THESIS
I

1

Date

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3 1293 01690 0726

This is to certify that the
thesis entitled ‘
FACTORS AFFECTING TRANSPORT OF
PSEUDOMONAS STUTZERI KC
presented by

Patrick D. Radabaugh

has been accepted towards fulfillment
of the requirements for

 

 

 

M. S . degree in Environmental Engineering i

 

kayak/1‘

Major professor

June 1998

MS U is an Affirmative Action/Equal Opportunity Institution

 

 

 

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FPKZTCNRSIKFFEKITHVC}TTLAPflflNCWCTCDF
IUHELHDCHMKZAL4SISTZITZIHRIBU:

By

Patrick D. Radabaugh

[\TTiESHS

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

INLASTTHK<DFT§3IEIQCIE

Department of Civil and Environmental Engineering

1998

ABSTRACT

FACTORS AFFECTING TRANSPORT OF
PS E UDOMONAS STUYZERI KC

By

Patrick D. Radabaugh

Bioaugmentation is the addition of nonnative microorganisms for bioremediation. In order
for this approach to be effective, the injected microorganisms must be transported to
specific locations where colonization can occur and contaminants can be degraded. Other
researchers have investigated methods of moving bacteria that are easily accomplished in the
laboratory, but are difficult to implement in the field. This thesis focused on simulation of
options that are practical for field use. Transport properties of Pseudamw stutzeri KC were
evaluated in column studies and modeled with clean bed filtration theory. The variables
tested include cell physiology, cell concentration, type of electron acceptor, and extent of
flocculation. Injection of strain KC as undiluted, non-flocculent, exponentially growing
inoculum resulted in the best transport. Diluted and flocculent cultures transported poorly.
The results demonstrate that inocula for bioaugmentation purposes can be prepared in a

manner that promotes cell transport, without resorting to expensive alternatives.

Patrick D. Radabaugh
1998

to Pygmy ruggers everywhere ......

iv

ACKNOWLEDGMENTS

I would first like to thank my advisor, Craig Criddle. His enthusiasm and dedication to
students is almost unfathomable. I have never seen another with such devotion to
knowledge and understanding principals in science and engineering. I wish you the best of

luck in your future endeavors, both personally and professionally.

I would like to thank the other members of my committee, Dr. Michael Dy’oas and Dr. Dave
Wiggert. Their insight was greatly appreciated. In addition, I would like to thank Mike for
countless hours teaching me all the microbiology I needed to know and about the

(in)correctness of conservative politics.

I would also like to thank my undergraduate advisor and fellow rugby player, Dr. Clint
Richardson, at New Mexico Tech. Not only did “Doc” introduce me to environmental
engineering, he also introduced me to rugby. I only hope that some of his diligence and

intelligence have rubbed off on me, as well as some of his rugby ability.

To my parents, I am forever grateful for all they did to give me all the opportunities in life
that I have had. I could write a book of thanks just to them. My folks always seemed to

believe I could do things, even when I wasn’t so sure. Patricia and John, thank you!

To the rest of my family, thank you for your concern about completing my thesis. Yes, I it is

now done. You never let me forget it was still unfinished.

To one of the most under thanked people in the world, Linda Steinman. Without your
work, no one would ever get anything done, or graduate.

To everyone else in the lab who helped me at various times including Lance Warnick, Randy
“superstar” Brown, Leslie Dybas, Mike Witt, Stanley Mathurarn, Cely Sepulveda, Blake Key,
Bob Solak, Stephen Callister, and the dishwashers. Thank you.

TABLE OF CONTENTS

List of Tables ix
List of Figures xi
List of Symbols xiii
CHAPTER 1 1
Introduction 1
Mechanisms of Deposition ....................................................................................... 4
Transport ............................................................................................................ 4

Adhesion ............................................................................................................ 4

Static System Models ................................................................................................. 5
Hydrophobicity ................................................................................................... 5

Surface Free Energy (Interfacial Tension) ........................................................... 5

DVLO (Derjaguin and Landau, Vervey and Overbeek) ....................................... 6

Isotherm Models ................................................................................................. 7

Modelling Systems .................................................................................................... 7
Hypotheses: ............................................................................................................ 14
CHAPTER 2 15
Materials and Methods 15
Organism ................................................................................................................ 15
Chemicals ................................................................................................................ 15
Groundwater .......................................................................................................... 15

Filters ...................................................................................................................... 16
Columns ................................................................................................................. 16
Methods .................................................................................................................. 16
Groundwater Medium ...................................................................................... 16

Analytical Methods ............................................................................................ 17

Growth of 1‘C-Iabeled strain KC ..................................................................... 17
Preparation of Packed Columns ........................................................................ 18

Transport Experiments ..................................................................................... 19

Data Analysis .................................................................................................... 19
CHAPTER 3 21
Influence of Cell Physiology on Transport in Porous Media 21
Introduction ............................................................................................................ 21
Materials and Methods ............................................................................................ 21
Results and Discussion ............................................................................................ 21
CHAPTER 4 31
Effects of Cell Concentration on Transport 31
Introduction ............................................................................................................ 31
Methods and Materials ............................................................................................ 31
Results and Discussion ............................................................................................ 32
CHAPTER 5 37
Effect of Flocculation on Transport in Porous Media 37
Introduction ............................................................................................................ 37
Materials and Methods ............................................................................................ 37

Results and Discussion ............................................................................................
CHAPTER 6
Engineering Application
7

Conclusions and Future Work Recommendations
Conclusions ............................................................................................................
Future Work Recommendations .............................................................................
REFERENCES
APPENDIX A
Chapter 3 Raw Data
APPENDIX B
Data from Chapter 4 Experiments
APPENDD( C
Data from Chapter 5

37
42
42
44
44
4.4
45
46
50
50
55
55
57
57

LIST OF TABLES

Table 1.1 Assumptions of clean bed filtration model

Table 1.2: List of variables

Table 3.1: Attachment efficiency and blocking factor

Table 3.2: Attachment efficiency and blocking factor with native flora

Table 5.1: Attachment efficiency and blocking factor for flocculating strain KC

Table A. 1: Effluent protein concentrations (jig/l) for transport experiments in figure 3.1
Table A2: Effluent protein concentrations (pg/l) for transport experiments in figure 3.2
Table A.3: Effluent protein concentrations (pg/l) for transport experiments in figure 3.3

Table A.4. Strain KC concentration in transport experiments represented in tables
A.1-A.3

Table A5: Cell associated 14C readings for transport experiments in figure 3.4 in CPM
(counts per minute)

Table A6: Cell associated 1‘C readings for transport experiments in figure 3.5 in CPM
Table A]: Cell associated 4C readings for transport experiments in figure 3.6 in CPM

Table A.8: Strain KC concentration in transport experiments represented in tables
A.5-A.7

Table A9: Cell "C readings for transport experiments in figure 3.7 in CPM
Table A.10: Cell 1‘C readings for transport experiments in figure 3.8 in CPM
Table A.11: Cell 1‘C readings for transport experiments in figure 3.9 in CPM

Table A. 12: Strain KC concentration in transport experiments (prior to dilution)
represented in tables A.9-A.11

Table B.1: Column effluent 14C readings for transport experiments in figure 4.1 in CPM

Table B.2: Column effluent 1‘C readings for transport experiments in figure 4.2 in CPM

ix

10

12

26

30

52

53

53

55

55

Table 3.3: Reactor growth for tables BI and B2

Table 8.4: Column effluent l‘C readings for transport experiments in figure 4.5 in CPM
Table 8.5: Reactor growth for table B.4

Table C.1: Column effluent 1‘C readings for transport experiments in figure 5.1 in CPM
Table C.2: Column effluent 14C readings for transport experiments in figure 5.2 in CPM
Table C.3: Column effluent 14C readings for transport experiments in figure 5.3 in CPM

Table C.4: Strain KC concentration in transport experiments represented in tables
AS-AJ

56

56

56

57

57

58

58

LIST OF FIGURES

Figure 1.1 Mass balance on particles in a control volume in colloid filtration theory. ............ 8
Figure 2.1 Radiolabeling of strain KC .................................................................................. 18

Figure 3.1 Bacterial breakthrough curves for an aerobic strain KC culture during mid-
exponential growth. ................................................................................................ 22

Figure 3.2 Bacterial breakthrough for an aerobic strain KC culture in early stationary phase.22
Figure 3.3 Bacterial breakthrough of an aerobic mid-stationary strain KC culture. .............. 23
Figure 3.4 Bacterial breakthrough of denitrifying culture during its first exponential phase. 24
Figure 3.5 Bacterial breakthrough of denitrifying culture during stationary phase. ............... 24
Figure 3.6 Bacterial breakthrough of denitrifying culture during its second stationary phase.25
Figure 3.7 Bacterial breakthrough of an aerobic late exponential phase strain KC .............. 27
Figure 3.8 Bacterial breakthrough of a diluted early stationary aerobic strain KC culture. ....27

Figure 4.1 Bacterial breakthrough curves of different concentrations of strain KC diluted
with pasteurized Schoolcraft groundwater. .............................................................. 32

Figure 4.2 Bacterial breakthrough curves for different percentage dilutions of strain KC
diluted with filter-sterilized supernatant from a strain KC culture ............................ 33

Figure 4.3 Cumulative number of strain KC cells transported for different percentage
dilutions prepared with pasteurized Schoolcraft groundwater. ................................. 34

Figure 4.4 Cumulative number of strain KC cells deposited for different percentage dilutions
prepared with pasteurized Schoolcraft groundwater. ............................................... 35

Figure 4.5 Breakthrough curves for a stationary phase culture of strain KC at different
percentage dilutions. ............................................................................................... 36

Figure 5.1 Bacterial breakthrough of a flocculating culture during mid-exponential growth. 38

Figure 5.2 Bacterial breakthrough of a flocculating culture at early stationary phase of
growth. ................................................................................................................... 38

Figure 5.3 Bacterial breakthrough of a flocculating culture at mid-stationary phase. ............ 39

LIST OF SYMBOLS

DVLO theory energy of interaction, joules

DVLO theory repulsive energy, joules

DVLO theory Van der Waals energy of attraction, joules
dielectric constant of water, unitless

radius of sand particles, meters

radius of bacteria, meters

zeta potential of sand, volts

zeta potential of bacteria, volts

inverse double layer thickness, meters1

distance, meters

Hamaker constant for sand and bacteria, joules

initial concentration of bacteria, #/1

concentration of bacteria, #/1

time, minutes

dispersion term in advection-dispersion equation, mz/ sec
groundwater pore velocity, m/ hr

upstream velocity (Q/ A), m/ hr

Q...

