Intrinsic and enhanced biodegradation of polyaromatic

hydrocarbons in aqueous and soil systems

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Authors Wang, Jiann-Ming

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INTRINSIC AND ENHANCED BIODEGRADATION OF POLYAROMATIC

HYDROCARBONS IN AQUEOUS AND SOIL SYSTEMS

by

Jiann-Ming Wang

A Dissertation Submitted to the Faculty of the

DEPARTMENT OF SOIL, WATER, AND ENVIRONMENTAL SCIENCE

In Partial Fulfillment of the Requirements

For the Degree of

DOCTOR OF PHILOSOPHY

WITH A MAJOR IN ENVIRONMENTAL SCIENCE

In the Graduate College

THE UNIVERSITY OF ARIZONA

1999
 UMI Number 9960243

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 2

THE UNIVERSITY OF ARIZONA 0

GRADUATE COLLEGE

As members of the Final Examination Committee, we certify that we have

read the dissertation prepared by Jiann-Mlng Wang

entitled Intrinsic and Enhanced Biodegradatlon of Polyaromatlc

Hydrocarbons in Aqueous and Soil Systems

and recommend that it be accepted as fulfilling the dissertation

requirement for the Degree of Doctor of Philosophy

M^k L. ^usseau
, / A / U
'/Raiha/H. Maier Date
^ y, i
Tar|^r^i^

!i/-E3 a?'!
Conklin Date

Pabl A. "Ferre Date

Final approval and acceptance of this dissertation is contingent upon

the candidate's submission of the final copy of the dissertation to the
Graduate College.

I hereby certify that I have read this dissertation prepared under my

direction and recommend that it be accepted as fulfilling the dissertation
requirement.

Dissertation Director Mark L. Brusseau Date

 3

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an

advanced degree at The University of Arizona and is deposited in the University Library to
be made available to borrowers under rules of the Library.

Brief quotations from this dissertation are allowable without special permission,
provided that accurate acknowledgement of source is made. Requests for permission for
extended quotation from or reproduction of this manuscript in whole or in part may be
granted by the head of the major department or the Dean of the Graduate College when in
his or her judgement the proposed use of the material is in the interests of scholarship. In
all other instances, however, permission must be obtained from the author.

SIGNED:
 4

ACKNOWLEDGEMENTS

I want to give my thanks to several people. First, I wish to express my appreciation

to my major advisor. Dr. Mark Brusseau, for giving me the opportunity to conduct
biodegradation studies. Second, I must give my profound thanks to my co-advisor, Dr.
Raina Maier, who has guided me into the spectacular "micro" world and strengthened my
knowledge in the environmental microbiology field. I would also like to thank my
committee. Dr. Janick Artiola, Dr. Martha Conklin, Dr. Roger Bales, and Dr. Paul Ferre.
They enlarge and deepen my thinking and understanding in soil chemistry, environmental
chemistry, and hydrogeology. I thank them for their time and assistance. In addition, 1 need
to express my appreciation to Dr. Yimin Zhang, who helped me to start my first
biodegradation experiment; Dr. Xiaojiang Wang, who showed me how to use HPLC; also
Julia Neilson, who helped to answer my various, broad microbiology questions and tolerated
me when I "invaded" their laboratory.
It's a wonderful experience to cooperate with other students in different projects.
For example, with Joe Piatt in the bioavailability of biotracer project; with Elizabeth
Marlowe in the HPCD-enhanced biodegradation study; with Joong-Hoon Cho in the
phenanthrene-hexadecane biodegradation study; with Susie Snyder In the benzoate
biodegradation kinetics experiments; and with Adria Bodour in the microbial succession
study. Special thanks to Asami Murao for isolating phenanthrene degraders in the microbial
succession study. We have piled up thousands of plates in five months. In addition, it has
been a good experience for me to help perform field experiments (AFP 44 and Hill AFB).
I have bad the chance to become acquainted with many field people, such as Jon Rohrer,
Nicole Nelson, John McCray, and Bill Blanford. Their activity has made a very deep
impression on me. It is a pleasure to know so many people in Raina's and Mark's groups.
Thanks for all of your Mendship and assistance.
Most importantly, I must thank my family and my wife's family for their patience
and constant support during these years. Especially, I must give my deep thanks to my
lovely wife, Ching-Hwa. She is always so considerate of me and gave up her job to take
care of our two children, who are the most valuable treasures for us. Thanks to my
daughter. Shining,and my son, Centric, for their seraphic smiles and irmocent words, which
encourage me to keep going toward the goal. Special thanks to James Callegary, Janet
Zamble, and Paul Kelso. They are not only our English tutors, but also our true friends in
Tucson. In addition, I must make mention the first reader of this dissertation, Brandolyn
Patterson, who has spent valuable time in helping me to improve my writing skills. I want
to give my thanks to her and I hope she has also gotten some benefit from the readmg and
revising.
dedicated to

my parents — Mao-Kai Wang and Yun-Hsiao Tsai

my wife — Ching-Hwa Chen

my daughter — Shining Wang

my son — Centric Wang

 6

TABLE OF CONTENTS

Page

LIST OF ILLUSTRATIONS 9

LIST OF TABLES 10

ABSTRACT 11

CHAPTER 1: INTRODUCTION 13

Explanation of Dissertation Format 13

Research Problem 15

CHAPTER 2: LITERATURE REVIEWS 17

Surfactants 17

Properties of Surfactants 17

Biodegradation of HOCs in the Presence of Surfactants in Batch

Aqueous Systems 17

Interactions of Surfactant and Soil 21

Soil-Flushing with Surfactants in Column Systems 21

Biodegradation of HOCs in the Presence of Surfactants in

Soil Systems 22

In-Situ Surfactant Flushing 24

Summary 24

Cyclodextrins 25

Properties of Cyclodextrins 25
 7

TABLE OF CONTENTS - Continued

Page

Detennining Factors for the Formation of an Inclusion Complex 27

Mechanisms 29

Release of Included Molecules 30

Application of Cyclodextrins 32

Biodegradation of Contaminants in the Presence of Cyclodextrins 33

Biodegradation Kinetics 36

Growth Kinetics 36

Monod Equation 37

Half-Saturation Constant 39

Modified Monod Equation 40

Limitations and Applications of Monod Kinetic Model 43

Microbial Succession 45

CHAPTER 3: PRESENT STUDY 48

Summary 48

APPENDDC A: CYCLODEXTRIN - ENHANCED BIODEGRADATION OF

PHENANTHRENE 54

APPENDDC B : THE INFLUENCE OF BACTERIAL POPULATION

HETEROGENEITY AND DYNAMICS ON BIODEGRADATION

AND TRANSPORT OF PHENANTHRENE IN SOIL 79

 8

TABLE OF CONTENTS - Continued

Page

APPENDIX C: SUPPLEMENTAL EXPERIMENTS 108

SUBDIVISION 1:

CHARACTERISTICS OF BENZOATE BIODEGRADATION .... 109

SUBDIVISION 2:

BIOAVAILABILITY OF BENZOATE AT THE AFP 44 SITE .... 127

SUBDIVISIONS:

THE MEASUREMENT OF BIODEGRADATION KINETIC

PARAMETERS FOR BENZOATE AND

2,4-DICHLOROPHENOXYACETIC ACID (2,4-D) 132

SUBDIVISION 4;

BIOAVAILABILITY OF PYRENE IN THE PRESENCE OF

HYDROXYPROPYL-p-CYCLODEXTRIN(HPCD) 153

SUBDIVISION 5:

ADHERENCE OF BACTERIAL CELLS TO HEXADECANE AND

PARTITIONING OF PHENANTHRENE BETWEEN WATER AND

HEXADECANE 158

REFERENCES 167
 9

LIST OF ILLUSTRATIONS

Page

Figure 1. Schematic of Cyciodextrins in 3-Diinensions 26

Figure 2. Structure of Hydroxypropyl-P-Cyclodextrins 26

 10

LIST OF TABLES

Page

Table 1. Summary of the Effects of Surfactants on the Biodegradation of Phenanthrene

in Aqueous Systems 19

Table 2. Physic Properties of Cyclodextrins 27

 11

ABSTRACT

Bioremediation is currently one of the most popular methods for remediating soil

and groundwater contaminated by organic compounds. However, it has been found that the

availability of the target contaminant to the microbial populations capable of degrading the

compound may serve as a limiting factor in many systems. Thus, there is interest in the use

of solubilization agents for enhancing bioavailability of organic contaminants. The impact

of hydroxypropyl-P-cyclodextrin (HPCD) on the biodegradation of polycyclic aromatic

hydrocarbons (PAHs) was investigated in a batch study. Results showed that analytical-

grade HPCD can significantly increase the apparent solubility of phenanthrene, which had

a major impact on the biodegradation rate of phenanthrene. For example, in the presence

of 10' mg L ' HPCD, the substrate utilization rate increased 5.5 times while the apparent

solubility was increased from 1,3 mg L ' (no HPCD) to 161.3 mg L '. As a result, only 0.3%

of the phenanthrene remained at the end of a 48-hour incubation for the highest

concentration of HPCD tested (10' mg L*'). In contrast, 45.2% of the phenanthrene

remained in the absence of HPCD. Based on these results, it strongly suggests that HPCD

can significantly increase the bioavailability, and thereby enhance the biodegradation, of

phenanthrene.

Biodegradation is often of great importance for the transport, fate, and remediation

of organic contaminants in the subsurface. When modeling biodegradation processes, it is

usually assumed, either implicitly or explicitly, that the microbial population responsible

for biodegradation is composed of a single species. However, this is unlikely to be true for
 12

many, if not most, field situations. The effect of multiple species of degraders on

phenanthrene biodegradation and transport in a saturated soil was evaluated with a series

of miscible-displacement experiments. Miscible displacement experiments were conducted

using a soil with a high phenanthrene sorption capacity and with an indigenous microbial

population consisting of 24 species of phenanthrene degraders. Brealcthrough curves

obtained for the non-sterile column experiment exhibited oscillations in microbial

populations as well as in oxygen and phenanthrene concentrations during the 6 months of

continuous injection of a constant-concentration phenanthrene solution. This behavior is

due to the response of the heterogeneous bacterial population to substrates and oxygen

availability, wherein population dynamics is hypothesized to be mediated by competition

and other multi-species interactions. The dynamics of heterogeneous microbial populations,

especially under growth conditions, should be considered when evaluating contaminant

biodegradation and transport in natural subsurface systems.

 13

CHAPTER 1; INTRODUCTION

Explanation of Dissertation Format

This dissertation consists of three major parts:the literature review, manuscripts, and

supplemental experiments.

There are four sections included in the literature review. In the first section, the

properties and applications of surfactants are reviewed in detail, along with the motivation

for the search for an alternative solubilization agent - cyclodextrin. The second section

introduces the characteristics and properties of cyclodextrins and their applications to the

environmental field. This section gives background information for the manuscript attached

in Appendix A. In the third section, the origin and development of biodegradation kinetics

are presented. Biodegradation equations and kinetic parameters are discussed in detail. The

last section elucidates the course of microbial succession.

Two manuscripts are presented in Appendices A and B. The paper in Appendix A

has been published in the peer-reviewed journal - Environmental Science & Technology.

It obtained a first place award, given by the Air & Waste Management Association - Grand

Canyon Section (AWMA-GCS) scholarship in 1998. The other paper, in Appendix B, will

soon be submitted to a professional journal.

Additional experiments are included in Appendix C. These experiments are

classified into five subdivisions: characteristics of benzoate biodegradation, bioavailability

of benzoate at the AFP 44 site, the measurement of biodegradation kinetic parameters.

 14

bioavailability of pyrene in the presence of cyclodextrins, and biodegradation of

phenanthrene in the presence of hexadecane. The work included in the first and third

subdivisions has contributed to two published papers.

I was responsible for designing and conducting almost all of the aforementioned

experiments and analyzing their results. There are two exceptions; one is the viable counts

and the identification of phenanthrene degrader, CRE7, in Appendix A which were done by

Elizabeth M. Marlowe (Ph.D. 1999) and Eileen M. Jutras (Ph.D. 1997), respectively; the

other is the identification of phenanthrene degraders in soil microcosms after microbial

succession in Appendix B which will be conducted by Adria Bodour (Ph.D. student).

 15

Research Problem

There is tremendous interest in using bioremediation techniques for the clean up of

contaminated soil and groundwater. However, biodegradation rates in the subsurface are

often constrained by a limited supply of electron acceptors and nutrients and by factors

related to bioavailability, such as solubility, dissolution rate, and sorption. It has been

proposed that solubilization agents, like surfactants, may be added to the system to enhance

the bioavailability of low solubility and highly sorptive compounds. However, there are

constraints to the use of surfactants. Recently, an altemative agent, cyclodextrin, has been

used in environmental applications to improve the remediation of contaminated soil and

groundwater. However, its efficacy on the bioavailability and biodegradation of

hydrophobic organic compounds (HOCs) has not yet been investigated for in-situ

environmental applications. One of the major objectives of the first study is to investigate

the influence of cyclodextrins on the enhancement of the biodegradation of polycyclic

aromatic hydrocarbons (PAHs).

PAHs, which are produced from the incomplete combustion or spill of fossil fuels

or organic chemicals, are ubiquitous in the environment (Andelman and Snodgrass, 1974;

Bossert and Bartha, 1984). Biodegradation is one of mechanisms for the removal of PAHs

firom the environment (Cemiglia and Heitkamp,1989). However, most PAH biodegradation

studies were conducted using a pure, single species of microorganisms isolated from soil,

sediment, sludge, or sewage by enrichment technology (Stucki and Alexander, 1987;

Bumpus, 1989; Culien et al., 1994; Wang et al., 1998). This is not the case in the real
 16

world. Although biodegradation experiments have been performed in soil columns, the

majority have been focused on either biotransformation or transport behavior of

contaminants and have not examined microbial dynamics (Jenkins and Lion, 1993; Dohse

and Lion, 1994; Devare and Alexander, 199S). In addition, most investigations have been

based on the use of one average specific growth rate to represent what is in actuality

multiple species in the soil. To date, few have examined the influence of multiple-species

populations and attendant effects (e.g., competition, predation, microbial succession) on

biodegradation and transport behavior of PAHs or other organic compounds in soil (Barker

et al., 1987; Madsen et al., 1991). Therefore, the second study was performed to address

this issue.
 17

CHAPTER 2: LITERATURE REVIEWS

Surfactants

Properties of Surfactants

Surfactants are well known "solubilization" agents which can increase solubility of

hydrophobic compounds by forming aggregates called micelles. Micelles are an

arrangement of surfactant molecules formed by the hydrophobic tail groups coming together

to create a thermodynamically favorable hydrocarbon (HC) pseudophase with a hydrophilic

exterior (Rouse et al., 1994). The surfactant concentration at which micelles are initially

formed is termed the critical micelle concentration (CMC). Because of the amphipathic

nature of surfactants, the hydrophobicity of organic compounds, which is indexed by

hydrophile-lipophile balance (HLB) number, is very important to evaluate the potential

efficacy of surfactants. The larger the HLB number, the more hydrophilic the molecule. In

addition, the solubilization capacity of surfactants is expressed by molar solubilization ratio

(MSR), which is defined as the number of moles of organic compound solubilized per mole

of surfactant added to solution (Attwood and Florence, 1983).

Biodepradation of HOCs in the Presence of Surfactants in Batch Aqueous Systems

Although surfactants have been shown to successfully enhance the apparent

solubility (Kile and Chiou, 1989; Edwards et al., 1991; Liu et ai., 1991) or dissolution rate

(Grimberg et al., 1994; 1995) of HOCs, the effects of surfactants on the biodegradation of
 18

poorly soluble compounds in aqueous solution may vary. Much research has concluded that

surfactants enhance the biodegradation of PAHs and long-chain alkanes by increasing the

apparent solubility (Guerin and Jones, 1988; Bury and Miller, l993;Tiehm, 1994; Churchill

et al., 1995; Liu et al., 1995; Volkering et al., 1995; Zhang et al., 1997) or dissolution rate

(Volkering et al., 1995; Grimberg et al., 1996). Conversely, research has also indicated that

surfactant efficacy is dependent upon the interaction among the surfactant structure,

substrate type, and cell property (Zhang and Miller, 1995). Operating conditions, such as

pH, shaking speed, and time may also influence the surfactant's biodegradation efficiency

(Zhang and Miller, 1992). Table 1 summarizes the effects of surfactants on the

biodegradation of phenanthrene in aqueous solution. Some surfactants, like SDS (sodium

dodecyl sulfate), inhibit biodegradation of phenanthrene. This inhibition is most likely due

to SDS's toxicity to bacteria (Tiehm, 1994; Descheneset al., 1995). Some surfactants, such

as Tween, have no effect on the biodegradation of phenanthrene (Guerin and Jones, 1988).

And yet other surfactants, such as Marlipal 013/90, can enhance the biodegradation of

phenanthrene but they are toxic to other PAH-degraders (Tiehm, 1994). Only a few

surfactants, monorhamnolipid, for example, can enhance the biodegradation of PAHs and

have a minimal effect on the environment (Zhang et al., 1997). Furthermore, it is difficuh

to compare surfactant studies to one another because every study uses a unique bacterial

species, inoculum density, surfactant type and concentration, substrate concentration, and

phase of substrate in the system.

Table 1. Summary of the Effects of Surfactants on the Biodegradation of Phenanthrene in Aqueous Systems.

So.ubiUea.io„Agen. Initial substrate Biodegradation Bacterial

Isolate/Consortium
Type Concentration (mg/L) Rate Extent (References)
(mM) (mg/hr) (%)•
Monorhamnolipid 0 165 2.6E-2 75 Pseudomonas putida Cre7
(Biosurfactant) 0.35 (surface limited 5.4E-2 54
3.5 coating) 1.6E-1 10 (Zhang et al., 1997)
Triton XlOO 0 0.8 5.7E-4 20 Mixed culture
(Nonionic 0.2 0,9 5.6E-4 15
surfactant) 1.2 4.1E-4 46 (Guha and Jaffe, 1996a,
4.8 3.3E-4 >91 1996b)
Triton N101 0.07 0.8 5.6E-4 22
(Nonionic 1.2 0.9 3.3E-4 48
surfactant)
Brij30 0.35 1.1 6.3E-4 25
(Nonionic 3.5 5.0E-4 47
surfactant)
Tergitol NP-10 0 l.I 7.2E-3 11 Pseudomonas stutzeri P I 6
(Nonionic 0.7 10 3.2E-2 100
surfactant) 1.5 20 4.0E-2 100 (Grimberg et al., 1996)
 Table 1. Continued.

1 Solubilization Agents Initial substrate Biodegradation Bacterial

Isolate/Consortium
Type Concentration (mg/L) Rate Extent (References)
(mM) (mg/hr) (%)•
SDS 0 200 (solubilized) NA" 12 Mixed culture
(Anionic 1.2 NA 29
surfactant) 1.6 NA 38 (Tiehm, 1994)
1 2.0 NA 83
1 Marlipal 013/90 10 120
1 (Nonionic (solubilized) 2.6 2
1 surfactant) (small crystal) 0.9 27
1 (large crystal) 0.3 69
1 Tween 0 NA NA NA Mycobacterium sp. strain
1 20 0.082 100 3.8E-1 >90 BGl
1 40 0.078 3.7E-1 >90
60 0.076 4.6E-1 >90 (Guerin and Jones, 1988)
80 0.076 3.8E-1 >90
85 0.053 3.8E-1 >90
(Nonionic
surfactant)

* The extent of biodegradation was reported as the relative concentration of substrate remaining after 30 hours incubation.
The only exception was the values of SDS which were reported after 213 hours incubation period.
•• NA = Not Available.
 21

Interactions of Surfactant and Soil

While soils are introduced components in an aqueous system, the interactions

between surfactants and soils must be taken into account. Ducreux et al. (1990) investigated

the phenomenon of surfactant retention in soil flushing experiments and surmised that an

anionic surfactant can react with minerals (especially with clay) through a series of

adsorption and precipitation processes. Adsorption behaviors were observed not only for

nonionic surfactants(Abdul and Gibson, 1991; Jahan et al., 1997) but also for biosurfactants

(Bai et al., 1997; Torrens et al., 1998). Adsorption of surfactants on minerals is an

important phenomenon in the study of enhanced biodegradation and soil-flushing

technologies because it limits the available amount of surfactant in the aqueous phase.

Thus, it reduces treatment efficiency and increases cost. However, the adsorption of

surfactant on porous materials does not have a deleterious effect in HOC immobilization

(e.g., Wagner etal., 1994)or surfactant recycling (e.g., Narkisand Ben-David, 1985). Some

surfactants can be removed from aqueous solution by highly adsorptive porous materials,

such as powdered or granular activated carbons and mineral clay. Ideally, the more a

surfactant sorbs, the more it can be recycled.

