 

 

’uﬂﬂﬂgﬂsﬂﬁ.‘

x!
‘ ‘x. .9..Vs

a,
I.\

in).

‘
A ”It.
‘7‘ X.

$“a
‘,v.
f” u
., ‘

. l v I
as!"

M‘Z:

.w

 

llfllJJHlllUllllHINHHIHIWJHIHIII fllilllillillil

31293 01417 2260

 

THESIS

“L ‘ LIBRARY
Michigan State 1
University

 

 

 

This is to certify that the

dissertation entitled

t: Homo deocowiocw
\leté
:D‘} . Kai/“0‘s" (Lt/A VM'EKY Tcé
C:ODr-O]§1 Wmf‘Do

presentedby

MMCOS ROW5 F7185

has been accepted towards fulﬁllment .
of the requirements for

PhD degree in C70? “MA SV\\ Q9415

 

 

5 42—95

Date

 

 

 

PLACE ll RETURN BOX to romovo this chockout from your record.
TOAVOID FINES roturn on or bdoro duo duo. '

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU IoAn Afﬂnnctlvo Action/Equal Opportunlty Intuition
Wanna-m

. ___V .—_ _._____..._. -7

MICHIGAN STATE UNIVERSITY
REDDRD OF DISSERTATION AND ORAL EXAMINATION REQUIREMENTS FOR DOCTORAL DEGREE CANDIDATE

We! error a son. sermons

 

 

 

 

 

 

 

and“. um mcos RUBENS nuns ' m Numb“ A11209766
1_ Oieeertstion m; DIVERSITY OF MONO AROIIATIC HYDROCARBOII DEGRADING BACTERIA AND THEIR
ICE CO—OXIDATION POTENTIAL
2. Dieeeertetionhesbeen:_x Accepted .__Rejected _Acceptede:ﬂecttorevietom(beyoMmInoreditorielchengee)
required by the ComrI'IIItee.
3. mmmmammmmmm 6-€.—.:75
The student _X__ Peeeed FeIIed: Reeeon

 

 

 

 

4. DMDQWNWMMWWMmy:

5. Subjecttomeeetlstectorycompletionotmrequirements. misstudenttsrecommendedtorthedegreeboctorot:

  

 

 

__._ Educetion __Musicel Ans
”m... , . m...
I. 1. DR JAMES H. r1932 6’9’75
h
2. 2, DR MICHAEL nus 4-3.15

 

a. /_;t; z 3_ DR STEPHEN BOYD 4.1-3"

D.
4. DR LEE JACOBS gzgés"
on.

 

 

 

 

 

 

 

,. /i

/
7. Revisiorteﬂzltenyepprovedﬁa.‘ %§: ”47/ 6:843,

 

 

 

AseocietelAssietentDeen
DISTRIBUTION:
emery—Dean
Pink-
BIue—GutdenceCouunIttee
. Goldenrod—SIM
mwnmumy

we? USU IS AN AFFIRMATNE ACTMOUAL OPPORTUNITY WSW

DiI arsi

Diversity of Mono Aromatic Hydrocarbon Degrading Bacteria
and Their TCE Co-oxidation Potential.

BY

Marcos Rubens Fries

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Crop and Soil Sciences

1 995

ABSTRACT

Diversity of Mono Aromatic Hydrocarbon Degrading Bacteria
and Their TCE Co-oxidation Potential.

BY

Marcos Rubens Fries

Trichloroethene (T CE) is the most frequently observed volatile organic
contaminant in ground water. Bacteria with certain aromatic mono— or
dioxygenases have the potential to co-oxidize TCE. A shallow anoxic aquifer
was amended with phenol + 02 and then toluene + 02 to determine the
effectiveness of these treatments in removing TCE. We isolated the phenol and
toluene-degrading populations after both treatments from the terminal positive
MPN dilution tubes using phenol and toluene as substrates. A high percentage
of the isolates could grow on both phenol and toluene regardless of the
substrate on which they were isolated . Half of the isolates were also
denitrifiers, which may have been a result of high nitrate and low oxygen in this
aquifer. When restricted genomic DNA from the isolates was probed in
Southern blots with DNA probes encoding genes for the ﬁrst steps of the five
different catabolic pathways for toluene metabolism, we found that the toluene
ortho-hydroxylase pathway was dominant among the isolates . Other organisms
with this pathway are known to be effective TCE co-oxidizers. ARDRA analysis
suggested similar community structures when phenol and toluene were the

primary carbon sources, consistent with the interpretation from analysis of the

dominant
DNA wer
did Isolat
were not
the pepul
were dras

rebound t

dominant isolates. The majority of bands observed in ARDRA from community
DNA were found in ARDRA for the most common isolates, suggesting that we
did isolate the major populations from the site and that unculturable populations
were not important in this study. When 1,1-DCE was injected into the aquifer,
the population of heterotrophs and monoaromatic degraders as well as diversity
were drastically reduced. Once 1,1-DCE was removed the microbial populations

rebound to the original density and composition.

iv

In memory of my grandfather

MILTON

who’s love and support made my career possible

 

The a
Dr. .'
COLR‘SE OI thIS

Drl

committee.

Lead

menge an

ACKNOWLEDGMENTS

The author expresses his sincere thanks to:
Dr. James Tiedje for his patience, guidance, and encouragement throughout the

course of this study and preparation of this dissertation. I could not have a better role model.

Dr. Michael Klug, Dr. Stephen Boyd and Dr. Lee Jacobs members of my guidance
committee.

Leadir, my wife and friend, Milton, Kalynka and Henrique, my beloved children, for their
patience and abnegation expressed during my training program.

My grandparents and family, for the loving support they have given to me.

Fellow lab colleagues, Joanne Ghee-Sanford, Robert Sanford, Jorge Santo Domingo,
lnez Taro-Suarez, Mary An ans, Klaus Nuesslein, Jim Champine, Suzane Thiem. Rick Ye,
Tamara Tsoi, Jim Cole, Jizhong Zhou, Serguei Bezborodnikov, Roberta Fulthorpe, Al Rhodes,
Greg Thom, Cindy Nakatsu, Cathy McGowan, Jong-ok Ka, Yuichi Suwa, Arturo MassoI-Deya,
Frank Loffler, Alenka Pn'ncic, Nanci Tonso, Yuichi Suwa, Amy Cascarelli, Grace Calabrese,
Kirsti for the friendliness they have given to me.

Mrs. Joyce Vlﬁdenthal, David Ham's, Keith Nelson and Hellen Cortew-Neuman for
laboratory help and friendship.

To the CME staff Ann. Lisa, Betty, Annette for their help and assistance.

Dr. Lany Fomey, Dr. Perry McCarty and Dr. Gary Hopkins for participating in this exciting project
and Dr. Wynne Lewis and Dr. Bill Holben for the Gene probe workshop.

To NSF for supporting CME.

The author is indebted to CME, CAPES and Departamento de Solos-UFSM, Brazil, for
providing the opportunity and ﬁnancial aid during the course of his training program and to the
Department of Crop and Soil Sciences-MSU for accepting him into its Graduate Program.

vl

LIST OF TAE.

 

LIST OF FIGLI

Chapter I ......

Introduction.
Overview .....

&ﬂomwm

 

TﬂChIOf0et|
Degradano
US! of Refers

Chapter II .......

modanon 0C(
'"Umuctro
Mmehal ar
Field 58mg
ChemICaI ll
“MEN'S.-.

mpIESf

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................................ x
LIST OF FIGURES ................................................................................................................... xii
Chapter I .................................................................................................................................... I
Introduction .............................................................................................................................. 1
Overview ................................................................................................................................. 1
Background ............................................................................................................................. 3
Sources of aromatic compounds and biodegraders in the environment ................................. 3
Biochemical pathways for aromatic hydrocarbon degradation ............................................... 8
Trichloroethylene biodegradation ........................................................................................ 12
Degradation of mono aromatic hydrocarbons under denitrifying conditions ......................... 22
List of References: ................................................................................................................. 23
Chapter II ................................................................................................................................. 30
Microbial populations of phenol and toluene degraders in an aquifer where successful TCE co-
oxidation occurs ..................................................................................................................... 30
Introduction ........................................................................................................................ 30
Material and Methods ......................................................................................................... 33
Field sample site description ................................................................................................ 33
Chemical introduction into the aquifer-1993
experiments ............................................................................................................................... 38
Samples for microbiological analysis. ................................................................................. 40
Microbial biomass .............................................................................................................. 41
Most probable number (MPN) population estimates ........................................................... 41
Isolation of dominant populations ....................................................................................... 45
Characterization of isolates ................................................................................................ 46
Standard strains ................................................................................................................. 47
Trichloroethylene co-oxidation ............................................................................................ 47
FAME analysis ................................................................................................................... 48
Molecular methods ............................................................................................................. 48
Analytical methods. ............................................................................................................ 52
Results .................................................................................................................................. 52
Population densities ........................................................................................................... 52
Isolation of populations ....................................................................................................... 57
Discrimination of isolates by REP-PCR analysis ................................................................. 58
Isolate distribution .............................................................................................................. 59
FAME/taxonomic and degradative diversity analysis .......................................................... 59
Diversity of toluene degradation genes ............................................................................... 70
TCE co-oxidation rates ....................................................................................................... 71
Discussion ....................... 8O
Acknowledgments .................................................................................................................. 84
List of References .................................................................................................................. 84
Appendix A ............................................................................................................................ 89
Appendix B ............................................................................................................................ 91

vii

  
 

Chapter III .

Chapter Iv . _ _.

Natumr SEIE
OXIUaiIOn'
‘merCIIOr
Material 3W

FIEId San

Mammal
031 mm
FAME-“
ISOIate Cf
MO"acme

AnaIYIICa
Resins .

Chapter III .............................................................................................................................. 100

Microbial succession during a ﬁeld evaluation of phenol and toluene as the primary substrates

for TCE co-oxidation. ........................................................................................................... 100
Introduction .......................................................................................................................... 100
Material and Methods .......................................................................................................... 102
Field sample site .............................................................................................................. 102
Field treatments and sampling ......................................................................................... 102
Analysis of microbial biomass and extradion of total microbial community DNA ............... 102
Species diversity index ..................................................................................................... 103
FAME ............................................................................................................................... 103
Molecular methods ........................................................................................................... 104
Analytical methods ........................................................................................................... 106
Results ................................................................................................................................ 107
Succession determined from studies of isolates ................................................................ 107
Comparison of the microbial groups from different aquifer samples .................................. 114
Community composition determined by ARDRA ............................................................... 117
Discussion ...................................................................................................................................... 124
Acknovuedgments ................................................................................................................ 128
List of references ................................................................................................................. 129
Chapter IV .............................................................................................................................. 132
Natural selection of denitriﬁers at a ﬁeld site amended with phenol and toluene for TCE co-
oxidation .............................................................................................................................. 132
Introduction .......................................................................................................................... 132
Material and Methods .......................................................................................................... 135
Field sample site .............................................................................................................. 135
Microbial strains ................................................................................................................ 135
Most probable number of heterotrophs and denitriﬁers ..................................................... 135
FAME ............................................................................................................................... 137
Isolate characterization .................................................................................................... 137
Trichloroethylene co-oxidation under aerobic versus anaerobic denitrifying conditions ..... 138
Molecular methods ........................................................................................................... 138
Analytical methods ........................................................................................................... 139
Results ................................................................................................................................ 140
Heterotrophic and denitrifying population densities ........................................................... 140
Denitriﬁcation products in MPN tubes... ........................................................................... 140
Characteristics of an unusual NO-producing denitriﬁer, MF-18 ......................................... 143
Strains that degrade toluene and phenol under denitrifying conditions .............................. 149
Discussion ........................................................................................................................... 151
Acknowledgments ................................................................................................................ 156
List of References: ............................................................................................................... 156
Chapter V. .......................................................................................................................... 161
Isolation, characterization, and distribution of denitrifying toluene degraders from a variety of
habitats ................................................................................................................................ 161
Materials and methods ..................................................................................................... 162
Enrichments and isolations ............................................................................................... 162
Characterization of isolates .............................................................................................. 163
Molecular methods ........................................................................................................... 163
Analytical methods ........................................................................................................... 164
Nucleotide sequence accession numbers .......................................................................... 164

viii

Results
Ennchm'
Charade

DISCUSSIori

Aclrnoweri;~

References

 

 

Results ................................................................................................................................ 164

Enrichments ..................................................................................................................... 164
Characteristics. ................................................................................................................ 165
Discussion ........................................................................................................................... 167
Acknowledgments ................................................................................................................ 169
References .......................................................................................................................... 169

ix

Table

Table 1.1. Ma.