0|

OZ .9-

>

attachment term in advection-dispersion equation, time'1
Detachment term in advection-dispersion equation, time'1
column porosity, unitless

Collision efficiency (number of collisions that result in attachment
compared to total number of collisions)

collector mass transfer efficiency (ratio of particles that strike a
collector compared to number that flow toward a collector - d/df)

accumulation of particles on filter, #/time

rate at which particles strike a collector, #/ time

rate at which particles flow toward a collector, #/ time
number of collectors in column

cross sectional area of column, m2

clean bed collision efficiency, unitless

Blocking factor (ratio of blocked area of cell to area occupied by the

cell), unitless

fraction of surface area covered (ratio of area covered by cells to the
total surface area of all the collectors in the column)

Correction factor in collector mass transfer efficiency equation to
correct for neighboring collectors (Happel, 1951), unitless

a function of porosity, unitless
Peclet number, unitless

London/ Van der Waals term, unitless

straining term, unitless

xiv

sedimentation term, unitless
diffusion constant of bacteria, mZ/s
density of bacteria, kg/rn3

density of growth medium, kg/rn3
gravitational constant, m/s2
viscosity of medium, kg/m*s
column length, meters

column hydraulic retention time, min
filtration constant, meters"

flow rate into column, cm3/ min
surface area available in a column, m2

diameter of columns, cm

mass transfer coefficient, 5

Owner!

INTRODUCTION

In recent years, interest in remediation of contaminated soils and aquifers has increased with
the increasing number of contaminated drinking water wells. This has led to a rise in the
number of treatment technologies. Present technology for the clean up of groundwater
includes various pump and treat schemes. However, in situ bioremediation offers a less
expensive alternative for sites contaminated with organic compounds. Two processes can be

employed for in situ bioremediation: biostirnulation and bioaugrnentation.

Biostimulation involves “stimulating” the growth of native or indigenous bacteria by
providing them with growth-limiting nutrients. A potential problem with use of indigenous
bacteria is lack of pathway control and the production of compounds that are more toxic or
persistent than the original compound. Biodegradation of carbon tetrachloride is an
example. Carbon tetrachloride, a suspected human carcinogen, has been used in various
industrial processes and as a fumigant in grain silos and railroad cars. In the laboratory and
field, chloroform is often produced during bacterial decomposition of carbon tetrachloride
(Criddle et al., 1990; Semprini et al., 1992; Vogel et al., 1987). Chloroforrn is a “probable

human carcinogen of low carcinogenic hazard” (EPA, 1998).

Bioaugrnentation involves introduction of non-native microorganisms. This process allows
for the use of an organism that has been well studied in the laboratory, whose growth
kinetics are known, and whose behavior can be accurately modeled. One potential problem

1

with this approach is that imported bacteria may be unable to compete with the indigenous

flora. Another potential problem is inability to transport the added microorganisms.

A carbon tetrachloride contaminated aquifer, located at Schoolcraft, Michigan, has been the
focal point of a combination laboratory/ field study for the development of an in situ
bioaugmentation remediation process. Psatdmrmas stutzeri KC (strain KC) degrades carbon
tetrachloride to carbon dioxide (COZ), formate (HCOO) (Dybas et al., 1994), and an
unidentified nonvolatile fraction, without the production of chloroform, under iron limiting,
denitrifying conditions (T atara et al., 1993;, Criddle et al., 1990). Dybas et al. (1994) found
that by increasing the pH of the system to 7.9 to 8.2, strain KC had a competitive advantage

over microflora in soil and groundwater, including microflora native to Schoolcraft aquifer

sediments. When the pH was reduced, strain KC numbers also drastically reduced.

Generally in the field of bioremediation, there is increasing interest in the formation and
maintenance of biofilms. In situ bioremediation designs typically involve a biofilm process.
A biofilm can be defined as an accumulation of biomass at any solid-water interface, where
biomass includes cells and cellular products. In systems with interfaces (soil, streams, etc.)
most of the bacteria are attached to surfaces as biofilms (Stotzky, 1972; Wuhrman, 1971;
Marshall, 1972). Biofilrns occur widely in nature on rocks in flowing rivers or streams, in
lakes, on boat hulls, and aquifer and soil systems, on the teeth and in the intestinal tracts of
animals, and in systems for the detoxification of hazardous and non-hazardous waste. At

Schoolcraft, an in situ biofilm process is being used to degrade carbon tetrachloride.

Prediction of biofilm formation and control of biofilms is of interest because of the many

beneficial and harmful roles of biofilms. Biofilms are used in wastewater treatment on

trickling filters, rotating biological contactors, fluidized bed reactors, and various anaerobic
processes. Biofilms have also been used in slow sand filters (the “schmutzdecke”), in
remediation, in the mining industry to help with ore leaching, and in microbial enhanced oil
recovery (MEOL) schemes. Biofilms are also responsible for decreased efficiency of heat
exchangers, increased drag on ships, cavities, infections in animals, corrosion, and increased
energy requirements for pumping. Despite their widespread use and occurrence, the nature

of the formation and maintenance of biofilms is not fully understood.

Because biofilms are ubiquitous, it is clear that attachment is advantageous. Some of the

advantages of biofilms include: (Criddle et al., 1991; Loosdrecht, 1990;, Rijarnts, 1994)

0 Reduced threat of predation. Attached cells are less prone to predation from
viruses, bdellovibrio, and protozoa because they have less exposed surface area.

0 Preservation. Some bacteria are known to attach in the presence of substrate,
thus securing themselves near a food supply.

0 Substrate availability. Numerous organic substrates accumulate at the solid-water
interface.

0 Benefits of a microenvironment. The biofilm community promotes conditions
that are beneficial to the growth and survival of its members. This may include
regulation of pH, reduced exposure to toxicants, protection from desiccation,

and enhanced capacity for degradation.

Mechanisms of Deposition

Bacterial deposition occurs in a stepwise fashion. The two primary steps are transport and
adhesion. Transport is dominated by hydrodynamic phenomena, while adhesion involves of
a variety of processes. After the initial deposition, bacteria may secrete exopolymers
(firnbrils, adhesins) and multiply. This, in turn, promotes the formation of attached

microcolonies and the development of a biofilm.

Transport

The transport step conveys the microorganism to the solid surface where an interaction may
occur. The more rapid the transport, the greater the number of microorganisms that come
in contact with the solid surface, and the greater the chances of adhesion. This statement
holds until the velocity in the medium is so high that shear stresses prevent adhesion. The
three major factors affecting cell transport are the diffusion of the organism, convective
transport, and motility. Rijnaarts et al. (1993) concluded that convection plays a larger role

in attachment than diffusion.

Adhesion

Adhesion refers to the process whereby the colloidal microorganisms attach to solid
surfaces. Forces known to affect adhesion include hydrophobicity of the cell, electro-
potential of the cell (measured as zeta potential), surface tension, Van der Waals forces,
surface protein ionization (pK), and physical forces as impaction, straining, and shear stress.
Because so many forces contribute to adhesion, there is no single model in the literature that

can fully account for the observed interactions between cells and substratum.

4

Static System Models

Hydrophobicity

Various researchers have developed simple laboratory assays of bacterial hydrophobicity and
capacity for attachment to solids (van der Mei, Weerkamp, and Busscher, 1987, Stenstrom,
1988, Dillon et. al., 1986, Mozes and Rouxhet, 1987, VanHaecke et. al., 1990). Tests used
for this purpose include the Bacterial Adherence To Hydrocarbons (BATH) (Rosenberg,
1980), hydrophobic interaction chromatography (van der Mei, 1987, Dillon et. al., 1986), salt
aggregation (Lindahl et. al., 1981), adherence to polystyrene (Dillon et. al., 1986), and contact
angle measurements (Busscher et. al., 1984). The data collected from these experiments was
used as relative measure of hydrophobicity between strains, although there was no

correlation between any of the tests (Dillon et. al., 1986).

Surface Free Energy (Interfacial Tension)

This equilibrium model relies on the formation and destruction of interfaces between water-
cells, cells-substratum, and substratum-water. Data can be collected in the lab by
determining the contact angle of bacteria and substratum for polar and non-polar liquids
with known surface tensions and by completing a force balance. This model does not

account for electrostatic or Van der Waals forces.

DVLO (Derjaguin and Landau, Vervey and Overbeek)
DVLO Theory describes repulsive and attractive forces between a colloid and a surface. It is
applied to bacteria under the assumption that bacteria act as colloids when interacting with

surfaces. The energy of attraction, VT, is described by:

V, = VR + VA (1)

where VR = repulsion energy (joules)
VA - attraction energy (joules)

Electrostatic repulsive forces are a function of the charge on the interacting bacteria and
surface (measured as zeta potential), the radius of the interacting species, dielectric constant
of the fluid, ionic strength (affects the inverse double layer thickness), and the distance

between the bacteria and the surface.

VR = %[7“f;)l(w. + M141 + 6'“ )+ (W. + my ln(1- 6"“) (2)

where r = radius of sand particles 0 and bacteria (1,), In
DC = dielectric constant
w - zeta potential of sand Q or bacteria (1,), V
8 - inverse double layer thickness, m"
x - distance between surfaces, m

Van der Waals forces act to draw the two particles together. The Van der Waals forces, V A,
are a function of distance between the particles, the Hamaker constant, and the radius of the

interacting particles.