Soil-Flushing with Surfactants in Column Systems

From the viewpoint of soil flushing, because surfactants have the ability to increase

the desorption rate of HOCs, it is not surprising that they can facilitate the transport of PAHs

(such as naphthalene and phenanthrene) and 1,1-bis (^hloro-phenyl)-2,2,2-trichIoroethane

(DDT) in soil (e.g., Kan and Tomson, 1990). Another characteristic of surfactants is that

they can decrease the interfacial tension between non-aqueous phase liquids (NAPLs) and

water. This reduction in surface tension allows surfactant solutions to displace oil drops

trapped in the soil pores; drops that could not be otherwise displaced by water (Miller and

Qutubuddin, 1987). Surfactants can also disperse and transport more oil through the

channels of porous media with the flowing water (Ang and Abdul, 1991). For example,

surfactant washing has been used to facilitate the removal of polychlorinated biphenyls

(PCBs) (Abdul and Gibson, 1991), tetrachloroethylene (PCE) pool (Fountain et al., 1991),

or residual PCE (Pennell et al., 1994; Okuda et al., 1996) from column systems, packed with

glass beads (e.g., Okuda et al., 1996) or sand media (e.g.. Fountain et al., 1991; Pennell et

al., 1994). Biosurfactants have also shown their ability to enhance the removal of residual

hexadecane from sand columns (Bai et al., 1997; Herman et al., 1997a; 1997b). However,

it was reported that the application of rhamnolipid (a biosurfactant) on a soil with high clay

content caused soil dispersion and column plugging (Torrens et al., 1998).

Biodeeradation of HOCs in the Presence of Surfactants in Soil Systems

From the viewpoint of enhanced bioremediation, the situation becomes more

complex after the addition of surfactants into a soil-water system. Theoretically, surfactants

can increase the bioavailability ofHOCs by increasing their apparent solubility or desorption

rate, thereby enhancing the bioremediation effects in soil. However, similar to the

biodegradation studies without soil, both enhancements and inhibitions of biodegradation

 23

of HOCs, in the presence of surfactants, have been reported. For instance, Graves and

Leavitt(199l) andThibaultetal. (1996) found that surfactants inhibited the biodegradation

of petroleum hydrocarbons and pyrene in soils, respectively. In contrast, Oberbremer et al.

(1990) reported that both the rate and extent of hydrocarbon degradation in a soil slurry were

increased in the presence of biosurfactants. It, therefore, appears that soil or surfactant type

may have an influence on HOC biodegradation. For example, Providenti et al. (1995)

observed that biosurfactants can enhance the mineralization of phenanthrene in creosote-

contaminated soil slurries but inhibits biodegradation in sandy loam and silt loam soil

slurries. Thai (1993) demonstrated that Corexit 0600 (a nonionic surfactant) has a positive

effect but Brij 35 (also a nonionic surfactant) has a negative elfect on octadecane

biodegradation in sand. Some researchers also indicated that the applied concentration of

surfactants has a great influenceon the biodegradation of contaminants in soil-water system.

For example, Laha and Luthy (1991) found that the mineralization of phenanthrene was

substantially inhibited in the presence of surfactants at CMC, while there was no effect on

phenanthrene mineralization when the concentration of surfactants was below the CMC.

Tsomides et al. (1995) showed that only one (Triton X-100) of seven nonionic surfactants

did not inhibit phenanthrene mineralization at concentrations above CMC. In comparison,

some studies showed that using surfactants at concentrations below the CMC can stimulate

biodegradation of sorbed PAHs (Aronstein et al., 1991; Aronstein and Alexander, 1992;

1993) or trapped hexadecane in soil (Herman et al., 1997b).

 24

In-Situ Surfactant Flushing

Although laboratory studies have shown that surfactants can aid in the washing out

of sorbed or residual contaminants from soil, the efficacy of in-situ surfactant flushing has

not been well established. Abdul et al. (1992) demonstrated a successful case for the

application of surfactants to a PCB-contaminated site. However, performance failures of

in-situ surfactant flushing at a site contaminated with JP-4 jet fuel, lubrication oils, and

chlorinated solvents have also been reported (Chawla et al., 1990). One possible

explanation for the failure operation of in-situ surfactant flushing is that surfactants can,

under some conditions, disperse small particles of clay minerals or other solids that interact

with petroleum products to form viscous emulsions (Abdul et al., 1992). This behavior

could lead to pore clogging and subsequent flow reduction.

Summarv

In summary, the use of surfactants in efforts to enhance the removal of contaminants

in environments has been of considerable interest for several years. Although surfactants

show they have the ability to improve the efficiency of biodegradation or soil flushing, the

aforementioned negative results have also been obtained. Therefore, it isa worthy endeavor

to look for an alternative solubilization agent which is not adsorbed to the soil, is nontoxic

to the environment, is easy to degrade, and can enhance apparent solubility.

 25

Cvclodextrins

Properties of Cvclodextrins

Cyclodextrins (CDs), sometimes called Schardinger dextrins, cycloamyloses,

cyclomaltoses, or cycloglucans, are a series of oligosaccharides obtained from the

breakdown of starch and related compounds by the action of Bacillus macerans amylase

(Bender and Komiyama, 1978; Bender, 1986). To date, it has been reported that

cyclodextrinases can be isolated from B. macerans (DePinto and Campbell, 1968) and B.

coagulans (Kitahata et al., 1983; Kitahata and Okada, 1985). In addition, mtracellular

amylases capable of splitting cyclodextrins may be isolated from Pseudomonas sp. (Kato

et al., 1975) and Flavobacterium sp (Bender, 1981).

Cyclodextrins, which have a lampshade-shaped structure (Figure 1), are composed

of a-(l,4)-linkages of a number of D-glucopyranose units (Figure 2). All the glucose units

in a CD molecule are in substantially undistorted CI (D) (chair) conformations. The

primary hydroxyl groups at the C-6 atom of a glucose unit are arranged in one of the open

ends of the cavity, whereas the secondary hydroxyl groups at the C-2 and C-3 atoms of a

glucose unit are located in the other open end. The interior of the cavity consists of a ring

of C-H groups, a ring of glucosidic oxygens, and another ring of C-H groups. Therefore,

cyclodextrins have a relative hydrophobic (apolar) cavity compared to water, which gives

them the ability to include hydrophobic molecules to form inclusion complexes. The CD

molecule is referred to as the "host molecule" and the included compound is referred to as

the "guest molecule".

Apolar Cavity Second^ Hydroxyls

Primary Hydroxyls

Figure I. Schematic of Cyclodextrins in 3-Dimensions.

(Adaptedfrom Szejtli, 1990)

Figure 2. Structure of Hydroxypropyl-P-Cyclodextrins.

(Adapted from Szejtli, 1990)
 27

Generally speaking, cyclodextrins may be classified into three groups: a-CD

(cyclohexaamylose), P-CD (cycloheptaamylose), and y-CD (cyclooctaamylose), which

contain 6, 7, and 8 glucopyranosyl residues in a molecule, respectively. Some physical

properties of the cyclodextrins are sununarized in Table 2 (Bender, 1986).

Table 2. Physical Properties of Cyclodextrins.

Properties a-CD p-CD Y-CD

Number of Glucose Residues 6 7 8

Molecular Weight, (g/mol) 972 1135 1297

Water Solubility at 20°C, (g/lOOml) 14.5 1.85 23.2

Specific Rotation, 150.5 ±0.5 162.5 ± 0.5 177.4 ±0.5

Diameter of Cavity, (nm) 5 ±0.3 6.2 ± 0.3 7.9 ±0.4

External Diameter, (nm) 14.6 ±0.4 15.4 ±0.4 17.5 ±0.4

Height, (nm) 7.9 ±0.1 7.9 ±0.1 7.9 ±0.1

Determining Factors for the Formation of an Inclusion Complex

Because of their different internal ring sizes, the CD molecules show different

degrees of inclusion formation with different sized guest molecules. The steric effect is one

of the determining factors in the formation of an inclusion complex. Cyclodextrins can only

include molecules of suitable size; those which can fit in their hydrophobic cavities. The
 28

effect of cyclodextrins can be observed from the increase of the apparent solubility of

hydrophobic molecules. The molar ratio of a guest to a host molecule is usually 1:1 or 1;2

for an inclusion complex formed in an aqueous solution (Bender and Komiyama, 1978;

Wang and Brusseau, 1995a). Nevertheless, for some long-chain aliphatics, such as

octadecane which has 18 carbon atoms in a row, the cavity size of one CD molecule is not

large enough to include the whole aliphatic molecule. Only part of the long-chain aliphatic

molecule is included in the cavity of the CD molecule; the remainder of the molecule resides

in the solution. This logic has been used to explain why the apparent solubility of long-

chain aliphatics does not dramatically increase in the presence of cyclodextrins. A potential

way to solve this problem would be to use threaded cyclodextrins which cross link adjacent

CD units to create a molecular tube (Harada et al., 1992; 1993).

Another special case is when the guest compound consists of a polar group and an

apolar group, such as benzenesulfonate (Bender and Komiyama, 1978). While the inclusion

complex is forming, the apolar group of the guest molecule will be included in the cavity

of cyclodextrin. The polar group of the guest molecule will stay in solution or form

hydrogen bonds with the hydroxyl groups of cyclodextrins. This type of structure is

extremely stable.

As well as the steric factor, the hydrophobicity of guest molecules is another

determining factor forthe formation of inclusion complex. The strongerthe hydrophobicity,

the higher the tendency to be included in the cavity of cyclodextrins.

 29

Mechanisms

Hydrophobic interactions occur when the non-polar groups prefer to interact with

each other, excluding polar groups. These interactions seem to be the most probable

mechanism for inclusion complex formation. Furthermore, it appears that the formation of

the inclusion complex can be divided into several steps. First, the hydrophobic compound

approaches a CD molecule. The structure of water molecules inside the CD's cavity breaks

down and some water molecules are removed from the cavity. Then, the interaction occurs

between the hydrophobic compound and the CD molecule, either inside the cavity or on the

rim of the CD molecule. Hydrogen bonds may also form between guest and host molecules.

Finally, water molecules become reconstituted around any guest molecules that remain

outside of the cavity.

From an energy viewpoint, the formation of an inclusion complex is associated with

a favorable enthalpy change (AH<0) and an unfavorable (or slightly favorable) entropy

change (AS<0). The primary interactions thought to cause an enthalpy change include van

der Waals interactions between guest and CD molecules, and hydrogen bonding between

the guest and the hydroxyl groups of CD molecules. The favorable enthalpy change is

thought to occur as the host and guest molecules interact during inclusion complex

formation. During this process, water molecules are excluded from the cavity and energy

is released. Additional energy is released when the guest molecule is included in the

hydrophobic cavity. The guest molecule causes the conformational strain energy of the

cyclodextrin to be released. Though this school of thought appears logical, the major
 30

binding force for inclusion complex formation still remains controversial (Bender and

Komiyama, 1978).

Release of Included Molecules

The inclusion complexes in solution are in dynamic equilibrium. Guest molecules

included in the cavity rapidly exchange with free guest molecules in solution. Assuming the

stoichiometry of guest to host molecules in inclusion complexes is 1:1, the formation of an

inclusion complex can be expressed as the following equilibrium equation;

CD + X»-CD-X [1]

Vf=k,[CD][X] [2]

v, = k,[CD-X] [3]

While at equilibrium, Vf = v,; therefore,

[CD-X] k,
[CD][X] kr "

where

kf = recombination rate constant (forward reaction rate constant),

k, = dissociation rate constant (reverse reaction rate constant),

Vf = forward reaction rate,

= reverse reaction rate,

K = equilibrium constant,

[CD] = concentration of cyclodextrin,

 31

[X] = concentration of guest molecule, and

[CD-X] = concentration of inclusion complex.

The concentration gradient and hydrophobicity of guest molecules are probably the two

primary driving forces for the inclusion complex formation. Bender and Komiyama (1978)

summarized the processes which may be involved in the release of guest molecules &om the

cavity of cyclodextrins. Those processes include (a) breakdown hydrophobic interactions

between CD and included molecules, (b) diffusion of guest molecules out of the cavity, and

(c) conformational change of cyclodextrin. So far, there is no conclusion as to which is the

dominant process.

The reaction rate constants, and kf, for the inclusion complex can be determined

by two well-defined methods, temperature-jump (Cramer et al., 1967) and ultrasonic

relaxation (Rohrbach et al., 1977). The concept of the temperature-jump method, which

may be applied over a wide time range, is very simple. If the temperature of a reaction is

changed quickly, any temperature-dependent equilibrium in the sample is thereby upset

(Turner, 1986). The concentrations of reactants and products must change to the values

required for equilibrium at the new temperature. If the temperature is changed faster than

the system can react, then the time dependence, or the relaxation of the concentration

changes, can be measured. This time dependence, which is typically exponential, can be

used to derive the rate constants at the final temperature for each chemical involved in the

reaction. Several techniques such as absorption, fluorescence, and optical activity can be

used to detect the concentrations of reactants and products. The reported relaxation times
 32

are from 10'^ to ^ 10^ seconds. The concept of the ultrasonic method is that the passage of

a sound wave through a medium provides a way to perturb a dynamic equilibrium by the

pressure and temperature variations that accompany the wave (Stuehr, 1986). The range of

ultrasonic frequency extends from about lO"* to 10' Hz. The corresponding relaxation times

span the range from approximately 10*" to 10*^ seconds.

Application of Cvclodextrins

The most unique property of cyclodextrins is that they have an apolar cavity which

can include hydrophobic molecules. Thus, CD molecules hold the promise to increase the

apparent solubility of hydrophobic compounds. Conventionally, cyclodextrins are widely

used in the pharmaceutical, food, cosmetic, and toiletry industries (Szejtli, 1982; Bender,

1986). Recently, the application of cyclodextrins has been expanded to solve environmental

problems. This is because they have the potential to improve the effectiveness of soil

flushing and enhance bioavailability. For example, hydroxypropyUP-cyclodextrin (HPCD)

has been shown to have the ability to significantly increase the apparent solubility of several

low-polarity organic compounds such as trichloroethene (TCE), chlorobenzene,

naphthalene, anthracene, and phenanthrene (Wang and Brusseau, 1993; Wang etal., 1998).

Carboxymethyl-P-cyclodextrin (CMCD), a modified cyclodextrin, displayed the capacity

to simultaneously increase the apparent solubility of HOCs, such as anthracene,

trichlorobenzene, biphenyl, and DDT, and complex with heavy metals, such as cadmium,

(Wang and Brusseau, 1995b). It was also reported that P-CD had the ability to remove
 33

phthalic acid esters (PAE) from aqueous solution by forming an inclusion complex (Murai

et al., 1998). In soil flushing operations, HPCD and methyl-P-cyclodextrin (MCD)

exhibited the ability to facilitate the transport of organic compounds, such as biphenyl,

naphthalene, anthracene, and pyrene, and chlorinated solvents, such as 2-chlorobiphenyl,

2,4,4'-trichlorobiphenyl, trichlorobenzene, trichloroethene, and tetrachloroethene (PCE),

through soil columns (Brusseau et al., 1994; Boving et al., 1999). One of the most

significant results showed that there was not any measurable sorption of HPCD by either the

sandy or the high organic-carbon porous media used in the experiments (Brusseau et al.,

1994). In addition to HPCD and MCD, CMCD has also demonstrated its ability to enhance

the desorption and elution of phenanthrene and cadmium from soil columns (Brusseau et

al., 1997). Furthermore, this innovative technology was applied in the field to enhance the

removal of non-aqueous phase liquids (NAPLs) from a jet fuel contaminated aquifer

(McCray and Brusseau, 1998). The results suggested that HPCD could be used as an

solubilization agent for iri'SUu soil flushing technology.

Biodeeradation of Contaminants in the Presence of Cvclodextrins

The applications of cyclodextrins in biotechnology have been reviewed by Szejtli

(1990). However, there are few studies which have correlated the biodegradation of

contaminants with the presence of cyclodextrins. Thus far, P-CD research suggests that the

use of P-CD decreases the toxicity of some pesticides and their decomposition products in

activated sludge systems (01^ et al., 1988). It has also been reported that P-CD has the
 34

ability to stabilize the emulsion of 4-chlorobiphenyl and consequently enhance the

biodegradation rate in a mixed culture (Hiramoto et al., 1989). Schwartz and Bar (1995)

used P-CD to remove or alleviate the toxicities of substrates (toluene and p-toluic acid) and

to further initiate or enhance biodegradation. The most direct evidence for cyciodextrins

enhancing the biodegradation of contaminants was exhibited by the author's research which

proved that HPCD significantly increased the apparent solubility as well as dissolution rate

of phenanthrene, and thereby enhanced the biodegradation rate of phenanthrene in aqueous

solution (Wang et al., 1998).

To date, no research has conclusively elucidated the mode of microbial uptake of

hydrocarbons in the presence of cyciodextrins. The following example can be used to depict

the complexity of this issue. First, consider the phase distribution of phenanthrene in a CD

aqueous system, there are two phases of phenanthrene (dissolved and complexed) when

phenanthrene is not in excess and three phases of phenanthrene (solid, dissolved, and

complexed) when phenanthrene is in excess. In the presence of cyciodextrins, bacteria may

use complexed phenanthrene as well as dissolved phenanthrene. In this case, if there is no

excess phenanthrene in the system, the biodegradation rate is determined by the apparent

solubility. In contrast, when phenanthrene is in excess, the biodegradation rate is

determined not only by the apparent solubility but also by the dissolution rate. As well,

bacteria may be using only dissolved phenanthrene as a food source. In this case, if

phenanthrene is not in excess, biodegradation rate is subject to the dissociation rate. In

contrast, when phenanthrene is in excess, the biodegradation rate is subject to both the
 35

dissociation rate and the dissolution rate. Although the dissolution rate was measured, the

dissociation rate constant of phenanthrene from HPCD was not measured for this study.
 36

Biodepradation Kinetics

Growth Kinetics

Considerable interest has been given to the kinetics of bacterial growth. In a

microbial growth curve, the most widely studied phase is the logarithmic or exponential

growth phase. In this phase, bacterial numbers increase exponentially with time. This may

occur, for instance, when a small amount of pure cells are inoculated into aqueous solution

with a fixed amount of carbon source and nutrients. The most common way to describe the

relationship between the biomass increase rate, dB/dt, and the biomass concentration, B, in

the exponential growth phase is a first order equation:

dB
[5]
dt

The proportionality factor, |i, which is often called the specific growth rate, i.e., the growth

rate per unit of biomass, is a constant and has a unit time '. To establish the relationship

between the substrate utilization rate (dS/dt) and the biomass growth rate (dB/dt), the yield

coefficient (Y) is introduced. The yield coefficient is defined as the mass of cells produced

in a unit time as the result of the mass of substrate utilized in the same reaction time (Gaudy

and Gaudy, 1980). The relationship between them can be written as the following form

[6]
dt ~ dt

where the negative sign indicates that the concentration of the growth-limiting substrate
 37

declines with time. Therefore, the substrate utilization rate can be expressed as a function

of specific growth rate (n), biomass concentration (B), and yield coefficient (Y):

[7]
dt Y ^

Monod Equation

There are several biotransformation models that have been proposed to represent the

biodegradation kinetics. Monod equation is one of the most well-known models used for

simulating microbial growth kinetics in batch systems. It was developed in a well-

controlled system (sufficiently available substrate, oxygen, and nutrients) comprising a

single pure culture. The basic assumptions include that the component being used as the

carbon or energy source is water soluble and nontoxic to specific degraders, that the

metabolite is not harmful for degraders, and that the system is well agitated. Monod (1949)

recognized that if there was a decrease in the substrate concentration, then there was a

reduction in the bacterium's specific growth rate. In other words, the specific growth rate

of the species was restricted by the concentration of the growth-limiting substrate. Monod

realized that the growth rate of bacterial cells will increase in proportion to the increase in

substrate concentration and further formulated a mathematical expression to adequately

describe these relationships. The Monod equation was developed empirically and written

commonly as
 38

where

|i = specific growth rate of biomass, [T'];

l^max = maximum specific growth rate of biomass, [T'];

S = substrate concentration, [ML^']; and

K, = the half-saturation constant, [ML^ ].

Equation [8] describes the growth of biomass in response to the availability of a substrate.

Mathematically, the Monod equation describes a hyperbolic function with ^ approaching

l^niax asymptotically as S increases. However, some researchers have pointed out that the

equation ignores the existence of the lag phase which is the period of time before

biodegradation is observed to commence. In other words, the Monod equation presumes

that there is no biodegradation for the initial lag period and it sets time "zero" at the end of

the lag period (Kelly et al., 1996).

Substituting for ^ inequation [7], by the Monod relationship, a differential equation

is obtained which describes the utilization of a single limiting substrate and the resulting

microbial growth by a pure culture of microorganism suspended in a liquid at a constant

temperature in a batch system;

dt Y

In the above equation, the rate of substrate utilization increases with the substrate
 39

concentration, S, but as S grows large, the rate asymptotically approachesa maximum value

given by Thus, the fastest possible substrate utilization rate depends on the

biomass concentration, the maximum utilization rate, and the yield coefficient. The

asymptotic approach to the maximum rate is controlled by the relative values of S and K,.

The above equation is very useful in solute transport because the dS/dt term can be

substituted into the mass loss term caused by biodegradation in advection-dispersion

transport equation.

The Monod equation can be rearranged by taking the reciprocal of both sides to give

a linear formula

1 , ,1 I
-=( )-* [10]

which predicts that a plot of l/^ versus 1/S yields a straight line. In practical terms this

means that and K, can be obtained from the ordinate intercept (l/|imax) the slope of

the regression line respectively.