->..
~~~~~

LIST OF TABLES

Table ’ Page
Chapter I
Table 1.1. Major genera of hydrocarbon degrading microorganisms ........................................... 6
Chapter II
Table 2.1. Time course of the four (I-IV) ﬁeld experiments showing the concentrations of
chemicals added to injected water and sampling schedule of glass beads. ......................... 39
Table 2.2. List of DNA probes used in this study. ..................................................................... 50

Table 2.3. Cellular, physiologic and genotypic properties of all isolates with different REP
patterns obtained from the Moffett Field aquifer, from the four sampling times. .................. 65

Table 2.4. Distribution of main characteristics observed for the 63 isolates with different REP
patterns from Moffett Field. ................................................................................................ 69

Table A1. Total number of heterotrophs , toluene, phenol and TCE degraders recovered using
different concentrations of substrates, and present on three different samples from the
Moffett Field aquifer ........................................................................................................... 89

Table 8.1. Original data on all isolates from Moffett Field. Isolates are ananged according to
common REP-PCR groups, source of isolate, and use of toluene and phenol are also shown.
........................................................................................................................................... 91

Chapter III

Table 3.1. Number of unique ARDRA bands for the indicated sample relative to the total bands
in that sample. The template DNA was extracted from glass beads sampled after three ﬁeld
treatments. ....................................................................................................................... 123

Table 3.2. Number of common ARDRA bands obtained from pure cultures thought to be the
most dominant members of the community compared to the ARDRA bands obtained from
DNA extracted from glass beads of samples I, II, III and IV. The number of bands seen in
community DNA but not seen in isolates tested is shown in parentheses. ......................... 125

Chapter IV
Table 4.1. Number of denitriﬁers and heterotrophs observed in a sample that is on the path of
carbon flow (ﬁlter) compared with a sample from the aquifer (sand) that has not been
subjected to treatments at the Moffett Field site ................................................................ 141

Table 4.2. An example of diversity of denitriﬁcation products in MPN tubes from a Moffett Field
ﬁltered water sample. ....................................................................................................... 142

Table 4-1 Cha
heme-Type

Table 4.4. Cha
atmow-mere

Table 4.5. 099'

Table 1. L151 0f

Table 2. SOUme

degradation
under denItr
sample .......

Table 3. Remox
strains in BE
Incubation...

Table 4. Summ.
degraders .........

Table 4.3. Characteristic denitriﬁcation products of Moffett Field isolates, hybridization to the
heme-type nitrite reductase gene probe, and capability of toluene or phenol degradation

under denitrifying conditions. ............................................................................................ 144
Table 4.4. Characteristic denitriﬁcation products of isolate MF-18 when grown under different
atmospheres ..................................................................................................................... 148
Table 4.5. Degradation of toluene under aerobic and anaerobic-denitrifying conditions and
percent removal of TCE by toluene—degrading denitriﬁer isolates from Moffett Field ......... 150
Chapter V
Table 1. List of DNA probes used in this study ........................................................................ 154
Table 2. Source of inoculum, number of enrichments with positive activity for toluene
degradation
under denitrifying conditions, and number of isolates obtained with this activity from each
sample .............................................................................................................................. 165

Table 3. Removal of different substrates by toluene-degrading isolates and well-known aerobic
strains in BS medium under aerobic and anaerobic (denitrifying) conditions after 2 weeks of
incubation ......................................................................................................................... 166

Table 4. Summary of characteristics of the different denitrifying toluene
degraders ................................................................................................................................ 166

xi

Figure

Figure 1.1

bI0r9r

Figure 1.2.
IITSI Str

Figure 1.3.
Methar
WaCke
I992)...

Figure 2.1. (

and New

degraders

FIQUre 2 3b N
' I

0Omalnlng
Samples tar

LIST OF FIGURES
Figure P890
Chapter I
Figure 1.1. Relationship of frequency of biodegraders in the community to application of
bioremediation (T iedje, 1993). .............................................................................................. 7

Figure 1.2. Pathways for the aerobic metabolism of toluene and available DNA probes for the
ﬁrst step. .............................................................................................................................. 9

Figure 1.3. Proposed aerobic pathways for TCE cD-metabolic biotransfonnation. (a)
Methanotrophic bacteria (Little et al., 1988); (b) M. trichospon’um 083D (Newman and
Wackett, 1990); (c) Pseudomonas putida F1, toluene dioxygenase system (U and Wackett,
1992) .................................................................................................................................. 15

Chapter II

Figure 2.1. Cross section viewof Moffett Field test site (modiﬁed from Roberts et al.,
1990) ................................................................................................................................. 35

Figure 2.2. Experimental protocol for the determination of the Most Probable Number of toluene
and phenol degraders, TCE co-oxidizers, total viable heterotrophs and for isolation of the
most dominant populations from the aquifer. ...................................................................... 42

Figure 2.3a. Effects of toluene and phenol concentration on total number of toluene and phenol
degraders detected by MPN on glass beads from aquifer well. The total number of
heterotrophs present in the same sample was also determined by MPN. ............................ 53

Figure 2.3b. Number of TCE cD-Dxidizers and primary substrate degraders estimated from vials

containing toluene or phenol as primary carbon sources. Data are from three different
samples taken from the aquifer at the ﬁrst sampling period. ............................................... 55

xii

Figure 2
th
left.

CIUS‘.
Figure 2 I

Figure 2 e
hybnc
Sp. JS

Flame 2]
dlﬂerer
was the

Source 6

Figure 2.4. REP-PCR ﬁngerprint patterns of Moffett Field isolates generated by using genomic
DNA or whole cells. Outside lanes show DNA size markers indicated (in base pairs) on the
left. Strains are ordered by similarity in band patterns according to the Ambis System
clustering program. ............................................................................................................ 60

Figure 2.5. Frequency distribution of isolates according to REP analysis. ................................. 64

Figure 2.6. RFLP proﬁles of genomic DNA from Moffett Field isolates digested with EcoRt and
hybridized with the gene probe encoding for the toluene ortho-hydroxylase of Pseudomones
sp. JS—150 .......................................................................................................................... 72

Figure 2.7. Pattern of toluene consumption (ﬁlled symbols) and TCE removal (open symbols) by
different strains representing the ﬁve known aerobic toluene degrading pathways. Toluene
was the primary carbon source. .......................................................................................... 75

Figure 2.8. TCE co-oxidation patterns of selected isolates. Toluene was the primary carbon
source and was totally consumed by all strains at 120 hours. ............................................. 77

Figure 2.9. TCE cD-oxidation proﬁle of selected isolates that show fast toluene degradation
rates ................................................................................................................................... 78

Figure 2.10. Distribution of TCE co-oxidation ability by Moffett Field isolates with or without
homology to the ortho-hydroxylase gene (TOM) canted by Burkholden'e cepecr'a strain G-4.
Evaluation at 15 days of incubation. ................................................................................... 79

Chapter III

Figure 3.1. (A) Populations of total heterotrophs, phenol and toluene degraders and TCE co-
oxidizers obtained from glass beads sampled following each of the four indicated
treatments, I, II, III, III. For the initial sample, the populations of toluene and phenol
degraders were obtained with 50 ppm carbon substrate. Populations for samples II, III, and
IV were obtained with 25 ppm of carbon substrate. (B) Species richness index calculated as
suggested by Pielou (1975) considering isolates from glass beads with different REP
sequences as different ”species”. .................................................................................... 108

xiil

Figure 3.2.
chrome:
for pane
indicate

Figure 3.3. 1
levels or

isolated

Figure 3 4. [
XXX wer

ﬁgure 35, A
aQuifer.
(B). and ,
stained w
IAQ: GB)
WEI (Fir
bfields fro

Figure 3.2. REP-PCR ﬁngerprint patterns of Moffett Field isolates generated by using
chromosomal DNA. Isolates that were observed to repeat overtime were run side-by-side
for pattem comparisons. Lanes 1, 15. 16 and 28 are size markers, with the base pairs
indicated on the left. ......................................................................................................... 111

Figure 3.3. REP genotypes persistent during the course of the experiment. The population
levels of each isolate is estimated using the most dilute tube from which the strain was
isolated. ........................................................................................................................... 113

Figure 3.4. Dendogram based on the fatty acid proﬁles of the isolates. FAME groups I through
)00( were deﬁned at a Euclidian distance of 20 (r m= 0.86) .............................................. 115

Figure 3.5. ARDRA analyses of aquifer community DNA and of pure cultures dominant in the
aquifer. The PCR ampliﬁed 16S rRNA gene product was digested with Hpe II (A), Hae III
(B), and Seu3A (C), electrophoresed on a 0.5XTAE (WN) NuSieve (FMC) agarose, and
stained with ethidium bromide. The lanes show PCR products from aquifer glass beads
(AQ. GB) of sample I, cells retained on the 0.22 pm ﬁlter (Filter 1) and cotton ﬁber ﬁltered
water (Filter 2) from sample I, glass beads from sample III (phenol amendment), glass
beads from sample IV (toluene amendment) and the indicated pure cultures. ................. 119

Chapter IV

Figure 4.1. Distribution among the 63 REP-PCR groups of the (a) characteristic denitriﬁcation
products. (b) capability to degrade phenol or toluene under denitrifying conditions and (c)
homology to the heme type nitrite reductase gene probe. . ............................................... 146

Figure 4.2. Cluster analysis of FAME data from toluene-degrading denitriﬁers from Moffett Field
together with well characterized Azoancus strains from different geographic regions

(rw=0.88) ......................................................................................................................... 152

Chapter V

Figure 1. REP-PCR ﬁngerprint patterns of toiuene-degrader denitriﬁer isolates generated by
using chromosomal DNA. Lanes 1 and 11 show size markers, with the base pairs indicated
on the left. ........................................................................................................................ 165

xiv

Figure 2. Ph
Isolate T
3 and To

 

Figure 3. So.
digested
heme nitr
(D). Hybr
Conditions

Figure 4. Phy
00'3“)qu
and Footer
0mildew,

Sequence:

Figure 2. Phase-contrast photomicrographs of isolates Td-2 (A). Td-3 (B), and Td-17 (C).
Isolate Td-2 was grown on BS-toluene liquid medium to late exponential phase. Isolates Td-
3 and Td-17 were grown on M-R2A solid medium for 48 h. Bars, 14 pm .......................... 167

Figure 3. Southem hybridization of genomic DNA from pure cultures of toluene degraders
digested with ECORI and hybridized with the following gene probe: Universal 235 rRNA (A),
heme nitrite reductase (B), toluene ortho-hydroxylase (C), and toluene meta-hydroxylase
(D). Hybridizations were done under high (A and B) and Iovv (C and D) stringency
conditions. Size markers (in base pairs) are indicated on the left. .................................... 168

Figure 4. Phylogenetic position of the toluene-degrading denitriﬁer (T d) isolates. This tree was
constructed by using the programs SEQBOOT, DNAPARS, and CONSENSE in PHYLIP 3.5
and rooted by reference to E. coli. The numbers under the nodes are the bootstrap
conﬁdence estimates on the branches in 100 replicates. All other 168 rRNA gene
sequences were obtained from the Ribosomal Database Project. ..................................... 169

XV

Thr
Hazardous
Microbial E
Research I
Environmen
PIOQram we
EHhanCe i
groundWEIEr

mnstltutivery

Chapter I

Introduction

Overview

Through the combined efforts of researchers within the Western Region
Hazardous Substance Research Center at Stanford University, the Center for
Microbial Ecology and the Great Lakes Mid Atlantic Hazardous Substance
Research Center at Michigan State University, and the EPA Gulf Breeze
Environmental Research Laboratory and associated collaborators a research
program was initiated in 1993, for the field scale study of bioaugmentation to
enhance and improve the in-situ bioremediation of TCE contaminated
groundwater. The organism proposed for bioaugmentation is Pseudomonas
cepacia G4 PR1, the nonrecombinant derivative of P. cepacia G4 that
constitutively expresses toluene ortho-monooxygenase (T OM), and is highly
effective in TCE degradation (Shields and Reagin, 1992).

This research was part of the main project and describes research
objectives aimed at answering fundamental questions regarding genotypic and
phenotypic patterns of natural biodegrading populations as well as to evaluate
community and population changes when the primary carbon source stimulating

the TCE degradation is switched from phenol to toluene. The general goal of

the isola
important
recognize
communit
dominatec
high level
determinin
were addn

group and

2

the isolate portion of the study was to understand which populations are the
important competitors of G4, what activity they have on TCE, and to learn how to
recognize, and differentiate them from G4 in the ﬁeld. The goal of the
community analysis portion of the study was to determine which populations
dominated the competition in the ﬁeld study. I also investigated if the natural
high levels of nitrate present in the low 02 aquifer water played a role in
determining which populations of denitriﬁers were selected. These objectives
were addressed in laboratory studies using samples collected by the Stanford

group and sent to MSU.

Overall this study was designed to answer the following questions:
Fundamental properties:
i. What portion of the C-selected phenol degraders also degrade toluene and vise-versa?
ii. How effective are the selected indigenous populations at TCE degradation and how does their
TCE oxidizing capacity compare to that of the well-studied strains, specially Pseudomonas
cepacia G-4'?
iii. What is the extent of diversity among the selected toluene and phenol degrading strains as
judged by REP, FAME, and biochemical methods?
iv. What is the effect of anoxic conditions on TCE co-oxidation?
Ecology of the site:
i. What is the biological limit (population size) of phenol and toluene degraders when compared
to the total heterotrophic population?
ii. How does the community structure respond to a perturbation, i.e., the change in the primary