 

VA=_HSb( rsrb ] (3)

Numerous studies have shown that increasing ionic strength over several orders of
magnitude can decrease the inverse double layer thickness increasing attachment of bacteria
to solids Oewitt et al., 1995; Rijnaarts et al., 1996a). Although sufficient in describing the
effects of ionic strength, the DVLO theory cannot account for hydrophobic effects and

localized charges on the cell and substratum that could affect attachment.

Isotherm Models

Loosdrecht et. al. (1989) used Langmuir and Volmer isotherms to estimate interaction free
energy between the bacteria and the substratum for separation distances greater than 1 nm.
In addition to assrunptions made when using isotherms, the authors make the assumption
that adhesion occurs at the secondary minimum in DVLO theory and that bacteria at this

level are free to migrate parallel to the surface.

Modelling Systems

Isotherm models using partition coefficients for the attachment and detachment of bacteria
have also been used in the advection-dispersion equation (W itt et. al., 1995, Peterson and

Ward, 1989, Corapcioglu and Haridas, 1984, Escher, 1987, and Sarkar et. al., 1994). A

ical one dimensional trans ort e ation is as follows:
P C111

2
%?=Da%x—€-VE%C—R0+Rd (4)

where C — concentration of a bacteria (#/1)
t = time (5)
Da - dispersion term (mz/s)
v - velocity term (m/s)
R, - attachment term
Rd - detachment term

All of the above—cited authors above use a linear equilibrium partitioning coefficient with the

exception of Sarkar et al., (1994) who used the following nonlinear equilibruim coefficientzz

AC .
C11) = 71) (5)
1+ BC",

 

where CD - dimensionless flowing bacterial concentration
Cm - dimensionless total bacterial concentration a (CI—C "’)/Cr
A,B, and C“ need to be experimentaly determined

Filtration theory is also derived from the advection dispersion equation. Filtration theory is a
mass balance over a control volume similar to equation (4), (see figure 1.1). The sink terrrr,

R,, becomes the filtration equation and R, is removed Rd is assumed negligible (Rijnaarts et

al., 1996).

Advective flux into

Advective flux out of
control volume

 

 

 

 

 

 

 

. . control volume
Accumulation in
CrunAAt liquid in the meunAAt
+ nAAzAC >
D An A dcJr control volume D An dCr+Ar
dx dx
Diffusive flux into Dif'fusive flux out of
control volume control volume

 

V

Accumulation on the Collectors

Figure 1.1 Mass balance on particles in a control volume in colloid filtration theory.

where u - upstream groundwater velocity, m/min
n =- porosity of column
A - cross sectional area of column, m2
D - diffusion coefficient of bacteria, mz/min
x - position, meters

If the accumulation of particles on collectors is defined as

a, = chAt (6)

where ds = the rate at which particles strike a collector = nunC 1trs2
or = the collision efficiency
1] = collector mass transfer efficiency (unitless)
Nc = the number of collectors in a control volume = A(1 -n)L / (41tr53/ 3)
rs - the radius of the sand particles in the column (m)

A mass balance equation can be written over the control volume in the form of equation (7).

3

’1

dCx ] _ ChmunAAt + DAnAt[£—Ai] = nAAzAC +
dx dx

 

 

CzunAAt — DAnA:( (1 _ "larICunAAzAt (7)

Dividing equation (7) by nAAxAt, taking the limit as Ax and At go to zero, substituting in
equation (6) for the acculumation on the collector term, and assuming dispersion and

accumulation in the liquid are negligible, yields.

dc 3 l—n
Ez‘Zl , law (8)

S

 

Several researchers have used colloid filtration theory to model bacterial deposition in
porous media including Martin et al. (1992), Harvey and Garabedian (1991), Logan et al.
(1995), Martin et al. (1996), Johnson and Logan (1996), and Rijnaarts et al. (1996b).

Assumptions for use of clean bed filtration theory are provided in Table 1.1.

Table 1.1 Assumptions of clean bed filtration model.

Assumptions for clean bed filtration modeling of bacterial deposition in porous media

 

bacteria are modeled as spherical colloids in the collector mass transfer efficiency equation
collectors are spherical and uniform
ideal plug flow in filter
particles do not interfere with each other
monolayer coverage of bacteria on collectors

steady state conditions

Rijnaarts et al. (1996b) found that bacterial transport in porous media could be modeled
using clean bed filtration theory where the collision efficiency is a function of cell-solid

interactions and cell-cell interactions as defined in equation (9) for monolayer coverage.

a = a. (1 - W) (9)

where one - the clean bed collision efficiency (dimensionless
B = dimensionless blocking factor (ratio blocked area per cell to cell area)

6 2 fraction of collector surface area covered

Cell-cell interactions occur when attached cells prevent the adhesion of more cells by

screening or blocking the collector surface (Rijaarnts et al., 1996a). The blocking factor, B,

and the fraction of surface covered, 9, are related by 9mam = 1/ B. Equation (9) states that
initially or=oro and 9=0 and that as surface coverage increases (9), the collision efficiency (or)

decreases.

10

Unlike isotherm models, which do not separate the contributing forces behind attachment,
filtration theory separates and quantifies the contributions of each identified factor that may
contribute to attachment. The collector mass transfer efficiency (71) is a “semiempirical”
summation of all the forces (equation 10) acting on a cell and substratum and is defined as
the probability of a particle colliding with the substratum (the collector) (Rajagopalan and

Tien, 1976; Tobiason and O’Melia, 1988; Logan et al., 1995).

77 = 4As'/3N,j:/3 + A_,N§N;78 + 0.0038A5N52Ngo" (10)

The first term in equation (8) is for the contribution of advective to diffusive forces. The
second term represents the contribution of London/ Van der Waals forces and the third
term, represents the physical forces of sedimentation and impaction. The variables that

compose the terms of n are defined in equations 11 to 15. As is a correction factor

determined by Happel (1958) to account for the effects of neighboring collectors.

 

: 20—125) (11)
‘ (2-3p+3p’-2p°)

US

where p - (1-n and n a the column porosity

The Peclet number (N I.) is a representation of the ratio between plug and diffusive flow.

 

210'
S
NP ""

. D. (12)

NU, represents the attraction of the bacteria and the sand by Van der Waals/ London forces.

NM = 9 H?
mutt

 

(13)
NR is the ratio between bacterial radius and the radius of the particle with which it is

colliding. It represents the effect of straining.

11

NR =_ (14)

NG represents the effects of sedimentation.

2 ——
N0 = 2rb (pl) p)g (15)
9w

The variables used in these equations can be found in Table 1.2.

Table 1.2 List of variables.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Variable Title Value Units

9 porosity 0.41 unitless

u velocity varies meters/ second (m/s)
rs radius of sand particle 0.0001 meters (m)

. . 4.1“10'7 (aerobic)
rb radius of bacteria 303,104 (denitrifj . 1 meters (m)
Diffusion coefficient of 3.65"10"3 2
D" bacteria (Rijnaarts, 1995) meters /sec (m/s)
* _22 ..

H Hamaker constant 2 10 13:3;m5’ joules (l)

p density of media 997.2 kilograms/meters3 (kg/m”)
pb density of bacteria 1000 (estimated) (kg/m”)

p. viscosity of media 9.99"'104 Kg/m*s

g gravitational constant 9.81 m/s2

 

 

 

If blocking is negligible, equation (9) can be integrated over the length of the column (L) to

produce
_ V

However, when blocking does occur, equation (8) can be solved and the effluent from a soil

column becomes a function of time as in equation (17) (T ien, 1989).

12

 

exp[m2ucona.flt']
c _ 4

_ _ - 2 .
co exp[ 3(1 :)s ”aux) + exp[ 7Z7", ucoZaoflt ]_1

 

(17)

 

 

S

where ti = actual time minus column hydraulic retention time.
A mass balanced performed on the liquid phase within a column yields.

dc
—=-kCa l-BB 18
d, .< > < >

where k is defined as the mass transfer coefficient (equation 19).
4r, n
This theory only accounts for single layer adhesion. Some modification is required for multi—

layering attachment (0t>1). Further modifications are suggested for this model in this thesis.

One of the goals of this thesis was to develop a method of conducting cell tranposrt
experiments that would realistically simulate field conditions. All experiments were
performed and designed so that strategies developed in the laboratory for improved cell
transport could be easily implemented in the field. The variables chosen for investigation
were those that could be easily modified in the field with mimirnal effort. Accordingly, the
inocula used in cell transport experiments were not modified following growth (previous
researchers have washed and resuspened their bacteria) nor was the sand washed (other

esearchers have performed transport experiments on sterile sand).

13

Hypotheses:

1.

A laboratory method can be developed in the laboratory that can mimic field
injection conditions without modifying the bacteria.

Cell growth stage affects transport. Cells that are exponentially growing are
more readily transported than older cells.

Flocculating cultures do not transport well. The result is filter ripening and
clogging.

Lower cell concentrations in the liquid enhances cell transport.

Electron acceptors influence cell transport properties.

14

W2

MATERIALS AND METHODS

mandate
Psatdamcms stutzen' KC (DSM deposit no. 7135, ATCC deposit no. 55595) was routinely

maintained on R2A agar (Difco) plates.

Chemicals

All chemicals used were ACS reagent grade (Aldrich or Sigma Chemical Co.). Radioactive

acetate with a specific activity of 6.7 mCi/mmol was obtained from Sigma.