Half-Saturation Constant

The half-saturation constant (K,), sometimes called the "affinity constant", is an

important biodegradation kinetic parameter. It defines the substrate concentration at which

the population grows at half the maximum specific growth rate, i.e., the value of S when (i.

is equal to (Alexander, 1994). Thus, it has the same units as substrate concentration.
 40

It is an index for the afSnity of the microbial population to the substrate; that is, the lower

the value of K,, the greater the microorganism's affinity for the specific-substrate and the

greater the capacity for microorganisms to grow rapidly in an environment with low growth-

limiting substrate concentrations (Lynch and Poole, 1979). Alexander (1994) summarized

that the K, values will extend over a wide range at different circumstances. For a single

bacterium, the K, value varies with different substrates. For example, Russell and Baldwin

(1979) reported that the affinity for glucose was much higher in B. ruminicola than affinities

for maltose, sucrose, and cellobiose. In addition, the K, value differs with the bacterial

species for a single substrate. For example, 8. fibrisolvens had a 500-fold greater affinity

for glucose than did S. bovis (Russell and Baldwin, 1979). It is then not surprising that there

are different K, values for a single substrate in pure culture and in microbial communities.

For example, the reported Kj value of glucose was 0.0071 mg L' (van der Kooij and Hijnen,

1981) for a pure culture and 26 mg L ' for river water (Larson, 1980). Moreover, the K,

value may depend upon the substrate concentration for one single microorganism. For

example, Ishida et al. (1982) reported that there were two different K, values, 29 and 1314

mg L*', for glucose at low and high concentrations, respectively.

Modified Monod Equation

Alexander(198S) proposed two categories of Monod kinetics: Monod-with-growth

and Monod-no-growth. The basic assumption used in the aforementioned models is a mass

balance of carbon. Equations [5]~[10] ignore bacterial decay and assume that the
 41

disappearance of substrate is linked directly to an increase in the number of cells (Simkins

and Alexander, 1984).

In the Monod-with-growth category, a relatively small biodegrading population is

present initially and the substrate concentration is above any threshold that might exist.

Under these conditions, the bacteria will grow but at a rate that falls constantly with the

diminishing and always limiting substrate concentration. Simkins and Alexander (1984)

used equation [9] to represent the governing equation for the Monod-with-growth category,

which can be transformed to one of two linear approximations based on the relative values

of S and K,. When S is much less than K, (S«K,) and biomass is under steady-state

conditions, equation [9] can be reduced to a first-order equation as

[11]

and its integrated form as

In5=-Ar ,f+In5„ [12]

where k, is the first-order rate constant [T']. The value of k, can be obtained by finding the

slope of the best fit line, on a plot of the natural log of substrate concentration versus time.

When S is much greater than K, (S»K,), equation [9] is reduced to a zero-order equation

as
 42

[13]

and its integrated form as

S=-kj^S^ [14]

wliere is the zero-order rate constant [T']. Again, the value of can be obtained by

finding the slope of the best fit line on a plot of substrate concentration versus time.

In the Monod-no-growth category, the initial bacterial concentration is high and

bacterial growth does not influence the biodegradation reaction rate. That is, there is no

appreciable increase in cell number either because there is a threshold or because the initial

cell number is too large. Thus, equation [9] can be converted to the following form

where is the initial bacterial cell concentration which is equal to S-S^+B; S„ is the initial

substrate concentration (expressed as the carbon concentration). Similar to the Monod-with-

growth category, the governing equation for Monod-no-growth category can be converted

to logarithmic or logistic kinetic models depending upon whether S»K5 or S«K5,

respectively. The only difference is that cell density (B) is a constant in the Monod-no-

growth case, instead of a variable as in the Monod-with-growth case. Moreover, Alexander

 43

(1994) summarized these six models (Monod-no-growth, zero-order, first-order, Monod-

with-growth, logarithmic, and logistic) in detail and presented their characteristic curves of

substrate disappearance.

Limitations and Applications of Monod Kinetic Model

Because the Monod kinetic model was derived from a well-controlled batch system

(e.g. single substrate, pure culture), it is not surprising that there are some obstacles in

applying it to complex systems. The Monod equation may not be appropriate for substrates

that are sorbed, insoluble, or toxic (Alexander, 1994). For instance, Scow et al. (1986)

reported that in their experiments the Monod equation could not adequately describe the

kinetics of mineralization of lowconcentration organic compounds added to soil,. Koch and

Wang (1982) indicated that the Monod equation was not suitable to describe the bacterial

growth for a soluble substrate, in pure culture, under their experimental design conditions.

The Monod equation has, however, been modified to incorporate the effect of cunent

substrate utilization or competitive inhibition in a batch system (Yoon et al., 1977;

Papanastasiou and Maier, 1982) and in a continuous flow, stirred tank reactor (CSTR)

(Papanastasiou and Maier, 1983). In addition, the Monod equation was further developed

to include the effect of endogenous decay of cells (Kledka and Maier, 1988). Thus far, it

has been established that Monod kinetic models have been successfully applied to mixed

cultures (Alexander and Scow, 1989), continuous-flow bioreactor systems (Gaudy and

Gaudy, 1980), and activated sludge reactors (Shamat and Maier, 1980). The application of
 44

Monod kinetics has also been extended to solute transport models to predict the mass loss

of contaminants (substrates) via biodegradation in the subsurface environment (Ardakani

etal., 1973; Sykesetal., 1982; Corapcioglu and Haridas, 1985; Borden and Bedient, 1986;

Molz et al., 1986; Bouwer and Cobb, 1987; Srinivasan and Mercer, 1988; Bosma et al.,

1988; Kindred and Celia, 1989; MacQuarrie etal., 1990; Chen etal., 1992). Althoughmany

solute transport models incorporating Monod-type biotransformation have been developed,

only a limited number of models have been tested and validated against results of

experiments (e.g., Estrella et al., 1993; Maier et al., 1996; Hu and Brusseau, 1998).

Brusseau et al. (1992) and Xie (1996) reviewed most of these coupled-process transport

models. Furthermore, the effects of and K, on solute transport in soil columns were

recently examined systematically (Brusseau et al., 1999a; 1999b).

 45

Microbial Succession

Many recalcitrant contaminants may undergo in-situ biodegradation processes

(Madsen, 1991;Hincheeetal,, 1994). The rate ofbiodegradationofa contaminant depends

on many biotic factors such as availability of microorganisms, microbial populations, and

microbial diversities, and abiotic factors such as contaminant concentration and structure,

electron acceptor availability, nutrient availability and concentration, sorption, temperature,

pH, salinity, pressure, and water activity (Ghiorse and Wilson, 1988; Leahy and Colwell,

1990). However, the most important step in the initiation of biodegradation in-situ is the

presence of specific microorganisms which have the ability to degrade the target

compounds. The "ability" can be obtained by exposure of soil microcosms to specific

compounds for a period of time (Spain et al., 1980). This process is referred to as

adaptation. Adaptation may occur by inducing or derepressing specific enzymes, required

to degrade specific compounds, that are not normally present or are present at low levels

(Aelion et al., 1987). Once the microorganism synthesizes these catabolic enzymes,

degradation of compounds can happen more quickly.

The microbial communities in soil consist of many species that can degrade different

kinds of pollutants (Paul and Clark, 1989). Adaptation processes can stimulate one species

to become dominant and control the progression of biodegradation. However, adaptation

is dependent upon time, the species present, and degradability of the contaminants.

Adaptation may proceed slowly. As time progresses, other species may also adapt and

develop a competitive advantage over the previous dominant degrading species (Fries et al..
 46

1997). In other words, it is possible that more than one species can adapt in response to

particular substrate at the same time or at different times. The shift in the dominant

microbial populations is referred to as microbial succession.

Microbial population succession can be caused by gene transfer as well as enzyme

induction. Intrinsic biodegradation requires the indigenous soil community to possess the

appropriate degradative genes. Those specific genes, encoding partial or complete

degradative pathways for a particular chemical, may be found on both chromosomal and

plasmid DNA (Sayler, 1991). They can be present within many members of the microbial

community and, moreover, may be transferred to other cells (Sayler, 1991). For example,

Neilson et al. (1994) demonstrated the transfer of plasmid pJP4 from Alcaligenes eutrophus

JMP134 to an introduced Variovoraxparadoxus strain in a well-controlled system. Gene

transfer has also been demonstrated in soil microcosms (Stotzky, 1989). For instance,

plasmid pJP4 which contains the genes for the degradation of 2,4-dichlorophenoxyacetic

acid (2,4-D) has been successfully transferred to indigenous soil microorganisms (recipients)

(DiGiovaimi et al., 1996). After gene transfer, not only did the populations of indigenous

recipients or transconjugants increase but the rates of 2,4-D degradation were enhanced.

Bioattenuation (or intrinsic biodegradation) is one of a number of proposed

bioremediation techniques. So far, most of our understanding of biodegradation is from

studies of single species in the laboratory. This does not always translate well to natural

environments because in-situ bioattenuation involves the biodegradation of contaminants

by multiple species. Because there are multiple species present in soil, it is likely that
 A1
microbial succession plays an important role in developing the capacity for biodegradation.

However, there remains a lack of information about the influence of microbial succession

on biodegradation and transport of contaminants ui soil.

 48

CHAPTERS: PRESENT STUDY

The methods, results, and conclusions of this study are presented in the appendices

attached to this dissertation. In general, most of the research combines methodologies in the

area of environmental microbiology with methodologies in contaminant transport (miscible

displacement techniques). The following is a sununary of the most important findings in

the dissertation, which contribute to the present understanding of the effect of cyclodextrins

on biodegradationof PAHs in aqueous system and the dynamics of heterogeneous microbial

populations on biodegradation during contaminant transport in soil.

Summary

The objective of the first study (Appendix A) of this dissertation is to investigate the

influence of hydroxypropyl-P-cyclodextrin (HPCD) on the biodegradation of phenanthrene

in a batch aqueous system. HPCD can increase the apparent solubility of phenanthrene by

forming inclusion complexes. Because one of the hypotheses in this study was that the

bioavailability of phenanthrene was constrained by the extremely low water solubility, the

implementation of HPCD was used to increase bioavailability. The biodegradation of

phenanthrene wasevaluated by the substrate disappearance and by the total cell counts. The

mass loss of phenanthrene was quantified by high performance liquid chromatography

(HPLC), which was used to determine the substrate utilization rate. The cell counts were

used to examine the cell growth in the system.

 49

Solubility studies confirmed that HPCD can significantly increase the apparent

solubility of phenanthrene. For 10^ mg L ' HPCD, solubility was increased 124 times

compared to the measured solubility of phenanthrene (1.3 mg L'). From the result of viable

counts, it indicated that HPCD does not inhibit cell growth during biodegradation of

phenanthrene and HPCD is not used as a carbon or energy source by this particular

phenanthrene degrader, CRE7. The bioavailability of phenanthrene in the aqueous solution

was directly correlated to HPCD concentration. Biodegradation rate and extent of

phenanthrene was determined by the substrate disappearance. The results clearly indicated

that HPCD can significantly enhance the rate and extent of phenanthrene biodegradation.

The substrate utilization rate for samples with 10^ mg L ' HPCD was 5.5 times larger than

for 0 mg L ' HPCD. A longer lag phase suggests that either the phenanthrene degraders

required some time to adapt to the presence of HPCD or they required time to adapt to the

high bioavailable levels of phenanthrene. The effect of inoculum density on phenanthrene

degradation in the presence of HPCD wasalso evaluated. The results showed that inoculum

density only affects the lag phase; it has no effect on the extent and rate of phenanthrene

disappearance. In addition, the impact of technical-grade HPCD on the biodegradation of

phenanthrene was examined. The results showed that the biodegradation rate and extent of

phenanathrene are influenced by the predominant impurity (propylene glycol, 3.2%, w/w)

in technical-grade HPCD. However, the biodegradation rate and extent for samples with

technical-grade HPCD are still higher than the samples without HPCD.
 50

When modeling biodegradation processes, it is usually assumed, either implicitly or

explicitly, that the microbial population responsible for biodegradation is composed of a

single species. However, this is unlikely to be true for many, if not most, field situations.

The effect of multiple species of degraders on phenanthrene biodegradation and transport

in a saturated soil was evaluated in the second study (Appendix B) presented in this

dissertation.

Miscible displacement experiments were conducted using a soil with a high

phenanthrene sorption capacity and with an indigenous microbial population. The

hydrodynamic properties of the column and the sorption behavior of the soil were

characterized by simulating the breakthrough curves of a nonsorbing, nonreactive solute

(e.g., PFBA) and a sorbing solute (e.g., phenanthrene), respectively, obtained in a sterilized

column. The simulations were performed by a non-linear, least-squares optimization

program (CFITIM3) with the local equilibrium assimiption (LEA)-based and

nonequilibrium-based transport model.

The breakthrough curve obtained for the non-sterile column experiment exhibited

oscillating concentrations during 6 months of continuous injection of a constant-

concentration phenanthrene solution. The evidence for biodegradation in the non-sterile soil

column included the mass loss of phenanthrene in the efHuent, the dissolved oxygen in the

effluent, the temporal distribution of phenanthrene degraders in the efSuent, and the spatial

distribution of phenanthrene degraders in the soil profile. At the end of experiment, 24

species of phenanthrene degraders, either in the effluent or in the soil, were identified with
 51

cultural and 16S rDNA polymerase chain reaction (PCR) analysis. The overall transport

behavior is due to the response of the heterogeneous bacterial population to substrate and

oxygen availability, wherein population dynamics is hypothesized to be mediated by

competition, microbial succession, and other multi-species interactions. The dynamics of

heterogeneous microbial populations, especially under growth conditions, should be

considered when evaluating contaminant biodegradation and transport in natural subsurface

systems.

In addition, several supplemental experiments are presented in Appendix C. In the

first subdivision of Appendix C, the inherent biodegradation potential of benzoate in Hill

AFB site's groundwater and aquifer materials was evaluated. The results suggested that two

different groups of benzoate degraders exist in the solid phase (aquifer material) and the

aqueous phase (groundwater). The degraders present in the aquifer material exhibited a

longer lag phase, higher biodegradation capacity, and higher bacterial activity. One possible

explanation for this phenomenon is related to the bacterial characteristics. The other

possibility is that there were some sorbed contaminants initially present in the aquifer

material that delayed the initiation of benzoate biodegradation. In addition, the other sorbed

contaminants may have served as carbon sources to facilitate the biodegradation of

benzoate. A biodegradation study was performed to evaluate the bioavailability of benzoate

in aerobic and anaerobic conditions. It showed that benzoate can be biodegraded under both

conditions. However, aerobic biodegradation was more efficient than anaerobic

biodegradation. A nutrient study was conducted to compare the biodegradation potential

 52

of benzoate in mineral salt media,synthetic groundwater, and real groundwater. The results

showed that the rate and extent of biodegradationare controlled by the presence of nutrients.

In the second subdivision of Appendix C, the bioavailability of benzoate in the

aquifer of AFP 44 site was evaluated. The results suggested that the benzoate degraders

existing in the aquifer material have the same characteristics as those present in

groundwater. However, the biodegradation capacity and bacterial activity of benzoate

degraders at the AFP 44 were lower than those at the Hill site. One possible explanation for

this phenomenon is related to the residual TCE in AFP 44's aquifer material and

groundwater, which may inhibit the activity of benzoate degraders.

In the third subdivision of Appendix C, a series of batchexperiments was performed,

not only to evaluate the biodegradation behavior of Na-benzoate and 2,4-D, but also to

measure their biodegradation kinetic parameters, including half-saturated constant (K,),

maximum specific growth rate, (//„„), and biomass yield coefficient (y). The results

showed that benzoate has a shorter lag phase than 2,4-D. The Y value of benzoate (0.65)

was higher than that of 2,4-D (0.29). It suggested that the chlorinated substrate (e.g., 2,4-D)

has a lower cell yield than the non-chlorinated substrate (e.g., benzoate). In other words,

benzoate was degraded more efficiently than 2,4-D. However, a lower K, and a higher

were observed for 2,4-D. This implied that 2,4-D degraders in an amended soil had higher

affinity for substrate (2,4-D).

In the fourth subdivision of Appendix C, a biodegradation study was conducted to

investigate the bioavailability of pyrene (4-rtng PAHs) in the presence of HPCD. At the end
 53

of experimental period (24 weeks), 14% (w/w) of the pyrene wasdegraded by Burkholderia

CRE7 in the presence of lO"* mg L"' HPCD. The degradation rate was equal to 0.015 mg of

pyrene per week. Although a long lag period was required for Burkholderia CRE7 to adapt

pyrene, the results suggested that HPCD has the potential to enhance the biodegradation of

pyrene.

In the last subdivision of Appendix C, the adherence of bacterialcells to hexadecane

was examined. The mass distribution of phenanthrene between the aqueous phase and

hexadecane was also measured. The effect of hexadecane volume on the distribution of

phenanthrene wasevaluated. From the results of adherence of bacterial cells to hexadecane,

it showed that hexadecane is not taken up by the cell. The cells, CRE7, still used

phenanthrene as the major carbon or energy source. In the partition experiment, it showed

that the partitioning ratio of phenanthrene increases with increasing the volume of

hexadecane for the systems with same amount of phenanthrene but different volumes of

hexadecane. Conversely, the partitioning ratio of phenanthrene increased with increasing

amounts of phenanthrene for the systems with same volume of hexadecane but different

amounts of phenanthrene. This suggests that the amount of phenanthrene and the volume

of hexadecane are the two factors determining the partitioning ratio of phenanthrene in the

system.
 54

APPENDIX A:

CYCLODEXTRIN - ENHANCED BIODEGRADATION OF PHENANTHRENE

Jiann-Ming Wang, Elizabeth M. Marlowe, Raina M. Miller-Maier, and

Mark L. Brusseau*

429 Shantz Bldg.

Dept. of Soil, Water, and Environmental Science

The University of Arizona

Tucson, AZ 85721

Published in:

Environmental Science and Technology

Volume 32 (13), pl907-1912, 1998

* Corresponding author
 55

Abstract

The effectiveness of in-situ bioremediation in many systems may be constrained by

low contaminant bioavailability due to limited aqueous solubility or a large magnitude of

sorption. The objective of this research was to evaluate the effect of hydroxypropyUp-

cyclodextrin (HPCD) on phenanthrene solubilization and biodegradation. Results showed

that analytical-grade HPCD can significantly increase the apparent solubility of

phenanthrene. The increase in apparent solubility had a major impact on the biodegradation

rate of phenanthrene. For example, in the presence of 10' mg L ' HPCD, the substrate

utilization rate increased from 0.17 mg hr' to 0.93 mg hr' while the apparent solubility was

increased fi:om 1.3 mg L"' to 161.3 mg L"'. As a result, only 0.3% of the phenanthrene

remained at the end of a 48-hour incubation for the highest concentration of HPCD tested

(10' mg L*'). In contrast, 45.2% of the phenanthrene remained in the absence of HPCD.

Technical-grade HPCD, which contains the biodegradable impurity propylene glycol, also

increased the substrate utilization rate, although to a lesser extent than the analytical-grade

HPCD. Based on these results, it appears that HPCD can significantly increase the

bioavailability, and thereby enhance the biodegradation, of phenanthrene.

 56

Introduction

There is tremendous interest in using in-situ bioremediation for the clean up of

contaminated soil and groundwater. However, biodegradation rates in the subsurface are

often constrained by a limited oxygen supply and by factors related to bioavailability, such

as solubility, dissolution rate, and sorption {1-4). Recent research has examined the

possibility of enhancing the bioavailability of low solubility and highly sorptive compounds

by adding a "solubilization" agent, such as a surfactant, to the system We have been

investigating cyclodextrin as an alternative agent for enhancing solubilization {14).

Cyclodextrins are cyclic, nonreducing maltooligosaccharides produced from the

enzymatic degradation of starch and related compounds by certain bacteria that contain the

cyclodextrin glycosyltransferases{IS). The most pertinent property of cyclodextrins is that

they have a hydrophilic shell and a toroidal-shaped, apolar (hydrophobic) cavity. Thus,

cyclodextrins have the ability to form water-soluble inclusion complexes by incorporating

suitably sized low polarity molecules in their cavities. Cyclodextrins are widely used in

pharmaceutical, food, and cosmetic industries {16). Through research for these applications,

it has been shown that cyclodextrins can aid the microbial transformation of water-soluble

compounds such as vanillin (/ 7), and lowsolubility compounds such as cholesterol {18) and

steroids {19).

Recently, cyclodextrins have been used in environmental applications to improve

the remediation of contaminated soil and groundwater. For example, it has been

demonstrated that cyclodextrins have the ability to increase the apparent water solubilities
 57

of low polarity organic compounds such as trichloroethene, naphthalene, anthracene,

chlorobenzene, and DDT (20). They can also reduce the sorption and facilitate the transport

of these compounds through soil{21). A recent field study demonstrated that a cyclodextrin

solution removed significant amount of multicomponent, immiscible-organic liquid

contamination firom an aquifer (22). It has been shown that ^-cyclodextrin can decrease the

microbial toxicityof some pesticides and aromatics for waste-water treatment and bioreactor

applications (23,24). The bioavailability and biodegradation of such compounds in the

presence of cyclodextrins has not yet been investigated for in-situ environmental

applications. The objective of this study was to evaluate the influence of hydroxypropyl-P-

cyclodextrin (HPCD) on the biodegradation of a selected nonionic, low-polarity organic

chemical (phenanthrene).