carbon source from phenol to toluene?
iii. ls dominance maintained during the ﬁeld experiment, i.e., do the same populations repeat
over time? ‘
iv. What is the role that denitriﬁcation plays in the ecology of this site? Does denitriﬁcation
confer a competitive advantage to certain microbial populations?

 

 

Sources 0

enter oxic

rllimary anc

 

have been

Structural pl
against inve
compounds,
Including I‘ly
hydroxybenz
complex We
(Harbome! 1

from 00n

3

Background

Sources of aromatic compounds and blodegraders In the environment.

Aromatic compounds from natural and anthropogenic sources readily
enter oxic or anoxic environments. Aromatic structures are found in many
rpimary and secondary plant metabolites and several thousand plant phenols
have been described (Harbome, 1980). They are an integral part of the
structural plant matrix, serve as ﬂower pigments, act as constitutive protection
against invading organisms, function as signal molecules, act as allelopathic
compounds, and affect cell and plant growth. Plants produce simple phenols,
including hydroxyquinone, gallic acid, salicylic acid, protocatechuic acid, and p-
hydroxybenzoic acid; more complex phenols, such as the ﬂavonoids; and
complex phenolic polymers, including Iignins, catechol, melanins, and flavolans
(Harbome, 1980). Phenolic units may also originate through microbial synthesis
from non aromatic carbon and energy sources. Numerous phenolic and
hydroxybenzoic acid compounds are synthesized by fungi and other
microorganisms. Cultures of Hendersonula toruloidea (Martin et al, 1972) and
Stachybotrys atra, Stachybotrys charfarum and Epicoccum nigrum (Martin and
Haider, 1969) synthesize dark colored, humic acid-type substances when
growing in glucose medium. They are formed in the culture media, in the cells,
or both, and have been referred to as Iignin-Iike because they resist degradation
in 72% sulfuric acid, as melanins because they are dark colored, or as humic

acids because they are soluble in alkali, precipitated by acid and have high

exchange
Compoun

indudedr

Although
the origin
vanous sc
such dep
toluene, e
anbnacenr
Consumed
1.5 million
“”PIOtecter
DOIential f,
P8troleUm
modem env
Due I.

IS not SUrpn-

4

exchange capacities (Martin and Haider, 1969 and Martin et al, 1972).
Compounds which could be separated from glucose -based culture medium
included phenolic acids and hydroxylated toluenes (Martin and Haider, 1969).

Aromatic structures are also present in large amounts in fossil fuels.
Although the detailed composition of crude petroleum deposits depends upon
the origin and location of the petroleum, considerable similarities exist among
various sources. Aromatic compounds comprise more than 50% by volume of
such deposits and include homocyclic aromatics, both nuclear (benzene,
toluene, ethylbenzene, xylenes) and polynuclear (naphthalene, phenanthrene,
anthracene) (Cole, 1994). About 65% of the petroleum used as a fuel is
consumed as gasoline, which is stored primarily underground in an estimated
1.5 million storage tanks. Almost all of the tanks installed prior to 1988 were
unprotected steel underground storage tanks that have leaked or have the
potential for leaking gasoline into the environment (Cole, 1994). Thus,
petroleum products have become an increasingly common substrate in the
modern environment.

Due to the ubiquitous nature of aromatic hydrocarbons in the environment,
is not surprising that microorganisms have evolved with the ability to degrade
these compounds. This activity can be considered as part of the normal process
of the carbon cycle. Bacteria and fungi are dominant members of most soil
communities and representatives of both groups are capable of hydrocarbon

degradation (Bossert and Bertha, 1984). The ability to degrade and/Dr utilize

hydrocarb I

Compilatlo

 

presented
genera fou
hydrocarbo
and 31 gen
Based,
the most frr
soil enviror
Bacillus, F
Among the
599. are ((-
are the m!
ﬁequehtly
VersUs fUI
EX‘lensiVel

hydrocarbon substrates is exhibited by several bacterial and fungal genera.
Compilation of reported genera for hydrocarbon degrading bacteria and fungi is
presented in Table 1.1. Hydrocarbon-degrading bacteria are represented in 25
genera found in the marine environment and 29 genera found in soil. Similarly
hydrocarbon-degrading fungi are found in 25 genera from marine environment
and 31 genera found in soil.

Based on the number of times that each genera was reported in this survey,
the most frequently isolated hydrocarbon degrading bacteria in both marine and
soil environments are Achromobacter, Acinetobacter, Alcaligenes, Arthrobacter,
Bacillus, Flavobacterium, Nocardia, Pseudomonas spp. and the coyneforms.
Among the fungi, Aureobasidium, Candida, Rhodotorula, and Sporobolomyces
spp. are the most common marine isolates and Tn'coderma and Mortierella spp.
are the most common soil isolates. Aspergillus and Penicillium spp. have been
frequently isolated from both environments. The relative contribution of bacteria
versus fungi in the biodegradation of hydrocarbons in nature has not been
extensively studied and may be a function of the ecosystem and local
environmental conditions. In a comparative study of hydrocarbon degradation by
bacteria and fungi in a sandy loam soil, 82% of the n-hexadecane mineralization
was attributed to bacteria and only 13% was attributed to fungi (Song et al.,
1986).

The fraction of monoaromatic (phenol and toluene) biodegraders in nature

is estimated to be in the order of 1 per every 100 total heterotrophic bacteria

Table 1.1. Major genera of hydrocarbon degrading microorganisms'.

 

 

 

 

 

 

Becterls W
III Germ b m '1‘
Cohen Sol “:43... 3°“ Breoldeh water
ureter . environment.
envirorunent
AW 4 3 Amnonlum 2
Acherobecter 3 4 Aleecherle 1
Actlnomyeee 2 Aspergillus 3 4
Amen. 2 Aureobeeldkrnr 2 3
Alceilgenee 3 3 Beeuverie 1
Arthrobecter 7 5 Botryde 1 2
Bacillus 2 5 Candide 2 5
Beneclree 1 CW 1
Brevlbectedtun 1 1 W 1
Chmobecterkn 1 Cledoeporiun 1 2
Corynebectertwn 5 Cochliobolue 1
Coryneronne 7 W 1
CW 2 Cytindmcupen 1
Envinle 1 1 Marines 1 2
FIevobecterknn 5 6 PM 1 1
m 1 aeobdchum 1
Lectobeclllue 1 mm 1
Leucothrlx 1 Darya-Hunt 1
W 3 0mm 2
male 1 I-I-Ieenuie 1
3 WWII 1
Nocardla 3 6 Manhole 1
Peptecoccm 1 lonlﬂe 1
Protein 1 W 3
Peeudom 12 8 Racer 1
Serelne 2 1 Oldiodendnun 1
Sell-rude 1 Peeerlomycee 1 1
Spherotﬂue 1 Penicillium 4 4
SplrIIIuIn 1 1 Phlelophora 2
Streptomyeee 1 1 Prime 1
We 1 7 Rhodapormun 2
mummies 2 1 Rhodotorule 1 4
Secchuomycee 2 2
mm 1
Seekcobeeldlum 2
Scopulclopele 1
Splccie 1
Sporobolornyoes 1 4
Sproulchum 1
Telypocledlrnn 1
Terulopeie 1 1
Madeline 6 1
THchoepomn 2
Verticilllum 2

 

'Dara compiled by Bossert, I., and Bertha, R., (1984) for soils and by Floodgate, e. D.,
(1 984) for marine and brackish water environment.

' Number of citations for each genera in different publications. There were 19
publications for marine and brackish water and 20 publications for soil data.

(Figure 1.1

 

compound:

microbial 9

Frequency

 

Example

APPlicaIIDI

:Mrch

FIgUl’e 1

7
(Figure 1.1.). Therefore, the focus for remediation technologies for this class of
compounds should be on insuring that the environmental conditions do not limit

microbial growth (T iedje, 1993).

 

 

memy Widespread Common Rte Urknown
>rrro2 1110‘ 11108 1110"
a a
Phenol W NTA am Nodortm
Examrlb Toltlne 2,4-o mm.» ﬁuorene TcDD
Tine to Application

4 >

Appljcdjon Short-range Long-range Explaetory

FoeusofReeelshorEvelustionbnportantbAppliostion

 

RWCII Envkorlnentel Organilnsl Moleouk
Focus

Figure 1.1. Relationship of frequency of biodegraders in the community to
application of bioremediation (Tiedje, 1993).

 

aromatic ht,

  
  

in general
1994). Dun
benzene rln
attack or ri'
hydr0xyl gr
PIOduce a
dlhydroxyb.

IIIUStfated f

Biochemical pathways for aromatic hydrocarbon degradation

The aerobic biochemical mechanisms by which microorganisms utilize
aromatic hydrocarbons have been studied in great detail in the last decades and
in general can be sub-divided in three parts (reviewed by VVIlliams and Sayers.
1994). During the ﬁrst set of reactions, substituent groups are introduced on the
benzene ring making possible alternative modes of biodegradation by side chain
attack or ring attack. For almost all aromatic substrates, the introduction of
hydroxyl groups onto the substrate is accomplished by mono or dioxygenases to
produce a dihydroxyaromatic metabolite, most commonly catechol (1,2-
dihydroxybenzene) or protocatechuate. The versatility of these reactions is best
illustrated for toluene metabolism (Figure 1.2.).

The formation of compounds carrying two hydroxyl groups seems to be an
important biochemical strategy to destabilize the chemically stable resonant
structure of the aromatic ring in order to facilitate its subsequent opening
(Dagley, 1986). The second set of reactions involves the opening of the
catechol ring and is accomplished by dioxygenases which break one of the
carbon-carbon bonds of the ring adjacent to the hydroxyl substitution by the
addition of molecular oxygen, producing an unsaturated aliphatic acid. One of
two different cleavage enzymes can be responsible for ring attack. An intradiol
(ortho) dioxygenase which produces cis-cis muconate or an extradiol (meta)
dioxygenase that yields 2-hydroxymuconate. Depending on the initial ring

substitutions, analogous products can be observed at this stage. After ring

 

 

I I .l.‘ n C “:10 ‘ I
U
IO.>DD.0 at: \ I so 321
i 3:02e0 o.o( c.0330 nausea-sign .2321 inﬁel

lololo

2
I0 2000 010 IOQIU

a... E: a: B. .82.. $3 3.3.3.. .23 2.3.2 .o 5.32% 032... 2.8.1.3.... .3 me o.

.85....- Essie-den...
.liueevolsxoeteszéucxot Tagged

.Iiusa..8s.§§o528:32a .oxastageeioged
.85. o§.83§oi§o§3a8£ 5235.33.35.36

72.8380 .8: 352.35.32.53 {ceases-8.82.3.6
.iiscgogigi :23... .55.. «3533885234

3.9.0.0

..:...\.e. ,M
52/..waA 50

 

v.03

 

 

cleavage.

 

compounds

Of majr
Toluene, Ihr
attack and .
degradatlor
to tricarbo;
reactions 3
A for the c
collectively
pathway e
(Plasmid p.
°deations
benZDate \

IOIIOMd b)

10

cleavage, a third set of enzymes converts the products to small aliphatic
compounds which can directly enter central metabolic routes.

Of major interest to this project, is the degradation of toluene and phenol.
Toluene, the simplest of these substituted benzenes, is biodegraded by both ring
attack and methyl group hydroxylation. There are ﬁve elucidated pathways for
degradation of toluene and they share similar reaction sequences from catechol
to tricarboxylic acid cycle intermediates, but each has a unique series of
reactions and distinctive intermediates, prior to catechols (Figure 1.2.). Pathway
A for the degradation of toluene is almost always encoded by large plasmids
collectively called the TOL plasmids (Assinder and Vtﬁlliams, 1990). The
pathway encoded on the TOL plasmid of Pseudomonas putida strain mt—2
(plasmid pWWO) involves the conversion of toluene to catechol by sequential
oxidations of the methyl group, through benzyl alcohol and benzaldehyde to
benzcate which is then converted to catechol in two steps, a dihydroxylation
followed by a dehydrogenation I decarboxylation. Catechol is then degraded by
enzymes of a meta cleavage pathway to 002, acetaldehyde and pyruvate
(Worsey and \erliams, 1975; Kunz and Chapman, 1981).

Pathway B, in Figure 1.2., characterized in Pseudomonas putida F1 by
Gibson and co-workers, is chromosomally encoded and involves a
multicomponent enzyme system, designated toluene dioxygenase, that
incorporates two atoms of molecular oxygen directly into the aromatic nucleus to

produce cis-toluene dihydrodiol (Zylstra and Gibson, 1989). The further

metabolisr
form 3-me
6—oxo-2,4-
dienoate

metabolite
Gibson, 1!
Three par
groups by
OI Pseudc
hIrdroxylar
Pitydroxy;
co”Wale r
Pamway t
PSeUdOmc

al, 1994).

pOSIIIOn O

11

metabolism of cis-toluene dihydrodiol involves a dehydrogenation reaction to
form 3-methylcatechol and meta cleavage at the 2,3 position yields 2-hydroxo-
6-oxo-2,4-heptadienoate , which is further metabolized to 2—hydroxypenta-2,4-
dienoate and acetate. Further reactions in the pathway, eventually yielding
metabolites that enter the TCA cycle, have not been investigated (Rogers and
Gibson, 1977).

Three pathways have been elucidated involving the incorporation of hydroxyl
groups by ring attack. Pathway C in Figure 1.2., was found to be characteristic
of Pseudomonas mendocina strain KR. In this pathway, toluene is initially
hydroxylated to p-cresol, followed by sequential oxidation of the methyl group to
p-hydroxybenzoate, which is hydroxylated to form protocatechuate and then
complete dissimilated by an ortho cleavage pathway (Whited and Gibson,1991).
Pathway D in Figure 1.2., was detected in Burkholden‘a pickettii PKO1 (formerly
Pseudomonas pickettir) and also has a ring hydroxylation mechanism (Olsen et
al, 1994). However, in this case hydroxylation of toluene occurs at a different
position on the aromatic ring to produce m—cresol. A subsequent hydroxylation
of meta cresol by a separately inducible enzyme yields 3-methyl catechol which
is completely mineralized by the meta ring cleavage pathway (Kukor and Olsen,
1 991). Pathway E in Figure 1.2., was ﬁrst demonstrated in Burkholden‘a cepacia
strain G4 (formerly Pseudomonas cepacia). In this strain , a nonspeciﬁc

monooxygenase hydroxylates toluene by two successive monooxygenations,

12

forming ﬁrst ortho-cresol and then 3-methyl catechol, which is further degraded

by enzymes of a meta cleavage pathway (Shields et al., 1989).

Trichloroethylene biodegradation

For many years, it was held that chlorinated aliphatic hydrocarbons
(CAH’s) could only be degraded anaerobically. In 1985, however, leon and
Wilson presented some of the ﬁrst evidence of aerobic degradation of TCE in
soil enriched with natural gas (77% methane). After their work, aerobic
degradation of CAH’s, such as trichloroethylene, has been widely demonstrated,
initially using methanotrophic bacteria. Unlike anaerobic degradation which can
produce toxic by-products such as vinyl chloride (Vogel and McCarty, 1985), the
products produced aerobically are thought to be relatively harmless for other
organisms (Little et al., 1988; Oldenhuis et al., 1989; Tsien et al., 1989). Many
studies have demonstrated that TCE can not be used as sole energy source by
microbial consortia or pure cultures leading to the suggestion that aerobic
degradation of TCE is a “co-metabolic” process. A deﬁnition proposed for
cometabolism by Dalton and Stirling (1982), states “ the transformation of a non-
growth substrate in the obligate presence of a growth substrate or another
transformable compound ". This deﬁnition however implies the concomitant
presence of a growth substrate and does not contemplate transformations that

occur after depletion of the primary growth and/or energy substrate.

13

Methanotrophs are bacteria that can oxidize methane for energy and
growth. In the conversion of methane to methanol, the initial oxidation is
catalyzed by the methane monooxygenase (mmo) enzyme system. Several
investigators have examined the degradation of TCE by this group of
microorganisms. A methanotrophic mixed culture transformed 1‘C-labeled TCE
to C02, cell biomass and nonvolatile or nonchlorinated compounds. Acetylene,
a known inhibitor of mmo activity, inhibited degradation indicating that methane
oxidizing bacteria probably initiated TCE oxidation (Fogel et al., 1986).

Working with a pure culture, strain 46-1, Little et al. (1988) demonstrated
degradation of TCE only when growth was in the presence of methane. These
studies were conducted using liquid cultures in inverted serum bottles.
Degradation stopped when methane was depleted and continued if additional
methane was added. These researchers concluded that TCE degradation is a
cometabolic process that provides little or no beneﬁt to methanotrophs because
strain 46-1 initiated the degradation of TCE but was unable to metabolize the
intermediates. Preliminary evidence indicated that glyoxylic acid and
dichloroacetic acid were the breakdown products. They also proposed a
mechanism of degradation, which is presented in Figure 1.3(a) and
hypothesized that TCE is first converted to its epoxide, which breaks down
spontaneously, yielding dichloroacetic acid, glyoxylic acid, fonnate and carbon