Groundwater

Groundwater and the aquifer core samples were obtained from a carbon tetrachloride-and
nitrate-contaminated aquifer Schoolcraft, MI. Groundwater samples were obtained from
wells in the delivery well gallery constructed for Phase 2 of the Schoolcraft Bioaugmentation
Project. These wells are screened from 30 to 80 feet below the ground surface.
Groundwater samples were collected in 20 gallon presterilized Nalgene ‘3 carboys and stored
at 4° C. Aquifer core samples were obtained from the grid for use in the column
experiments. Once in the lab, the core material was placed in a presterilized food grade 5

gallon food grade bucket submerged in unpasteurized Schoolcraft groundwater adjusted to

pH 8.2. Aquifer material was kept at 4° C until use.

15

Filters

Radiolabeled cells were removed from their growth medium by filtration through 0.2 pm
polycarbonate membranes (Corning Nucleopore Track-Etch Membranes catalog no.

110606).

Columns

Column studies were performed using 2.5 cm diameter chromatography columns (Kontes
catalog no. 420400-2510 for columns 10 cm in length and catalog no. 42400-2520 for 20 cm
columns) fitted with flow adapters (Kontes catalog no.420415-2500). The flow adaptors

allowed the packing of the columns to be different lengths.

Methods

Groundwater Medium

For aerobic growth, Schoolcraft groundwater was supplemented with 1600 ppm acetate (as
sodium salt). For growth of denitrifying cells, groundwater was supplemented with 1600
ppm acetate and nitrate (both as sodium salts) prior to pasteurization. Supplemented

groundwater was pasteurized by placing it in a 60°C oven for 8 hours (Knoll, 1994).
Pasteurized groundwater was stored at 4°C. Just prior to use, 10 ppm of phosphate (1’04")

was added and the pH of the groundwater was adjusted to 8-8.2 using sterilized 1 M

hydrochloric acid (HCI) or 1 M sodium hydroxide (NaOH). When radiolabeled acetate was

used, it was added just prior to inoculation of the groundwater with strain KC.

16

Analytical Methods

All ions (nitrate, nitrite, phosphate, bromide, acetate, sulfate) were measured by ion
chromatography (Dionex model 2000i/ SP ion chromatograph with suppressed conductivity
detection and a Dionex Ionpak AS4-A anion exchange column). Protein samples were
measured using the modified Lowry protein assay (Markwell et al., 1981). pH was measured
using a Orion pH probe and meter. Radiation samples were placed in scintillation vials
along with 10 ml scintillation cocktail and measured in a Packard 1500 Tri-Carb scintillation
counter. Each radiation sample was counted for 5 rrrinutes. Spectrophotometric samples

were measured on a Shimadzu UV-160 spectrophotometer.

Growth of 1‘C-Labeled strain KC

An important criterion for cell transport experiments was that the growth and injection
conditions mimic field conditions. Numerous researchers have studied bacterial transport in
columns using washed cells harvested in either the late exponential or early stationary phase
and resuspended in a buffer to the desired concentration. Such procedures are generally not

representative of field methodologies and were therefore avoided in this study.

Because realistic growth conditions were desirable, one goal of this research was to develop a
measurement technique that did not alter growth conditions, was easy to measure, and was
representative of field conditions. Prior to inoculation of the groundwater meditun with

strain KC, 20 uCi of MC-labeled acetate ("Cl-13COONa) was added to the culture. Acetate

served as the electron donor and carbon source in all experiments. Strain KC was labeled as

it incorporated acetate into its cell mass. This is illustrated in Figure 2.1.

17

 

1“vCHsOOO
8L12CH3COO

 

 

 

Electron donor

 

 

 

 

 

 

 

 

 

 

Fraction of
electrons to
biomass, carbon
assimilated in . .
biomasg _ Radio acuve
r o
Biomass
Fraction of
electron that 3° to Biomass removed by filtration (02 um filter)
electron acceptor, Filter counted in scintillation coturtcr
caron converted to
C01 ‘ ’
lzCOz, 14CO2’
& H20
Figure 2.1 Radiolabeling of strain KC.

Preparation of Packed Columns

A radioactivity balance was
performed by counting a sample
from the reactor (labeled
bacteria, unused acetze, and
bicarbonate), counting the fikered
biormss solids, and counting a
filtered liquid sample, cornining
unused acetate and bicarbonate.

Columns were wet packed with Schoolcraft aquifer sand to a porosity of 0.41. For each

experiment, a set of three columns were packed to approximate lengths of 3, 6, and 9 cm.

Because the columns and their retention times were short, growth of the inoculum within

the columns is negligible.

Transport Experiments

For transport experiments, Psadamms stutzeri KC was grown in glass reactors using
pasteurized Schoolcraft groundwater medium at pH 8.2. Groundwater medium was
inoculated with a 24 hour old culture of strain KC grown in nutrient broth (Difco). A 0.6%
(by volume) inoculum was added. Approximately 400 ml of the reactor were removed
aseptically for transport experiments at 18, 24, and 30 hours for aerobic cultures and 18, 30,
and 48 hours for denitrifying cultures. One hundred milliliter syringes were filled with
culture, which was then pushed through the packed columns with syringe pumps. Effluent
samples were collected with a fraction collector and then analyzed for either tracer or

suspended radioactivity.

Data Analysis
Bacterial breakthrough curves were plotted with time. A filtration coefficient, A, can be
found taking a linear regression of the breakthrough curves and integrating equation (9) to

form equation (20).

C

145-] = —2.L (20)

0

where Co = influent concentration of bacteria
L = column length

l. = filtration constant (see equation 21)

The collision efficiency, a, can then be calculated from the filtration constant, it, as in

equation (21).

19

4 r

3 1-
A = —[—n]a77 (21)
The collision efficiency calculated in equation (21) is substituted into equation (9) and the
clean bed collision efficiency, a0, and the blocking factor, B, can be determined for each

column by a simple linear regression procedure. The other variable in equation (9), the

fraction of surface covered, 6, can be calculated from equation (22).

0(t) = [% ((1— %)dt]7;—:— (22)

where Q - flow rate into the column, m3/ min
Am, - surface area of the porous media, mz/m3

The surface area in the column, Acol, can be calculated from equation (23).

 

r

3

Am, = 3‘1"“) 4512:) (23)

where dC = the diameter of the column in meters

20

Onpter3

INFLUENCE OF CELL PHYSIOLOGY ON TRANSPORT IN POROUS MEDIA

Introduction

For field application, it is desirable to be able to control cell transport properties with
minimal effort. If cell transport properties are controlled by the cell physiology, culture
growth stage could be a valuable control variable in injection schemes for bioaugrnentation
cleanups. The goal of this chapter is to investigate the influence of the growth of the culture

on its transport properties.

Materials and Methods

Details of the methods used for these experiments can be found in chapter 2. For this
experiment, column effluent was monitored by cellular protein as measured in the modified
Lowry protein assay (Markwell et al., 1981). The flow rate was set at 2 ml/min for the
aerobic culture and 3.5 ml/ min for the denitrifying culture, which corresponded to pore

velocities of 0.60 and 1.04 m/ hr, respectively.

Results and Discussion

The bacterial breakthrough curves for an aerobic strain KC culture in mid-exponential
growth (culture grown for 18 hours from a 0.6% inoculum) are provided in Figure 3.1.

C/Co is the ratio of effluent to influent concentration of strain KC.

21

 

 

 

 

LM
I
I
0.80
0. . I
I
. I
0.60 , I.
‘ I
5 A
D O
‘ I
A Column Lengths
0.40
I
‘. u 3.3 cm
. .5 8
. cm
020 :
A A90 cm
‘I
0.00 ‘ ~ . A
0 2 4 6 8 10
Port: Volumes

Figure 3.1 Bacterial breakthrough curves for an aerobic strain KC culture during mid-

exponential growth.
Figure 3.2 illustrates bacterial breakthrough curves for an aerobic strain KC culture in early

stationary phase (24 hours).

 

 

 

 

1.00
0.80 «»
I
I
0.60 .
o I
u I '
”(3 o 0 0 .
A
0.40 A . .‘ I 001mm [truths
‘ I
‘- . I 3.4 cm
A
. 06.2 cm
I
020 . A 10 1 cm
A ‘ '
‘I
0.00 H
0 2 4 6 8 10
Pore Volumes

Figure 3.2 Bacterial breakthrough for an aerobic strain KC culture in early stationary phase
22

Figure 3.3 illustrates results for a 30 hour aerobic culture in mid-stationary phase.

 

 

 

 

1.00
0.80
I I
0.60 I g
I
. I
8 J
0 no
A
0.40 . i O
‘ . Column Lengths
I
’ .3] cm
030 ‘ . 06.0 cm
10.0
a‘ ‘ “n
0 00 fl. .
0 2 4 6 8 10
Time (urinates)

Figure 3.3 Bacterial breakthrough of an aerobic mid-stationary strain KC culture.

Additional transport experiments were performed with nitrate (N 03') as the electron
acceptor. While denitrifying, strain KC has two exponential growrh phases separated by a
short stationary phase (Knoll, 1994). The first exponential growth phase utilizes nitrate as
the electron acceptor and the second utilizes nitrite (NOZ’) (Knoll, 1994). The bacterial
breakthrough curves can be found in figures 3.4, 3.5, and 3.6. Figure 3.4 contains the results

from a denitrifying culture during its first exponential phase.

23

 

 

 

 

1.00
. I
. I .- A I I
0.80 I I '
A A ‘ ‘
o
o
A

0.60
(3 "- Column Lengths
\
U

A
0.40 .11 cm
06.0 cm
‘92 cm
020 ..
A
0.00 33.!
0.00 4.00 8.00 12.00 16.00
Pore Volumes

Figure 3.4 Bacterial breakthrough of denitrifying culture during its first exponential phase.

Figure 3.5 illustrates the breakthrough of strain KC during its short stationary phase between

its two exponential phases.

 

 

 

 

1.00
0.80
I
I . ' '
0.60 o g o o
C
'0
Q e
U ' A A A ‘ A
0.40 0 ColurmLengths
‘ I4.0crn
°1° ‘ 0 06.4cm
I
59.00!)
0.00...
0.00 4.00 8.00 12.00 16.00

Pore Volumes

Figure 3.5 Bacterial breakthrough of denitrifying culture during stationary phase.
24

Figure 3.6 illustrates the breakthrough of a strain KC culture early in its second stationary

phase.