Materials and Methods

Materials

Analytical-grade hydroxypropyUP-cyclodextrin (HPCD) (purity >99%) and

technical-grade HPCD (with 3,2%(w/w) propylene glycol) were supplied by Cerestar USA,

Inc. (Hammond, IN). Phenanthrene (purity >98%) was purchased from Aldrich Chemical

Co. (Milwaukee, WI). [9-'''C]Phenanthrene (specific activity, 13.3 mCi/mmol, reported

purity >98%) was purchased from Sigma Chemical Co. (St. Louis, MO). The actual purity

of [9-'''C]phenanthrene, determined by HPLC, was 96.3%. The mineral salts medium

(MSM), specifically Bushnell-HAAS (Difco Laboratories, Detroit, MI), consisted of 0.2 g

 58

MgS04,0.02 g CaClj, 1 g KH2PO4,1 g (NH4)2(HP04), I g KNO3, and 0.05 g FeClj per liter

of distilled water. Agar (Bacto-Agar) and RjA agar employed in bacterial cultivation, were

also purchased from Difco Laboratories (Detroit, MI). Propylene glycol was purchased

from Aldrich Chemical Company, Inc. (Milwaukee, WI). ScintiVerse BD scintillation

cocktail was purchased from Fisher Scientific Chemical (Fair Lawn, NJ). Chloroform, used

to dissolve or extract phenanthrene, was purchased from Mallinckrodt Baker, Inc. (Paris,

KY). Methanol (HPLC-grade), used to dilute the extracts of phenanthrene, was purchased

from J.T. Baker, a division of Mallinckrodt Baker Inc. (Phillipsburg, NJ). Acetonitrile UV,

employed as a carrier for HPLC analysis, was purchased from Burdick & Jackson Inc.

(Muskegon, MI).

Solubilitv of Phenanthrene

The solubility of phenanthrene in HPCD solutions was determined by use of '"'C-

labelled phenanthrene. Two ml of a mixture of phenanthrene and '''C[phenanthrene] in

chloroform was added to 250-ml flasks and allowed to evaporate, which produced a 12.5-

mg coating of phenanthrene (specific activity, 0.082 mCi/mmol) on the bottom of the flasks.

A 25-ml aliquot of aqueous HPCD solution was then added to each flask. Samples were

prepared in triplicate for 7 HPCD concentrations (including zero) and placed on an orbit

shaker (Lab-Line Instruments, Inc., Model 3527, Melrose Park, IL) at 200 rpm for three

days at room temperature. A 50-^1aliquot of each sample was added to 5 ml of ScintiVerse

BD and the radioactivity was determined using a liquid scintillation counter (Packard Tri-

Carb Co., Model 1600 TR, Meriden, CT).

 59

Phenanthrene-Degrading Isolate

A phenanthrene-degrading isolate was obtained from an estuary sediment sample.

In the enrichment procedure, 10 g of estuary sediment and 50 ml of MSM were added to

flasks coated with phenanthrene. The enrichment culture was shaken on an orbit shaker at

200 rpm at room temperature for one month, and then was transferred to fresh flasks coated

with phenanthrene and containing Bushnell-HAAS MSM. The culture was incubated for

2 weeks. After several such transfers, the incubation period was decreased gradually from

2 weeks to 3 days until a single phenanthrene degrading isolate was obtained. The isolate

was maintained on Bushnell-HAAS agar plates spray-coated with phenanthrene.

The phenanthrene degrader used in this study was characterized as a gram negative

rod, which carries a plasmid of approximately 83kb. The isolate was identified by 16S

rDNA using polymerase chain reaction. Polymerase chain reaction with the primers

forward 5' AGA GTT TGA TCC TGG CTC AG 3' and reverse 5* ACG GTT ACC TTG

TTA CGA CTT 3* for Eubacterial 16S rDNA (synthesized by IDT, Inc., Coralville, IW)

were used to amplify a 1.5 kb fragment from isolate DNA. Reaction conditions were as

follows: Primer mix of forward and reverse I pM, 200^M dNTP's (Pharmacia Biotec,

Uppasala, Sweden), IX Pfii buffer, 2.5 U of Pfii DNA polymerase enzyme (Stratagene Inc.,

La Jolla, CA), and 10-50 ng of template DNA. Cycling was done in the GenAmp PCR

System 9600 (Perkin Elmer Cetus Corp., Norwalk, CT). One cycle of 95°C for 1 minute

denaturation, followed by 30 cycles of 94''C 15 seconds denaturation, 55''C 15 seconds

annealing, ITC 30 seconds extension, finishing with a 72°C 2 minutes final extension.
 60

The PGR products were then cloned and sequenced. Either the Invitrogen pCR™

2.1 (Invitrogen, San Diego, CA) or Stratagene pCR-Script™ SK(+) (Stratagene, La Jolla

CA) vector was used to clone the 1.5 kb ribosomal sequence following manufacturers'

directions. Selection of white colonies from LB, IPTG, X-gal, and ampicillan plates were

used to grow 25 ml cultures in SOC. Plasmids were extracted using the QiaFilter midiprep

system (Qiagen Inc., Chatsworth, CA). Sequencing was performed using the ABI 377

automated sequencer (LSME, Tucson, AZ). Whole, 1.5 kb sequences were aligned and

assembled on GCG (Genetics Computer Group Inc., Madison, WI). FASTA was used to

search GenEMBL for homology. This procedure resulted in two possible isolate identities.

Burkholderia sp. isolate CRE 7 (Accession #U37340) showed 95.4% homology, and

Burkholderia cepacia (Accession #X87275) showed 94.6% homology, to the 16S rDNA

sequence of the phenanthrene degrader.

Biodegradation Studies

The biodegradation of phenanthrene was quantified in two ways: (1) direct

measurement of phenanthrene loss, used to determine substrate utilization, and (2) cell

counts, used to evaluate cell growth. For the substrate utilization experiments, which were

conducted in triplicate, 12.5 mg of phenanthrene were dissolved in chloroform and added

to 250-mL flasks. The solvent was allowed to evaporate overnight, leaving a thin coating

of phenanthrene covering the sides and bottom of the flasks. Purified and analytical-grade

HPCD in Bushnell-HAAS broth (25 ml) were added in various concentrations. One set of
 61

samples was inoculated with 0.5 ml aliquots of the phenanthrene degraders from late-log

precultures to achieve a final cell density of approximately 10^ CFU ml''. A second set of

samples was inoculated with diluted late-log precultures of phenanthrene degraders to a

final cell density of 10'* CFU ml"'. Samples were incubated at 200 rpm on a gyratory shaker

at room temperature.

Periodically, a set of triplicate samples was sacrificed and the contents of each flask

were serially extracted three times with 50 ml of chloroform. Samples from the first 2 days

had higher levels of phenanthrene. Therefore, the first extract was diluted 100-fold with

HPLC-grade methanol. The second and third extracts were concentrated with a Rotavapor

Evaporation System (Biichi Co., Switzerland) to 1 ml, after which the volume was adjusted

to 5 ml with HPLC-grade methanol. The amount of phenanthrene in both the first extract

and in the combined second and third extracts was quantified by high performance liquid

chromatography (Waters, Milford, MA). The total remaining phenanthrene was the sum of

these determinations. For samples sacrificed after 2 days, all extracts were combined for

analysis as described above.

HPLC analysis was performed isocratically using a mobile phase of 5% water

(HPLC-grade) and 95% acetonitrile UV (HPLC-grade) and a reverse phase column,

Adsorbosphere UHS C18 5U (Alltech, Deerfield, IL), with LD. 4.6 mm and length 150 mm.

The flow rate was 1 ml/min and the wavelength used for detection of phenanthrene was 254

nm.
 62

Cell growth was measured by viable plate counts on R2A agar. The culture was

serially diluted and triplicate plates were inoculated at each dilution ratio. Phenanthrene

degraders were enumerated after incubation for two days at room temperature.

Cell growth on propylene glycol, which is a major impurity in technical-grade

HPCD, was determined by protein content analysis based on the Lowry method (25). In this

experiment, flasks containing propylene glycol as the only carbon source were inoculated

with 10' CFU ml*' late-log precultures of phenanthrene degraders. The concentrations of

propylene glycol were 160 and 3200 mg L"', which were equivalent to the amount that

would be present in 5000 and 10^ mg L ' technical-grade HPCD, respectively. Analysis of

protein content was carried out by UV-Visible spectrophotometry (Hitachi, Model U-2000)

at a wavelength of 650 nm.

 63

Results and Discussion

Solubility

The biodegradation of PAHs is limited by their low bioavailability resulting from

extremely low water solubility and high sorption. It is hypothesized that cyclodextrins can

increase the apparent solubility of phenanthrene by forming inclusion complexes, thereby

enhancing the bioavailability of phenanthrene. Apparent solubility is defmed as the amount

of phenanthrene in solution in the presence of HPCD. This hypothesis is supported by

results from this study, which showed a linear relationship between the apparent solubility

of phenanthrene in HPCD solution and the concentration of HPCD from 0 to 10^ mg L*'

(Figure 1).

These results indicate that HPCD significantly increased the apparent solubility of

phenanthrene. For example, the apparent solubility of phenanthrene in a 10^ mg L ' HPCD

solution was 161.3 mg L ', an increase of 124 times compared to the measured aqueous

solubility of 1.3 mg L"'. The larger apparent solubility means more substrate is directly

available in solution for supporting bacterial activity at equilibrium conditions. In addition

to the magnitude of solubility, the rate of dissolution may also influence the rate at which

bacteria utilize substrate (2,S). All systems essentially approached equilibrium within 30

minutes (including 10^ mg L*' HPCD) in the preliminary study (data not shown).
 64

Biodegradation

Substrate Disappearance

The impact of analytical-grade HPCD on biodegradation of phenanthrene is shown

in Figure 2. Samples containing higher concentrations of HPCD exhibited more rapid

biodegradation of phenanthrene. Thus, more mass of phenanthrene was biodegraded at the

end of the experiment. The amount of phenanthrene remaining was used to evaluate the

extent of biodegradation (Table 1). For example, in the presence of 10^ mg L ' HPCD, the

phenanthrene remaining after two days was 0.3%(w/w) of the initial amount. In contrast,

I2.3%(w/w) and 45.2%(w/w) of the phenanthrene remained after two days in the samples

with 10"' mg L ' HPCD and without HPCD, respectively. At the end of the 7-day

experiment, phenanthrene was completely degraded in the samples with 10^ mg L"' HPCD.

However, 7.2%(w/w) of the phenanthrene remained in the samples without HPCD. These

results clearly indicate that HPCD can significantly enhance the rate of phenanthrene

biodegradation.

Although phenanthrene degradation was more rapid in the presence of HPCD, the

systems with 10^ mg L"' HPCD exhibited a longer lag phase (about 12 hours) than the

systems with lO"* mg L"' HPCD or without HPCD. A longer lag phase suggests that either

the phenanthrene degraders required some time to adapt to the presence of HPCD or they

required time to adapt to the high bioavailable levels of phenanthrene. However, once

adapted, the degraders benefitted from the presence of HPCD as indicated by the increased

growth rate discussed below.

 65

The effect of inoculum density on phenanthrene degradation in the presence of

analytical-grade HPCD is shown in Figure 3. Clearly, the lower inoculum density caused

the lag phase to increase from 0.5 days (10' CFU ml"') to 1.3 days (10^ CPU ml ') for

samples with 10^ mg L ' HPCD. However, the extent and rate of phenanthrene

disappearance were essentially identical for the two inoculum densities.

For control samples that contained phenanthrene and HPCD but no degraders, the

added phenanthrene was completely recovered, even after 7 days. For control samples that

contained phenanthrene and MSM but no degraders, 100% and 97.5% of the added

phenanthrene was recovered after one day and 7 days, respectively. Also, 100% of added

phenanthrene was recovered after chloroform evaporation. The results of these conu-ol tests

confirm that first, HPCD did not have any adverse effect on the efficacy of chloroform

extraction. Second, no mass loss of phenanthrene occuned during overnight evaporation

of chloroform; and third, all mass loss wascaused by biodegradation due to inoculation with

the phenanthrene degraders. This third result was confirmed by the results of preliminary

'^COi evolutionexperiments, which showed that '"'C-phenanthrene was mineralized to '•'CO,

in the presence of the degraders (data not shown).

Cell Growth

The effect of HPCDon phenanthrene biodegradation was confirmed by determining

viable counts as a measure of cell growth (Figure 4). In this system, the lag phase before

significant cell growth occurred was approximately 5 hours, which is sufGcient for
 66

phenanthrene solubilization to reach equilibrium. Therefore, the bioavailability of

phenanthrene in the aqueous solution should be directly correlated to HPCD concentration.

The results presented in Figure 4 also show that HPCD did not inhibit cell growth during

biodegradation of phenanthrene. Finally, the results demonstrate that HPCD was not used

as a carbon or energy source during the experiment. This is consistent with the fact that the

genus Pseudomonas, some species of which were recently reclassified as Burkholderia,

produces very low amounts of amylolytic enzymes, which are required to hydrolyze HPCD

{26).

Substrate Utilization Rate

The substrate utilization rate was calculated for phenanthrene degradation in the

presence and absence of HPCD. This was done by evaluating the slope of the substrate

utilization curves during exponential growth (Figure 2). The calculated substrate utilization

rates, which are the maximum slopes of the curves in Figure 2, are presented in Table 1.

The substrate utilization rate for samples with 10^ mg L"' HPCD was 1.6 times greater than

the utilization rate in the presence of the lO** mg L"' HPCD, and was 5.5 times larger than

for 0 mg L ' HPCD. Because a higher substrate utilization rate requires more bioavailable

substrate, substrate utilization rate should be directly related to the dissolution rate, which

is dependent on HPCD concentration.

For samples with HPCD, there are three phases of phenanthrene (solid, dissolved,

and complexed) when excess phenanthrene is present, and two phases of phenanthrene
 67

(dissolved and complexed) when phenanthrene is not in excess. It is not clear whether

bacteria can biodegrade complexed phenanthrene directly. Addressing this issue requires

knowledge of the dissociation rate of phenanthrene-HPCD complexes. If dissociation is

instantaneous, as is generally assumed, the enhanced rate of phenanthrene biodegradation

in the presence of HPCD could be explained by the biodegradation of only dissolved

phenanthrene. If the dissociation process is not instantaneous, the enhanced rate of

phenanthrene biodegradation could indicate that both dissolved and complexed

phenanthrene are used directly.

Influence of Impurities in HPCD

The use of analytical-grade HPCD for field applications would generally be

prohibitively expensive. It is much more likely that technical-grade forms would be used.

In such cases, it is important to evaluate the potential impact of HPCD impurities on

contaminant fate. Therefore, an experiment was conducted to examine the impact of

propylene glycol, which was the predominant impurity (3.2%(w/w)) in technical-grade

HPCD, on phenanthrene degradation.

The biodegradability of propylene glycol by the phenanthrene degrader used in these

experiments was evaluated by determining protein as a measure of cell growth. Propylene

glycol was added as sole carbon source in amounts equivalent to that found in SOOO and 10^

mg L ' technical-grade HPCD. Results showed that propylene glycol was degraded and

therefore could possibly compete with phenanthrene as a carbon source (data not shown).
 68

The impact of technical-grade HPCD on the biodegradation of phenanthrene is

shown in Figure 5, where in is presented the degradation of phenanthrene in the presence

of both lO"* mg L ' technical-grade and analytical-grade HPCD. An inoculum of 10^ CPU

ml ' was used for these experiments. Phenanthrene degradation was somewhat slower in

the presence of technical-grade HPCD as compared to the analytical-grade. For example,

after two days, 12,3%(w/w) phenanthrene remained insamples with analytical-grade HPCD,

whereas 29.0%(w/w) remained in samples with technical-grade HPCD. Substrate utilization

rates were calculated to be 6.4 mg day' for technical-grade HPCD and 14.4 mg day ' for

analytical-grade HPCD. These results indicate that impurities (e.g., propyleneglycol) in the

technical-grade HPCD competed with phenanthrene as a carbon source. However, the

substrate utilization rate in the presence of technical-grade HPCD was still higher than the

utilization rate in the absence of HPCD, which was 4.1 mg day'. While this might in some

cases be a drawback, e.g., presence of high microbial numbers, it should be noted that in

situations where there are low numbers of microbes present, e.g., a subsurface environment,

the HPCD impurity may help build up a phenanthrene degrading population.

The impact of surfactants, a widely used solubilization agent, on biodegradation of

organic compounds is under investigation. In aqueous systems, some surfactants appear to

enhance biodegradation of phenanthrene, such as monorhamnolipid (13), an anionic

biosurfactant. Conversely, the biodegradation of phenanthrene was inhibited by some

anionic synthetic surfactants, such as SDS (sodium dodecyl sulfate) (9), and by some

nonionic synthetic surfactants, such as Tergitol NP-10 ((J), Triton NlOl (//), Triton XlOO
 69

{6,12), and Brij 30 (6,12). It is difficult, however, to compare these results directly given

the variety of experimental designs and initial/control conditions used.

The results of this study indicate that HPCD can enhance the solubility and

dissolution rate of phenanthrene, which increases its bioavailability for degradation by

bacteria. Our previous research has shown that HPCD can also enhance the desorption and

dissolution of similar compounds (27, 22). Thus, cyclodextrins hold promise for use as an

agent to increase the performance of in-situ bioremediation systems.

Acknowledgements

This research was supported by grants provided by the U.S. Department of Energy

Subsurface Science Program and the National Institute of Environmental Health Sciences

Superfund Basic Sciences Research Program. We thank Yimin Zhang and Xiaojiang Wang

for their assistance. We also thank Eileen M. Jutras for identifying the phenanthrene

degrader.
 70

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 72

Table 1; Substrate Utilization Rate and Remaining Amount of Phenanthrene

HPCD Substrate Phenanthrene Remaining (%)

conc. Utilization
(mg/L) Rate
(mg/day) (after 2 days) (after 5 days) (after 7 days)

10' 22.35 0.3 O.l 0

10^ 14.35 12.3 0.9 0.1
0 4.07 45.2 13.2 7.2
 73

Figure Captions

Figure I. The relationship between the apparent solubility of phenanthrene and the

concentration of hydroxypropyl-p-cyclodextrin (HPCD).

Figure 2. Effect of HPCD (analytical-grade) on phenanthrene disappearance with

time. (Inoculum density was 10' cfu/ml.)

Figure 3. Effect of inoculum density on biodegradation of phenanthrene in the

presence of analytical-grade HPCD.

Figure 4. The effect of analytical-grade HPCD on phenanthrene biodegradation as

measured by viable counts. (Inoculum density was 10^ cfli/ml.)

Figure 5. Comparison of the impact of technical-grade HPCD (lO** mg L*') and

analytical-grade HPCD (10"* mg L"') on the biodegradation of phenanthrene.

(Inoculum density was 10' cfti/ml.)

180

160

140

120

100 y=1.6211x+1.3182
(R^=0.9962)

0 20 40 60 80 100 120

HPCD Concentration (g/L)

Figure 1. The Relationship between the Apparent Solubility of Phenanthrene and

the Concentration of HPCD
~-j
 •A-
• '

10 mg/LHPCD
lO'* mg/L HPCD
-A- 0mg/LHPCD
~0~ Control for 10^ mg/L HPCD
"B— Control for 10^ mg/L HPCD
-A— Control for 0 mg/L HPCD

± I I I I-

0 3 4 5 8

Time (days)

Figure 2. Effect of Analytical-grade HPCD on Phenanthrene

Disappearance with Time
110

100,

^ 10 cfii/ml, 10 mg/LHPCD
80 10^cfWml, lO'*mg/LHPCD
O- lO^clWinl, lO^mg/LHPCD
^ lO^cfii/ml, lO'*mg/LHPCD

20

10 t
0 1 2 3 4 5 6 7 8

Time (days)

Figure 3. Effect of Inoculum Density on Biodegradationof Phenanthrene

in the Presence of Analytical-grade HPCD
 le+9

S le+8
500 mg/L phen., 10 mg/LHPCD
500 mg/L phen., 5000 mg/L HPCD
500 mg/L phen., 0 mg/L HPCD
S— 0 mg/L phen., 10^ mg/L HPCD
0 mg/L phen., 5000 mg/L HPCD

le+7 -

0 10 20 30 40 50 60

Time (hrs)

Figure 4. The Effect of Analytical-grade HPCD on Phenanthrene

Biodegradation as Measured by Viable Counts
-J
110

100|

90 Analytical-grade HPCD
Technical-grade HPCD
80

70

60

50

40

30

20

10

0
00 0.5 1.0 1.5 2.0

Time (days)
Figure 5. Comparison of the Impact of Technical and Analytical-grade
HPCD on the Biodegradation of Phenanthrene
 79

APPENDIX B:

THE INFLUENCE OF BACTERUL POPULATION HETEROGENEITY AND

DYNAMICS ON BIODEGRADATION AND TRANSPORT OF

PHENANTHRENE IN SOIL

Jiann-Ming Wang, Adria Bodour, Raina M. Maier, and Mark L. Brusseau*

429 Shantz Bldg.

Dept. of Soil, Water, and Environmental Science

The University of Arizona

Tucson, AZ 85721

Prepared for:

Environmental Science and Technology

* Corresponding author
 80

Abstract

Biodegradation is often of great importance for the transport, fate, and remediation

of organic contaminants in the subsurface. When modeling biodegradation processes, it is

usually assumed,either implicitly or explicitly, that the microbial population responsible for

biodegradation is comprised of a single species. However, this is unlikely to be true for

many, if not most, field situations. The prime objective of this research is to investigate the

influence of bacterial population heterogeneity, (i.e., multiple species of degraders) on

biodegradation and transport of phenanthrene in a saturated soil. Miscible displacement

experiments are conducted with a soil whose indigenous microbial population consists of

24 species capable of degrading phenanthrene. The breakthrough curve obtained for the

non-sterile column experiment exhibits oscillating concentrations during the 6 months of

continuous injection of a constant-concentration phenanthrene solution. This behavior is

due to the response of the heterogeneous bacterial population to substrate and oxygen

availability, wherein population dynamics is hypothesized to be mediated by competition,

microbial succession, and other multi-species interactions. The dynamics of heterogeneous

microbial populations, especially under growth conditions, should be considered when

evaluating contaminant biodegradation and transport in natural subsurface systems.