monoxide. The end products are completely metabolized to CO; by

heterotrophic bacteria in mixed cultures.

 

sy:

mo

Net
ln'cl
mon
hydr
trichl
Little
and

Pseui

Itducr

substr

14

Henschler et al. (1979) evaluated TCE-epoxide reactivity in aqueous
systems and found that the epoxide decomposes to form formate, carbon
monoxide, glyoxylic acid and dichloroacetic acid. The amount of each product
was pH dependent and at lower pH’s, fewer one carbon products were observed.
Newman and Wackett (1991) proposed a new mechanism for Methylosinus
tn'chosporium OB3b. Four different methanotrophs expressing soluble methane
monooxygenase produced 2,2,2-trichloroacetaldehyde (chloral hydrate). Chloral
hydrate was showm to be biologically transformed to trichloroethanol and
trichloroacetic acid. Figure 1.3. shows a combination of pathways proposed by
Little et al. (1988) and Newman and Wackett (1991) for methanotrophic bacteria
and by Li and Wackett (1992) for the toluene dioxygenase system of
Pseudomonas putida F1.

The methane monooxygenase enzyme system requires a source of
reducing power to carry out the transformations of TCE. Vtﬁien a growth
substrate, such as methane or any one of the metabolites of methane is
oxidized, reducing power is regenerated. When mmo oxidizes a non-grth
substrate, such as TCE, reducing power is not regenerated, and the oxidation
will stop. The effect of an alternative energy source addition on the
transformation capacity of a mixed metanotrophic culture was tested by Alvarez-
Cohen and McCarty (1991b). \NIth the addition of 20 mM of forrnate and TCE at
a concentration of approximately 20 mglL, the transformation capacity of a

culture increased from 0.036 to 0.073 mg TCE/mg of cells. However, signiﬁcant

15

..umm. £9.35 ecu ..: same 3835:. 88.9
.E was... 885.88g. .3 .68. amass Eu 55302. £8 E38805... .3. .e .68. ..a .o 1......
2.203 010058050523 00.55.980.53 200808.00 m9. .9. £03.00 050.00 03000.... .mé 0.32“.
admdﬁﬂ

o: N+ :oIHI: All cm: a

003.53 of Wlalgn

 

 

manta; H .500:

0
33.98 209.0

 

 

 

 

Iu/z “m0 zUtu/x
.ﬂaaamﬁuﬂg IONSIHIU All +
«m0 <5
3.50 / o/ 4 \0 moEﬂxﬂdHWMQSEE
a 33.33393”. al./o 4/ JIM uzm..>Em0¢0..:0.E
220/ +\ ID
(are. 1. T)? Vwr
a. a
"a 0A0 “ﬂaws-mum“.
4/ 4%... 8... .0/0
7H as AMI ..\/ l\u/u

 

16

declines in methane conversion rates following exposure to TCE were observed
for formate fed cells, suggesting toxic effects caused by TCE or its
transformation products.

Even when methane is provided, reducing power can still be depleted.
Using a pure culture, Henry and Grbic-Galic (1991b) showed that carbon
monoxide competitively inhibited methane oxidation until formate was added as
an exogenous electron donor. She also showed that formate addition to the
mixed culture MM1 did not enhance degradation rates at low TCE
concentrations and assumed this was because MM1 methanotrophs possess
lipid storage granules which can serve as an alternate source of electrons.

Henry and Grbic-Galic (1991a) also tested the effect of formate addition on
the rates of TCE transformation during methane starvation. At a TCE
concentration of 30-60 [lg/L, formate addition did not increase rates for the
mixed culture MM1. However, the rates were enhanced for the ﬁrst ten hours of
methane starvation when the pure culture MM2 was incubated with 2 mM
formate. Men the culture was incubated without formate and the formate was
added simultaneously with TCE, transformation rates remained signiﬁcantly
enhanced throughout 62 hours of testing.

Among the groups of bacteria that produce oxygenases and have so far
been demonstrated to be capable of transforming TCE and other CAHs by co-
metabolism include not only the methane oxidizers(Alvarez-Cohen and McCarty,

1991 a; Alvarez-Cohen and McCarty, 1991b; Alvarez-Cohen et al., 1992; Fogel

17

et al., 1986; Henry and Grbic-Galic, 1990; Henry and Grbic-Galic, 1991a; Little
et al.1988; Oldenhuis et al.,1989; Tsien et al., 1989); but also propane oxidizers
(Wackett et al., 1989; Malachowski et al., 1994); propylene oxidizers (Ensingn et
al.,1992); ammonia oxidizers (Arciero et al., 1989; Rasche et al., 1991);isoprene
oxidizers(Ewers, et al.,1990); isopropylbenzene oxidizers (Dabrock et al., 1992);
and monoaromatic hydrocarbon (toluene, phenol, or cresols) degrading
organisms (Folsom et al., 1990; Harker and Kim, 1990; Nelson et al., 1987,
Nelson et al, 1986, Nelson et al, 1988; Wackett and Gibson, 1988).

The ﬁrst report of a pure culture capable of TCE degradation under non
methanotrophic conditions was presented by Nelson et al. (1986). Studies with
“C-labelled TCE resulted in 60% of the total “c-TCE converted to 002 and 35%
remained as an unidentifed nonvolatile product. They also showed that an
unidentiﬁed compound present in the water, from which the strain was isolated,
and oxygen were required for TCE degradation. Later research using the
isolated strain resulted in the identiﬁcation of the required component present in
the water as being phenol (Nelson et al., 1987). This was the ﬁrst report
involving an aromatic degradation pathway in the cometabolism of TCE. Later
toluene, o-cresol and m-cresol were shown to stimulate TCE degradation.

Five pathways differing in the ﬁrst step have been elucidated for the
degradation of toluene. These ﬁrst step enzyme systems vary from oxidation of
the methyl group to mono- or dioxygenations of the ring and seem to be the

enzymes implicated in the cometabolic transformations of TCE. Pseudomonas

18

putida F1 is the strain studied that contains the toluene dioxygenase enzyme
system. A mutant strain defective in the todC gene, which encodes me
oxygenase component of the toluene dioxygenase, failed to degrade TCE; but a
mutant strain defective in todE encoding the 3-methyl catechol 2,3-dioxygenase,
an enzyme dowstream in the pathway for toluene degradation, oxidized TCE as
rapidly as the wild type (Wackett and Gibson, 1988). A spontaneous revertant
selected from a todC culture regained simultaneously the ability to oxidize
toluene and to degrade TCE. Evidence for the involvement of the ﬁrst step
enzyme on TCE degradation is also provided by the fact that in Pseudomonas
mendocina KR, the toluene monooxygenase system (TMO), inserts a single
atom of oxygen at the para-position of toluene to form p-cresol, however, p-
cresol was not an inducer of TCE degradation (Vthter et al., 1989).

In methanotrophic bacteria at least two classes of methane monooxygenase can
be differentiated on the basis of their intracellular localization: a particulate form
associated with the cell membrane (pmmo) and a soluble form (smmo) (Dalton et
al., 1984). Soluble methane monooxygenase has a broader substrate range.
Lower copper levels cause derepression of its synthesis increasing TCE
oxidation (Oldenhius et al, 1989; Tsien et al., 1989; Alvarez-Cohen et al, 1992).
Since TCE seems to be more efficiently degraded by the smmo form, practical
applications based on this group of bacteria will have to be based on selective

conditions for expression of this type of enzyme.

19

Little is known about the efﬁciency of TCE co-oxidation by the different
monoaromatic hydrocarbon degrading pathways or if the presence of a speciﬁc
enzyme system would be favorable for TCE degradation. Also, when one
considers biodegradation of TCE by a pure culture containing catabolic
oxygenases more than one oxygenase is probably induced for the degradation
of the primary substrate. Independent monooxygenases involved in the TCE
degradation were observed in an Alcaligenes strain degrading 2,4-
dichlorophenoxyacetic acid (Harker and Kim, 1990). The toluene dioxygenase
pathway of Pseudomonas putida F1 is one of the best studied enzyme systems
for toluene degradation and was shown to be the only enzyme system required
for TCE degradation (Wackett and Gibson, 1988). Working with toluene induced
cells of Pseudomonas putida F1, Wackett and Gibson (1988) showed that the
initial rate of TCE oxidation was linear with respect to substrate concentration
over the range of 8 to 80 uM of TCE. Initially, over the ﬁrst 20 minutes, a fairly
rapid rate of 1.8 nmoI/min per mg of protein was obtained using an initial TCE
concentration of 80 uM, but the rate decreased rapidly over the next six hours of
the experiment. No TCE oxidation was observed at an initial TCE concentration
of 320 uM.

This rapid decrease in activity was attributed to toxic effects of TCE or TCE
metabolites. The same toxic effects were attributed to the decrease in growth
rates from 0.463 hr’1 to 0.139 hr’1 observed for Pseudomonas putida F1 growing

in the presence of TCE (Wackett and Householder, 1989). Recombinant studies

53.

20

using the toluene dioxygenase genes and E. coli JM109 as the host, showed a
slower initial biodegradation but a prolonged linear rate of TCE disappearance
when compared to the toluene dioxygenase genes expressed in the original
donor Pseudomonas putida F1. The observation that TCE oxidation rates are
more sustained in E. coli could be an indication that different hosts respond in
different ways to cytotoxic effects from TCE or metabolites (Zylstra et al., 1989).

Folsom et al. (1990), evaluated the kinetic parameters for phenol and TCE
degradation by Burkholden’a cepacia G4, a strain that carries the toluene ortho-
hydroxylase pathway. Whole cell studies showed that this enzyme has an
apparent Kmand Vm for phenol oxidation of 8.5 M and 466 nmollmin per mg of
protein, respectively. The Kmand Vm for TCE were 3uM and 8 nmollmin per
mg of protein. As both substrates are oxidized by the same enzyme system,
competitive inhibition was likely to occur since the Km values for both substrates
are similar. Folsom et al. (1990) also showed where in experiments with equal
concentrations of phenol and TCE, a decrease of about 50% in the rate of
phenol degradation was observed.

The third type of toluene monooxygenase (toluene para-monooxygenase)
studied for the kinetic parameters of TCE biodegradation is present in
Pseudomonas mendocina strain KR which oxidizes TCE when grown in the
presence of toluene (\Nlnter et al., 1989). Recombinant E. coli strains containing
the genes encoding this enzyme oxidized TCE at a rate of 1-2 nmollmin per mg

of protein, which also conﬁrmed the role of this enzyme system in the oxidation

21

of TCE. For the fourth type of toluene degrading enzyme system, the toluene
meta-monooxygenase, identiﬁed in Burkoldhen'a pickettii (Olsen et al., 1994), no
kinetic parameter data is avaliable for TCE oxidation. The ﬁfth type of enzyme
system involved in the biodegradation of toluene is present in Pseudomonas
putida PaW1 and involves the hydroxylation of the methyl group of toluene to
form benzoate(Worsey and \Mlliams, 1975). This toluene degrader did not
oxidize TCE (Nelson et al., 1988) and suggests that ring oxygenases are the
enzyme systems efﬁcient for the biodegradation of TCE.

The pathway for TCE degradation by enzymes involved in the
biodegradation of aromatic compounds seems to be similar to the pathway
proposed for methanotrophs, except for the dioxygenase system where TCE
epoxide, the proposed universal intermediate for most monooxygenase enzyme
systems, does not appear to be present. Metabolic studies and chloride release
experiments during the degradation of TCE by Pseudomonas mendocina KR,
resulted in non chlorinated water soluble products putatively identiﬁed as formic
acid and glyoxylic acid, carbon dioxide and cellular constituents (Vthter et al.,
1989). Li and Wackett (1992), working with puriﬁed toluene dioxygenase
obtained from E. coli recombinant strains and 1“C-TCE, found formic acid and
glyoxylic acid as the major oxidation products for this enzyme system. None of
the other intermediates identiﬁed for the methane monooxygenase system
(Figure 1.3.) were detected indicating major differences in the mechanism of

TCE oxygenation from those previously proposed. This also eliminates the

22

possibility that the dioxygenase system functions as a monooxygenase adding
oxygen across the double bound. In one possible mechanism proposed by the
authors, an iron bound dioxygenated intermediate might rearrange on the
enzyme surface to yield formate from both carbon atoms as proposed in Figure
1. 3.

Determining the distribution of aromatic degrading organisms is important
to the overall understanding of diversity and ecology of biodegrading
communities. For the methane monooxygenase systems, where differences in
TCE cometabolic transformation is well established, aromatic pathways may
differ in their efﬁciency for cometabolic transformation. When this is the case,
selection and introduction (i.e. bioaugmentation) of robust strains may increase
the degradation rates of the pollutant in the ﬁeld. I investigated the co-metabolic
efﬁciency of different pathways involved in the degradation of toluene present in
well-studied laboratory strains and compared those with isolates from a ﬁeld site
where successful TCE degradation occurs. To further investigate the
relationship between pathway and strains, I used gene probes to determine the

distribution of pathways in the new isolates.

Degradation of mono aromatic hydrocarbons under denitrifying
conditions.
The availability of oxygen, due to its low solubility in water and inefﬁcient

transport through saturated porous matrices such as soil and sediments, is

23

usually the rate limiting parameter for BTEX removal from contaminated sites. It
has only been in recent years that anaerobic degradation of these compounds
has been conclusively established. Of the BTEX class of compounds, toluene
seems to be the most easily degraded under anaerobic conditions . The
degradation of toluene has been reported under denitrifying conditions (Chee-
Sanford et al., 1992; Dolfing et al., 1990; Evans et al., 1991; Evans et al., 1992;
Kukor and Olsen, 1990; Schocher et al., 1991), methanogenic conditions (Grbic-
Galic and Vogel, 1987; VVIlson et al., 1986), sulfate reducing conditions
(Edwards et al., 1993; Rabus et al., 1993) and ferric iron reducing conditions
(Lovley and Lonergan., 1990; Lovley et al., 1989). Very little was known about
the organisms responsible for toluene degradation under denitrifying conditions
and how widely they are distributed in nature. Hence one of my goals was to

isolate such organisms and describe their physiological features.

List of References

Alvarez-Cohen, L. and P. L. McCarty. 1991a. Product toxicity and cometabolic
competitive inhibition modeling of chloroform and trichloroethylene by
methanotrophic resting cells. Appl. Environ. Microbiol. 57:1031-1037.

Alvarez-Cohen, L. and P. L. McCarty. 1991b. Effects of toxicity, aeration, and
reductant supply on trichloroethylene transformations by a mixed
methanotrophic culture. Appl. Environ. Microbiol. 57:228-235.

Alvarez-Cohen, L. , P. L. McCarty, E. Boulygina, R. S. Hanson, G. A.
Brusseau, and H. C. Tsien. 1992. Characterization of a methane urilizing

bacterium from a bacterial consortium that rapidly degrades
trichloroethylene and chloroform. Appl. Environ. Microbiol. 58:1886-1893.

0a

Do.

Edy

Ensj

24

Arciero, D. T. T. Vanelli, M.Logan, and A. B. Hooper. 1989. Degradation of
trichloroethylene by the ammonia-oxidizing bacterium Nitrosomonas
europaea. Biochem. Biophys. Res. Commun. 159:640-643.

Assinder, SJ. and PA. Williams. 1990. The Tol plasmids: determinants of the
catabolism of toluene and the xylenes. Advan. in Microb. Physiol. 31: 1-69.

Bossert, l., and R. Bartha. 1984. The fate of petroleum in soil ecosystems, p.
434-476. In R. M. Atlas (ed.), Petroleum microbiology. Macmillan
publishing Co., New York.

Chee-Sanford, J., M. R. Fries, and J. M. Tiedje. 1992. Anaerobic degradation
of toluene under denitrifying conditions in bacterial isolate Tol-4, abst. Q-
233, p.374. Abstr. 92th Annu. Meet. Am. Soc. Microbiol. 1992.

Cole, G. M. 1994. Assessment and Remediation of Petroleum Contaminated
Sites. Lewis Publishers, Boca Raton, Florida.

Dabrock, 8., J. Riedel, J. Bertram, and G. Gottschalk. 1992.
lsopropylbenzene (cumene) a new substrate for the isolation of
trichloroethylene-degrading bacteria. Arch. Microbiol. 158:9-13.

Dagley, S. 1986. Biochemistry of aromatic hydrocarbon degradation in
Pseudomonas, p.527-555. In J.R.Sokatch and L.N.Omstron(eds.), The
Bacteria. Academic Press, New York.

