5.515 0.0 0.636 0.0 Sthv 7.54E+06 9.88E-10
96 80-5 85.1 0.0 3.7 0.0 3.40E+06 2.00E+05
96 80-6 81.5 0.0 5.1 0.0 3.36E+06 3.20E+05
96 Avg 83.3 0.0 4.4 0.0 G.Mean 3.38E+06 2.53E+05
96 Sthv 2.546 0.0 0.990 0.0 Sthv 2.83E+04 8.54E+04
140 80-7 84.6 0.0 6.4 0.0 1.00E+05 1.00E+05
140 80-8 80.9 0.0 6.3 0.0 2.00E+05 0.00E+00
140 Avg 82.8 0.0 6.4 0.0 G.Mean 1.41E+05 1.00E+05
140 Sthv 2.616 0.0 0.071 0.0 Sthv 7.17E+04 1.46E-11

 

 

 

 

 

 

 

 

 

 

97

Table B-15: Mixed-strain experiment, initial pH 8.2, KC starter medium: SGW

medium, initially 6.8:1 (PKC:Schoolcraft)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(CFUImL)
Time Sample Acetate Nitrate Phos. Nitrite PKC Aquifer
(fire)
0 82-1 151.0 66.4 4.8 0.0 6.00E+07 7.00E+06
0 82-2 150.2 66.1 3.5 0.0 2.40E+07 2.80E+06
0 82-3 149.3 66.9 3.7 0.0 3.90E+07 9.00E+06
0 Avg 150.2 66.5 4.0 0.0 G.Mean 3.83E+07 5.61E+06
0 Sthv 0.850 0.404 0.700 0.0 Sthv 1.84E+07 3.27E+06
24 82-1 93.6 0.0 5.5 5.9 1.36E+07 4.00E+05
24 82-2 86.9 0.0 4.4 0.0 2.72E+07 8.00E+05
24 Avg 90.3 0.0 5.0 3.0 G.Mean 1.92E+07 5.66E+05
24 Sthv 4.738 0.0 0.778 4.17 Sthv 9.76E+06 2.87E+05
48 82-3 85.3 0.0 3.3 0.0 1.12E+07 1.00E+05
48 82-4 87.4 0.0 3.5 0.0 1.88E+07 3.20E+06
48 lflg 86.4 0.0 3.4 0.0 G.Mean 1.45E+07 5.66E+05
48 Sthv 1.485 0.0 0.141 0.0 Sthv 5.42E+06 2.68E+06
96 82-5 85.5 0.0 3.1 0.0 8.20E+06 1.20E+06
96 82-6 85.6 0.0 3.0 0.0 3.50E+06 2.00E+05
96 Avg 85.6 0.0 3.1 0.0 G.Mean 5.36E+06 4.90E+05
96 Sthv 0.071 0.0 0.071 0.0 Sthv 3.40E+06 7.67E+05
140 82-7 89.4 0.0 5.2 0.0 3.00E+05 0.00E+00
140 82-8 83.0 0.0 6.0 0.0 3.00E+05 1.00E+05
140 Avg 86.2 0.0 5.6. 0.0 G.Mean 3.00E+05 1.00E+05
140 Sthv 4.525 0.0 0.566 0.0 Sthv 1.16E-10 1.00E+05

 

 

 

 

 

 

 

 

 

 

 

DATA RECORDED FROM CT TRANSFORMATION ASSAYS

Table C-1: Percent CT remaining, Schoolcraft acclimated culture, pH 8.2

APPENDIX C

 

 

 

 

 

 

 

 

 

 

Time Control 308:1 32:1
(hours) Average Sthev Average Sthev Average Sthev
0.0 100.0 12.5 100.0 9.2 100.0 10.8
12.0 90.4 9.6 94.1 6.4

21.5 66.8 7.6
42.5 81.0 12.6 88.9 14.3
50.0 51.8 6.7
65.5 51.5 5.3
77.0 67.2 15.6 83.2 3.0
156.5 30.2 9.3
171.5 26.7 11.8

 

 

 

 

 

 

 

Table C-2: Percent CT remaining, SGW, pH 8.2

 

 

 

 

 

 

 

 

 

 

 

 

Time Control 160:1
(hours) Average Sthev Average Sthev
0.00 100.00 4.15 100.00 3.33
0.75 90.86 4.33 87.45 3.87
3.58 104.90 8.51 86.37 2.81
5.08 93.96 3.79 76.74 3.52
115.20 1.98 1.98

120.00 60.10 3.95

 

 

98

 

 

99

Table C-3: Percent CT remaining, SGW, pH 8.0

 

Time Control 18:1
(hours) Average Sthev Average Sthev
0 100.00 5.89 100.00 5.98
21 93.56 4.92 6.43 6.19
46 83.79 4.20 3.62 4.54

 

 

 

 

 

 

 

 

 

 

APPENDIX D

DATA RECORDED FROM DISINFECTION EXPERIMENTS

Table D-1: Groundwater disinfection with hydrogen peroxide (CF UlmL)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Time No Geo Std Geo Std Geo Std Geo Std

(min) H20, Mean Dev 1.5 % Mean Dev 3 °/o Mean Dev 0.1% Mean Dev
354000 354000 354000 354000

0 322000 314539 40855 322000 314539 40855 322000 314539 4085 322000 314539 40855
273000 273000 273000 5 273000
1 26000

30 1 1 0 29000 26254 2518
1 24000
1 12400

60 156000156000 1 1 1 1 0 10800 10059 2457
1 7600
1 1 3400

120 203000 203000 1 1 0 1 1 0 6900 5173 1824
1 1 5900
1 1 1370

180 187000 187000 1 1 0 1 1 0 2370 2077 725
1 1 2760

 

100

 

101

Table D-2: Groundwater disinfection with bleach (CF U I mL)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

'fime
(min) No Geo Std 2 ppm Geo Std 10 ppm Geo Std
Bleach Mean Dev Mean Dev Mean Dev
0 234000 234000 120000
0 237000 230213 9075 237000 230213 9075 143000 118918 22569
0 220000 220000 98000
5 256000 95000
5 216000 227166 24352 98000 104337 14821
(6.5 for 50ppm) 212000 122000
10
10 196000 196000 1
10
15 210000 99000
15 212000 207266 6430 89000 129064 88655
15 200000 244000
20
20 199000 199000 1
20
30 101000
30 189000 189000 1 100000 1 78000 93573 14255
30 104000
60 117000
60 213000 213000 1 100000 1 54000 88308 34845
60 109000

 

 

 

102

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table D-2 (cont)
Tlme
(min) 100 ppm Geo Std 25 ppm Geo Std 50 ppm Geo Std
Mean Dev Mean Dev Mean Dev
0 234000 148000 115000
0 237000 230213 9075 146000 141817 8721 53600 83368 31621
0 220000 132000 94000
5 6900 22900 9300
5 5500 6003 758 23300 23099 23100 9900 10367 1476
(6.5 for 50ppm) 5700 12100
10 5600
10 5600 5463 231
10 5200
15 4800 19300 9600
15 5700 4823 804 18700 18414 1027 16500 12819 3474
15 4100 17300 13300
20 8100
20 5800 5728 2076
20 4000
30 4100 24000 7600
30 4200 4099 100 20800 21746 1909 1 1800 9135 2221
30 4000 20600 8500
60 24900 1 1000
60 22000 22996 1620 17200 12882 3513
60 22200 11300

 

 

 

 

 

 

 

 

 

 

 

 

 

nICHIcaN STATE UNIV. LIBRARIES
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