 81

Introduction

Biodegradation is an important mechanism in the study of the transport, fate, and

remediation of organic contaminants in the subsurface. When modeling biodegradation

processes, the biodegradation term is usually represented with the first-order or the Monod

equation (Ardakani et al., 1973; Sykes et al., 1982; Mironenko and Pachepsky, 1984;

Corapcioglu and Haridas, 1985; Borden and Bedient, 1986; Molz et al., 1986; Bouwer and

Cobb, 1987; Srinivasan and Mercer, 1988; Bosmaetai., 1988; Lassey, 1988; Kindred and

Celia, 1989; MacQuarrie et al., 1990; Chen et al., 1992; Brusseau et al. 1999a). No matter

which models are applied, an average specific growth rate is used to represent what is in

actuality growth due to multiple species in the soil (e.g., Angley et al., 1992; Estrella et al..

1993; Kelly et al. 1996; Maier et al., 1996; Reddy and Ford, 1996; Hu and Brusseau, 1998;

Langner et al., 1998; Brusseau et al. 1999b). In other words, it is assumed that either the

microbial population responsible for biodegradation is comprised of a single species, or that

the multiple species present will exhibit uniform composite behavior.

Although many biodegradation experiments have been conducted using soil

columns, the majority have been focused on either biotransformation or transport behavior

of the contaminants and have not involved specific examination of microbial dynamics.

Most investigations have been based on the use of a small input pulse, a well-identified

species, or a readily degradable substrate. However, this is unlikely to be true for many, if

not most, field situations. In the field, multiple species of bacterial populations normally
 82

exist in the soil. So far, little effort has been made to examine the influence of multiple-

species populations (bacterial population heterogeneity) and attendant effects (e.g.,

competition, predation, microbial succession) on biodegradation and transport behavior of

PAHs or other hydrophobic organic compounds. Therefore, the primary objective of this

research is to investigate the influence of bacterial population heterogeneity and dynamics

on biodegradation and transport of phenanthrene in a saturated soil.

Materials and Methods

Materials

Phenanthrene (purity >98%) and pentafluorobenzoic acid (PFBA) were purchased

from Aldrich Chemical Co. (Milwaukee, Wl). PFBA served as a nonsorbing, nonreactive

tracer to characterize the hydrodynamic properties of the column. Chloroform, used to

dissolve or extract phenanthrene, and calcium chloride (dihydrate), used as an electrolyte,

were purchased from Mallinckrodt Baker, Inc. (Paris, KY). Methanol (HPLC grade), used

to dilute phenanthrene samples after concentration and employed as a carrier for HPLC

analysis, was purchased from J.T. Baker (Phillipsburg, NJ). The soil used in this study has

the following properties: sand, 77.7%; silt, 18.1%; clay, 4.2%, and organic matter content

2.7%(Estrellaetal., 1993).

The mineralsalts broth(MSB) used for phenanthrene degrader cultivation consisted

of l.O g KH2PO4, l.O g Na2HP04,0.5 g NH4NO3, 0.5 g (NH4)2S04,0.02 g CaCl2-2H,0,

0.002 g FeCls, 0.002 g MnS04-2H20, and 0.2 g MgS04*7H20 per liter of distilled water
 83

(Hernian et al., 1997). Phenanthrene plates were prepared by mixing one liter of MSB with

15 g purified agar (BBL) which was purchased from Becton Dickinson Microbiology

Systems (Cockeysville, MD). RjA agar used in bacterial cultivation was purchased from

Difco Laboratories (Detroit, MI).

Miscible-Displacement Experiments

The apparatus and methods employed for the miscible-displacement studies were

similar to those used previously (Hu and Brusseau, 1998; Brusseau, et al., 1999b). An

autociaved precision-bore stainless steel chromatography column (Alltech Associates Inc.

Deerfield, IL) with 2.1 cm inner diameter (i.d.) and 15.0 cm length was used in the miscible-

displacement experiments. Both ends of the column had stainless steel distribution plates

to help ensure uniform flow and 0.5-|im stainless steel porous plates to serve as bed

supports. In addition, the fittings and tubings were all made of stainless steel to prevent any

sorption of phenanthrene and were sterilized with 2% (v/v) bleach.

Each column was prepared by packing it with air-dried, sieved (^ 0.5 mm) soil in

a manner to obtain uniform bulk density (1.41 g cm'^) and porosity (0.48). The packed

columns were slowly wetted with electrolyte solution (0.01 N CaCy from the bottom to

establish saturation. The pore volume, 25 ml, was determined by measuring the mass of

water retained in each column after saturation. The column was connected to a single-piston

LC pump (Scientific Systems, Inc., SSI Model 300, State College, PA) to generate flow.
 84

The influent phenanthrene stock solution was generated by pumping autoclaved

CaCU solution through a chromatographic column (ACE Glass Inc., Louisville, KY) which

was packed with phenanthrene-coated glass beads. The phenanthrene concentration was

analyzed using fluorescence spectroscopy (Hitachi Ltd., Model F-2000, Tokyo, Japan) with

EX 2S0 nm and EM 36S nm. The average concentration of phenanthrene in the influent

solution was 1.2±0.1 mg L '.

The hydrodynamic characteristics of the packed columns were determined by

conducting tracer tests with 250 mg L ' sterile PFBA solutions. A flow-through, variable-

wavelength UV detector (Gilson, Model 115, Middleton, WI) with wavelength 243 nm was

used to continuously monitor the PFBA effluent concentration.

A colunm experiment was conducted with sterile soil to examine the influence of

sorption on transport. A pulse of phenanthrene/0.01 N CaCl, solution (390 pore volumes),

followed by sterilized 0.01 N CaCl, solution, was injected at a flow rate of 1.86 ml min*',

which is equivalent to a pore water velocity of 67.1 cm hr"'. For the non-sterile miscible-

displacement column experiment, phenanthrene solution was injected continuously for 6

months, equivalent to 11648 pore volumes. The average hydrodynamic residence time was

0,5 hours for the first 2383 pore volumes and 0.2 hours for the remainder of the experiment.

Dissolved oxygen m the influent and effluent was monitored by use of an oxygen meter

(Microelectrodes, Model OM-4, Bedford, NH) equipped with a MI-730 micro-oxygen

electrode.
 85

Residual Phenanthrene in the Column

After the non-sterile column experiment was completed, the soil was removed from

the column,subsampled, and extracted with 30 nJ of chloroform. The samples were placed

on an orbital shaker (Lab-Line Instruments, Inc., Model 3527, Melrose Park, IL) at 200 rpm

for one day at room temperature and then sonicated (ETL Testing Laboratories, Inc.,

Aqusonic Model 250D, Cortland, NY) for 15 minutes. The extract was collected after

centriftiging the sample (Beckman Instruments Inc., Model GP, Palo Altd, CA) for 10

minutes at 3000 rpm. The aforementioned extraction procedure was repeated three times.

Triplicate extracts were combined together and then concentrated with a Rotavapor

Evaporation System (Biichi Co., Switzerland). The above extraction procedure was

developed in a separate experiment. It confirmed that chloroform plus sonication can

achieve the best recovery (approximate 97%) of phenanthrene from the soil.

The amoimt of phenanthrene in the concentrate was quantified by high performance

liquid chromatography (HPLC) (Waters, Milford, MA), which was performed isocratically

using a mobile phase of 5% water (HPLC grade) and 95% methanol (HPLC grade) and a

reverse phase column, LUNA 5^ C18 (Phenomenex, Torrance, CA), with i.d. 4.6 mm and

length 150 mm. The flow rate was I ml min"' and the wavelength used for detection of

phenanthrene was 250 nm.

Distribution of Bacteria in the Effluent and in the Soil Profile

Phenanthrene degraders in the effluent were monitored from 5364 to 11648 pore

volumes. The samples were collected from the outlet of the system and transferred to plates
 86

wherein phenanthrene served as the sole carbon source. The plates were counted after

incubation for 3 days and after 14 days. The distribution of bacteria in the soil profile at the

end of non-sterile column experiment was evaluated by viable counts using both RjA agar

plates and phenanthrene plates. Total viable bacteria and viable phenanthrene degraders

were enumerated after incubation at room temperature for 2 days and 6 days, respectively.

Phenanthrene degraders, either in the effluent or in the soil, were identified to the species

level using ERIC- and 16S rDNA PCR analysis.

Data Analvsis for Sorption Kinetics

A basic advective-dispersive transport model was employed to examine the

hydrodynamic properties of the flow domain (van Genuchten, 1981). The breakthrough

curve (BTC) of PFBA was simulated with a non-linear, least-squares optimization program

(CFITIM3) using the local equilibrium assumption (LEA)-based transport model to

determine the fitting parameter P, referred to as the Peclet number, which represents the

contribution of hydrodynamic dispersion to transport. The retardation factor (R), which

represents the effect of sorption on transport, was determined by moment analysis. The size

of the solute pulse (TJ was known from measurement.

Conversely, a sorption-related nonequilibrium-based model was used to fit the BTC

for phenanthrene for the sterile column. Two fitting parameters, p and m, were obtained

from the simulation. The value of p, defined as [l+F(Pb/0)kJ/R, is the fraction of

instantaneous retardation, where F is the fraction of sorbent for which sorption is

 87

instantaneous. The Damkohler number (o), defined as k2(l-P)RL/v, is the ratio of the

hydrodynamic residence time to the characteristic time for sorption, where k2 is the

desorption rate coefficient in a rate-limited sorption domain. Parameters P and CD specify

the degree of nonequilibrium existing in the system which decrease as either of the two

increase in magnitude (Brusseau et al., 1991).

Results and Discussion

Transport in Sterile Colunm

The breakthrough curve for PFBA transport is shown in Figure 1. It is synunetrical

and relatively sharp, indicating that the soil was homogeneously packed and that flow was

ideal. The Peclet number (P), which represents the relative contributions of advective and

dispersive flux to transport, obtained firom optimization was 73.4. This corresponds to a

dispersivity of 0.2 cm.

The breakthrough curve obtained for phenanthrene transport in the sterile soil is

presented in Figure 2. In contrast to PFBA, the breakthrough curve for phenanthrene is

noticeably asymmetrical, suggesting that its transport is influenced by rate-limited sorption.

A large retardation factor (R=362), which conesponds to an equilibrium sorption coefficient

(K^ of 123 ml g*', indicates that the soil used herein possessed very strong sorption capacity

for phenanthrene. The high sorption capacity was presumably due to the high organic

carbon content in the soil.

 88

The calibrated simulation produced with two-domain rate-limited sorption

incorporated into the transport equation provides a relatively good match to the measured

data (Figure 2). Values for fitting parameters, p and o, were 0.45 and 1.41, respectively,

which were used to calculate values for F and k,. The F value, 0.45, suggests that 55% of

sorption observed in the system was caused by rate-limited processes. The small k, value,

0.03 (hr ') indicates that the desorption rate of the phenanthrene from the rate-limited

domain was very slow. Both F and k, values are reflected in the breakthrough curve as the

early arrival of the breakthrough front and the long tailing in the elution front. From

moment analysis, 98.3% of the applied phenanthrene was recovered in the sterile column

effluent, indicating that only a very small portion of the applied phenanthrene was lost

during the experiment.

Biodegradation and Transport of Phenanthrene in Non-Sterile Column

The breakthrough curve for phenanthrene in the non-sterile soil column exhibited

a damped-oscillation pattern as presented in Figure 3. Phenanthrene was first detected in

the effluent at the 2028"* pore volume. The concentration then oscillated through three

cycles. The first, largest wave had the highest peak and occurred from 2028 to 4680 pore

volumes. The second wave had a slightly lower peak and spanned from 4680 to 7007 pore

volumes. The third, smallest wave had the lowest peak and started at 7007 pore volumes

and extended to the end of experiment (11648 pore volumes). The late breakthrough of

phenanthrene was probably caused by coupled biodegradation and sorption. As

 89

demonstrated in the sterile coltimn experiment, the soil used herein has a very strong

sorption capacity. The evidence of biodegradation in the non-sterile column will be

presented in the next section.

Biodegradation Rate and Mass Balance of Phenanthrene

The most direct evidence of biodegradation in the non-sterile soil column was the

mass loss of phenanthrene. A total of 40.7 mg of phenanthrene was eluted from the effluent,

which was equivalent to 11.4% of the total mass applied to the column. Only a very small

portion of phenanthrene (1.6 mg, 0.5%) remained in the soil column at the end of the

experiment. From the mass balance, 88.1% of the input substrate was degraded by

phenanthrene degraders.

In order to clearly elucidate the biodegradation processes that happened, the

breakthrough curve of phenanthrene was classified into four stages. As mentioned

previously, the average residence time during the first 2383 pore volumes was 0.5 hours;

then, it was adjusted to 0.2 hours until the end of the experiment. Therefore, the first stage

was defined from the beginning, 0, to 2383 pore volumes. The second stage was from 2383

to 4680 pore volumes, which corresponds to the first wave of the breakthrough curve. The

third and the fourth stages coincided with the second and third waves.

A mass distribution concept was applied to analyze the data. The basic assumptions

were that the column was always under saturated conditions and that biodegradation was

the primary mechanism for the mass loss of phenanthrene in the column. The total mass of
 90

phenanthrene (Mq) applied in the system can be subdivided into four portions: the mass

eluted in the effluent (M^n), the mass sorbed in the soil (Mjort,), the mass retained in the pore

water (Mp^J, and the mass biodegraded in the column (MbJ. A mass distribution equation,

defined as Mbio=Mo-M5ort,-Mpo„-Meff, was applied to derive the Mbjo term. The MSOH, term was

obtained from the linear sorption isotherm with Kj = 123 ml g '. The only exception was

the fourth stage because the mass of phenanthrene retained in the soil including Mj^i,

and Mpo,e> was directly measured at the end of experiment. Table 1 lists the mass

distribution of phenanthrene in each of the four stages and the corresponding biodegradation

rates. The average biodegradation rate for each stage was reported as mg of phenanthrene

degraded per pore volume.

Table 1 shows that the average biodegradation rate of the fourth stage, 0.03 mg PV',

was higher than the third (0.019 mg PV') and the second (0.015 mg PV') stages. The

increased biodegradation rate is reflected in the decrease in the amplitude of the wave. At

the end of the third wave, phenanthrene was no longer detected in the effluent. Examined

by mass distribution, 96.0% of the input phenanthrene was degraded by bacteria at the

fourth stage, compared to 63.9% and 53.8% at the third and second stages, respectively.

These results suggest that not only were the phenanthrene degraders becoming more active

with time, but also die biodegradation capacity was increasing.

The analysis also shows that a significant amount of phenanthrene (80.9%) was

degraded at the first stage, corresponding to a biodegradation rate of 0.015 mgPV '. This

suggests that indigenous phenanthrene degraders existed in the soil microcosms and
 91

confirmed the previous deduction that the delay of phenanthrene brealcthrough resulted from

biodegradation. However, the brealcthrough of phenanthrene at about 2000 pore volumes

may indicate that a change m conditions deleterious to the phenanthrene degraders took

place at that time. Because the flow rate of the first stage was different &om the remaining

stages, it is difficult to compare the biodegradation rate of the first stage with the others.

Elution of Phenanthrene Degraders

To further examine phenanthrene biodegradation in the non-sterile column, the

elution of phenanthrene degraders was monitored. The temporal distribution of

phenanthrene degraders in column effluent firactions is presented in Figure 4. Elution of

degraders exhibited dynamic characteristics. During the early stages of effluent monitoring,

Day 3-Colonies (those observed on phenanthrene plates after 3-days incubation) were the

dominant species degrading phenanthrene in the column. Later on, Day 14-Colonies began

to appear and coexisted with Day 3-CoIonies. This suggests that Day 14-Colonies obtained

the "ability" to degrade phenanthrene and gradually out-competed Day 3-Colonies to

become the dominant degrading species.

A comparison between the phenanthrene breakthrough curve (Figure 3) and the

dynamic curve of the phenanthrene degraders (Figure 4) suggests that the size of the second

oscillatory wave of the phenanthrene breakthrough curve was mainly determined by the

degradation capacity of Day 3-Colonies and the size of the third oscillatory wave was

created by the combined action of both Day 3- and Day 14-Colonies. The damped
 92

oscillation property of the phenanthrene breakthrough curve implies that the biodegradation

capacity increased in the column by an increase in either the number or the diversity of the

phenanthrene degraders. Evidence of an increase in the diversity of phenanthrene degraders

in the effluent will be presented in the last section.

From moment analysis, there were 1.8x10"^ mg and 1.4x10'^ mg of biomass

produced per pore volume for Day 3-Colonies and Day 14-Colonies, respectively. During

the same time period, there were 0.043 mg and 0.021 mg of phenanthrene biodegraded per

pore volume by Day 3- and Day 14-Colonies, respectively. Therefore, the yield coefficients,

defined as the mass of cells produced in a unit time as the result of the mass of substrate

utilized in the same time, were 4x10'^ and 7x10"^ for Day 3- and Day 14-Colonies,

respectively. That is, Day 14-Colonies seem to use phenanthrene more efficiently than Day

3-Colonies.

Dissolved Oxveen Curve

Bacterial activity was reflected in the fluctuation of dissolved oxygen concentration

in the column from 3464 to 9186 pore volumes (Figure 3). The oxygen concentration in the

column effluent had oscillatory behavior that mimicked the degradation breakthrough curve

although it lagged slightly behind. In the current experiment, the only oxygen available was

the dissolved oxygen supplied by the influent solution (-8 mg L '), which was sufficient to

mineralize phenanthrene in the column. However, comparing the dissolved oxygen curve

with the temporal distribution of phenanthrene degraders in the effluent, a similar

 93

fluctuation tendency is observed. It suggests that the variation of dissolved oxygen

concentration was influenced by the growth of phenanthrene degraders, which was linuted

by the bioavailable levels of phenanthrene in the column.

Spatial Distribution in Soil Profile

The spatial distributions of total viable bacteria and viable phenanthrene degraders

in the soil core are shown in Figures 5 and 6, respectively. Neither the total bacteria nor the

phenanthrene degraders were distributed uniformly in the colunm. Both showed a tendency

to decrease with depth. This type of spatial distribution may be related to the distribution

of substrate and dissolved oxygen in the column, coupled with growth.

Figure 7 exhibits the residual phenanthrene concentration profile in the non-sterile

soil column at the end of experiment. It shows that most of the remaining phenanthrene

resided in the area adjacent to the inlet of the column. The decrease was very significant in

the first 5 cm depth, which means that the phenanthrene degraders were very active in this

area.

Heterogeneous Population and Microbial Succession

The species of phenanthrene degraders in the effluent and in the soil profile were

analyzed by ERIC-PCR and identified by 168 PGR. A total of 24 species of phenanthrene

degraders, named Species AtoS and / to 5, were identified firom the Day 3-Colonies, Day

I4-Colonies, and soil samples taken afier the experiment was completed. Those species
 94

were classified into attached bacteria and suspended bacteria. For phenanthrene degraders,

the dynamic variation in the effluent and the heterogeneous distribution in the soil profile

are included in Figures 4 and 6. Table 2 lists the species of phenanthrene degraders that

were present in the effluent and in the soil.

At the end of the experiment, 19 out of 24 species of phenanthrene degraders existed

in the effluent (Species 1,2, A, G, K, 0, R) and in the soil (Species B, E, F, H, /, J, K, L, M,

N, P, Q, and 3). This suggests that the increase in the biodegradation rate of phenanthrene

could be contributed by the diversity of the degraders.

Summary

The dynamics of heterogeneous microbial populations, especially under growth

conditions, should be considered when evaluating contaminant biodegradation and transport

in natural subsurface systems. The results of this experiment show that one reason

biodegradation rates increase with time is that a succession of degrading species occurs,

resulting in a fmal population that is more capable of degradation than the initial one.

Acknowledgements

This research was supported by grants provided by the U.S. Department of Energy

Subsurface Science Program and the National Institute of Environmental Health Sciences

Superfimd Basic Sciences Research Program. We thank Julie W. Neilson for her advice and

Asami Murao for her cordial assistance in isolating pure phenanthrene degraders. Special

thanks to Brandolyn Patterson for her help in reviewing the manuscript.

 95

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in Soil Columns. p255-273. In M.F. Wheeler (ed.) Environmental Studies: Mathematical,
Computational, and Statistical Analysis. Springer-Verlag, Inc., New York, NY.

Mironenko, E.V. and Y.A. Pachepsky. 1984. Analytical Solution for Chemical Transport
withNonequilibriimi MassTransfer, Adsorption, and BiologicalTransformation. J. Hydrol.
v70. pl67-175.
 97

Molz, F.J., M.A. Widdowson, and L.D. Benefield. 1986. Simulation of Microbial Growth
Dynamics Coupled to Nutrient and Oxygen Transport in Porous Media. Water Resour. Res.
v22. pl207-1216.