Dalton, H., S. D. Prior, D. J. Leak, and S. H. Stanley. 1984. Regulation and
control of methane monooxygenase, p.75-82. In L. R. Crawford and R. S.
Hanson (ed.), Microbial growth on C1 compounds. American Society for
Microbiology, Washington, DC.

Dalton, H., and DI. Stirling. 1982. Co-metabolism. Phil. Trans. Soc. Lond.
8. 2972481496.

Dolﬁng, J., J. Zeyer, P. Binder-Eicher, and R. P. Schwarzenbach. 1990.
Isolation and characterization of a bacterium that mineralizes toluene in the
absence of molecular oxygen. Arch. Microbiol. 154:336-341.

Edwards, E. A., L. E. Wills, D. Grbic-Galic, and M. Reinhard. 1993. Anaerobic
degradation of toluene and xylene under sulfate-reducing conditions. Appl.
Environ. Microbiol. 58:794-800.

Ensingn, S. A., M. R. Hyman, and D. J. Harp. 1992. Cometabolic degradation
of chlorinated alkenes by alkene monooxygenase in a propylene-grown
Xantobacter strain. Appl. Environ. Microbiol. 58:3038-3046.

 

25

Evans, P. J.,W. Ling, B. Goldschmidt, E. R. Ritter, and L. Y. Young. 1992.
Metabolites formed during the anaerobic transformation of toluene and o-
xylene and their proposed relationship to the initial steps of toluene
mineralization. Appl. Environ. Microbiol. 58:496-501.

Evans, P. J., D. T. Mang, K. S. Kim, and L. Y. Young. 1991. Anaerobic
degradation of toluene by a denitrifying bacterium. Appl. Environ. Microbiol.
57:1139- 1145.

Ewers, J., D. Freier-Schroder, and H. J. Knackmuss. 1990. Selection of ‘
trichloroethene(T CE) degrading bacteria that resist inactivation by TCE.
Arch. Microbiol. 154:410-413.

Floodgate, G. D. 1984. The fate of petroleum in marine ecosystems, p. 355-
397. In R. M. Atlas (ed.), Petroleum microbiology. Macmillan publishing
Co., New York.

Fogel, M. M., A. Taddeo, and S. Fogel. 1986.Biodegradation of chlorinated
ethenes by a methane-utilizing mixed culture. Appl. Environ. Microbiol.
51:720-724.

Folsom, B. R., P. J. Chapman and P. H. Pritchard. 1990. Phenol and
trichloroethylene degradation by Pseudomonas cepacia G4: kinetics and
interactions between substrates. Appl. Environ. Microbiol. 56:1279-1285.

" Grbic-Galic, D. and T. M. Vogel. 1987. Transformation of 'toluene and benzene
by mixed methnogenic cultures. Appl. Environ. Microbiol. 53:254-260.

Harbome, J. B. 1980. Plant phenolis, p.329-402. In E. A Bell and B. V.
Charlvvood (ed.), Secondary plant products. Springer-Verlag, New York.

Harker, A. R. and Y. Kim. 1990. Trichloroethylene degradation by two
independent aromatic independent pathways in Alcaligenes eutrophus
JMP134. Appl. Environ. Microbiol. 56:1179-1181.

Henry, S. H. and D. Grbic-Galic. 1990. Effect of mineral media on
trichloroethylene oxidation by aquifer metanotrophs. Microb. Ecol. 20: 151-
169.

Henry, S. H. and D. Grbic-Galic. 1991a. Inﬂuence of endogenous and
exogenous electron donors and trichloroethylene oxidation toxicity on - 1‘
trichloroethylene oxidation by methanotrophic cultures from a groundwater
aquifer. Appl. Environ. Microbiol. 57:236-244.

26

Henry, S. H. and D. Grbic-Galic. 1991b. Inhibition of trichloroethylene
oxidation by the transformation intermediate carbon monoxide. Appl.
Environ. Microbiol. 57:1770-1776.

Henschler, D,. W. R. Hoos, H. Fentz, E. Dallmeir and M. Meltzler. 1979.
Reactions of trichloroethylene epoxide in aqueous systems. Biochemical
pharmacology. 28:543-548.

Kukor, J. J., and R. H. Olsen. 1990. Diversity of toluene degradation following
long term exposure to BTEX in situ, p. 405-421. In D. Kamely, A.
Chakrabarty and G. S. Omenn, (ed.), Biotechnology and Biodegradation,
Gulf Publishing Co., Houston, Texas.

Kukor, J. J., and R. H. Olsen. 1991. Genetic organization and regulation of a
meta cleavage pathway for catechols produced from catabolism of toluene,
benzene, phenol, and cresols by Pseudomonas pickettii PKO1. J. Bacteriol.
173:4587-4594.

Kunz, D. A., and P. J. Chapman. 1981. Catabolism of pseudocumene and 3-
ethyltoluene by Pseudomonas putida (arvila) mt-2: evidence for new
functions of the Tol (pWWO) plasmid. J. Bacteriol. 146:179-191.

Li, S. and PI. L. Wackett. 1992. Trichloroethylene oxidation by toluene
dioxygenase. Biochem. Biophys. Res. Comm: 185:443-451.

Little, C. D., A. V. Palumbo, S. E. Herbes, M. E. Lidstrom, R.L. Tyndall, and
P. J. Gilmer. 1988. Trichloroethylene biodegradation by a methane-
oxidizing bacterium. Appl. Environ. Microbiol. 54:951-956.

Lovley, D. R., M. J. Baedecker, D. J. Lonergan, I. M. Cozzarelli, E. J. P.
Phillips, and D. l. Segal. 1989. Oxidation of aromatic contaminants coupled
to microbial iron reduction. Nature 339:297-300.

Lovley,. R., D. J. Lonergan. 1990. Anaerobic oxidation of toluene, phenol, and
p-cresol by the dissimilatory iron reducing bacterium GS-15. Appl. Environ.
Microbiol. 56:1858-1864.

Malachowsky, K. J., T. J. Phelps, A. B. Teboli, D. E. Minnikin and D. C.
White. 1994. Aerobic mineralization of trichloroethylene, vinyl chloride, and
aromatic compounds by Rhodococcus species. Appl. Environ. Microbiol.
60:542-548.

27

Martin," J. P., and K. Haider. 1969. Phenolic polymers of Stachybotrys aha.
Stachybotrys chartarum and Epicoccum nigrum in relation to humic acid
formation. Soil Sci. 107:260-270.

Martin J. P., K. Haider, and D. Wolf. 1972. Synthesis of phenols and phenolic
polymers by Hendersonula toruloidea in relation to humic acid formation.
Soil Sci. Amer. Proc. 36:311-315.

Nelson, M. J. K.,S. O. Montgomery, E. J. O’Neil and P. H. Pritchard. 1986.
Aerobic metabolism of trichloroethylene by a bacterial isolate. Appl.
Environ. Microbiol. 52:383-384.

Nelson, M. J. K.,S. O. Montgomery, E. J. O’Neil and P. H. Pritchard. 1987.
Biodegradation of trichloroethylene and involvement of an aromatic
biodegradative pathway. Appl. Environ. Microbiol. 53:949-954.

Nelson, M. J. K.,S. O. Montgomery, and P. H. Pritchard. 1988.
Trichloroethyleene metabolism by microorganisms that degrade aromatic
compounds. Appl. Environ. Microbiol. 54:604-606.

Newman, D., and L. P. Wackett. 1991. Fate of 2,2,2-trichloroacetaldehyde
(chloral hydrate) produced during thrichloroethylene oxidation by
methanotrophs. Appl. Environ. Microbiol. 57:2399-2402.

Oldenhuis, R., R. L. J. M. Vink, D. B. Jansen, and B. Witholt. 1989.
Degradation of chlorinated aliphatic hydrocarbons by Methylosynus
trichosporium OB3b expressing soluble methane monooxygenase. Appl.
Environ. Microbiol. 55:2819-2826.

Olsen R. H., J. J. Kukor , and B. Kaphammer. 1994. A novel toluene-3-
monooxygenase from Pseudomonas pickettii PKO1. J. Bacteriol. 17613749-
3756.

Rabus, R., R. Nordhaus, W. Ludwig, and F. Widdel. 1993. Complete oxidation
of toluene under strictly anoxic conditions by a new sulfate-reducing
bacterium. Appl. Environ. Microbiol. 59:1444-1451.

Rasche, M. E., M. R. Hayman, and D. J. Harper. 1991. Factors limiting
aliphatic chlorocarbon degradation by nitrossomonas europaea: cometabolic
inactivation of ammonia monooxygenase and substrate speciﬁcity. Appl.
Environ. Microbiol. 57:2986-2994.

Rogers, J. E. and D.T. Gibson 1977. Puriﬁcation and properties of cis-toluene
dihydrodiol dehydrogenase from Pseudomonas putida. J. Bacteriol.
130:1117-1124.

 

waCket

28

Schocher, R. J., B. Seyfried, F. Vazquez and J. layer. 1991. Anaerobic
degradation of toluene by pure cultures of denitrifying bacteria. Arch.
Microbiol. 157:7-12.

Shields, M. 8., S. O. Montgomery, P. J. Chapman, S. M. Cuskey, and P. H.
Pritchard. 1989. Novel pathway of toluene catabolism in the
trichloroethylene-degrading bacterium G4. Appl. Environ. Microbiol.
55:1624-1629.

Shields, MS. and M. J. Reagin. 1992. Selection of a Pseudomonas cepacia
strain constitutive for the degradation of trichloroethylene. Appl. Environ.
Microbiol. 58:3977-3983.]

Song, H. G., T.A. Pedersen, and R. Bartha. 1986. Hydrocarbon mineralization
in soil: relative bacterial and fungal contribution. Soil Biol. Biochem. 18:109-
1 1 1.

Tiedje. J. M. 1993. Bioremediation form an Ecological Perspective, p110-120. In
In Situ Bioremediation . When does it work? National Academy Press.
Washington , D.C.

Tsien, H. C., G. A. Brusseau, R. 5. Hanson and L. P. Wackett. 1989.
Biodegradation of trichloroethylene by methylosinus 083b. Appl. Environ.
Microbiol. 55:3155-3161.

Vogel, T. M. and P. McCarty. 1985. Biodegradation of tetrachloroethylene to
trichloroethylene, dichloroethylene, vinyl chloride , and carbon dioxide under
methanogenic conditions. Appl. Environ. Microbiol. 49:1080-1083.

Wackett, L.P., G. A. Brusseau, S. R. Householder and R. S. Hanson. 1989.
Survey of microbial oxygenases: trichloroethylene degradation by propane-
oxidizing bacteria. Appl. Environ. Microbiol.55:2960-2964.

Wackett, L.P., and D. T. Gibson. 1988. Degradation of trichloroethylene by
toluene dioxygenase in whole cell studies with Pseudomonas putida F1.
Appl. Environ. Microbiol.5421703-1708.

Wackett, L.P., and S. R. Householder. 1989. Toxicity of trichloroethylene to
Pseudomonas putida F1 mediated by toluene dioxygenase. Appl. Environ.
Microbiol.55:2723-2725.

29

Whited, G. M., and D.T. Gibson. 1991. Toluene-4-monooxygenase, a three-
componentenzyme system that catalizes the oxidation of toluene to p-
cresol in Pseudomonas mendocina KR. J. Bacteriol. 173:3010-3016.

Williams, P. A. and R. R. Sayers. 1994. The evolution of pathways for aromatic
hydrocarbon oxidation in Pseudomonas. Biodegradation 5:195-217.

Wilson, B. H., G. B. Smith, and J. F. Rees. 1986. Biotransformation of
selected alkylbenzenes and halogenated aliphatic hydrocarbons in
methanogenic aquifer material: a microcosm study. Environ. Sci. Technol.
20:997-1 002.

Wilson, J. T. and B. H. Wilson. 1985. Biotransformations of trichloroethylene
in soil. Appl. Environ. Microbiol. 37:242-243.

Winter, R. B., K. Yen and B. D. Ensley. 1989. Efﬁcient degradation of
trichloroethylene by a recombinant Escherichia coli. Bio/Technology 7:282-
285. ,

Worsey, MJ. and P.A. Williams. 1975. Metabolismof toluene and Xylenes by
Pseudomonas putida(an/illa) mt-2: evidence for a new function of the Tol
plasmid. J. Bacteriol. 124:7-13.

Zylstra, G. J. and D. T. Gibson. 1989. Toluene degradation by Pseudomonas
putida F 1: Nucleotide sequence of the todC1CZBADE genes and their
expression in Escherichia coli. J. Biol. Chem. 264:14940-14946.

Zylstra, G. J., L. P. Wackett and D. 1'. Gibson. 1989. Trichloroethylene
degradation by Escherichia coli containing the cloned Pseudomonas putida
F1 toluene dioxygenase genes. Appl. Environ. Microbiol. 55:3162-3166.

Chapter II

Microbial populations of phenol and toluene degraders in an aquifer where
successful TCE co-oxidation occurs.

Introduction

Contaminated water is a problem facing many communities throughout the
world. Trichloroethylene (T CE), an Environmental Protection Agency priority
pollutant, is widespread in the environment. TCE is an efﬁcient industrial
solvent and degreaser that can be transported into the environment by
inadequate disposal techniques, accidental spillage, leaking storage tanks, and
landﬁll Ieachates. TCE is relatively resistant to biodegradation in soil and the
subsurface since microorganisms can not use this compound as a sole source of
carbon or energy.

In 1985, Wilson and VVIlson presented some of the ﬁrst evidence of aerobic
degradation of TCE in soil enriched with natural gas (77% methane). After their
work, aerobic cometabolic degradation of TCE has been widely demonstrated,
initially using methanotrophic bacteria. Unlike anaerobic degradation which can
produce toxic by-products such as vinyl chloride (\Iogel and McCarty, 1985), the
products produced aerobically by methane oxidizers are thought to be relatively
harmless for other organisms (Little et al., 1988; Oldenhuis et al., 1989; Tsien et
al., 1989). The ﬁrst report of a pure culture capable of TCE degradation under

non-methanotrophic conditions was presented by Nelson et al. (1986). Among

30

 

31

the groups of bacteria that produce oxygenases and have so far been
demonstrated to be capable of transforming TCE and other CAHs (chlorinated
aliphatic hydrocarbons) by co-metabolism include not only the methane
oxidizers (Alvarez-Cohen and McCarty, 1991 a; Alvarez-Cohen and McCarty,
1991b; Alvarez-Cohen et al., 1992; Fogel et al., 1986; Henry and Grbic-Galic,
1990; Henry and Grbic-Galic, 1991a; Little et al., 1988; Oldenhuis et al., 1989;
Tsien et al., 1989); but also propane oxidizers (Wackett and Householder, 1989;
Malachowski et al., 1994); propylene oxidizers (Ensingn et al., 1992); ammonia
oxidizers (Arciero et al., 1989; Rasche et al., 1991); isoprene oxidizers (Evvers et
al., 1990); isopropylbenzene oxidizers (Dabrock et al., 1992); and monoaromatic
hydrocarbon (toluene, phenol, or cresols) degrading organisms (Folsom et al.,
1990; Harker and Kim, 1990; Nelson et al., 1987, Nelson et al., 1986, Nelson et
al., 1988; Wackett and Gibson, 1988).

Initial attempts to stimulate TCE co-oxidation in the field were done by
injecting methane and an oxygen source, but recently the injection of phenol and
toluene along with oxygen have been investigated since they appear to result in
more rapid TCE removal and are easier to engineer. The aerobic metabolism of
toluene is known to be initiated by a variety of oxygenases that result in ﬁve
different pathways leading to toluene mineralization. Phenol metabolism also
involves an oxygenase attack, and its pathway of metabolism converges with
some pathways of cresol metabolism that result from monooxygenase attack on

toluene. Hence, toluene and phenol additions may be stimulating very similar if

32

not the same monooxygenases active in TCE co-oxidation. How similar the
populations are that are stimulated by these two substrates or whether they are
equivalent in their cooxidizing ability is unknown. The best studied strains with
the ﬁve different toluene degrading pathways are very different in the TCE
cooxidizing ability, varying from completely inactive to the most active known
organism. Hence the success of phenol or toluene stimulated TCE cooxidation
treatments would appear to depend on which type of population ( and
oxygenase) is stimulated at a contaminated site.

In this study we characterized the phenol and toluene degrading
populations that grew in response to toluene, phenol and TCE additions to the
Moffett Field aquifer. The isolates were taken from two sources: glass beads
added to the aquifer in the ﬂow path of the injected substrates so that only those
organisms that grew in situ were obtained, and from ﬁltered water pumped from
the same hole so that any unattached organisms could also be obtained. Our
objectives were: (I) to evaluate the sensitivity of the populations to toluene and
phenol concentrations, (ii) to determine which populations were more frequently
isolated, (iii) to elucidate major characteristics of the isolate collection such as
gram reaction, frequency of use of both phenol and toluene, TCE cooxidation
ability and hybridization to the toluene pathway probes , and (iv) to compare the
rates of toluene use and TCE cooxidation among the more commonly selected

aquifer strains and with the previously well—characterized toluene degraders.

33

Material and Methods

Field sample site description. The experimental site is located at the Moffett
Federal Airﬁeld (formerly the Moffett Naval Air Station), Mountain View,
California, a few miles from Stanford University. It is located on the lower part of
the Stevens Creek alluvial fan, approximately 3 km south of the southwest
extremity of San Francisco Bay. The surface elevation at the site is 8.5 m above
mean sea level. The test zone is a shallow, semiconﬁned aquifer, consisting of
ﬁne to coarse-grained sands and gravels. The alluvial sediments contained in
the aquifer were deposited in the last 5000 years. This aquifer is about 4.5m
below ground surface, and is about 1.4 m thick. Above the aquifer is
approximately 4 m of silt and clay of a brownish-black to olive gray color, and a
layer of dark greenish-gray silty clay about 7 m thick underlines the aquifer. The
aquifer is spatially heterogeneous, with the composition varying appreciably over
short distances. The test zone appears to have the structure of a buried stream
channel containing sand and gravel in some areas and only sand in others
(Roberts et al., 1990).

Pumping test results suggest that this is a leaky conﬁned aquifer system, with
a hydraulic conductivity of 100 m/day. Groundwater velocity in the test zone is