 

 

 

 

1.00
I
I
0.80 . ' '
I . o
o
o
. o
.0
0.60
0
° A
8 ° . ‘
I ‘ ‘
°~4° commits
e A
A .3Jcm
06.0cm
020 ‘
‘9Jcm
I
A
0ng. - A
0.00 4.00 8.00 12.00 16.00

Time (minutes)

Figure 3.6 Bacterial breakthrough of denitrifying culture during its second stationary phase.

Table 3.1 gives the results of clean bed filtration modeling for figures 3.1-3.6. For one, the

averages of the three columns are indicated and ,for B, the results are stated for each column.
Rijnaarts (1996b) observed a similar trend of increasing blocking factor with an increase in
column length and suggests that blocking becomes independent of length with columns of
length greater than 7.5 cm (Rijnaarts, 1996b). A likely explanation for the increase in
blocking is multilayer adhesion and ripening of the filter (sand column) in the shorter
columns. The data of Table 3.1, along with data presented in Chapter 4, suggests that aging
cultures of strain KC have altered their cell surface facilitating deposition and attachment to

solids.

25

Table 3.1 Attachment efficiency and blocking factor.

 

 

 

 

 

 

 

 

 

 

 

 

 

Electron Acceptor Culture Phase (10 ii
Short Medium Long
2 mid-exponential 0.44:0.07 2020 2280 2980
2 early stationary 0.281000 45.3 82.7 134
2 mid-stationary 0.32i0.00 49.6 80.4 134
NO; exponential 0.64i0.02 1240 2050 2690
NO,‘ &NO,‘ stationary 0.57:0.00 38.9 62.3 87.6
NC); stationary 0.78i0.00 59.8 96.9 156

 

In order to prove that increased attachment in aged cultures was a function of cell age and
not cell concentration, another experiment was performed in which cell concentrations were
all the adjusted to the same level (approximately 2*107 cfu/ ml) Figure 3.4, 3.5, and 3.6 are
the bacterial breakthrough curves for the mid-exponential, early stationary, and mid-
stationary cells, respectively. A fraction of early stationary and mid-stationary phase culture

was removed aseptically from the reactor and diluted using pasteurized Schoolcraft

groundwater previously adjusted to the same ionic strength and pH as the medium used to

grow the culture.

26

 

 

 

 

 

1.1”
0.80 . I
I
I g -
r 0 0 . I
. I I .
0.60 .. . ‘
‘ A A 4

o
O ‘0
\
O

0.40 » Column Lengths

A
. I3] cm
05.8 cm
0.20
‘ A 11.2 cm
I
0.00 3“ .
0.00 4.00 8.00 12.00 16.00

Pore Volumes

Figure 3.7 Bacterial breakthrough of an aerobic late exponential phase strain KC

Figure 3.8 illustrates transport of stationary phase cells diluted approximately 1:5 to achieve a
concentration of cells equivalent to the late exponential phase cells in Figure 3.7

(approximately 2*107 cfu/ ml) Note the change in scale on the y-axis.

 

 

 

 

0.10
. I I
I
0.08
I I
I
I
0.06
o
8 I I I .
U
. 001mm Lengths
0.04 4. D A
A 5*
‘ 0 I32cm
' A
06.5 cm
0.02 I
A A 10.2 cm
I
O
0.00 :. .:
0.00 4.00 8.00 12.00 16.00

Pore Volumes

Figure 3.8 Bacterial breakthrough of a diluted early stationary aerobic strain KC culture.

27

The breakthrough curves in Figure 3.9 are from for a culture removed from the reactor
during mid-stationary phase and diluted to about approximately 2*107 . Note the scale on the

y axis.

0.10

 

 

 

Column Lengths
0.08
I3.0crn
063cm
0.06 A10.4cm
Q
0
0.04 . . I '
I I
I
o I 0
0.02 0 °
I I
I
I A A
I A ‘
A

 

000 4.00 80) 12.00 16.00

Figure 3.9 Bacterial breakthrough of diluted stationary phase cells.

The experiments with diluted cells clearly demonstrate the dependence of cellular transport
on cell physiology. Escher (1986) reported a difference in attachment rate to smooth solids
for cells of varying growth rates. In addition, Grasso et al. (1996) reported that that the
“partition” coefficient for a Psaldmnras aemgbrosa on dolomite was a function of
physiological state of the cells as was the cell surface free energy. This observation suggests
that surface properties of bacterial cells change during growth. A plausible hypothesis is that
they exist in a planktonic or dispersed state in rich media permitting more efficient use of
nutrients in solution, but when nutrients are limited survival is enhanced by modification of
the cell surface to enable deposition and attachment.

28

The blocking factor listed for strain KC in Table 3.1 is two orders of magnitude higher than
those obtained by Rijnaarts (1996b) for Psadonmas panda. There are several possible
explanations. One of the more plausible is blocking by native Schoolcraft aquifer flora,
which are present on the aquifer solids at a concentration of approximately 3*10" CFU/ g.
Table 3.2 contains the results of modeling when the native flora are accounted for in the
blocking term. The only difference between Table 3.1 and 3.2 is inclusion of native flora in
the latter table. The magnitudes of the blocking factor in Table 3.2 are similar to those
obtained by Rijnaarts (1996b). The porous media used by Rijnaarts (1995, 1996a, 1996b)
and others (Camper, 1993, and Sarkar et al., 1994, Martin et al., 1996, Johnson and Logan,
1996, Jewett et al., 1995) is free of microorganisms prior to the experiment. Another
distinctive aspect of this work is that the strain KC cells used in these experiments were not
washed as others have done (Loosdrecht et al., 1989;, Johnson and Logan, 1995; Camper,
1993; Sarkar et al., 1994; Gross and Logan, 1995;; Gannon et al., 1991; Fontes et al., 1991;
Martin et al., 1996; and Wan et al., 1994). Washing may affect the magnitude of the blocking
factor by altering cell surface properties, although this was not proven in the present study.
To mimic a field injection, the column experiments in this work were performed with fresh,

active, growing strain KC cells, not cells that were resuspended in buffer.

Table 3.2 Attachment efficiency and blocking factor with native flora.

 

 

 

 

 

Electron Acceptor Culture Phase 0.0 is
Short Medium Long
O2 mid-exponential 0.44i0.07 399 153 132
O2 early stationary 0.28:0.00 23.6 30.9 36.1
02 mid-stationary 0.32:0.00 27.5 35.0 42.3

 

 

 

 

 

 

 

29

These studies suggest that a better understanding of kinetics of bacterial growth could be
very beneficial to the success of bioaugrnentation activities in subsurface environments.
Manipulation of growth conditions may have a profound affect on the extent to which
bacteria can be transported away from an injection well. If long travel distances are required,
injection of excess nutrients along with the bacteria may promote both growth and

transport.

30

0W4

EFFECTS OF CELL CONCENTRATION ON TRANSPORT

Introduction

In this chapter, the effects of cell concentration are investigated to determine the effect of
dilution. Intuitively, one might expect that a more dilute culture would bbe more readily
transported in as much as less clogging would be expected. On the other hand, dilution may
affect the blocking factor. The results presented here were used to design the full scale

inoculation scheme for the Schoolcraft Field Bioaugmentation Project.

Methods and Materials

Eight 3 cm long sand columns (hydraulic retention time = 1.7 minutes) were packed with
the sand as described in Chapter 2. Four of these columns were used for column transport
experiments using pasteurized Schoolcraft groundwater as the dilution medium. The
remaining four columns were used for dilution experiments with supernatant from strain KC
culture grown as the dilution medium. To obtain supernatant, a culture of strain KC was
grown for 24 hours, centrifuged at 5000G for 20 minutes, decanted. The supernatant was

then filtered through a 0.2 um filter and then stored frozen at -20°C until use. Experiments

to test the effects of dilution medium on cell transport were conducted with l4C-radiolabeled

culture of strain KC in the exponential growth phase. Dilutions were prepared at 10, 25, 50

31

and 100% of the original concentration. All experiments were performed at a pore velocity

of 1.04 m/hr.

Results and Discussion

Figure 4.1 illustrates bacterial breakthrough curves for different concentrations of strain KC
with pasteurized Schoolcraft groundwater as the dilution medium. The percentages on the
chart represent the percent (by volume) of dilution water. It should be noted that CO
changes for each column depending on the dilution used. For example, the influent to each

column can be calculated by subtracting the fraction of dilution medium from one and

multiplying that number the reactor concentration (5.6*107 cfu/ ml)

 

 

 

 

LW
0%(undiluted) I
........ 41
.A--'
0.75 1» '._A"
41% .
.5.
.. m. l
,A
2 050
U
1".
i 4»
88% fl . _
. 4’
.' 7 _ .
0.25 . r- - ~ ' ’f '—
CA-
0.00 / t : a t -
0.00 3.11) 60') 9.00 12.00 15W

Time (minutes)

Figure 4.1 Bacterial breakthrough curves of different concentrations of strain KC diluted
with pasteurized Schoolcraft groundwater.

32

From the same reactor, a separate set of columns was operated under identical conditions

using supernatant from a culture of strain KC as the dilution medium. Figure 4.2 illustrates

the bacterial breakthrough curves obtained.

 

0.75

0.25

 

 

0.00 1

 

 

Figure 4.2 Bacterial breakthrough curves for different percentage dilutions of strain KC
diluted with filter-sterilized supernatant from a strain KC culture.