Reddy, H.L. and R.M. Ford. 1996. Analysis of Biodegradation and Bacterial Transport:
Comparison of Models with Kinetic and Equilibrium Bacterial Adsorption. J. Contam.
Hydrol. v22. p271-287.

Srinivasan, P. And J.W. Mercer. 1988. Simulation of Biodegradation and Sorption

Processes in Groundwater. Groundwater. v26, p475-487.

Sykes, J.F.,S. Soyupak, and G.J. Farquhar. 1982, Modeling ofLeachate Organic Migration
and Attenuation in Groundwaters Below Sanitary Landfills. Water Resour. Res. vl8.
pl35-l45.

van Genuchten, M.Th. 1981. Non-equilibrium Solute Transport Parameters from Miscible
Displacement Experiments. Research Report no. 119. USDA Salinity Laboratory and
Department of Soil and Environmental Science. University of California, Riversides.
 98

Table I: Mass Distribution and Biodegradation Rate of Phenanthrene at Different Stages.

Stages Pore Volume M;' Biodegradation

(PV) (mg) (mg) (mg) Rate (mg PV ') (%)
First 0 ~ 2383 73.68 2.63 59.64 0.025 80.9
Second 2383-4680 66.02 19.07 35.55 0.015 53.8
Third 4680 ~ 7007 70.73 14.13 45.19 0.019 63.9
Fourth 7007-11648 144.80 4.83 139.00'" 0.030 96.0

•'.M, = E(V,A)m^ = 355.2 mg;

where

Vo, is the influent volume at the / stage;

Co is the influent concentration.

Meff = = 40.7 mg;

where

Vg, is the effluent volume at the i stage;

Ce, is the effluent concentration at the / stage.

• ^bio ^o'^sorb'^pore'^eff'

where Mp^^ is 0.03 mg; M„rt, is 11.4 mg based on Kd=123 ml g '.

The M„rt, and Mpo„ terms of the fourth stage were determined from the

measured residual phenanthrene in soil, which was 1.6 mg.

 99

Table 2: Identification of Phenanthrene Degraders Found in the Effluent or Soil

Group ID Present in Present in Microorganisms Identification

Effluent Soil
A X Acinetobacter j'mii
B X N/I
C X Burkholderia', Pseudomonas
D X Pseudomonas oleovorans
E X N/I
F X N/I
G X Asticcacaulis\ Caulobacter, Sphingomonas
H X X Sphingomonas sp.
I X Uncultured proteobacterium 0CS7
J X X Acinetobacter junii
K X Pseudomonas-, Burkholderia
L X Burkholderia sp. Isolate N2P5
M X Leptothrix sp. MBIC 3364
N X Bacillus pumilus
0 X Bacillus pumilus
P X N/I
Q X X Rhizobium giardinii
R X Arthrobacter globiformis
S X N/I
1 X N/I
2 X Arthrobacter globiformis
3 X Bacillus subtilis
4 X Methylobacterium sp.
5 X Methylobacterium sp. (stain F18)

(N/I: Non-Identified)
 100

Figure Captions
Figure 1. Breakthrough Curve of Conservative Tracer.

Figure 2. Phenanthrene Breakthrough Curve and Its Simulation in Sterilized Soil

Column. (A pulse of phenanthrene solution - 390 pore volumes)

Figure 3. Phenanthrene Transport and Dissolved Oxygen Profile in a Non-Sterilized Soil

Column. (A continuous pulse of phenanthrene solution; Residence time: 0.5

hours for the first 2383 pore volumes and 0.2 hours for the remainder of the

experiment.)

Figure 4. Temporal Distribution of Phenanthrene Degraders in the Effluent.

(Degraders were monitored firom 5364 to 11648 pore volumes.)

Figure 5. Distribution of Total Viable Bacteria in the Soil Profile.

(Set zero point at the inlet)

Figure 6. Distribution of Viable Phenanthrene Degraders in the Soil Profile.

(Set zero point at the inlet)

Figure 7. Residual Phenanthrene in the Soil Profile at the End of Experiment.

(Set zero point at the inlet)

 • Measured Data
1.0-. Simulation

(Peclet Number = 73.4)

0.8

o
U

0.4--

0.2. .

0.0
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

Pore Volume

Figure 1. Breai(through Curve of Conservative Tracer

 1.00

0.90. •

Simulation
0.80 • •
• Measured Data
0.70

0.60 • •

O
U
U
0.40..

0.30

0.20

O.iO

0.00
0.00 500.00 1000.00 1500.00 2000.00 2500.00

Pore Volume

Figure 2. Phenanthrene Breakthrough Curve and Its Simulation

in Sterilized Soil Column
 1st Stage 2nd Stage 3rd Stage 4th Stage
1.2
(0-2383) (4680-7007) (7007-11648)

I.O..

0.8 • •
o
U
U
0.6 • •
Oo

0.4
Phenanthrene
Dissolved Oxygen
0.2. •

0.0
0 2000 4000 6000 8000 10000 12000

Pore Volume

Figure 3. Piienanthrene Transport and Dissolved Oxygen

Profile in a Non-Sterile Soil Column
 sooo.o

4500.0 • •
- [Day 3-Colonies]
4000.0 • • (Day 14-CoIonies)

3500.0 • •

3000.0 -1
1 2500.0 - •
u
2000.0 • I

1500.0 • I

1000.0 • I

500.0 • I

0.0 - I I I I

0.0 2000.0 4000.0 6000.0 8000.0 10000.0 12000.0

5 P.V.

Figure 4. Temporal Distribution of Phenanthrene Degraders

in the Effluent o
•1^
 I.OE+IO

•t

s
^ I.0E+08-.

-I 0 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16

Depth (cm)

Figure 5. Distribution of Total Viable Bacteria in the Soil Profile

O
I.OE+08

I.OE+07..

1.0E+06-.

I.OE+05 -H -I-
-1 10 11 12 13 14 15 16

Depth (cm)

Figure 6. Distribution of Viable Phenanthrene Degraders

in the Soil Profile
0.16

- 1 0 I 2 3 4 5 6 7 8 9 10 I ! 12 13 14

Depth (cm)

Figure 7. Residual Phenanthrene in the Soil Profile

at the End of Experiment
o
-J
 APPENDIX C:

SUPPLEMENTAL EXPERIMENTS
 109

SUBDIVISION 1

CHARACTERISTICS OF BENZOATE BIODEGRADATION

Contributed to the paper titled as

A Biotracer Test for Characterizing the In-situ Biodegradation Potential

Associated with Subsurface Systems

by

Mark L. Brusseau*, Joseph J. Piatt, Jiann-Ming Wang, and Max Q. Hu

Prepared for:

Field Testing of Innovative Subsurface Remediation and Characterization Technologies

An American Chemical Society Symposium Series Book

Published in 1999

* Corresponding author
 no

Objectives

The objectives of this study were to examine the inherent biodegradation potential

of benzoate for a Hill AFB site and the biodegradation tendency of benzoate under aerobic

and anaerobic conditions. The nutrients effect on the biodegradation of benzoate was also

evaluated in Hill AFB groundwater, synthetic groundwater, and mineral salts medium.

Materials and Methods

Materials

Sodium Benzoate (99%), used as a biotracer in field sites, was purchased from

Aldrich Chemical Co., Inc. (Milwaukee, WI). [Ring-UL-''*C]Benzoic acid (specific activity,

13.3 mCi/nwnol, reported purity >98%) and toluene (HPLC grade, purity >99.8%), used to

dissolve [UL-'"'C]benzoic acid, were purchased from Sigma Chemical Co. (St. Louis, MO).

Scintillation cocktails, ScintiVerse BD and Oxosol C'"*, were purchased from Fisher

Scientific Chemical (Fair Lawn, NJ) and National Diagnostics (Atlanta, GA), respectively.

Acetonitrile UV (HPLC grade), employed as a carrier for HPLC analysis, was purchased

from Burdick & Jackson Inc. (Muskegon, MI).

The mineral salts medium (MSM), specifically Bushnell-HAAS (Difco Laboratories,

Detroit, MI), consisted of 0.2 g MgS04,0.02 g CaClj, I g KH2PO4,1 g (NH4)2(HP04), 1 g

KNO3, and O.OS g FeClj per liter of distilled water. Agar (Bacto-Agar) and R2A agar

employed in bacterial cultivation were also purchased from Difco Laboratories (Detroit,
 Ill

MI). Either groundwater or aquifer material collected from a jet-fliel contaminated site

located at Hill Air Force Base in Layton, Utah, served as inoculum herein.

Viable Cell Counts

The presence of benzoate degraders at the Hill site was determined by use of the

viable plate count method. Benzoate plates were prepared by adding 0.1%(w/v) of the Na-

benzoate as the sole carbon or energy source to sterilized Bushnell-HAAS-agar broth. Two

grams of moist Hill aquifer material (moisture 14.1%, w/w), 1-ml groundwater, or 1-ml

mixture (after mineralization experiment) was added to 9-ml sterilized distilled water to

make a series of 1;10 dilution ratio. For each dilution ratio, a 100-/^1 aliquot of the mixture

was transferred to the benzoate or RjA plates. Triplicate plates were applied in each dilution

ratio. The plates were incubated upside down at room temperature for 2 days and then

enumerated.

Mineralization

The mineralization associated with the ability of bacterial populations to biodegrade

benzoate was quantified by the measurement of evolved '"'COj and '"'C-volatile compounds.

The benzoate stock solution, 376 mg L"', was prepared by dissolving Na-benzoate in

sterilized (autoclaved) Bushnell-HAAS broth. The concentration of Na-benzoate used

herein was close to the original amount applied in the field. A IS-ml aliquot of sterilized

benzoate stock solution was transferred to modified 125-ml micro-Fembach flasks

 112

(Wheaton, Milville, N.J.), designed for the collection of '"'CO, and '""C-volatile compounds.

These flasks were then spiked with uniform ring labelled ['''CJbenzoic acid dissolved in

toluene to obtain a specific activity of 0.89 ^Ci/mmol. After mixing, one set of triplicate

flasks was inoculated with either 1 ml of groundwater or 2 g of moist aquifer material. The

flasks that contained the same amount of either sterilized groundwater or sterilized aquifer

material served as controls. Non-inoculated flasks served as blank controls. Both control

sets were in duplicate.

All samples were incubated at room temperature on a gyratory shaker (Lab Line

Orbit Shaker, Model 3527, Melrose Park, IL) at 200 rpm. A flushing-tree technique was

employed to collect '''CO, and '''C-volatile organics periodically. Two vials containing

ScintiVerse BD cocktail and two vials containing Oxosol C'"* cocktail were connected in­

line to trap evolved ''*C-volatile organics and '""CO,, respectively. The radioactivity of the

evolved '^CO, and '"'C-volatile organics was determined by liquid scintillation counter

(Packard Tri-Carb Co., Model 1600 TR, Meriden, CT).

Mass Loss of Benzoate

A series of experiments was conducted to evaluate the magnitude and rate of mass

loss of benzoate under aerobic and anaerobic conditions. For aerobic experiments, the stock

solutions were prepared by dissolving Na-benzoate m autoclaved real groundwater,

synthetic groimdwater, or Bushnell-HAAS MSM. Conversely, only autoclaved real

groundwater was applied for the preparation of benzoate stock solution in anaerobic
 113

experiments. In both aerobic and anaerobic experiments, the concentration of benzoate

stock solutions was kept at 376 mg L ' and non-sterilized groundwater served as the

inoculum. To provide enough oxygen for biodegradation of benzoate, 2S-nil glass vials

with caps were employed in the aerobic experiments. For the aerobic experiments, 1-ml

aliquot of non-sterilized groundwater was added to 2S-ml glass vials containing 9 ml of

sterilized stock solution. Samples with sterilized synthetic groundwater and real

groundwater were incubated for 8.8 and 20.8 days, respectively. Subsequently, an even

amount of Bushnell-HAAS broth was added to those systems to facilitate the biodegradation

of Na-Benzoate. For the anaerobic experiment, 5-ml glass vials with Teflon septa were

used. Before inoculation, the sterilized benzoate stock solution was sparged with nitrogen

gas for more than two hours to remove dissolved oxygen. Dissolved oxygen was monitored

by a MI-730 Micro-Oxygen Electrode connected to an OM-4 Oxygen Meter

(Microelectrodes, Bedford, NH). After non-sterilized groundwater was induced, the amount

of inoculum was kept at 10% (v/v) and no head space existed in the vial. Sterilized stock

solution with sterilized groundwater was used as a control set in each experiment.

The vials were incubated at 200 rpm on an orbit shaker at room temperature. Two

or three vials were sacrificed at each sampling time. A 1.5-ml sub-sample was transferred

to a micro-centrifuge tube and preserved with 0.1-ml aliquot of sodium azide (0.1 M).

Samples were centrifliged for 10 minutes in a microcentrifuge (Brinkmann Instruments, Inc.,

Model 5415C, Westbury, NY). The supernatant was then passed through a 0.2 |im sterilized

syringe filter (Gehnan Sciences, Model Supor Acrodisc 13, Ann Arbor, MI) to screen out
 114

any potential suspended particles. After centrifligation and filtration, the concentration of

benzoate was quantified by high performance liquid chromatography (HPLC) (Waters,

Milford, MA) using a reverse phase column, Adsorbosphere UHS C18 SU (Alltech,

Deerfield, IL), with I.D. 4.6 mm and length 150 mm. The flow rate was 1 ml min*' and the

wavelength was 235 nm. The elution was isocratic using a mobile phase of 60% phosphate

buffer (pH=3.2) and 40% acetonitrile.

Results and Discussion

Inherent Biodegradation Potential

The inherent biodegradation potential of benzoate at the Hill AFB site was evaluated

using either the viable plate count or mineralization method. Both methods were performed

with use of groundwater and aquifer material as inocula. The results of the initial viable

counts in benzoate plates showed that the benzoate degraders existed at the site at levels of

(1.0±0.3)x10"* colony forming units (CFU) per ml of groundwater or (3.2±0.8)x10^ CFU per

gram of dry aquifer material.

The presence and character of benzoate degraders in the specific site was confirmed

by mineralization experiments. In Figure 1, the evolution of ''•CO, derived fi:om the

mineralization of benzoate, clearly demonstrated that benzoate degraders existed in the

indigenous bacterial populations in the field. However, two different types of mineralization

curves suggested thatthere were at lease two different groups of benzoate degraders existing

either in the solid phase (aquifer material) or in the aqueous phase (groundwater). Both
50

45

40

35

30

25

20 Aquifer Material
15 Groundwater

10 Control

5 Blank

0 ^1—
25 50 75 100 125 150 175 200 225

Time (hrs)
Figure 1. Characteristic Curve of Benzoate with
Two Different Inocula
 116

contributed to the biodegradation of benzoate. Another factor which may have caused the

difference in biodegradation was the "soil" effect. The aquifer material which served as

inoculum may contain sorbed contaminants which could delay the initiation of

biodegradation.

The characteristics of the benzoate degraders were examined by the lag phase,

biodegradation capacity, and biodegradation activity. The lag phases for samples inoculated

with aquifer material and groundwater were approximately 27 and 20 hours, respectively.

The longer lag phase indicated that the degraders in the solid phase needed more time to be

adapted for benzoate. Once adapted, they exhibited higher biodegradation capacity and

activity.

The biodegradation capacity was revealed on the plateau of the mineralization curve

which corresponded to the stationary phase of bacteria growth. Figure 1 shows that samples

inoculated with aquifer material had a relatively higher plateau (44±3%) versus samples

inoculated with groundwater (32±3%). A higher plateau represents that more benzoate was

mineralized to CO^. In other words, the degraders in the solid phase had a higher

biodegradation capacity than the degraders in the aqueous phase. The biodegradation

activity, or biodegradation rate, is reflected in the slope of the mineralization curve. A

steeper slope referres to a higher biodegradation rate or a higher bacterial activity. In Figure

1, samples inoculated with aquifer material had a steep slope during the exponential growth

phase. That means the activity for the degraders in the solid phase was higher than that of
 117

the degraders in the aqueous phase. One explanation for this is that there was an extra

carbon source in the aquifer material which would facilitate cometabolism of benzoate.

After the mineralization experiment, a small portion of the mixture from each

sample inoculated with aquifer material or groundwater was transferred to benzoate and RjA

plates. After 2-days incubation, the RjA viable count reached levels of (3.9±0.8)xl0' CFU

per ml of mixture or (2.1±0.1)xl0' CFU per gram of dry aquifer material for samples

inoculated with groundwater or aquifer material, respectively. Conversely, the number of

benzoate degraders enumerated in the benzoate plates was (2.4±l.l)xI0* CFU per ml of

mixture or (6.5±0.4)x10^ CFU per gram of dry aquifer material for samples inoculated with

groundwater or aquifer material, respectively. Compared to the initial viable plate counts,

the population of benzoate degraders increased four orders of magnitude in groundwater-

inoculated samples as well as aquifer material-inoculated samples. This suggests that the

benzoate degraders were not only existing at the site, but that they were also stimulated to

grow readily.

Groundwater Analvsis

Table 1 lists the analytic composition of groundwater at the Hill AFB site. The

accuracy of the groundwater analysis can be examined using the law of electroneutrality

(Snoeyink and Jenkins, 1982); the total amount of positive charge must equal to the total

amount of negative charge in an aqueous solution. Hence, the result of the Hill AFB

groundwater analysis was assessed by conducting the charge balance between cations and
 118

Table 1: Composition of Hill AFB Groundwater and Charge Balance Analysis

Cations Cone.* Mole Weight Charge Ion Normality

(mg/L) (mg/mmol) (mmeq/mmol) (meq/L)

Na" 116 23 1 5.04

K' 27.4 39.10 1 0.70

Ca-* 67.7 40 2 3.38

Mg-* 48.8 24.3 2 4.02

Total (© charge) = 13.14

Anions Cone.* Mole Weight Charge ion Normality

(mg/L) (mg/mmol) (mmeq/nmiol) (meq/L)
F- 0.959 19 1 0.05

cr 176 35.5 1 4.96

Br <0.2 79.9 1 <0.0025

NO,- <0.1 46 1 <0.0020

N03- 0.766 62 1 0.012

P04'- 2.73 95 3 0.087

so^^- 145 96 2 3.02

C03-- <1 60 2 <0.017

HCO3- 374 61 1 6.13

Total (0 charge) = 14.26

* The groundwater analysis was conducted by Soil, Water, Plant Analysis Lab, Dept. of Soil, Water, and

Environmental Science, University of Arizona.

 119

anions. This assessment is also illustrated in Table 1. A slight deviation (about 8%)

between cations and anions was found and indicated that the composition obtained from the

groundwater analysis may not be representative of the "true" components of the Hill AFB

groundwater. It implied that some of the ions which may be important to biodegradation

processes are disregarded in the groundwater analysis.

Because it was not practical to use Hill AFB groundwater through out all of the

investigations, it was necessary to create a synthetic groundwater that mimicked Hill AFB

groundwater characteristics. This task was particularly difGcult as the results of the Hill

AFB groundwater analysis were not thought to be entirely accurate. Creation of the

synthetic groundwater was based on the ionic species reported in the Hill AFB groundwater

analysis (Table 1). The amount of each species used was adjusted, such that a charge

balance between anions and cations was obtained. The final composition of the synthetic

groundwater and its charge balance are shown in Table 2. The formulated ingredients of the

synthetic groundwater are summarized in Table 3. Although the synthetic groundwater is

balanced ionically, some ions such as F*, Br", NOj", COj*', which existed in the Hill AFB

groundwater in very small quantities, were not included. In addition, it is quite possible that

these ions, or other ions not detected in the Hill AFB groundwater analysis, are necessary

for biodegradation processes to occur.

Table 4 displays thedifferences in composition among Bushnell-HAAS MSM, Hill

AFB groundwater, and the synthetic groundwater. It was very obvious that there weresome

ions which were abundant in Bushnell-HAAS MSM but that were either lacking or present
Table 2: Composition of Synthetic Groundwater and Charge Balance Analysis

Cations Cone. Charge Ion Normality

(mmol/L) (mmeq/mmol) (meq/L)

Na" 5.04 1 5.04

r 0.70 I 0.70

Ca-* 1.69 2 3.38

Mg=^ 2.01 2 4.02

Total (0 charge) = 13.14

Anions Cone. Charge ion Normality

(mmol/L) (mmeq/mmol) (meq/L)

cr 5.01 1 5.01

NOj- 0.012 1 0.012

S04-- 1.51 2 3.02

HCOj- 5.04 1 6.13

Total (0 charge) = 13.14

 121

Table 3: Formulated Ingredients of Synthetic Groundwater based on the Hill AFB

Groundwater Analysis

Species Molar Cone. (mM) MW (mg/mmol) Mass Cone. (rag/L)

MgSO^ 1.51 120.4 181.7

KNO3 0.012 101 1.21

MgCl, 0.50 95 47.5

CaCU 1.69 111 187.56

NaHCOj 5.04 84 423.36

K2HPO4 0.029 174.2 5.05

KCl 0.63 74.6 46.97

 122

Table 4: List of Components of Bushnell-HAAS MSM, Hill AFB GW, and Synthetic GW

Cations Bushnell-HAAS MSM Hill AFB GW Synthetic GW

(mmol/L) (mmol/L) (mmol/L)

Na" 0 5.04 5.04

r 17.25 0.70 0.70

Ca-* 0.18 1.69 1.69

Mg2* 1.66 2.01 2.01

Fe-* 0.39 0 0

NH4" 15.16 0 0

Anions Bushnell-HAAS MSM Hill AFB GW Synthetic GW

(mmol/L) (mmoI/L) (mmoI/L)

F" 0 0.05 0

cr 1.14 4.96 5.01

NO," 0 < 0.002 0

NOj- 9.9 0.012 0.012

HiPO^- 7.35 0 0

HP04-- 7.58 0 0

P04^- 0 0.029 0.029

SO4-- 1.66 1.51 1.51

HCOj- 0 6.13 5.04

COj^- 0 <0.017 0

Br- 0 <0.0025 0
 123

only in small amounts in Hill AFB or synthetic groundwater, for example, NH/, Fe*"^,

NO3*, H2PO4*, and HPO4-". These ions might be growth-required elements for benzoate

degraders.