relatively high, 1 m/day, based upon both bromide tracer tests and estimates
from the measured hydraulic gradient across the ﬁeld of 0.0032 and an

estimated porosity of 0.33. The natural gradient moves in a south to north

 

 

34

direction, that is from the injection well towards the extraction well (Roberts et
al.1990)

The organic matter content of bulk aquifer samples is 0.11 percent.
Sorption of TCE to aquifer material is rather high, as indicated by measured
retardation factors with respect to groundwater movement of 8 to 12, which is
consistent with sorptive properties measured in the laboratory. The groundwater
is anoxic, of low salinity and near neutral pH. It is somewhat contaminated from
other sources with chlorinated solvents, although the one of primary interest in
this study, TCE, is not present. The only chlorinated aliphatic
hydrocarbon(CAH) of signiﬁcance is 1,1,1-trichlorethane, which is at a
concentration of about 30 ug/L. These contaminants have posed no difficulties
in the experiments conducted at the site, as they are either too low in
concentration to be of signiﬁcance, or else are not reactive biologically under the
experimental conditions. Chemical analysis of water from the sampling site
revealed the following concentrations: phosphate less than 0.5 mglL, nitrate, 25
mglL, sulfate, 700 mg/L (Hopkins et al., 1993).

For the phenol injection experiments conducted during 1991-1992, a new
experimental leg was installed adjacent to the original one installed in 1986 for
other studies. The injection and extraction wells consist of standard 2 inch PVC
pipe, installed using a hollow stem auger. These wells contain 5-foot slotted
screens that fully penetrate the aquifer zone. The sampling wells (Figure 2.1.)

are located at 1(SSE1), 1.6(SSEGB), 2.2(SSE2), and 4 m(SSE3) from the

35

.82: ..a .o 9.33. ES. BEBE. we» .8. 22". sates. so 3%, 8:08 890 .5 230E

:5 .mwm =e>> 59.— 8:530

 

_
Na nmmm wam «mmw

 

 

 

 

 

.0230 “En team

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

30
._. i. i I... a
in; f . ﬂu;
20:05.5 0.1m; 02:22.6 20:02.2.

 

(w) eoeyns punOJs M0|aq mdaa

36

injection well, and consist of 125-inch stainless steel wire wound sand points
with 2-foot screens, installed in the middle of the aquifer test zone. An additional
sand point well with a 4.5-foot screen was placed 7m (extraction well-P2) from
the injection well. Normally water was extracted from the aquifer at a flow rate of
10 Umin. creating a localized ﬂow of groundwater towards the extraction well .
Water was injected at a flow rate typically of 1.5 Umin. From bromide tracer
tests, the mean time of travel of injected water is approximately 4,12, and 30
hours to the SSE1, SSE2, and SSE3 wells respectively.

Extracted water was treated by air stripping and ﬁltration to remove volatile
and suspended contaminants, respectively. A portion was used for the injection
water , and the remainder was discharged into the Moffett Field drainage
system. The test zone has many favorable features: high permeability, suitable
groundwater chemistry, shallow depth, conﬁned system providing two-
dimensional ﬂow, desirable size for ﬁeld experiments, well controlled
groundwater flow, and the ability to withdraw all chemicals or other materials
added to the system. Details of the on-line ﬁeld analytical system have been
provided elsewhere (Roberts et al., 1990). An automated data acquisition and
control system (DAC) was devised at the test site to implement the ﬁeld
experiments. Water samples for analysis were obtained from the monitoring well
locations and from injected and extracted water by automated pumping to the
analytical system. The automated analytical system perrnited the continuous

measurement of the principal chemical constituents for a given experiment,

 

 

37

namely phenol, toluene and TCE, 1 ,1-DCE, c-DCE and t-DCE. The instruments,
of interest for the ﬁeld part of this project, operated by the DAC system are: a
reverse phase high performance liquid chromatograph (HPLC) for phenol and
toluene analysis, a gas chromatograph equipped with an electron capture
detector (GC-ECD) and a Hall conductivity detector (GC-Hall) for CAC analysis.
TCE was analyzed by GC using a purge and trap system described by Roberts
et al., 1990. The gas chromatograph was equipped with a J&W Scientific
(Folsom, CA) 30 meter, DB-624, megabore capillary column, and a Tremetrics
(Houston, TX) Hall conductivity detector. Calibrations were made using an
external standard. Phenol and toluene were analyzed by reverse phase HPLC,
with separation on a Spherisorb ODS-2 column (Altech, Dearﬁeld, IL) with 50%
methanol in water as eluent, and detection using a Linear Model 200 UV
detector. Chemicals were introduced into the injected water in an automatic
programmed manner. The extracted water used for injection was treated before
chemicals were added by ﬁltration through a nominal 1pm ﬁlter, and UV
disinfection. Chemicals introduced in the past were oxygen, phenol, bromide,
TCE, cis and trans-1,2-dichloroethylene. Concentrations of the CAHs have
generally been in the 40 to 150 ug/I range and, TCE concentrations up to 1000
ug/I were injected during the year of 1992. CAHs were added to injection water
by continuous pumping of water solutions saturated with the particular CAH of

interest. The injection water and sampled waters all pass through stainless-steel

38

tubing, which prevents passage of gases and CAHs through tubing walls. Thus,
excellent mass balances of all chemicals can be maintained.

Chemical introduction into the aquifer - 1993 experiments. The ﬁeld
experiment was conducted by Gary Hopkins of Stanford University. For the
experiments described in this work corresponding to the year of 1993, a
summary of the injection concentrations for the chemicals of interest over this
study period is given in Table 2.1. Before chemical augmentation, the
groundwater was ﬁltered with a nominal 1 um cotton ﬁlter, and UV disinfected. It
was then passed through a gas absorption column where molecular oxygen was
used to purge the column, producing a dissolved oxygen (DO) concentration in
the groundwater of about 32 mglL. Phenol and toluene were added in pulses
during their respective test periods, once every 8 hours as controlled by a timing
clock. For phenol, a solution was prepared, and an aliquot yielding 9 g phenol
was added during each 10 min or less pulse, thus producing a 12.5 mglL time-
averaged concentration. Toluene was added in a similar fashion as phenol, but
as pure solute: 6.5 g was added over a 30 min period to yield a time-averaged
concentration of 9 mg/L for the eight hour cycle. A pair of static mixers with 24
elements was used to mix the toluene into the injection ﬂow stream. Although
the toluene was not perfectly dissolved within the contact time of less than 1s, it
was nevertheless very ﬁnely dispersed upon exiting the static mixer, with an
approximate pulse concentration of 200 mglL (toluene solubility is approximately

600 mglL). As in previous years, there was a period of 3 days in which the

1 I17? 0. D ‘haﬁtihn

 

39

08> 80. 20 8. 300509.... .0 00.0.03 .5 .- 0325... 0 as: e
0.05 05.00! 00 05 52. es... .003 80.0.00 .
0:20-5:35". 0009.30.05... .

08.5.80: 00. 2.0.00: .00... 000.00: .

 

03.8 v 0.0.000 00 000.

.0 0 0 000 «n 0 0 002-003

0 0 0 000 «0 0 0 003-003

0 0: 0 08 an 0 0 «03-0.2

0 0 0 000 «0 0 0 22.000 .2
09.5 0 0.0.000 00 000

0 0 0 000 «0 0 0.2 000.000 .5
09.8 0 0.0.000 00 000

0 0 00 000 an 0 0.0. 000-000

0 0 3: 03 «a 0 0.0. 000-000

0 0 0 000 u» 0 0.0. 000-00 .=
09.5 0 0.00.0900 30-

0 0 0 0 0 0 0 0.020- g
E.

..: 0.: ..: 0.0 ..: 0.0 30.0 30.5 30.5 30.5 00:00
000.. 000-0 000-... 000 00 m.0000...» m. .002... 05:

 

:80? 00000? 00 00000 5000:0050

 

.a 00000 000.0 00 2:00:00
0:20:00 0:0 00003 00.00? 0. 00000 0.00205 .0 0:000b:00:00
05 0530.? 00:05:00.8 0.0: $2. :00. 05 ..0 00.500 05:. . Va 030...

40

microbial population was prestimulated with phenol (6.5 mg/L time-averaged)
and oxygen (32 mglL) addition. This was done to prevent potential biofouling of
the extraction well and to provide time to correct the usual startup problems of
the injection and analytical systems. At time zero, the chemical augmentation of
TCE and bromide began along with the increase in the phenol concentration to
a time-averaged 12.5 mglL (Table 2.1). After TCE removal approached steady-
state, the 1.1-DOE augmentation and evaluation began. After the termination of
the 1,1-DCE addition and the quasi-steady state conditions were again
achieved, the primary substrate was changed from phenol to toluene.
Approximately one week later, augmentation with c-DCE began, followed by the
augmentation with t-DCE.

Samples for microbiological analysis. The 1993 study followed 8 months of
inoperation of the field site. At about -604 hours before the beginning of the
chemical augmentations for the 1993 season, three types of samples were sent
to MSU for microbiological analysis of the phenol and toluene-degrading
populations initially present on the ﬁeld (sample 1). The samples consisted of
microbial bioﬁlms on glass beads (present in the ﬁeld for 8 months) extracted
from the SSEGB well (Figure 2.1.), and two ﬁltered water samples. The ﬁrst
water sample resulted from ﬁltering 500 ml of water through a 0.2 pm ﬁlter after
extracting approximately 60% of the glass beads from the well (this sample is
termed 0.22 gm ﬁlter sample in this work). A cotton ﬁlter (Micro-wynd ll, J.N.

Fauver Company, F lint, MI) was used to process all water extracted during the

41

removal of the glass beads from the well, approximately 150 L, and is termed
here as the cotton ﬁlter sample. Samples 2, 3 and 4 consisted of glass beads
deposited in a screen-fabric bag (approximately 100 9 glass beads/bag) and
hung in the fully penetrating well (SSEGB, Figure 2.1.). At indicated times
(Table 2.1.), the bags of glass beads were removed from the well and sent on
ice by Federal Express to MSU for microbiological analysis. New bags were
deposited in the wells. For samples 2, 3 and 4, the period of microbial
colonization of the glass bead bags in the ﬁeld was 20-26 days.

Microbial biomass. To collect the biomass from the glass beads or ﬁlters,
these samples were shaken with sterile saline solution, pH 7, in sterile 250 ml
Nalgene plastic bottles. The supernatant was transferred to a clean, sterile
bottle, followed by centrifugation. The pellet was retained and the supernatant
discarded. At least eight extractions were performed in this manner from each
sample. The ﬁnal pellet was resuspended in saline to yield a biomass
concentration correspondent to 10 9 glass beads/ml of saline. The ﬁlter samples
were resuspended to a simmilar visual turbidity as the resultant glass beads
samples. This resuspended turbid solution was used for MPN determinations,
direct isolation of microbial populations and total microbial community DNA
extraction.

Most probable number (MPN) population estimates. For the MPN

determinations the basic experimental protocol presented in Figure 2.2. was

followed. The estimated population density and the 95% conﬁdence intervals

42

-

U-h‘ule-I ~00. a. \ 60.04. 6)::Inva cassebnt ups- :sev\5 sun; 5.

AwO(a 52v mad-.0“. m( Opp-Sn. mum; mwmbh watmOu wbDJE hmOS

.60: w ..o 0.0 .0 E0: on ..o 3
me». + .5sz

 

.50.. 00 ..o 0. 0020::

m
W
E

E3: 0 :0 0.0 4 Eng 00 ..o 8
wok + wzwaaob

-mi

 

A600 on :o o . wzwDJOh

-MI

(NC-2 056...

 
 

00.. w
_

Ea“... @@@@®@®@®

«or

a. 0W®®©®®®®®

........_§.
0-00-00-00-00

.58 .E 00 e. .e 0 B 20.5.... 3000/
38.80.080.055}: 2: 0.5-68.0.3 80330083058333... 82305553380850

-00e '- f‘t

 

 

 

80.338080100008805;

mZO_._.<._Ow_ DZ< ZO....<Z=2N_m._.wo Zn;— ”.0“. 2.0.me ._<._.Zm2_mmmxw

52:00 05 So...— 0:000.:000 0:05.000 .00.: 05 3 500.00. :8
000 00000000000 0.00.0 .009 6.00.0398 000 .20000000 .0000... 000 0000.8 .0
00:52 0.000000. 000.2 0.0 00 5005:0800 05 :2 00.32.00 .0029... 30:05:00.6 .~.~ 00.6.0.

00000320085030.0200 00.02000 03$.

.08.: 0000.008 00.03.00. 000000 00.0000 can 0.000000 000 .2020»
0.50 0000.00 .0083 0

0000.530 0800020 000 jet-.0200 00...0 000 .0000 000805.000
.030: 0.2.0.3208 00... B 0000.200

4 0...... 00 9 0200.008 :03
0000000 000 10000.0. 5.3 030.00. :00 0.3 000000 :03 080.00. .0303 3.000.. 000002000
0000000000003» 00.000. («c-3.0080080 :080000000000003003080000805000850

.

.0...” mW®®®mW®®mw®
.3...“ @mwmwnwmwmwmmemW
a. @mWQmewmwmwmme

 

44

were calculated by standard methods (Alexander,1982). Resuspended material
collected from glass beads and ﬁlters were serially diluted and 1 ml of each
dilution was transferred to sterile 20 ml vials. Five tubes were inoculated from
each serial dilution. The samples were incubated without shaking after addition
of 9 ml of basal salts (BS) medium (Owens and Keddie, 1969) amended with 5
mM KN03 and sealed with Teﬂon—lined stoppers. For sample 1, basal salts
medium containing toluene or phenol at 5 or 50 ppm concentrations without or
with TCE at 0.5 or 1 ppm, respectively, was added. For samples 2, 3 and 4.
basal salts medium containing toluene or phenol at 25 ppm without or with TCE
at 0.5 ppm was added. A vial was assumed to be positive for biodegradation of
the primary substrate and TCE co-oxidation when more than 50 and 95 % of the
TCE and toluene or phenol, respectively, had disappeared.

For the determination of total heterotroph population the vials were
inoculated with liquid M-RZA medium and analyzed for visible turbidity. Modiﬁed
R2A (M-RZA), is based on the original carbon composition provided by Difco
(Detroit, MI), combined with a low phosphate plus trace salts mixture. M-RZA

had the following composition per liter: salt mixture (SM) consisted of KH2PO4,
0.259; K2HPO4, 0.4g; KN03, 0.5059; CaCI2.2l-l20, 0.0159; M90l2.6H20,
0.029; FeSO4.7l-l20, 0.0079; Na2804, 0.0059; NH4CI, 0.89; MnCl2.4H20, 5mg;
H3803, 0.5mg; ZnCl2, 0.5mg; CoCl2.6H20, 0.5mg; NiSO4.6H20, 0.5mg;
CuClz.2H20, 0.3m9; NaM002.2H20, 0.01 mg. For solid medium 15 g of Bacto

agar (Difco, Detroit, MI) was added. The pH was adjusted to 7.0 before

aul
Pei
soc
30:
150
PM
2.2
ML
phe
orig
san
phe
hell
hell

oxic

Aha

add,

45

autoclaving. The carbon sources were per liter: yeast extract, 0.59; Proteose
peptone, 0.59; casamino acids, 0.59; Dextrose, 0.59; soluble starch, 0.59;
sodium pyruvate, 0-59- The incubation temperature throughout this work was
30°C.

Isolation of dominant populatlons. To isolate most of the culturable
population‘s present on the aquifer we followed the protocol presented in Figure
2.2. We used the most dilute MPN tubes shovm'ng disappearance of phenol and
toluene with or without TCE from all samples, to isolate the most dominant
phenol and toluene degraders. For samples 2, 3, and 4, we also used the
original dilution tubes with samples used for MPN and direct plated these
samples for microbial isolations because in sample 1 the number of toluene and
phenol degraders were below the expected levels in relation to the total
heterotrophic population and we attempted to verify if the most dominant
heterotrophs were capable of toluene or phenol degradation and TCE co-
oxidation. Samples from MPN tubes were serially diluted and plated on three
different solid media: 1) BS + 5 mM N03' + toluene vapors, 2) BS + 5 mM N03'
+ phenol and 3) Solid M- RZA Glass Petri dishes were used for phenol and
toluene-vapor based growth. Toluene was added in a small vial inside an
incubation jar. The amount of toluene added was calculated based on the total
volume of agar in the incubation jar to provide a ﬁnal concentration of 50 ppm.
After one week of incubation sufﬁcient toluene to provide 50 ppm was again

added. The jar used in the phenol and toluene vapor experiments was sealed

46

with a Teﬂon-lined aluminum sheet (Cole-Palmer, Chicago, II) to prevent phenol
or toluene absorption by the rubber sealer of the jar. The plates were incubated
under aerobic conditions. The selection strategy employed for isolation was
based on bacterial colonies obtained on the different media (toluene and phenol
vapors and M-RZA) and selecting all colonies that had differences in colony
morphology, size and pigmentation. For puriﬁcation of isolates, single colonies
from different media and from all dilutions showing positive-distinct-colony-
growth were selected and puriﬁed at least three times on M-RZA For very small