It is evident from the data in figures 4.1 and 4.2 that higher influent bacterial concentration
resulted in improved cell transport. This observation seems counterintuitive given that
lower cell concentration would result in fewer collisions and less congestion in the pore
spaces. Two possible explanations for the effect of dilution might be: (1) that something
secreted by the cells enhances their transport or (2) the solids have some finite capacity for
bacterial attachment and once the solids are saturated, additional cells remain are transported
further in the bulk solution. Figures 4.1 and 4.2 illustrate that the nature of the dilution
medium did not effect cell transport, indicating that a secreted factor is not responsible for
the observed dilution effect. The most logical conclusion is that the solids themselves must

33

have a finite “bacterial demand” and that once the attached cells have achieved a saturating
concentration, any additional bacteria entering the system do not attach, but remain in a

planktonic state.

Figure 4.3 shows the cumulative number of strain KC cells transported over time from each

column in Figure 4.1.

25E +09

 

2.0E+09 4

1.5E+09 »

Total PK

1.0E +09

5.0E +08

 

 

0.0E +00
0.00

 

 

Figure 4.3 Cumulative number of strain KC cells transported for different percentage
dilutions prepared with pasteurized Schoolcraft groundwater.

An important aspect of figure 4.3 is that this the results shown are for only 8.7 column
retention times. The differences between different dilutions will become greater as time

increases. Figure 4.4 shows the total number of strain KC cells deposited over time.

34

9.05 +08

 

8.0E +08 /

7.0E+08 «

U! 0*
8 '8
s s

bl b
'91 R
0 s

NumberofnninKCoellsdeposite

 

 

 

 

Time (airlines)

Figure 4.4 Cumulative number of strain KC cells deposited for different percentage dilutions
prepared with pasteurized Schoolcraft groundwater.

The decreasing of the slope in figure 4.4 for the culture diluted by 41% dilution and for the
undiluted culture suggests that the solids were saturated at a cell concentration of
approximately 3*107 strain KC cells/g (referred to .s solids loading capacity). This
conclusion is also supported by the steepness of the bacterial breakthrough curves in figure
4.1. Breakthrough of strain KC occured after this capacity is achieved. The two major
variables influencing the solids loading capacity were growth state and pore velocity of the
medium. Although the culture shown here was tested in mid-exponential phase of growth,
the same phenomenon was observed for stationary stage cells, as shown in Figure 4.5. The
stationary stage culture exhibited even larger differences in transport between dilutions, with
considerably less breakthrough of undiluted cells.

35

 

0J5

 

 

Q
8050 0%(urxiiluted)

o

........... 0

55% ...... t
025 .... ‘ .....
...I' """

...... ..---" 74% ’

..... ‘.-" - W

- x . ,- — o ., ~71»

0.00 5- 0"“;

 

 

Figure 4.5 Breakthrough curves for a stationary phase culture of strain KC at different
percentage dilutions.

No solids loading capacity could be determined from the data for the stationary phase

culture because the solids loading capacity was not achieved in the time of the experiment.

The data presented in this chapter establishes that inoculation of cells should be performed
using dense cultures without dilution. Use of a younger culture (exponential growth) only

partially offsets the effect of cell concentration on cell transport.

36

EFFECT OF FLOCCULATION ON TRANSPORT IN POROUS MEDIA

Introduction

Because strain KC is known to occasionally grow as a flocculent or clumpy culture, the

effect of this state was examined in a series of cell transport experiments.

Materials and Methods

To evaluate the effects of flocculation on cell transport, flocculent and non-flocculent
cultures were evaluated. The flow rate for these flow rates was 4.00 ml/ min, corresponding
to a pore velocity of 1.19 m/hr.. In flocculent cultures, flocculation, or clumpiness was not

visible at 18 hours, barely visible at 24 hours, and obvious at 30 hours old.

Results and Discussion

Figures 5.1-5.3 illustrate bacterial breakthrough curves from a flocculating culture in order
from youngest (exponential growth) to oldest (mid-stationary). Note the change in the y-

axis on Figures 5.2 and 5.3.

37

 

0.75

050

C/C

0.25

 

0.00 I

Figure 5.1 Bacterial breakthrough of a flocculating culture during mid-exponential growth.

1.”, -136 rrn'n (2.7 cm)

  
 

.
o.-
.“
I"

.. ..... 4 stnmis cm)

   
 
   

21 24 27

 

 

 

 

 

 

0.50
0.40
0.30
‘H -l.6lrn'n(3.2cm) —/
U A : v
U ..... I ------- ' """" '
”.I ------- "" 2.47min(4.9crn)
020 a - , e c c -
4.43 rmn(8.8 cm)
A '...
0.. I
0.10 i
0.00 r
0 9 12 15 18 21 24 27 30

Figure 5.2 Bacterial breakthrough of a flocculating culture at early stationary phase of

growth.

38

 

020

 

0.15 e,“ -151 min(2.6cm)

. ......... .I-"i
2.67 min (5.3 cm)

8 0.10. l.‘ ' '1
U 0
4.58min(9.lcm)

0.05 ,

 

 

0.00

 

Figure 5.3 Bacterial breakthrough of a flocculating culture at mid-stationary phase.

Figures 5.1-5.3 illustrate breakthrough patterns suggestive of filter ripening. Filter ripening is
a state in which, multilayer bacterial adhesion causes increased removal rates. This results in
large removal efficiencies because of decreaseing pore size over time. In addition, there is a

significant increase in head loss.

To better understand the nature of the flocculent state, both flocculating and non-
flocculating cultures of strain KC were observed under a microscope. The visual

observations were:

1. Flocculant cultures were not as motile as non-flocculent cultures. Studies have

shown motility to be a factor in transport Gewitt et al., 1995; Camper, 1993).

39

2. Clumps of cells within the flocculent culture increase in size with cell age. Mid-
stationary phase clumps are visible to the naked human eye and settle by gravity

in the absence of mixing.

The flocculation of cells is apparently triggered by nutrient stress. An experiment was
performed in which 6 Erlenmeyer flasks were inoculated with a culture of strain KC. To 3
of these vials, 10 ppm phosphate was added No phosphate was added to the other three
cultures. The three cultures with no phosphate added flocculated, while the three containing
phosphate did not. Dybas (1998) performed a similar experiment with filter-sterilized
Schoolcraft groundwater and established that, for this medium, flocculation was triggered by

a limitation in trace metals.

The results of clean bed filtration theory modeling of figures 5.1-5.3 can be found in table

5.1.

Table 5.1 Attachment efficiency and Blocking factor for flocculating strain KC.

 

 

 

 

 

Physiological State 0ro B
Short Medium Long
mid—exponential 0.521001 231.08 399.98 605.44
early stationary 1.08i‘0.00 17.49 26.78 48.09
mid-stationary 1.261000 14.83 30.23 51.90

 

 

 

 

 

The trends for the blocking factor are similar to those reported in Chapter 3 for a well-

dispersed culture of strain KC Most of the deposition occurs in the first few centimeters, as

evidenced by the breakthrough curve of figure 5.3. All three columns had almost the
40

 

identical breakthrough profiles despite their different lengths. This suggests that sorting by
cell age is occurring within the columns. A batch culture, like the one used in this
experiment, is composed of cells with a myriad of cell ages, distributed around some average
Cells with the highest at and the lowest {3 are the first to deposit. The young cells are not as
likely to attach and are smaller in clump size. As a result, they are more likely to pass

through the column, while the older, larger cells more likely to attach and be filtered out at

the column entrance.

When flocculent culture breakthrough curves are compared with breakthrough curves for
non-flocculent cultures lower breakthrough levels are observed for flocculent cells and
transport is nearly twice as retarded. This is probably related to the solids loading capacity
and cell sorting. In a flocculent culture, most of the cells are associated with clumps.
Planktonic cells are in the minority. Because the clumps are removed in porous media near
the well head, the distal end of the column “sees” fewer cells and it takes longer for the
bacterial demand of the solids to be achieved. Breakthrough of cells is therefore retarded
when compared to the breakthrough of a well dispersed culture.

It is critical that flocculent cultures be avoided in field applications where effective cell
distribution is needed. Flocculating cells have significantly impaired properties for transport.
If a flocculent culture is injected, the expected outcome is deposition of a large number of

bacteria near the well head, which will result in an increase in head loss. This could cause

problems for the injection pumps or the pipes carrying the substrates.

41

W6

ENGINEERING APPLICATION

In most field applications, biostirnulation is the preferred method of bioremediation.
However, there is now increasing interest in bioaugmentation. Two obstacles to the use of
bioaugrnentation are competition with indigenous microflora and transport of the added
microorganism. Competition can be facilitated by modifying the environment to favor the
added microorganisms, but cleanup can only occur if the added organisms are transported to

the desired location.

Past researchers have proposed many methods to increase transport of microorganisms in
porous media. Suggested methods include: use of low ionic strength cell suspensions
(Fontes, 1991; Rijnaarts, 1996; Loosdrecht et al., 1989; Jewitt, et al., 1995; Martin et al.,
1996; Sarkar et al., 1994; Martin et al., 1992; Gross and Logan, 1995), use of ultra micro
bacteria Gang, 1983), injection of non-attaching mutants (Camper et al., 1993), injection of a
surfactant to decrease attachment Oohnson and Logan, 1996; Gross and Logan, 1995; Sarkar
et al., 1994), pH modification of inoculum (Rijnaarts et al., 1995; Jewitt et al., 1995),
decreasing the concentration of cells (Sarkar et al., 1994; Escher, 1986), and increasing
velocity of the cell suspension (Sarkar et al., 1994; Rijnaarts, 1996b; Escher, 1986). Some of
the studies provide basic mechanistic understanding of bacterial transport. However, the
proposed strategies are often difficult to implement at the field scale. In this research,

laboratory studies were designed to simulate field operations, so that the strategies developed

could be readily implemented.
42

 

Data presented in this thesis indicates that some common practices in previous studies, such
as washing and resuspending bacteria, could be detrimental to cell transport. The results
also indicate that transport is dependent upon growth state and medium. The data also
suggest that native microorganisms already present on the collectors probably play a role in

attachment of the injected microorganism.