Characteristics of Benzoate Biodecradation in Aerobic and Anaerobic Conditions

Figure 2 showed the mass loss of benzoate in Hill AFB groundwater under aerobic

and anaerobic conditions. A long lag period and less mass loss of benzoate were obtained

under anaerobic conditions. The biodegradation of benzoate under anaerobic conditions was

substantially slower than under aerobic conditions. However, the results suggested that the

biodegradation of benzoate could proceed in both aerobic and anaerobic conditions. These

observations were similar to the results of other studies reported by Kuhn et al. (1988) and

Chapelle (1993). As would be expected, the lack of nutrients was thought to be the cause

of the low level of benzoate biodegradation, and oxygen appeared to be one of the factors

limiting biodegradation efficiency.

To elucidate the effect of nutrients on the biodegradation of benzoate in aerobic

conditions, the mass loss of substrate in the presence of MSM, Hill AFB groundwater, and

synthetic groundwater has been presented in Figure 3. Benzoate samples containing

Bushnell-HAAS MSM exhibited very rapid biodegradation. Samples in Hill AFB

groundwater showed an almost identical biodegradation rate, after a relatively short lag

phase, as samples in MSM. However, after 25% (w/w) of benzoate was degraded, the

substrate disappearance rate gradually slowed down. This implies that the nutrients required
110

100

90

80

70

60

50

40
•Anaerobic Incubation |
30 •Anaerobic Control |
•Aerobic Control
20 !
- Aerobic Incubation
10

0 •
H—• ' •—'—I—'—^ I I I I I I I I 1 • « ' '
) 2.0 4.0 6.0 8.0 10.0 12.0 14.0

Time (days)

Figure 2. Mass Loss of Benzoate in Hill AFB GW under Aerobic and

Anaerobic Conditions
NJ
-1^
110
Add IMSM
100 Hill AFB GW

90-- -O- Synthetic GW

80-- MSM
70-- Add MSM

60--

50-.

40-.

30--

20--

10--

0.0 5.0 10.0 15.0 20.0

Time (days)
Figure 3. Mass Loss of Benzoate in Hill AFB GW, Synthetic GW, and
Bushnell-HAAS MSM
 126

for the biodegradation of benzoate were gradually exhausted from the Hill AFB

groundwater. Because biodegradation was not constrained by oxygen herein, the declining

biodegradation rate might have resulted from the total consumption of existing nutrients or

the lack of growth-required elements. As well, the benzoate mass loss was less in the

synthetic groundwater than in the Hill AFB groundwater (Figure 3). One may deduce from

this phenomenon that some important growth-required ions were missing from the synthetic

groundwater. Ions that were either present in small quantities in the Hill AFB groundwater

and were not included in the synthetic groundwater, or ions that were not detected in the Hill

AFB groundwater, and thus also not included in the synthetic groundwater.

Those deductions were verified by adding Bushnell-HAAS MSM to the samples in

Hill AFB groundwater and in synthetic groundwater after a 20.8- and 8.8-day incubation

period, respectively. After the MSM was added to the system, the benzoate biodegradation

increased dramatically (Figure 3) in the rate and extent. This result suggests that the

decrease in the biodegradation rate in Hill AFB groundwater and in synthetic-groundwater

samples was due to the absence of nutrients. It confirmed that the NH4\ Fe-*, NOj",

H2P04', and HPOj*' ions, which were abundant in Bushnell-HAAS MSM but lacking or

insufficient in the Hill AFB and synthetic groundwater, were the growth-required ions for

benzoate degraders. However, it is difGcult to judge which ions control the biodegradation

rate of benzoate directly. To address this issue, further experiments should be conducted.
 SUBDIVISION 2

BIOAVAILABILITY OF BENZOATE AT THE AFP 44 SITE

Contribution to ongoing research

 128

Objective
The objective of this study was to examine the bioavailability of benzoate to

indigenous microorganisms in a trichloroethene (TCE)-contaminated aquifer.

Materials and Methods

Materials

Benzoic acid (99.5%) was purchased from Aldrich chemical Co., Inc. (Milwaukee,

WI). Both groundwater and aquifer material collected from a TCE-contaminated superfund

site, Air Force Plant 44 (AFP 44), in Tucson, Arizona, served as inocula. The other required

materials were the same as those discussed in Characteristics of Benzoate Biodegradation

studies.

Mineralization

The presence of indigenous benzoate degraders at the AFP 44 site was evaluated by

the benzoate mineralization. First, benzoate and [''*C]benzoate were mixed together in

toluene. The radiolabeled mixture was then added to the modified 125-ml micro-Fembach

flasks (Wheaton, Milville, N.J.), designed for the collection of '"'COz and '"'C-volatile

compounds. After evaporating the solvent for at least one day, it was assumed that all the

radiolabeled benzoate (1.5 mg) remained in each flask. Before injecting 1-mI groundwater

or I -gram aquifer material as inoculum, a 15-ml of Bushnell-HAAS broth was added to each

flask. Consequently, the concentration of benzoate in each flask was maintained at 100 mg
 129

L ' and the specific activity was 7.56 /zCi/mmoi. For control samples, sterilized aquifer

material (1 gram) or groundwater (1 mL) was added to the flasks containing 100 mg L ' of

benzoate solution.

For each concentration, triplicate samples were incubated at room temperature on

a gyratory shaker (Lab Line Orbit Shaker, Model 3527, Melrose Park, IL) operating at 200

rpm. A flushing-tree technique was employed to collect '''COj and '''C-volatile organics

periodically. Radioactivity was determined by liquid scintillation counter (Packard Tri-Carb

Co., Model 1600 TR, Meriden, CT).

Results

The presence and character of benzoate degraders at AFP 44 site were evaluated by

a mineralization experiment. In Figure 1, the evolution of '''COj from samples, inoculated

either with aquifer material or with groundwater, demonstrated that benzoate degraders

existed in the indigenous microbial community. Basically, the degraders in aquifer material

and in groundwater showed identical lag periods, matched slope lines in the exponential

growth period, and ahnost the same biodegradation capacity. Therefore, both inocula

possessed the same ability to degrade benzoate. However, compared with the Hill site

benzoate mineralization study, much less benzoate was converted to CO, (~20%) and the

slope in the exponential growth phase was found to be smaller. In other words, the

biodegradationcapacity and the bacterial activity were lowforthe benzoate degraders in this

specific site. This could bedue to the residual TCE in the aquifer material and groundwater,
 Aquifer Material

30-- Groundwater

Controls

i;" 20--

10--

0 10 20 30 40 50 60 70 80 90 100 110 120 130

Time (hrs)
Figure 1. Mineralization Curve of Benzoate with
Two Different Inocula
U>
O
 131

which may have a deleterious effect on the biodegradation of benzoate. To address this

issue, more complex experiments are required.

 SUBDIVISION 3

THE MEASUREMENT OF BIODEGRADATION KINETIC PARAMETERS

FOR BENZOATE AND 2,4-DICHLOROPHENOXYACETIC ACID (2,4-D)

Contributed to the paper titled as

Biodegradation during Contaminant Transport in Porous Media.

2. The Influence of Physicochemieal Factors

by

Mark L. Brusseau*, Max Q. Hu, Jiann-Ming Wang, and Raina M. Maier

Published in:

Environmental Science and Technology

Volume 33 (1), p96-l03, 1999

* Corresponding author
 133

Objectives
The objectives of this study were to evaluate the biodegradation behavior of target

compounds and to determine the biodegradation kinetic parameters, such as half-saturation

constant (K,), maximum specific growth rate (jd^), and biomass yield (I^, which were used

in the analysis of solute transport in the column. The target compounds used in this study

were benzoate, a common intermediate by-product of biodegradation, and 2,4-

dichlorophenoxyacetic acid (2,4-D), a well-known biodegradable herbicide.

Materials and Methods

Sodium Benzoate (99%) was purchased from Aldrich chemical Co., Inc.

(Milwaukee, Wl) and analytical grade 2,4-dichlorophenoxyacetic acid (CgH^CUOj) was

purchased from Sigma Chemical Co. (St. Louis, MO), [Ring-UL-'''C]Ben2oic acid

('•'CgHjCOOH, specific activity, 13.3 mCi/nrniol, reported purity >98%) and radiolabeled

2,4-D (2,4-Cl2'''CgH30CH2C00H, specific activity, 18.2 mCi/nunol, reported purity >98%)

were purchased from Sigma Chemical Co. (St. Louis, MO). Toluene (HPLC grade, purity

>99.8%), used to dissolve [UL-''*C]benzoic acid, was also purchased from Sigma Chemical

Co. (St. Louis, MO). Scintillation cocktails, ScintiVerse BD and Oxosol C'"* were

purchased from Fisher Scientific Chemical (Fair Lawn, NJ) and National Diagnostics

(AUanta, GA), respectively.

The mineral salts medium (MSM) used in the benzoate study, specifically Bushnell-

HAAS (Difco Laboratories, Detroit, MI), consisted of 0.2 g MgS04, 0.02 g CaClj, 1 g
 134

KH2PO4,1 g (NH4)2(HP04), I g KNO3, and 0.05 g FeClj per liter of distilled water. Agar

(Bacto-Agar) and R2A agar employed in bacterial cultivation, were also purchased from

Difco Laboratories (Detroit, MI). The 2,4-D EMB indicator medium consisted of 0.112 g

of MgS04-7H20,0.005 g of ZnS04-7H20,0.0025 g of Na2Mo04-2H20,0.218 g of K2HPO4,

0.014 g of CaCl2-2H20,0.00022 g of FeCl3-6H20,0.5 g ofNH4Cl, 0.08 g of Eosin B dye,

0.013 g of Methylene Blue dye, and 20 g of noble agar per liter of distilled water. After

autoclaving, it was supplemented with filter sterilized yeast extract solution 0.5 ml (50 mg

L ') and 2,4-D solution 50 ml (500 mg L"'). The pH was adjusted to 7. Noble agar and yeast

extract were purchased from Difco Laboratories (Detroit, MI). Inoculum used in the

benzoate study was the aquifer material from a jet-fuel contaminated site located at Hill Air

Force Base in Utah, which was amended with benzoate. An 80:20 (w/w) mixture of the

Mexican sandy loam soil (which had a reported 2,4-D implementation history) and Mount

Lemmon soil, amended with 2,4-D solution, served as inoculum in the 2,4-D study.

Viable Plate Counts

The existence of benzoate degraders and 2,4-D degraders either in the Hill site's

aquifer material or in the Mexican-Mount Lemmon mixture soil, before and after

stimulation, were verified by using the viable plate count method. Benzoate plates were

prepared by adding 0.1%(w/v) of the Na-benzoate as the sole carbon source to autoclaved

Bushnell-HAAS-agar broth. Plates containing 2,4-D as the sole carbon or energy source and

eosin-methylene blue agar (EMB) have the ability to indicate individual 2,4-D degrading
 135

bacteria which will produce dark red colonies with a typical metallic sheen (Loos, 197S).

Triplicate plates were applied in each dilution ratio. The numbers of benzoatedegraders and

2,4-D degraders were enumerated after 5-days and 3-weeks incubation at room temperature,

respectively.

Mineralization

The K, values of Na-benzoate and 2,4-D were determined by quantification of the

evolved '''CO,, that was produced during the mmeralization process. In order to stimulate

and enrich the population of benzoate degraders and 2,4-D degraders, the Hill site's aquifer

material and Mexican-Mount Lemmon soil mixture were first exposed to 200 mg L ' ofNa-

benzoate solution and 2,4-D solution for 34 days and 2 months, respectively, at room

temperature. The moisture content of the soil and the number of degraders were checked

regularly during the stimulation period.

Stock solutions of benzoate (500 mg L"') and 2,4-D (500 mg L"') were prepared by

dissolving Na-benzoate and 2,4-D in Bushnell-HAAS mineral salt broth, respectively. The

stock solutions were sterilized by passing them through a 0.22-^m cellulose acetate filter

(Coming Costar, Coming, NY). A series of benzoate and 2,4-D concentrations (100,30,

10,1, 0.1, and 0.01 mg L'') was established by diluting the stock solution with Bushnell-

HAAS broth under aseptic conditions. Each concentration was individually radiolabeled

by spiking with [lJL-'''C]benzoic acid or '''C-2,4-D. For each concentration, a 15-ml aliquot

of radiolabeled benzoate or 2,4-D solution was transferred to a modified 125-ml micro-

 136

Fembach flask (Wheaton, Milville, NJ.), designed for the collection of '"'CO, and '''C-

volatile compounds. Two grams of stimulated, moist Hill site aquifer material (final

moisture 10.1%, w/w) or Mexican-Mount Lemmon soil mixture (final moisture 23.8%,

w/w) were added to each flask as inoculum. Samples were prepared in duplicate for seven

solute concentrations (0.01, 0.1,1,10,30,100, and 500 mg L ') and placed on a gyratory

shaker (Lab Line Orbit Shaker, Model 3527, Melrose Park, IL) at 200 rpm at room

temperature.

The evolved '"'COi and ''*C-volatile compounds were collected periodically by a

flushing-tree technique. Two vials containing ScintiVerse BD cocktail and two vials

containing Oxosol C'"* cocktail were connected in-line to trap evolved '"'C-labeled volatile

organic compounds and '''CO,, respectively. Radioactivity was determined by a liquid

scintillation counter (Packard Tri-Carb Co., Model 1600 TR, Meriden, CT).

Data Analysis
Initial Biomass Concentration

In order to obtain the initial biomass concentration, the viable plate counts (CFU per

gram dry soil) were first multiplied by the unit cell mass, 9.5x10*'^ gram per CFU, to obtain

the total mass of cells in a unit mass of dry soil. This mass was then multiplied by the

amount of dry soil added to the system, and then divided by the volume of solution in the

system, which was 15 ml herein. Thus, the initial biomass concentration was converted to

units of mg per liter.

 137

Yield Coefficient (Y\

It was assumed that benzoate and 2,4-D were completely degraded and converted

to either CO, or cell mass, represented by the formula C5H7NO2 (Gaudy and Gaudy, 1970).

The mineralization curve was obtained first, by plotting evolved '''CO2 versus time. From

the mineralization curve, the mole number of C in substrate, converted to either CO, or cell

mass, was calculated. Then, the amount of cell mass, in mmol C unit, was converted to

mass, in terms of mg C5H7NO2, which was then substituted for the cell mass term in the

yield coefficient. The substrate term in the yield coefficient was the amount of substrate

utilized during the experimental period.

Half-saturation Constant (K^ and Maximum Specific Growth Rate (uj

Using the following biodegradation equations, the substrate (benzoate or 2,4-D)

utilization over time could be determined from the evolved CO, term for each initial

substrate concentration:

[2]
2
 138

Thus, the substrate utilization curve for each concentration was able to be established by

plotting the remaining substrate amount versus time. The specific growth rate (^) could be

calculated by applying the following equation

[3]

where mj is the maximum slope for each substrate utilization curve. The values of

K, were obtained by taking the reciprocal of both sides of Monod equation to give a linear

formula

[4]
w
"
u
"max
5 "max
u

which predicts that a plot of 1/n versus 1/S yields a straight line. The slope of which is

with ordinate mtercept

Results and Discussion

Effects of Stimulation

Initially, the number of benzoate degraders was 880 CFU per gram of dry Hill

aquifer material. After 34-days stimulation, the number of benzoate degraders increased to

1.4x10' CFU per gram of dry soil; this is about 5 orders of magnitude higher than the

original number. However, after 2-months stimulation, the number of 2,4-D degraders was

raised to only 7.3x10^ CFU per gram of dry soil, which was less than the number of
 139

benzoate degraders (1.4x10^). This may be caused by CI" ions, which are produced in the

biodegradation of 2,4-D (Equation [2]). The Bushnell-HAAS mineral salt media did not

provide sufficient buffer capacity to neutralize the acidity from CI" ions. Thus, the presence

of CI" ions maybe inhibited by the increase of 2,4-D degraders.

Biodegradation Kinetic Parameters

Biodegradation of benzoate and 2,4-D were studied by conducting batch experiments

using a series of concentrations. Figure 1 and Figure 2 suggest that benzoate and 2,4-D

were mineralized completely by the end of each experimental period. Both benzoate and

2,4-D had a lag phase present. In general, the lag phase can be attributed to enzyme

induction, random mutation, or an increase in the number of organisms capable of

biodegradation (Aelion et al., 1987). Because the soils used in these experiments had been

exposed to either benzoate or 2,4-D for a long time, the lag phase was most likely due to an

initial small population of degraders rather than to enzyme induction or random mutation.

As shown in Figure 1 and Figure 2, benzoate samples exhibited a relatively shorter lag phase

than 2,4-D samples. The longer lag phase reflected that 2,4-D, a chlorinated aromatic

compound, was more difflcuh to degrade than benzoate, an aromatic compound. For both

benzoate and 2,4-D samples, a very short lag in growth was observed at the lowest substrate

concentration. However, the lag phase appeared to increase with increasing substrate

concentration, possibly due to toxicity effects.

 30 ppm

100 ppm

500 ppm

10 20 30 40 50 60 70 80

Time (hrs)
Figure 1. Mineralization Curve of Benzoate at Different
Concentrations
80

70

60

50

40 0.01 ppm
0.1 ppm
30 1 ppm
10 ppm
20 30 ppm
100 ppm
10 500 ppm

0
100 200
Time (hrs)

Figure 2. Mineralization Curve of 2,4-D at Different Concentrations

 142

The mineralization curves of benzoate and 2,4-D are exhibited in Figure 1 and

Figure 2, respectively. Because the substrate utilization rate (dS/dt) is related to several

biodegradation kinetic parameters, such as K,, Y, and X^, these kinetic parameters are

calculated as described in the Data Analysis section. Figure 3 and Figure 4 show the plots

of l/^ versus 1/S for benzoate and 2,4-D which were used to find the and Kj values.

Table 1 and Table 2 sununarize the biodegradation kinetic parameters of benzoate and 2,4-

D, respectively, which include initial biomass concentration (X„), half-saturation constant

(K,), maximum specific growth rate (jt„), average biomass yield (Y), and initial substrate

concentration (S„) with its corresponding specific growth rate (ji) value.

In the batch systems, the initial biomass concentration (XJ of benzoate (1.6 mg L')

was higher than that of 2,4-D (0.073 mg L"'). This was consistent with the fact that the

population of benzoate degraders (1.4x10^ CFU/gdry soil) was higher than 2,4-D degraders

(7.3x10^ CFU/g dry soil) after stimulation. Further, if the batch data are desired to

incorporate with column experiments, the initial biomass concentration needs to be

multiplied by the factor, p/0, where p is the bulk density of porous medium [ML^"] and 0 is

the fi-actional volumetric water content (Brusseau et al., 1999b). Both p and 0 parameters

are fi-om column experiments.

Cell yield {Y), or the amount of cell mass produced, reflects the amount of energy

obtained by a microorganism in the oxidation of a substrate. Therefore, cell yield is

dependent on the carbon source as well as on the microorganism. As shown in Table 1 and

Table 2, the yield coefficient of benzoate, 0.65, was higher than that of 2,4-D, 0.29. The
 100.0

90.0

80.0

70.0

60.0
I
3
50.0 y = 8.8319x +0.4338
R^ = 0.9997 ^
40.0

30.0

20.0

10.0

0.0
0.00 2.00 4.00 6.00 8.00 10.00 12.00

1/S (L/mg)

Figure 3. Linear Regression of Benzoate Data to Determine Monod

Equation Parameters - u^aax and ^
140.0

120.0

100.0

80.0

y= 1.2061X +0.287
60.0 = 0.9999^

40.0

20.0

0.0
0.00 20.00 40.00 60.00 80.00 100.00 120.00

1/S (L/mg)
Figure 4. Linear Regression of 2,4-D Data to Determine Monod
Equation Parameters - Umax ^ ^
 145

Table 1. Summary of Biodegradation Kinetic Parameters of Benzoate.