colonies at least three individual colonies were transferred to new plates to
assure the maintenance of these morphotypes because our experience has
shown that many isolates can be lost at this stage.

Characterization of isolates. Gram stain, catalase and oxidase tests were
performed on pure cultures of isolates by standard methods (Smibert and Krieg,
1981). Conﬁrmation of phenol and toluene degradation was done by

transferring a heavy inoculum of each isolate to sterile 20 ml vials after which 5

ml of aerobic BS + NO3‘+ 25 ppm phenol or toluene medium was added. The

vials were sealed with sterile butyl rubber Teflon-lined septa and incubated for at
least 2 weeks before phenol or toluene disappearance was evaluated. Controls
from the same batch of medium without cells were incubated at the same time to
determine the non-biological phenol or toluene loss. Positive degradation

activity was deﬁned as at least 95% loss of toluene in the headspace as

47

measured by GC analysis and at least 95% loss of phenol in the supernatant of
the growth media as measured by HPLC analysis.
Standard strains. Laboratory standard strains of Pseudomonas putida F1,
Pseudomonas putida PaW1, Pseudomonas mendocina KR, Burkholderia
cepacia G-4, Burkholderia pickettii PK01 and Pseudomonas sp. JS-150 were
kindly provided by Dr. Ronald Olsen, University of Michigan.

Trichloroethylene c0-0xidati0n. To test for the isolates’ capabilities to co-
oxidize TCE during degradation of toluene or phenol, inocula were grown on

liquid M-RZA under aerobic conditions. lnocula (112 ml) were transferred to 20
ml sterile auto sampler vials and 4.5 ml of BS + N03' + 25 ppm of the aromatic
substrate and 0.5 ppm TCE was added to the vials. The vials were sealed with
Teﬂon-lined stoppers. Positive degradation activity was determined by TCE
disappearance in the headspace after 15 days of incubation as measured by
GC analysis, and after comparison to non-inoculated controls. For the TCE co-
oxidation rate studies, the cultures were grown on BS + N03” + 50 ppm
toluene. lnocula (10 %) was transferred to 160 ml serum bottles containing 130
ml of BS + N03' + 50 ppm of toluene and 1 ppm TCE. This culture was
subdivided in 5 ml portions and transferred to 20 ml sterile auto sampler vials
and duplicate Hungate tubes and sealed with Teﬂon-lined stoppers. Three vials
were sacriﬁced by adding 0.1 ml of 10 N HCI and the vials were frozen at - 20°C
for later analysis of initial toluene and TCE concentrations. Growth in Hungate

tubes was followed by optical density at 600 nm; at different time intervals vials

48

were sacriﬁced and preserved as before, for later analysis of remaining toluene
and TCE.

FAME analysis. The isolates analyzed for cellular fatty acids (FAME) were
precultured on M-R2A and inoculated into Erlenmeyer ﬂasks containing 0.3%
(wlv) tryptic soy broth (T SB), (Krieg, 1981). Cells were harvested by
centrifugation. The cell pellet was placed into a screw-capped test tube and
stored at -20°C until prepared for analysis. Saponiﬁcation, methylation and
extraction were performed using the procedure described in the MIDI manual
(Sasser, 1990). Gas chromatography data was compared to a fatty acid
identiﬁcation library version 3.8 (Microbial Identiﬁcation Systems, Inc., Newark,
Del.) for possible isolate identiﬁcation.

Molecular methods. Repetitive extragenic palindromic REP- PCR patterns
were obtained from cells using Rep-1 and Rep-2 primers and the polymerase
chain reaction (de Bruijn, 1992). Ampliﬁcation was performed using a Model
9600 Perkin-Elmer Cetus Thermocycler. Products (10 pl) were separated by
electrophoresis in 1.5% agarose gels and stained with ethidium bromide.
Ampliﬁcation was primarily done using individual colonies grown on M-R2A, but
for isolates with poor or no ampliﬁcation, DNA extracted from cells was used as
the template for PCR ampliﬁcation. Half of the ampliﬁed REP products were run
in a prelliminar gel that was scanned for band patterns using the Ambis System.
Band pattern cluster analysis of sets of isolates that could include up to 130

strains revealed which isolates had possible close REP patterns. The remaining

49

REP ampliﬁed product were re-run side-by-side as suggested by the computer
cluster analysis to eliminate siblings from further characterizations. For isolates
with very close (not identical) REP patterns, the same stock of primers and
reagents were used in the analysis to conﬁrm differences.

The gene probes used in this study are listed in Table 2.2. E. coli cultures
carrying the plasmids with probes were grown for plasmid ampliﬁcation in the
presence of the appropriate antibiotic. Plasmids were extracted by standardy
protocol (Maniatis et al., 1982). The probes were isolated as restriction

fragments from their respective vectors in 1% low melting point agarose, puriﬁed

with the Gene clean kit (Bio 101, Inc. La Jolla, CA) and labelled with alpha-[32F]
dCTP (3,000 CilmM; Dupont, NEN Research Products, VVrlmington, DE) using a
random hexamer priming kit from Boehringer Mannheim Biochemicals. Labelled

probes were separated from unincorporated nucleotides prior to use with a spun

column (Maniatis et al., 1982). The probes were used at approximately 106
cpmlml of hybridization ﬂuid.

Genomic DNA was obtained by standard methods (Elmerich et al., 1982)
from pure cultures of isolates and selected strains grown on M-RZA broth under
aerobic conditions. Restriction endonuclease digestion of DNA was performed
according to manufacturer speciﬁcations. Digested DNA was size fractioned by
electrophoresis in 0.7% agarose gels and transferred to nitrocellulose
(polyester-supported BAS 68380; Schleicher & Schuell, Keene, NH.) as

previously described (Maniatis et al., 1982) using 20 X SSPE (1 XSSPE is

Table 2.2. List of DNA probes used in this study.

 

Organism (pﬁrobe source)

 

Pseudomonas puﬂda PaW1

Pseudomoneeputldeﬂ

Pseudomonaemendoclne KR

Burkholderia pickettii PKO‘i

Pseudomonas ep J8-150

Burkholderia cepacia 64

Gene encoded and size
of the probe

Plasmid

 

 

Methyl monooxygenase

(hydroxylase and NADi-i-

terredoxin reductase).
2.35 Kb (Sal lIHlnd Ill).
Toluene
dioxygenaseﬂarge and
small subunits of the
oxygenase, ferredoxin
and part of the
reductase). 3.5 Kb (Eco
RIIBgl II)

Toluene
parahydroxyiase
(monooxygenase and
ferredoxin), 3.8 Kb (Eco
RIIEco RI)

Toluene
metahydroxylase (a
subunit of
monooxygenase). 0.88
Kb (Ape IlAve I).
Toluene

orthohydroxylase, 2.2Kb

(Eco RVIHlnd ill).
Toluene

orthohydroxylase, 2.7Kb

(EcoRlIl-llndil).

p05i-l2838

poroem

pMY421

pAB14AAva l

pROZM 1s

8. l-iarayama

D. Gibson

M. DeFiaun

lR. Olsen

R. Olsen

 

‘ Laboratories from which the clones were obtained from.
This strain was originally provided by J.C.8pain.

51
0.18 M NaCl, 10 mM NaH2PO4, pH 7.04, and 1 mM EDTA). The DNA on the

ﬁlters was cross-linked by U.V. light (Stratagene, La Jolla, CA). The solutions
used for DNA hybridization analysis have been described elsewhere (Holben et

al., 1988). The membranes were prehybridized for at least 24 h in heat sealed

bags containing 100 pl of prehybridization ﬂuid per cm2 of ﬁlter.
Prehybridization ﬂuid contained 5 X Denhardt solution, 5 X SSPE, 50%
formamide, and 200 pg of sonicated and denatured salmon sperm DNA per ml.

The hybridization solution was the same as for prehybridization but included

10% (wlv) of dextran sulfate, and was added at 50 pl per cm2 of ﬁlter.
Membranes were incubated for at least 24 h. Two hybridization temperatures
were used: low stringency at 30°C and high stringency at 42°C. After
hybridization, the ﬁlters were washed once for 15 min with agitation at 30°C with
2 X SSC (1 X SSC is 0.15 M NaCl plus 0.015 M sodium citrate), plus 0.1% SDS.
For low stringency hybridization a second wash of 30 min with 0.5 X SSC plus
0.1% SDS was performed. For high stringency hybridization a second wash of
15 min with 0.5 X SSC plus 0.1% SDS followed by a third wash with 0.1 X SSC
plus 0.1% SDS was performed. This was followed by a ﬁnal wash at 55°C for 30
min with the latter solution. After washes, hybridization signals were visualized
using the Betascope radioactive blot analyzer (Betagen Corp., Waltham, MA) or
by autoradiography using Kodak X-Omat AR ﬁlm (Kodak, Rochester, NY)
exposed at -70°C with a Quanta Ill (Sigma, St. Louis, MO) intensifying screen.

Exposure times were 1 to 3 days depending on the intensity of the radioactive

52

signal. For reuse of the same blot for another probe, the blots were stripped by
washing for 10 min in boiling water and 0.1% SDS at least two times, depending
on the signal left on the blot after evaluation with the Betascope. A ﬁnal wash
with 2 X SSPE + 0.1% SDS for 10 min completed the stripping protocol.

Analytical methods. Toluene and TCE were measured by gas chromatography
equipped with a ﬂame ionization detector(GCIFlD), a DB-624 capillary column
(J&W Scientiﬁc, Folson, CA) and a headspace sampler. The autosampler vials
were equilibrated at 30°C, the column at 90°C and injector and detector at
200°C. He was the carrier gas. Phenol was analyzed by reverse-phase HPLC
with a Hibar RT C13 column (E. Merck), with a ﬂow rate of 1.5 mein of 66:33:01

Hzo-CH3CN-H3PO4 and a W detector set to 218 nm.

Results

Population densities. The effect of toluene and phenol concentrations on
population densities of toluene and phenol degraders and TCE co-oxidizers
were evaluated. The mean initial population density of microorganisms able to
degrade toluene was 7.2x102 cells/g of glass beads from sample 1 when the
concentration of toluene in the medium was 50 ppm and 4.9x104 cells/g of glass
beads when the concentration of toluene in the mediium was 5 ppm (Figure
2.3a). This trend of a greater than one log unit increase in populations of
toluene degraders detected at 5 ppm versus 50 ppm was also noted for the

water populations obtained from the two ﬁlters (2.4x105 and 3.5x10s cells/ml of

 

lIllllIllHill"Will!!!"IlIlllllllllll

llllHllll"H"Hmlll”NIIIUHHUIWHHH é

 

 

 

(spmmﬁﬁlmomreﬁ Imam)“: uremia “HO 501

54

slurry at 5 ppm versus 3.3x103 and 3.3x10‘ cells/ml of slurry at 50 ppm for the
0.22 um and cotton ﬁlters respectively, Appendix A The presence of TCE at 0.5
ppm or 1 ppm together with 5 ppm or 50 ppm toluene, respectively, had no effect
on MPN estimates of toluene degraders for the glass beads sample (Figure
2.3a) or for the the two ﬁltered water samples (Appendix A).

Phenol concentrations had less of an impact on MPN numbers of phenol
degraders than toluene (Figure 2.3a) but there still was a signiﬁcant increase in
numbers, nearly one log when the phenol concentration was decreased from 50
to 5 ppm (Figure 2.3a). Based on the sensitivity of the aquifer population to
higher toluene and phenol concentrations, isolates from samples 2, 3, and 4
where enriched in MPN tubes at 25 ppm, the lower concentration feasible for
evaluation of TCE cooxidizers.

The number of toluene and phenol degraders on the glass beads and
ﬁlters was compared to the number that could cooxidize 1 ppm of TCE in 2
weeks. A vial was assumed to be positive for co-oxidation when more than 50%
of TCE had disappeared. When toluene was the primary carbon source
7.0x103, 7.9x103 and 2.4x104 cells of TCE cooxidizerslml of slurry was observed
for the glass beads, 0.22am ﬁlter and cotton ﬁlter samples, respectively (Figure
2.3b). For the same samples, when phenol was the primary carbon source
7.9x10",5.4x105 and 3.5x105 cells/ml of slurry were obtained, indicating a one
log greater number of TCE co-oxidizers growing on phenol than on toluene. At

50 ppm of toluene or phenol, the population density of TCE degraders was

oozed mEEEon .2: 2: um 52.3.5 2: So: :38 noEEoe
«cent... no.5 Eot eh Sea .3050» :onﬁo EuEtn no .98.... ..o 0:022 mEEmEoo

n_n_> th goEuno nee—enact Baboon» EoEto one 98535860 mOh co .onEaz .nn.~ 2:9...

3:: :oﬁoo

 

.52: E: «N6

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

W

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

28.26% m2. D

 

 

women 320

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

End 3 .285... I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Egg on cacao... W

 

 

 

or

(Aunls |l|i l swsgueﬁio) Jeqlunu lepauoeq to 601

identical to that of toluene degraders and phenol degraders, suggesting that
most organisms able to degrade toluene or phenol produced the necessary
enzyme to degrade at least half of the TCE present. The same experiment, but
done at 5 ppm of the primary carbon source, did not yield consistent data. The
low concentration of the carbon substrate may not have resulted in enough
enzymes for TCE co-oxidation (Appendix A) or may indicate that TCE at a 10%
concentration may be toxic for some populations.

The total numbers of heterotrophs detected for the three samples were
4.3x107, 2.4x108 and 2.4x108 cells/ml of slurry for the glass beads, 0.22um ﬁlter
and cotton filter samples, respectively (Figure 2.3a and Appendix A). Using
these values, toluene degraders represent 1.8%, 0.1% and 0.15% of the total
heterotrophs, and phenol degraders represent 12.6%, 2.2% and 13.8% of the
total heterotrophic population present on the the glass beads, 0.22 um ﬁlter and
cotton ﬁlter samples, respectively. The populations of toluene or phenol
degraders is likely higher than this since some isolates selected from direct
plating on M-RZA of the most dilute tube were positive for toluene and/or phenol
degradation. This results further indicates, that the conventional MPN method
can understimate the population of phenol or toluene degraders. We attribute
this to toxicity of these compounds, even at concentrations as low as 5 ppm.
Lower concentrations of substrates could be used, but the detection method,
especially for toluene would be compromised, when working with the number of

samples required for MPN estimations.

Isolation of populations. To examine a broader spectrum of culturable
populations capable of degradation of the primary substrates and in doing so
gain a greater understanding of the entire community, we attempted to go
beyond enriching for the best competitors in the laboratory. We used the most
dilute MPN tubes showing disappearance of phenol and toluene with or without
TCE from all field treatments, to isolate the most dominant phenol and toluene
degraders. We also plated the original dilution series samples 2, 3, and 4 on M-
R2A and selected isolates from these plates. For sample 2, isolates were
obtained only on M—RZA medium since no MPN tube was positive. The isolate
obtained from sample 3 on M-RZA as the most dominant heterotroph, MF-H-3—3,
was not retrieved from the MPN tubes when phenol or toluene were the carbon
sources. (For strain nomenclature, MF= Moffett Field; H, if present indicates
heterotrophic isolate from M-R2A, the next digit indicates the sample number (1,
2, 3, or 4), and the last digit is the strain number in the sample series). One
isolate, MF-H-3-5 obtained by direct plating on M-RZA was also isolated from
MPN tubes (e.g. MF-301). As expected, some isolates obtained as heterotrophs
from M-R2A plates were incapable of toluene or phenol degradation. For sample
4, some degraders obtained from MPN tubes, namely isolates MF-416, MF-421
and MF-441 were also isolated as heterotrophs: MF-H-4-2, 4, and 6,
respectively. interestingly, the most dominant isolate from this sample, MF-H-4-

5, was also not isolated from dilution tubes and does not grow on toluene as the

primary carbon source, but it does grow on phenol. These results suggest that
toluene and phenol were toxic at the concentrations used in the very dilute MPN
tubes (low cell number/unit of substrate) and that in the most dilute MPN positive
tubes, these strains were unable to compete for the carbon subtrates with the

other populations present in the MPN tube and hence were not isolated.

Discrimination of isolates by REP-PCR analysis. We obtained 348
numerically dominant isolates from the Moffett Field experimental site from July
1993 through November 1993. These represent bacterial populations present
after an untreated 8 month period before the start of the 1993 ﬁeld experiments
(sample 1) or after treatment with phenol or toluene at different times in 1993
(samples 2, 3,4). These isolates were further screened for their ability to