Results presented in this thesis demonstrate that simple steps may be taken to increase cell
transport. Transport of strain KC can be controlled by cell physiology under both aerobic
and denitrifying conditions. These observations are probably also true for other cell types.
Dilution of the inoculum resulted in a decrease in transport and that flocculent cultures were

transported poorly.

This thesis demonstrates that there are cost-effective variables that can be manipulated to
promote cell transport. For application of bioaugrnentation, the feasible transport distance
of the organism may be an essential design parameter. The feasible transport distance
depends on the concentration of the microorganisms required for colonization, inoculum
volume, injection time, and cell physiology. At the Schoolcraft Bioaugmentation Site, closely
spaced wells (15 wells 1 meter apart) and a relatively small inoculum volume (approximately
6.7 gallons inoculum per linear foot of well screen) were used to inoculate the biocurtain
currently in use at the site. The inoculum was injected undiluted when the culture was in
exponential growth. Early evidence indicates that this injection strategy allowed strain KC

to colonize the space between the wells to form a continuous biocurtain capable of efficient

degradation of carbon tetrachloride.

43

CONCLUSIONS AND FUTURE WORK RECOMMENDATIONS

Conclusions

1. Radiolabeling of strain KC with 1‘C-acetate allows strain KC to be grown and
used in transport experiments in the laboratory as it would be grown and injected

into the subsurface in the field.

2. Clean bed filtration theory can accurately model transport of strain KC in

repacked Schoolcraft sediments.

3. Cell physiology is a variable in attachment. Cells that are exponentially growing
display high blocking factors and are easily transported, while cells in stationary

phase have low blocking factors and are not easily transported.

4. Transport properties of cells grown under aerobic and denitrifying conditions

were similar.

5. Dilution of a strain KC culture resulted in decreased transport of the cells in

Schoolcraft aquifer sediments.

6. Schoolcraft aquifer sediments display a capacity for cells that are exponentially
growing. Strain KC is readily transported, once saturation of the solids is

achieved,.

7. Flocculent strain KC cells are non-motile. Dispersed cultures of strain KC are

composed of very motile cells.

8. Flocculent cultures of strain KC are poorly transported and promote filter

ripening.

9. Clurnp size in flocculent strain KC cultures increases with age of the culture.

Future Work Recommendations

1. Determine the effect on transport of the native Schoolcraft flora present on the

aquifer sediments used for the work in this thesis.

2. Evaluate how the different mineral components of Schoolcraft aquifer sediments

influence cell attachment.

3. Determine the effect of velocity on attachment. In addition, attempt to quantify

the effect of a radial flow field.

4. Examine effects of growth medium. This includes metals, pH, and surfactants.

45

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carbon tetrachloride by Pseudomonas sp. strain KC under denitrification conditions.
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Chemical Factors Influencing Transport of Microorganisms through Porous Media.
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Grasso, D., B.F. Smets, K. A. Strevett, B.D. Machinist, C. J. Van 055, RP. Giese, and W.
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Beds of Spherical Particles. AICHE J. vol. 4, no. 2. 197-201.

Harvey, R.W., and SP. Garabedian. 1991. Use of Colloid Filtration Theory in Modeling
Movement of Bacteria through a Contaminated Sandy Aquifer. ESBCT, 25, 178-185.

Harvey, R.W., D.W. Metge, N. Kinner, and N. Mayberry. 1997. Physiological
Considerations in Applying Laboratory-Determined Buoyant Densities to
Predictions of Bacterial and Protozoan Transport in Groundwater: Results in IN-
Situ and Laboratory Tests. ES8LT, 31, 289-295.

Jang, L.K., P.W. Chang, J.E. Findley, and TE. Yen. Selection of Bacteria with Favorable
Transport Properties Through Porous Rock for the Application of Microbial
Enhanced Oil Recovery. Appl. Environ. Microbiol, 46, 1066-1072.

Jewitt D.G., Hilbert, T.A., Logan B.E., Amold, R.G., and RC. Bales. 1995. Bacterial
Transport in Two Porous Media Systems: Influence of Ionic Strength and pH on
Collision Efficiency. Water Research. 29, 1673-1680.

Knoll, WH 1994. Factors Influencing the Competition Advantage of Pseudomonas sp.
strain KC for Subsequent Remediation of a Carbon Tetrachloride Impacted Aquifer.
Master’s Thesis at Michigan State University, East Lansing, MI

Lindahl, M., Paris, A., Wadstrom, T., and Hjerten, S. 1981. A new test based on ‘Salting
Out’ to measure relative surface hydrophobicity of bacterial cells. Biochim. Ciophys.
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Markwell, M.A., SM. Haas, N.E. Tolbert, and LL. Bieber. 1981. Protein Determination in
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Theory to Bacterial Deposition in Porous Media. ES8CT, 30, 1053-1058.

Mueller, R.F., W.G. Characklis, W.L. Jones, and J.T. Sears. 1992. Characterization of Initial
Events in Bacterial Surface Colonization by Two Psezdrmras Species Using Image
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Peterson, TC. and RC. Ward. 1989. Development of Bacterial Transport Model for
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Rijnaarts, H.H.M., W. Norde, E.J. Bouwer, J. Lyklema, and A.J.B. Zehnder. 1996a.
Bacterial Deposition in Porous Media: Effects of Cell-Coating, Substratum
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47

Rijnaarts, HH M., W. Norde, E.J. Bouwer, J. Lyklema, and A.J.B. Zehnder. 1993. Bacterial
Adhesion under Static and Dynamic Conditions. Appl. Environ. Microbiol, 59, 15-
25.

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Bacterial Transport in Porous Media. Doctoral Thesis. Wageningen Agricultural
University, Wageningen, The Netherlands.

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2195-2202.

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Media: 1. An Experimental Investigation. Biotech. Bioeng., 44, 489—497.

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Media: II. A Model for Convective Transport and Growth. Biotech. Bioeng., 44,
499-508.

Scholl, MA. and RW. Harvey. 1992. Laboratory Investigations on the Role of Sediment
Surface and Groundwater Chemistry in Transport of Bacteria Through a
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Crit. Rev. Microbiol. 2:59-126.

Tatara, G.M., M.J. Dybas, and CS. Criddle. 1993. Effects of Medium and Trace Metals on
Kinetics of Carbon Tetrachloride Transformation by Psaxbnmas sp. Strain KC.
Appl. Environ. MicrobioL, 59, 2126-2131.

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48

Witt, M.E., M.J. Dybas, R.L. Heine, S. Nair, C.S. Criddle, and DC. Wiggert. 1995.
Bioaugrnentation and Transformation of Carbon Tetrachloride in Model Aquifer.
Presented at 3rd International Symposium on In Situ and On Site Bioreclamation.

49

AppendixA

CHAPTER 3 RAW DATA

shown in figures 3.1, 3.2, and 3.3, respectively.

Table A.1., Table A.2., and Table A.3 contains the results from the column experiments

Table A1: Effluent protein concentrations (pg/l) for transport experiments in figure 3.1

 

 

 

 

 

 

 

 

 

 

 

 

 

Tune 3.3 cm 5.8 cm 9.0 cm

(minutes) column column column
0.00 0.00 0.00 0.00
2.40 20.31 5.66 4.99
5.40 29.97 17.31 12.99
8.40 34.96 22.64 14.32
11.40 46.95 31.97 25.64
14.40 56.61 34.96 29.97
17.40 61.60 37.63 34.30
20.40 60.60 44.62 33.63
23.40 61.93 54.94 40.62
26.40 66.60 57.61 42.62
29.40 68.59 61.93 43.62

 

 

 

 

Table A2: Effluent protein concentrations (pg/l) for transport experiments in figure 3.2

 

 

 

 

 

 

 

 

 

 

 

 

 

Time 3.4 cm 6.2 cm 10.1 cm

(minutes) column column column
0.00 0.00 0.00 0.00
2.40 21.62 6.03 5.32
5.40 32.25 18.43 13.82
8.40 40.40 24.10 15.24
11.40 41.11 34.02 27.29
14.40 52.45 37.21 31.90
17.40 53.16 40.05 36.51
20.40 56.35 47.49 41.82
23.40 59.19 48.56 43.95
26.40 65.92 47.85 45.37
29.40 73.01 49.97 42.88

 

 

 

 

50

 

 

Table A.3: Effluent protein concentrations (pg/l) for transport experiments in figure 3.3

 

 

 

 

 

 

 

 

 

 

 

 

 

Time 3.7 cm 6.0 cm 10.0 cm
(minutes) column column column
0.00 0.00 0.00 0.00
2.40 15.59 3.19 0.00
5.40 30.13 16.30 13.47
8.40 55.64 37.57 17.37
11.40 62.02 45.37 29.77
14.40 69.11 51.75 40.05
17.40 77.97 56.00 44.30
20.40 82.23 62.02 51.39
23.40 81.52 68.05 57.42
26.40 88.25 70.88 59.54
29.40 87.90 74.43 62.02

 

 

 

 

 

The concentration of strain KC cells in these three experiments represented in tables A.1.,
A.2., and A.3. can be found in table A.4.. Strain KC concentrations were determined by

serial dilution and plating on R2A agar.

Table A.4. Strain KC concentration in transport ex eriments represented in tables A.1-A.3

 

 

 

 

Reactor Protein Strain KC
Table Age Growth Phase concentration concentration
(hours) (ug/ l) (cfu/ ml)
A.1. 18 mid exponential 77.7 9.6E+06
A2. 24 early stationary 101.2 8.5E+07
A3. 30 mid stationary 136.1 1.1E+ 08

 

 

 

 

 

 

 

Tables A.5, A6, and A] contain the solid 1‘C scintillation readings from the column

effluents shown graphically in figures 3.4, 3.5, and 3.6.