Parameters Values Units

Number of benzoate degraders (1.4±0.1)xl0' CFU g"' dry soil

Unit cell mass 9.5x10'^ gCFU-'

Amount of moist soil as inoculum 2 g

Water content 10.1 %, (w/w)

Initial biomass concentration (XJ 1.6 mg L-'

Average biomass yield coefficient (Y) 0.65 mg mg"'

Max. specific growth rate (n^ax)

for substrate conc. 0.1~500 mg L ' 2.3 hr'
for substrate conc. 1~30 mg L ' 7.2 hr'
for substrate conc. l~100 mg L*' 9.6 hr'
for substrate conc. 1-500 mg L ' 12.6 hr-'

Half-sauration constant (KJ

for substrate conc. 0.1~500 mg L ' 20.4 mg L"'
for substrate conc. 1~30 mg L ' 75.3 mg L-'
for substrate conc. 1~I00 mg L ' 100.0 mg L"'

for substrate conc. 1-500 mg L"' 131.6 mg L*'

 146

Table 1. Continued.

Initial substrate concentration (mg L ') Specific growth rate, (hr ')
O.Ol 0.0009

0.1 0.011

1 0.095

10 0.79
30 2.44
100 6.67
500 30.2
 147

Table 2. Summary of Biodegradation Kinetic Parameters of 2,4-D.

Parameters Values Units

Number of benzoate degraders (7.3±0.5)xl0^ C F U g - ' d r y soil

Unit cell mass 9.5x10'^ g CFU'

Amount of moist soil as inoculum 2 g

Water content 23.8 %, (w/w)

Initial biomass concentration (XJ 0.073 mg L"'

Average biomass yield coefficient (Y) 0.29 mg mg '

Max. specific growth rate

for substrate conc. 0,01~500 mg L ' 3.5 hr-'

for substrate conc. 1~30 mg L ' 36.0 hr'
for substrate conc. I~100 mg L ' 45.1 hr'
for substrate conc. 1~500 mg L ' 58.1 hr-'

Half-sauration constant (KJ

for substrate conc. 0.01~500 mg L ' 4.2 mg L'
for substrate conc. 1~30 mg L ' 57.4 mg L-'
for substrate conc. 1-100 mg L"' 72.2 mg L"'
for substrate conc. 1~500 mg L ' 93.5 mg L"'
 148

Table 2. Continued.

Initial substrate concentration (mg L*') Specific growth rate, |a (hr ')
0.01 0.0083
0.1 0.075
1 0.62
10 5.54

30 11.4
100 35.1
500 147
 149

higher the cell yield, the more efficiently a substrate is degraded. In other words, benzoate

was degraded more efficiently than 2,4-D. That also means degradation of benzoate yields

more energy than 2,4-D. In general, biodegradation of non-chlorinated compounds will

generate more energy than chlorinated compounds. The more chlorine ions present in the

ring, the lower the cell yield that is produced.

The half-saturation constant (K^) is an important index that describes the affinity of

the microbial population to the substrate. The value is dependent upon the chosen

substrate concentration. For example, the K, value of benzoate at a concentration range

1~500 mg L ' was 132 mg L ', at a range of 1~100 mg L ' was 100 mg L ', and at a range of

1-30 mg L"' was 75 mg L"'. Conversely, the K, value of 2,4-D at concentration range of

1~500 mg L ' was 94 mg L"', at a range of 1~100 mg L ' was 72 mg L ', and at a range of

1~30 mg L ' was 57 mg L"'. Theoretically, the lower the value of K,, the greater the

microorganism's affinity for the specific-substrate and the greater the capacity for

microorganisms to grow rapidly in an environment with low, growth-limiting substrate

concentrations (Lynch and Poole. 1979). The range of the maximum specific growth rate

(jt^) for benzoate, which was from 7 to 13 hr ' (Table 1), was lower than for 2,4-D, which

was from 57 to 94 hr"' (Table 2). The low value suggests a low availability of substrate.

As shown in Table 1 and Table 2, the specific growth rate (ji) increased with the increase

in substrate concentration for both benzoate and 2,4-Dsamples, which is consistent with the

observation obtained by Monod (Monod, 1949).

 150

Effect of Water Content

In addition, the number of benzoate degraders and water content during the

stimulation period are list in Table 3. Low moisture content in soil was associated with a

considerable decrease in benzoate degrader numbers. Figure S clearly shows that the

number of benzoate degraders has a positive correlation with the amount of water in soil.

Actually, some researchers have reported that microorganisms have an optimum value of

water availability at which growth occurs most rapidly (Christian, 1980; Mugnier and Jung,

1985; Walter et al., 1987). As the moisture content is reduced from the optimum value,

there is not only a reduction in the specific growth rate but also a decrease in the maximum

microbial community size (Kangetal., 1969; Harris, 1981; Wheeler, 1988). In other words,

water content plays an important role in controlling the number of microorganisms.

Furthermore, it will affect the biodegradation rate of contaminants in soil.

 151

Table 3. List of Soil Moisture and Viable Plate Count Values During the Stimulation
Period.

Days Moisture Number of benzoate degraders

(w/w, %) (CFU/g dry soil)

0 0.34 (8.8±0.4)xl0-

10 20.7 (2.3±0.4)xl0''

15.5 16.2 (5.0±0.6)xl0'

34 lO.l (1.4±0.1)xl0'
o
CO

1.0E-K)7

•o

a
Q

l,0E+02
0 0.05 0.1 0.15 0.2 0.25

Water Content

Figure 5. Effect of Water Content on the Number of

Benzoate Degraders
 SUBDIVISION 4

BIOAVAILABILITY OF PYRENE IN THE PRESENCE OF

HYDROXYPROPYL-P-CYCLODEXTRIN (HPCD)

Jiann-Ming Wang, Raina M. Maier, and Mark L. Brusseau*

429 Shantz Bldg.

Dept. of Soil, Water, and Environmental Science

The University of Arizona

Tucson, AZ 85721

Prepared for:

Biodegradation

' Convsponding Author

 154

Objective

The objective of this study was to examine the bioavailability of pyrene in a pure

culture in the presence of hydroxypropyl-P-cyclodextrin (HPCD).

Materials and Methods

Materials

Analytical-grade hydroxypropyl-P-cyclodextrin(HPCD) (purity >99%) was supplied

by Cerestar USA, Inc. (Hammond, IN). Pyrene (purity >98%), a four-ring PAH, was

purchased from Aldrich Chemical Co. (Milwaukee, WI). The mineral salts medium

(MSM), specifically Bushnell-HAAS, employed in the biodegradation study, was purchased

from Difco Laboratories (Detroit, MI). Chloroform, used to dissolve or extract pyrene, and

methanol (HPLC grade), used to dilute the extracts of pyrene, were purchased from

Mallinckrodt Chemical Co. Acetonitrile UV, employed as a carrier for HPLC analysis, was

purchased from Burdick & Jackson Inc. (Muskegon, MI). The inoculum used in this study

was Burkholderia sp. isolate CRE 7 (Wang et al., 1998). The VOC vials and the PTFE

septa were purchased from Kimble Glass Inc. (Illinois) and National Scientific Company,

respectively.

Mass Loss of Pvrene

The biodegradation of pyrene was quantified by direct measurement of pyrene loss.

In this experiment, 0.5 mg of pyrene dissolved in chloroform was added to 20-mL VOC
 155

vials. The solvent was allowed to evaporate overnight, leaving a thin coating of pyrene

covering the sides and bottomof the vials. Purified HPCD inBushnell-HAAS broth(10 ml)

was added in lO"* mg L ' concentration. Another set of samples, containing only 10 ml

Bushnell-HAAS broth, was prepared. Both sets of samples were inoculated with 0.2 ml

aliquots of inoculum fi:om late-log precultures to achieve a final cell density of

approximately 10' CFU ml''. One set of samples, containing Bushnell-HAAS broth and

pyrene but no Burkholderia CRE 7, served as a control. Each vial was tightly capped with

a PTFE septum and had a half-volume of head space. The head space provided sufficient

oxygen for the pyrene biodegradation process. Samples were incubated at 200 rpm on a

gyratory shaker (Lab-Line Instruments, Inc., Model 3527, Melrose Park, IL) at room

temperature.

Periodically, a set of duplicate samples was sacrificed and the contents of each vial

were serially extracted three times with 20 ml of chloroform. The three extracts were

combined and concentrated witha Rotavapor Evaporation System (Bttchi Co., Switzerland)

to 1 ml, after which the volume was adjusted to 5 ml with HPLC-grade methanol. Pyrene

was quantified by high performance liquid chromatography (Waters, Milford, MA). HPLC

analysis was performed isocratically using a mobile phase of 15% water(HPLC grade) and

85% acetonitrile UV (HPLC grade) and a reverse phase column, Adsorbosphere UHS C18

5U (Alltech, Deerfield, IL), with i.d. 4.6 mm and length 150 mm. The flow rate was 1.5 ml

min*' and the wavelength used for detection of pyrene was 235 nm.
 156

Results

The results of the pyrene biodegradation conducted in the presence of analytical-

grade HPCD are shown in Figure 1. After 14 weeks incubation, biodegradation of pyrene

in the samples containing IC mg L ' HPCD and Burkholderia CRE 7 was initiated. At the

end of experimental period (24 weeks), 86.5%(w/w) of the pyrene remained in the samples

with 10'' mg L"' HPCD. In contrast, the set of samples without HPCD but with MSM and

Burkholderia sp. isolate CRE 7 maintained the initial amount of pyrene throughout the

entire experiment demonstrating that evaporative or photolytic loss did not occur. These

results suggest that HPCD increased the bioavailability of pyrene. This is supported by the

fact that HPCD has been reported to enhance the solubility of pyrene (Wang and Brusseau,

1993). It is also supported by experimental results that show bacteria have difficulty in

utilizing solid pyrene as a food or energy source because pyrene is essentially insoluble in

water (0.13 mg L"') (Mackay et al., 1992). The long lag phase reflected that the

Burkholderia sp. isolate CRE 7, a very efficient phenanthrene degrader, required time to

adapt to pyrene. This adaptation may have been caused by structural differences between

phenanthrene and pyrene or by toxicity caused by higher dissolved pyrene in the presence

of HPCD. However, once adapted, the degraders achieved a rate of degradation equal to

0.015 mg of pyrene per week. In general, this experiment exhibited that HPCD may have

the efficacy to enhance the biodegradation of high-ring PAHs, such as pyrene.

 -•-1% HPCD and CRE7

" -A-MSMandCRE7.

-A-MSM

- ' • • • I • • • • I • • • • I • • • • I • • • '
0 5 10 15 20 25

Time (weeks)

Figurl. Bioavailability of Pyrene in the Presence of HPCD

 158

SUBDIVISION 5

ADHERENCE OF BACTERIAL CELLS TO HEXADECANE AND

PARTITIONING OF PHENANTHRENE BETWEEN WATER AND HEXADECANE

Contributed to the paper titled as

Biodegradation of Phenanthrene in the Presence of

Non-Aqueous Phase Liquid (Hexadecane)

by

Joong-Hoon Cho^, Jiann-Ming Wang^ Ji-Won Yang^, Raina M. Maier*^

Prepared for:

Biodegradation

* Korea Advanced Institute of Science and Technology

* The University of Arizona

' Corresponding Author

 159

Introduction

In a contaminated soil or aquifer, spilled or leaking fuels and industrial solvents are

often found in the form of non-aqueous phase liquids (NAPLs). The presence of NAPLs

may have positive or negative effects on the biodegradation of hydrophobic compounds.

A NAPL might suppress or inhibit the biodegradation of compounds dissolved within it if

the NAPL was toxic to the bacteria (Alvarez and Vogel, 1991) or the NAPL was

biodegraded preferentially by the bacteria (Pimik et al., 1974). Conversely, a NAPL might

also promote biodegradation of compounds by bacteria. Cometabolism is one way to

enhance biodegradation (Alvarez and Vogel, 1991). Cell adhesion, which is an index of cell

wall hydrophobicity (van Loosdrecht et al., 1987), is used to depict the potential attitude for

bacteria to attach to the interface between the aqueous and NAPL phases. It is a very

important property to determine the relationship between the biodegradation of target

compounds and the presence of NAPLs phase (Rosenberg and Rosenberg, 1981; Zhang and

Miller, 1994).

The properties ofNAPLs, such as solubility, density, viscosity, and structure, might

affect the partitioning of the compounds in a NAPL-water system. The mass transfer rate

and the volume ofNAPLs may have an influence on the biodegradation of hydrophobic

compounds in the presence ofNAPLs (Efroymson and Alexander, 1991; 1994; Carroquino

and Alexander, 1998). One of the major hypotheses in this study was that the mass

distribution of apolar compounds in different phases has a very close relevance to

mineralization, which is used to evaluate the extent of biodegradation. The objectives of

 160

these experiments, which were part of a series of studies, were to examine the adherence of

bacterial cells to hexadecane, to measure the mass distribution of phenanthrene between the

aqueous phase and hexadecane, to test whether the extent of mineralization would increase

with increasing volume of solvent, and to examine the effect of the hexadecane volume on

the distribution of phenanthrene.

In this study, phenanthrene was chosen as a target compound to represent the PAH

compounds and hexadecane was used to represent tiie n-alkane compounds present in

NAPLs. Hexadecane which has a density of 0.773 g mL"', is lighter than water and is

characterized as a light (/)-NAPL. Its viscosity at 25°C is 3.03 centipoise (cP) (Lide, 1997).

The polarity of hexadecane is represented as logP, which is defined as the logarithm of the

partition coefficient of hexadecane in a octanol/water two-phase system. The reported \ogP

value for hexadecane is from 8.8 (Rezessy-Szabo et al., 1987) to 9.16 (Carroquino and

Alexander, 1998). Hexadecane is characterized as a very hydrophobic solvent which is, to

say, insoluble in water (Lide, 1997) or has very low water solubility, about 1.25x10 ' mol

L*', based on the calculation by fitting the logP value (Carroquino and Alexander, 1998).

Materials and Methods

Materials

Hexadecane (purity >99%) and phenanthrene (purity >98%) were purchased from

Aldrich Chemical Co. (Milwaukee, WI). N-[l-'''C]-hexadecane (specific activity, 2.2

mCi/mmol, purity >98%) and [9-''*C]phenanthrene (specific activity, 13.3 mCi/mmol,

 161

reported purity >98%) were purchased from Sigma Chemical Co. (St. Louis, MO).

Chloroform, used to dissolve phenanthrene, and acetone, used to wash out any residual

['••Clphenanthrene in micro-Ferabach flasks (Wheaton, Milville, N.J.) or separator funnels,

were purchased from Mallinckrodt Baker, Inc. (Paris, KY). ScintiVerse BD scintillation

cocktail was purchased from Fisher Scientific Chemical (Fair Lawn, NJ). The mineral salts

medium (MSM) used to culture phenanthrene degrader, consisted of 1.0 g of KH2PO4,1.0

g of Na2HP04, 0.5 g of NH4NO3, 0.5 g of (NH4)2S04, 0.2 g of MgS04-7HA 0 02 g of

CaCl2'2H20,0.002 g of FeClj, and 0.002 g of MnS04'2H20 per liter of distilled water. R2A

agar employed in bacterial cultivation, was purchased from Difco Laboratories (Detroit,

MI). The phenanthrene degrader used in this study was a Burkholderia sp., referred to as

CRE7(Wang,etal., 1998).

Adherence of bacterial cells to C,^

The adherence of ['''CJhexadecane to bacterial cells was measured by radioactivity

assay. For the preparation of the preculture, 5 mg of phenanthrene was dissolved in

chloroform and added to 125-ml flasks. The solvent was allowed to evaporate overnight,

leaving a thin coating of phenanthrene covering the sides and bottom of the flasks. After

10 ml of MSB was added to each flask, a loop of CRE 7 was transferred to it. The sample

was incubated on an orbit shaker (Lab-Line Instruments, Inc., Model 3527, Melrose Park,

EL) at 200 rpm for 3 days at room temperature. Two percent (v/v) of the culture was
 162

transferred again to phenanthrene-coated flasks with 10 ml fresh MSB. The preculture was

ready to use afrer one day of incubation at room temperature at 200 rpm.

Ten ml of MSB solution was addedto a 50-ml glass test tube which had been coated

with S mg of phenanthrene on the bottom. An aliquot of 130 ^l of a mixture of hexadecane

and ['''Clhexadecane was then added to each tube. The specific activity of['"CJhexadecane

in each tube was 1.17 |iCi/mmol. The system was inoculated with 2% (v/v) of preculture

and incubated on an orbit shaker at 200 rpm for 2 days at room temperature.

Cell growth was evaluated by RjA viable plate counts. A 0.1 ml aliquot of 2-days

culture was serially diluted 10-fold. Triplicate plates were prepared for each dilution ratio.

Viable cells were enumerated after two days incubation at room temperature. Tlie

remaining 2-days culture was transferred to a 40-ml glass centrifuge tube and centrifuged

to pellet the cells at a centrifuge (Beckman Instruments, Inc., Model J2-21, Palo Alto, CA)

at 5000 rpm for 10 minutes. After removing the supernatant, the cells were then washed

twice with 10 ml fresh MSB. Vortexing and centrifliging were executed during each wash

procedure. Finally, the washed cell pellet was taken up in 1 ml MSB and placed into a

scintillation vial with 10 ml ScintiVerse BD cocktail. The radioactivity was determined

using a liquid scintillation counter (Packard Tri-Carb Co., Model 1600 TR, Meriden, CT).

Mass distribution of phenanthrene between phases

The partitioningof ['^CJphenanthrene betweenhexadecane (NAPL phase)and MSB

(aqueous phase) was also measured using a radioactivity assay. A O.OS or 0.5-ml aliquot of
 163

a mixtuFe ofphenanthrene and ['''Cjphenanthiene inchlorofonn was added to 12S-ml micro-

Fembach flasks. The solvent, chloroform, was allowed to evaporate, which produced a O.S-

mg (specific activity, 12.62 ^Ci/mmol) or 5-mg (specific activity, 1.23 (iCi/mmol) coating

ofphenanthreneonthebottomofthe flasks,respectively. A 10-ml aliquotofMSB aqueous

solution was then added to each flask. For samples containing O.S mg of radiolabeled

phenanthrene, two different volumes of hexadecane (0.13 and 1.3 ml) were added to the

flasks. For samples containing 5 mg of radiolabeled phenanthrene, three different volumes

of hexadecane (0.13,1.3, and 13 ml) were added to the flasks. Samples were prepared in

triplicate and placed on an orbit shaker at 200 rpm.

After 24 hours of mixing at room temperature, the flush tree method was employed

to capture any potential radiolabeled volatile organic compounds. Samples were then

separated into aqueous and NAPL (hexadecane) phases based on their different densities (1

and 0.773, respectively) in separator funnels. Each separated phase of samples was placed

into a glass vial. For the aqueous phase, a O.S-ml aliquot of sample was added to 10 ml

ScintiVerse BD cocktail. For vials containing 1.3 or 13 ml of a 0.1-ml aliquot of C,6

was removed carefully and placed into a scintillation vial filled with 10 ml ScintiVerse BD

cocktail. For vials containing 0.13 ml of C,6, a 10-ml ScintiVerse BD cocktail was added.

The amount of radioactivity in the hexadecane was determined by the difference in the

sample containing aqueous phase only and the sample containing both aqueous phase and

C,£. The radioactivity was determined using a liquid scintillation counter.

 164

Results

For samples incubated for two days, the total viable count value was (5±0.5)xl0^

CFU per ml of aqueous culture. After washing the cells three times, there was only

0.13±0.03 % of [''*C]hexadecane incorporated in the cell pellets. This suggests that

hexadecane was not taken up by the cell. Thus, the cells, CRE7, still used phenanthrene as

the major carbon source.

Table 1 shows the mass distribution of phenanthrene in different volumes of

hexadecane in the system. For systems with same amount of phenanthrene but with

different volumes of hexadecane, the partitioning ratio increased with an increasing volume

of hexadecane. This result appeared in both 5 mg and 0.5 mg phenanthrene systems. A

higher volume of hexadecane means that more hexadecane molecules exist in the system.

Phenanthrene molecules can better contact hexadecane when there is a larger surface area

of hexadecane available. Therefore, phenanthrene molecules have more of a chance to

partition into hexadecane. For systems with the same volume of hexadecane but with

different amounts of phenanthrene, the partitioning ratio increased with increasing amounts

of phenanthrene. This result was very obvious in the system with 1.3 ml hexadecane and

with both 0.5 mg and 5 mg phenanthrene. In this case, a higher partitioning ratio represents

that a higher amount of phenanthrene molecules had more chance to contact with a fixed

amount of hexadecane. Table 1 also points out that less phenanthrene exists in the aqueous

phase while more phenanthrene partitioned to hexadecane.

 165

Table 1. Distribution of Phenanthrene (C,4H,o) between Aqueous and Organic

(Hexadecane, C16H34) Phases.

Systems % of Phenanthrene Partitioning Ratio

(C,4H,(/C,6H34)
in aqueous phase in C,6 phase
0.5nig/0.13ml 3.81 55.65 14.6
0.5mg/1.3nil 3.12 74.31 23.8
5mg/0.l3ml 3.49 44.26 12.7
5mg/l.3nil 2.28 88.68 38.9
5mg/13ml 1.66 93.60 56.3
 166

In this system, hexadecane was suspended on the surface of the aqueoussolution and

phenanthrene was distributed uniformly on the bottom of the flasks because of the specific

manner in which the flasks were coated. Thus, shaking was an important extemal force

used to facilitate the transformation of phenanthrene to hexadecane. The results also reflect

the fact that in addition to internal mechanisms, which include the properties of hexadecane

and phenanthrene, the amount of phenanthrene and the volume of hexadecane are two other

factors that determine the partitioning ratio of phenanthrene in the system.

 167

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