51

(counts per minute)

Table A5: Cell associated 14C readings for transport experiments in figure 3.4 in CPM

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Time 3.1 cm 6.0 cm 9.2 cm
(minutes) column column column
0 0.00 0.00 0.00
2 457.73 35.95 40.79
5 1339.54 485.69 74.01
8 2002.27 1343.75 399.07
11 2052.94 1660.18 1097.12
14 2084.27 1722.42 1399.89
17 2133.47 1990.91 1568.51
20 2125.90 2063.66 1823.13
23 2230.61 2038.01 1818.72
26 2284.01 2155.55 1847.10
29 2280.86 2124.85 1874.22

 

Table A.6: Cell associated 1‘C readings for transport emeriments in figure 3.5 in CPM

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Time 4.0 cm 6.4 cm 9.0 cm
(minutes) column column column
0 0.00 0.00 0.00
2 451.14 36.19 49.03
5 1265.67 623.47 416.42
8 1719.75 1487.03 941.83
11 1791.71 1608.44 1327.11
14 1897.76 1689.03 1431.69
17 1984.67 1799.92 1432.11
20 2084.41 1907.02 1444.10
23 2030.12 1873.99 1401.39
26 2054.95 1909.13 1434.00
29 2117.02 1909.55 1535.42

 

Table A]: Cell associated 4C readings for trans

ort experiments in figure 3.6 in CPM

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Time 3.7 cm 6.0 cm 9.7 cm
(minutes) column column column
0 0.00 0.00 0.00
2 371.21 90.08 37.29
5 1693.27 1173.75 131.56
8 2358.39 1820.43 688.58
11 2545.04 2019.23 1104.41
14 2813.18 2401.54 1176.47
17 2879.59 2632.40 1524.22
20 3005.28 2622.55 1610.74
23 3048.02 2689.80 1733.70
26 3106.25 2702.37 1848.92
29 3264.21 2811.09 1946.54

 

52

 

 

 

Table A8 contains the reactor concentrations of the initial cell 1“C and strain KC.

Table A.8: Strain KC concentration in transport experiments represented in tables AIS-A.7

 

 

 

 

 

Reactor Age Initial Cellij Strain KC
Table (hours) concentration concentration
(CPM/ ml) (cfu/ml)
A5 20 2478.3 1.0E+ 07
A6 30 3108.9 6.5E+07
A.7 48 3682.5 9.7E+07

 

 

 

 

 

Tables A.9., A10, and A.11 contain the solid 1‘C scintillation readings from the column

effluents represented in figures 3.7, 3.8, and 3.9, respectively.

Table A9: Cell 1‘C readings for transport experiments in figure 3.7 in CPM

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Time 3.7 cm 5.8 cm 11.2 cm
(minutes) column column column
0 0.00 0.00 0.00
2 210.48 46.96 34.17
5 1172.93 649.88 44.44
8 1280.05 1025.34 267.71
11 1317.79 1231.84 735.41
14 1434.98 1274.18 976.58
17 1422.61 1305.84 1067.90
20 1485.08 1372.29 1101.44
23 1470.62 1377.95 1094.52
26 1535.18 1369.78 1112.13
29 1560.55 1381.73 1141.69

 

 

 

53

 

Table A.10: Cell 14C readin s for transport experiments in figure 3.8 in CPM

 

 

 

 

 

 

 

 

 

 

 

 

 

Time 3.2 cm 6.5 cm 10.2 cm
(minutes) column column column
0 0.00 0.00 0.00
2 19.60 2.00 0.00
5 36.10 4.70 0.00
8 45.30 14.60 10.80
11 49.80 22.50 18.80
14 52.60 26.60 21.70
17 53.30 28.80 27.30
20 60.50 30.90 26.80
23 63.50 35.90 27.60
26 64.90 36.10 28.90
29 64.90 35.40 28.60

 

 

 

 

 

Table A.11: Cell 1‘C readin s for transport experiments in figure 3.9 in CPM

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Time 3.0 cm 6.3 cm 10.4 cm
(minutes) column column column
0 0.00 0.00 0.00
2 5.97 0.00 0.00
5 15.27 2.97 1.37
8 18.47 8.07 1.37
11 27.37 10.17 0.37
14 30.97 15.87 1.47
17 31.07 18.77 3.27
20 34.47 19.17 5.47
23 35.47 20.87 6.77
26 36.97 21.07 8.17
29 37.37 21.67 8.37

 

 

 

Table A.12 contains the reactor concentrations of the initial cell 1‘C and strain KC.

Table A.12: Strain KC concentration in transport experiments (prior to dilution) represented

 

 

 

 

in tables A.9-A.11
Reactor Initial Cell 1‘C Strain KC
Table Age Dilution concentration concentration
(hours) (CPM/ ml) (cfu/ml)
A.9 18 N/ A 2478.3 4.E+07
A.10 24 1:4.54 3108.9 8.E+07
A.11 30 1:5.00 3682.5 1.E+08

 

 

 

 

 

 

54

 

Appaxiz'xB

DATA FROM CHAPTER 4 EXPERIMENTS

Table B.1 contains the cell associated l4C column effluent CPM for exponentially growing

strain KC inoculum diluted with Schoolcraft groundwater represented in figure 4.1.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table B.1: Column effluent 14C readin s for transport experiments in fi e 4.1 in CPM
Time 0.12 Dilution 0.31 Dilution 0.59 Dilution 1.00 Dilution
(rrrrnutes)
0.00 0.0 0.0 0.0 0.0
1.00 9.4 17.8 35.7 46.1
3.00 38.3 119.6 293.7 1771.4
5.00 71.3 278.5 656.5 2666.0
7.00 78.1 390.1 918.7 2771.9
9.00 82.3 445.2 1064.5 2958.6
11.00 93.3 462.5 1230.2 2940.8
13.00 100.7 488.2 1340.3 2974.4
15.00 108.5 498.2 1369.7 2995.3

 

 

Table B.2 contains the cell associated 1‘C column effluent CPM for a exponentially growing

strain KC inoculum diluted with supernatant from a strain KC culture represented in figure

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4.2.
Table B.2: Column effluent 1‘C readin s for transport experiments in fi e 4.2 in CPM
Time 0.12 Dilution 0.31 Dilution 0.55 Dilution 1.00 Dilution
(minutes)

0.00 0.0 0.0 0.0 0.0
1.00 5.2 14.2 22.0 29.4
3.00 26.2 93.3 373.4 1703.2
5.00 52.4 199.3 652.3 2203.5
7.00 67.1 333.9 805.2 2647.1
9.00 81.8 402.1 997.2 2674.4
11.00 82.9 484.4 1285.5 2672.3
13.00 86.0 492.2 1329.9 2665.0
15.00 100.7 500.8 1406.4 2674.4

 

55

 

Table B.3: Reactor growth for tables BI and B2

Table B.3 contains the information about growth of the inoculum.

 

 

 

 

 

 

 

 

Reactor Age Strain KC Cell assimilated “C
(hours) (cfu/ ml) (CPM/ ml)
0 4.7E+ 06 0.00
18 1.0E+ 07
24 5.6E+07 3127.5
30 2.8E+ 08

 

 

Note: The experiments were performed at a reactor age of 24 hours.

Table B.4 contains the cell associated “C column effluent CPM for a stationary phase strain

KC inoculum diluted with Schoolcraft groundwater represented in figure 4.5.

 

 

 

 

 

 

 

 

 

Table B.4: Column effluent 1‘C readin s for transport experiments in E e 4.5 in CPM
Time 0.13 Dilution 0.26 Dilution 0.45 Dilution 1.00 Dilution
(mmutes)

0.0 0.0 0.0 0.0 0.0
2.0 4.0 19.5 78.6 118.1
5.0 9.1 31.4 141.7 373.0
8.0 12.1 43.1 195.0 855.4
11.0 13.9 48.6 236.8 1132.8
14.0 15.3 54.4 314.4 1455.2
17.0 15.9 75.5 384.6 1529.6
20.0 17.0 81.7 406.5 1636.6

 

 

 

 

 

 

Table B.5 contains the information about growth of the inoculum.

Table B.5: Reactor growth for table B.4

 

 

 

 

 

 

 

 

Reactor Age Strain KC Cell assimilated “C
(hours) (cfu/ ml) (CPM/ ml)
0 6.2E+ 05 0.00
18 8.6E+ 06
24 8.8E+ 07 2678.9
30 9.6E+ 07

 

 

56

Note: The experiment was performed at a reactor age of 24 hours.

 

Appaxiz'xC

DATA FROM CHAPTER 5

Tables C.1, C2, and C3 contain the solid 14C scintillation readings from the column

effluents shown graphicallyin figures 5.1, 5.2, and 5.3.

Table C.1: Colurrm effluent 11C for ' 5.1 in CPM

  

113 .53

1147.51

1

1233.61 11 956.36
1121.19 1.41

Table C.2: Column effluent 14C readings for transport experiments in figure 5.2 in CPM

 

 

 

 

 

 

 

 

 

 

 

 

Time 3.2 cm 4.9 cm 8.8 cm
(minutes) column column column
0 0.00 0.00 0.00
2 92.52 39.89 39.89
5 494.76 165.20 89.80
8 791.74 569.32 224.72
11 932.93 690.03 380.94
14 994.12 904.31 445.06
17 1103.55 997.88 536.53
20 1145.32 1016.26 614.01
23 1154.93 1056.35 696.09
26 1164.75 1080.37 723.24
29 1215.91 1074.52 734.31

 

 

 

 

 

 

57

in 5.3 in CPM
cm

   

Table C.3: Column effluent “C for

0.00

 

Table C.4 contains the reactor concentrations of the initial cell "C and strain KC.

Table C.4: Strain KC concentration in transport experiments represented in tables A.5-A.7

 

 

 

 

 

 

 

Reactor A e Initial Cell 1‘C Strain KC
Table (hours) g concentration concentration
(CPM/ ml) (cfu/ ml)
Cl 18 1809.8 7.7E+06
(:2 24 4418.8 5.6E+07
C3 30 6050.4 6.7E+ 07

 

 

 

58

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