degrade phenol or toluene; 273 isolates were shown to be positive for
degradation of at least one of the carbon substrates. Degraders of toluene or
phenol were ﬁrst grouped by similar colony morphology as much as possible.
To focus the further characterization, we eliminated very closely related isolates
or siblings using REP-PCR patterns. Visual comparison of patterns on several
gel pictures is a difﬁcult task so we used the Ambis system to scan and cluster
isolates with similar patterns. New gels were then run with closely related
strains side-by-side. This confirmed differences or similarities in REP patterns.
Isolates with identical REP-PCR patterns were considered the same and only

one representative of that pattern was analyzed further. The REP-PCR analysis

(Si

is

of the isolates revealed that the 273 degraders produced 63 different REP
ﬁngerprint patterns (Figure 2.4). Isolate MF-88, produced two different unstable
(reverting spontaneously to the original appearance) colony morphologies on M-

R2A medium, but the REP pattern remained the same (Figure 2.4).

Isolate distribution. One dominant REP group (MP-19) contained 15% of the
isolates (41 strains). Analysis of isolates obtained from glass beads,
representing the attached population and isolates obtained from the ﬁltered
water, representing the planktonic community (Figure 2.5.) shows that ﬁve of the
6 types most frequently isolated, MF-19, MF-63, MF-11 and MF-175 were
retrieved from both sources. Isolates MF-163, MF-180, MF-415 and MF-419
were isolated only from glass beads and isolates MF-185 and MF-168 only from

filtered water, but these types were less frequently recovered.

FAME/taxonomic and degradative diversity analysis. The isolates obtained
during the course of this experiment were analyzed for their cellular fatty acid
composition. Species identiﬁcation based on total fatty acids was poor for most
of the isolates since the analysis gave in many cases a low similarity index by
Using the MIDI database (Table 2.3). From the 63 bacterial isolates with unique
REP-PCR patterns, 16 isolates were assigned to 11 different genera and 15
different species with proﬁle similarity index of 3 0.5 (range, 0 to 1.0). The

taxonomic designations suggested were: Acinetobacterjohnsonii (strain MF-24),

60

Figure 2.4. REP-PCR ﬁngerprint patterns of Moffett Field isolates generated by
using genomic DNA or whole cells. Outside lanes show DNA size markers indicated
(in base pairs) on the left. Strains are ordered by similarity in band patterns

according to the Ambis System clustering program.

61

-3 7" I :i l
.: II.;:‘I.'lilil
: II l

 

é
,.
c
._

62

 

‘53

 

O
V

E

35
(DE
EE
iii.
33
eU

 

O O O
to N 1-

lhwuwis deal males! sewn :0 JOCIWNN

9'7'H‘JW

ZWdW
WSW
i911"
877'!"
977'!“
V it’d"
299:!"
69 PAW
29 P!"
St mm
6 i Pd"
es-aw
MAN
99'!"
39'!"
Edw
993W
Q‘JW
391W
8 PdW
OQ’JW
”5"
i it‘dW
099%!
”39‘
«2'5“
i3 PSW
[.0 P39“
ZO'JW
(33W
WAN
$':lW
VZ’dW
§dW
gov-aw
lot-aw
W P:iW
B i PdW
ZS PdW
so PAW
89'!“
[3M

E ”'3“
S lZ'M
Z 2'5"
Ed"
9 PdW
S PdW
99 P5“
£61!“
83 Pd"
vrz-aw
99 P3”
89 PM
6 W'JW
S WEI”
EZ'dW
09 P3“
9L PSW
i PM
Q'JW
6 PM

isolate identiﬁcation code

Figure 2.5. Frequency distribution of isolates according to REP analysis.

 

c822 02

 

'33

 

 

>. as a w «it:

 

 

 

+

.3... 5.6.... Sergei... + .s on. a p 5.:
.3... . 8.3.8.. £2.82 . + + a 3...:
8...... .3328... 3:12.321... . z. + . 8 B 5.:
«he... 0:93.520. 0.88.500 + u + i ON Nana—E
522...: + . + >. a 3...:
2...... £3... 8...... . . + + a... 8...:
«8... 33...... 53.8.5. + . + >. «a 8...:
.2... 3......1. 3.....o . .s + >+ a 33...:
3a... 38...... 3......3 . z. + >+ 8 8...:
1:6 eeuctae eecoaabaeet + .1. 3 . up 8...:
3 ..e 3.5.8.... .Pgael: + z. z. a. 8...:
3.... .353... .?§=.§EI + . . h . 2...:
«8... 3.5.3.... .9382}... + .s I 2 2...:
539.5 02 .. + ... up 00...:
.8... .838: .9332}... + + + 3 8...:
.8232 . + + 2 3...:
3...... .gl.u.ioe.u21£ + .5 z. - 2 3...:
2e... 8...... 3.88%....8 . - + + : «3.:
.2... 3x82... asset... + + + . 3 8...:
8.... 2.8.51 .3853 . . + a 3...:
8...... 3.5.3... .Pioegne. + - . 3-..:
3.96 2.29633 3.35250 ... + h 3...:
3.... .23.. 83.38.... + + o 2-..:
5.232 + ++ I a 2d:
..«u... alien. 33.3....3 + 3 + . n 5.:
5.8.2 + z. n :-..:
an... .5328... .9331: - + . u 5...:
2.... 8.8.8.... 8%.... + I + >. . 3.:

. 5.8
o. ..e. wastage... .8... o:o» 8.3.6 8.3.0 8.5 an... .828.

 

.eoEu aEEEon ..:o. 2: Eot £0.53 20... unto—2 05 Eo... uoESno

3.8.3 .51 59.05.. 5.3 noun-on. =u ..o encased 2.52.3 use 06229:... £22.00 .nd sin...

 

 

esegobaeem easy}

66

«8... 2. - + - 8 ..:-..:
8...... .558... 55:82:... a. . - . .2. .84.:
«8... 1.58.... 8......3 .2. + + - .8. 53.:

5.... 2. .2. + + . 8 2...:
as... 835er Satan ..: + + ... 3.". a 3...!
2.... 38...... 85.8.23 + + .s >- 8 ..:-..:
8.... 2...... 85.5.3 e. + .s - 8 23.:
8.... 3.5.... 85523 + + .s - «a 2...:
.8... 58...... 5:58.50 2. - + + 8 . 8...:

£22.. 2. - - ... - on 83...:
«an... 88551.8 8:25:30 + I. + >- At 3.1.—E
.8... 8.8.2... 5.5.8.. .2. - + - E 93...:

:82: o: - - + + 33 32...!
.8... 3.826.335. 8...... + + + 8 «8...:
8.... 5...... 8.88.8.8 - + + - 8 8...:
8.... 8...... 8555.... - + + >- 8 «8...:
.8... 55sec... 85.55.. + - + E ......-..:

5.2.32 - + + 8 2.3.4.:
.2... 885.5... 8.88:5... . - + + 8 2..«...-..:
2...... 8.38.5... 5.38.53 - + + - 8 3.24.:
8...... 5......58... 8.5.35: - - + >+ 8 1.8.:
2.... 8...... 88.3.... .2. + + 8. ..u-z...:

5.... e2 - - + + .. ..:-..:

5.5.2 - + + - e. ..:-..:
.8... 558555.... + + + - 8 «2...:
«8... 1:5... 8.....n - + 8 2-..:
2.... 5.8.2... 85.55.... + - 8 8...:
8.... 8818555.... + - 8 ..2-..:
2.... .88.... 3.3.5.3 + + + - 8 2...:

5.2.5... + + + - 8 8...:

5...: oz - - + + 8 8...:
2.... 85.5... 533.325.. + z. + - 8 5...:
8.... 8585823 + + + - .. 8...:

5.2.52 - + + 8 ..:-..:

 

dug—5:03 .«.N 03¢...

67

.8... :o....>. E2...» .9: .5 5 us... .0. 332... o... 0. ..:..=E_. .0 .83..
.82 .:......m. 2...... ..:...
.87....- ..:oEou:... E... 2...... o..:.u...8o:oE .5... 23...... .5 55... 5:38.53 .0... 52......» E2. 253:.

65......

.mm c.2023 223.5... a 8 39:5... 2. .3523... 2.38: .mm .53.... 335...... c. 32...... .39.: EB... mom-um:

 

6:32:82. .:..:.Eo.. on :82... ..:-.52.... 225 ..:: E202:
(«m-s. :o :8... ..:. ..:E3 .35.... .5 .o 2.... 5.3.... ..:.E... «>23. .5 E9: .35.... 22> ..:-1-..... 90E... .82....

.mOh + 225...: 2.2:... 2.. ..:. 2.52.... 29>? «BE... 81. ..3 an :2an3: 2.3.0..

do... + 225...... 3:2... 2.. .2... .358... 225.. o_.Euu. can. ..on a. :2an..: «8......
do... + HE ..J ..:. 2253: 3:2... .5 .8... .358... 28> .u «BE... cau- ....u 2. 3.23:: «8......—
...2 .o 5... o... .8 2...
2.. o. 3...... 22> 2.2.-o. ..:: 3:2... 5.3 8:252... 05 20.2. .3583: 0.2: c. 3:... 8 w- 2... an 3.3.5.: .82....-

 

vou... 23:03:08. 0?. ..: - - - C v. 0.1-1....
.3. .22.... .5555: 2. + + - .2. VII:
3... 2.1%.... 8.....n - - + + .3. «.12.:
QFV.O IE ‘gﬁﬁl I + + I 8 Du'uzlls
«3... 3.8.8.2. 5.0.6.3.. - .5 + - 2.. 3.1.2

:82: o: ..: - .5 + «a «3...:
on»... 2.28.23... .233.:..< ..: - + - 5 vow-.5.
can... 08.3.8:- -.b.:ooz - - + + .3. 03...:

:22: o: - - + + ..o 57.:
.3... 2.38.. 2.22.02 ..: - + + on 3.1.2
3.... 23:32: 2.2.80 ..: - + + on 3.7.2

:82: o: ..: .I. + - 5 3.7.2

 

€852... ...« o...»

H ydrogenophaga pseudoﬂava (strain MF-62), Bacillus subtilis (strain MF-89),
Nocardia asteroides (strain NIP-108), Comamonas acidovorans (strain MF-92),
Janthinobacterium Iividum (strain MF-118), Bacillus pumilus (strain MF-181),
Staphylococcus haemolyticus (strain MF-212), Pseudomonas putida (strain MF-
304), Bacillus amyloliquefaciens (strain MF-342), Acidovorax delaﬁeldii (strain
MF-413 and MF-415), Acidovorax facilis (strain MF-414), Nocardia restricta
(strain MF-450), Burkholderia pickettii (strain MF-H-4-4) and Van'ovorax
paradoxus (strains MF-39) (Table 2.1). Of the remaining 47 isolates, 30 isolates
presented identiﬁcation proﬁles with a similarity index below 0.5, 15 isolates had
no match at all to the MIDI database and two isolates did not grow on TSB
medium, the medium recommended for comparison to the database. MIDI
speciﬁes similarity indices of > 0.5 as having a high probability of being correct
and those between 0.6 and 1.0 as being excellent matches. The low similarity
indices observed for most of these isolates of environmental origin may be a
reﬂection of the fact that the MIDI-FAME data base is primarily composed of
clinical isolates. Gram stain results were in agreement with the identiﬁcation
Provided by FAME (Table 2.3). Overall, 30% of the isolates were gram positive
and 70% gram negatives (Table 2.4).
Analysis of the collection with different REP patterns revealed that 65% of
the isolates can degrade toluene, 90% can degrade phenol and 57% can
degrade both carbon substrates. Only 8% of all isolates could degrade toluene

exclusively and 35% could degrade phenol exclusively (Table 2.4). Gram

69

6:95.00 50..

500:: 030000: 0.0 000.500 50.00 50.. 00: .05 3.0.0»... .0050... :0030 an 000: .05.... ..0 0:020...-

 

 

 

 

 

 

 

 

 

 

9.000490
3 2. 3 50 2... 0:028 0:0 .0:0:.. Q.
00 03 an 00.. 8.. 8000500 .05.... 0..
no on 00., Q 00.. 9.000.600 0:020... .x.
mm 2. 2. o o 9.0.3.. sop .x.
on 2. on «v «N 8000500 NO... .x.
L055. .0520... «3:0»... .0520...
.80... 002.30: 50.0 002200.. 80:0
8 m... m 8 ﬂouuaulu 000 0..
ﬂ 3 v mm 90.3.. .200 .x.
3 R «v 2. 93000: EEO .x.
Q 8 a on 25.8.. 520 .x.
5 mm o .50... .0 .x.
0:020... .80...
0:0 3:05. 2:0 3:09.“. 2:0 0:020... .0 .x
00.000000 530.0

 

.20.“. 02.0.2 :6: «53.8.
mm: .5.ch 5.3 8.28. 8 as .8 02:23.0 83:38:20 50:. .0 5.50.20... .3 030...

70

positives isolates were more frequently exclusive-phenol degraders (53%)
compared to gram negatives (27%). No gram positive isolate was an exclusive
toluene degrader, whereas 12% 0f gram negatives could only degrade toluene.
Of the gram positive isolates, 100% are phenol degraders and 47% are toluene
degraders. For gram negative isolates that can degrade phenol, 70% can also
degrade toluene and for gram negatives that can degrade toluene, 88% can also
degrade phenol. Analysis of the cometabolic TCE transformation potential
showed that 60% of the isolates were TCE co-oxidizers. Gram positives (42%)
and gram negatives (70%) were more effective TCE co-oxidizers when phenol
was the primary carbon source, where only 22% and 56%, respectively, were

effective TCE cometabolizers (T able 2.4).

Diverslty of toluene degradation genes. Chromosomal DNA isolated from
isolates and ﬁve well studied aerobic toluene degraders was hybridized to
various toluene pathway probes to determine which strains carried similar
sequences. Probes for the ﬁrst steps in all ﬁve aerobic toluene degrading
pathways were used. In a preliminary search, slot blots were used to analyze for
the presence of homology between DNA from each isolate and the ﬁve different
Probes. Under low stringency conditions the results were inconsistent because
many isolates hybridized to all probes, probably as a result of the low speciﬁcity
inherent to the hybridization conditions. Under high stringency conditions only

the probe for the ortho-hydroxylase gene showed signiﬁcant signals. We then

71

used Southern blot analysis for a more speciﬁc evaluation of hybridization. Only
the probes for the ortho-hydroxylase from Pseudomonas sp. JS-150 genes
showed hybridization under low and high stringency conditions (Figure 2.6)
corroborating the results from slot blots. On the basis of the hybridization signal
intensity under high-stringency conditions, different levels of sequence
homology were observed for the different isolates (Figure 2.6). Fifty-ﬁve percent
of the tested isolates showed homology to the gene encoding the toluene ortho-
hydroxylase probe (Table 2.4). Only 18% and 4% of the exclusive phenol 0r
toluene degraders, respectively, showed homology to the TOM probe, whereas
78% of toluene and phenol degraders were positive for the presence of this
gene sequence (Table 2.4). Hence, these results demonstrate that DNA highly
homologous to the genes encoding toluene ortho-hydroxylase is present in the
majority of the bacterial strains tested that catabolize both phenol and toluene.

Interestingly, no gram positive isolate tested showed homology to this gene.

TCE co-oxldatlon rates. The rate of TCE degradation by ﬁve different well
known aerobic toluene degraders that harbor different ﬁrst step mechanisms for
toluene attack is shown in Figure 2.7. TCE was metabolized at a high initial rate
that decreased over time for strains F1, KR, PKO1 and G-4. Toluene was
degraded at a very fast rate by strain PaW1, but no co-oxidation of TCE was
Observed for this organism. For strains F1, KR, PK01 and G-4, the onset of

TCE degradation was delayed until a substantial fraction of toluene had been

72

Figure 2.6. RFLP profiles of genomic DNA from Moffett Field isolates digested with
Ecol-i1 and hybridized with the gene probe encoding for the toluene ortho-
hydroxylase of Pseudomonas sp. JS-150.

 

74

 

00.000 :0900 208.... 0:. 003 0:020... 03350..
0200.000 0:022 0.00.00 :305. 02. 05 022000.00. 0:.000 20.0.20 >0
3002.0 :000. .2650. mo... 0:0 .2008: 00...: 5:083:00 0:022 B 50:00 KN 0.50...

 

 

050...:
au— 8. 8 8 3 00 o
. . . . . P
0.0 : ..- no I. o o. - 0
..

o... M.

0.. / . /. 1
) / ... 4.. 2 m.
m 0.0 4 4 4 4 4 4 4 4 4 4 a
w 4... . 0 . as w
m . U .............. O / /./ I «O.
m 0.0 O ...... W ............ 0.: ............. . / /p I v, 0” m
9 . 0:0: . .. .

.. z 0% in...” ....... m .... , ..
w a... 028 ...-235.44 x /.0. .4 3 W

m ...-as . \l
"—0.. a6 a” w 1.3.00 .50.” / ...Q'//.. .Adu
T . P m

_,. ...... ....-.r w ..

I HI...“

u... n

 

 

8

76

metabolized, notably for strain F 1. At 45 h from the beginning of the experiment
toluene was consumed by all strains, but G-4 was the only strain to show
extensive cometabolism of TCE (to below detection).

The isolates from Moffett Field showed variable rates of TCE co-
oxidation, and for many strains TCE was cometabolized at a high initial rate that
decreased over time (Figure 2.8). The extent of TCE degradation was strain
dependent. At 120 h, toluene was totally consumed by all strains but less than
50% of TCE was co-oxidized by strains MF-7, MF-163 and MF-H-3-3, whereas
more than 50% was co-oxidized by strains MF-23, MF-15, MF-168, MF-182, MF-
183, MF-179 and MF-80. Burkholderia cepacia strain G-4, used as a control in
this experiment, showed complete removal of TCE and had the fastest co-
oxidation rate of any strain in this group. Some strains degraded toluene at
rates that are not signiﬁcantly different from those of Burkholderia cepacia G-4
(Figure 2.9.). However, extensive degradation (to below detection) of TCE was
only observed with strain G-4.

The relative change in TCE concentration between inoculated and
uninoculated controls measured at 15 days of incubation was used to evaluate
the relative TCE transformation capacity of the entire collection. Isolates varied
markedly in their ability to co-oxidize TCE. The rank ordered cometabolic
transformation capacity proﬁles for isolates grown on phenol and toluene are
illustrated in Figure 2.10. Isolates with homology to the toluene ortho-

hydroxylase gene (TOM) were distinctive in both the proportion of isolates with

% TCE co-oxIdIzed

 

H MF-168
OO MF-15
u MF-‘ITO
" MF-163

.9 MF-23

 

 

 

 

 

 

D ............ Q
a“ i‘
1 ...A
............. A
v
0.0 - V v v v v
I I T l T I
0 20 40 60 80 100 120

Time (hr)

Figure 2.8. TCE co-oxldation patterns of selected Isolates. Toluene
was the primary carbon source and was totally consumed
by all strains at 120 hours.

Toluene concentration ( ppm)

78

 

 

 

 

O. Toluene 64
OO TCE 64

DD TCE MF=-180
AA TCE MF-H-3-3

()0 Tee NIP-80

VV TCE MF-163

 

I. Toluene MF-180

M Toluene MFR-34

O. Toluene RIF-80

W Toluene MF-163

é """""" (3'3 ........
é»;
'i‘<>.
D. -
‘2 ﬁght:
%§......D ..... D
§ ........ 9

1.5

I
d
e
°

.2
_ OI
(mdd ) uouenuesuoo 391

 

 

I

20 40 60 80 100 120

ﬁmemﬂ

fast toluene degradation rates

140

Figure 2.9. TCE co-oxldatlon profile of selected Isolates that show

79

60000002 .0 0>00 m. .0 :00020>m .70 2000
0.00000 0.00.05.05 >0 02.200 :20... 0:00 000.38.030.03 0... 0. >020 500
0005.3 .0 5.3 00.0.00. 0.0.“. 02.0.2 >0 32.00 :00000800 mo... .0 5000.005 .0...~ 0.00.“.

>0:0000.. 0200.00.00

 

       

oé ad 06 4.: N6 06
. _ . F . .
i 6

3020002000 02.000: 5.0.. 5.,

350002000 300002 .20... .0 . 2.”. I. l or
0520002000 020.000 .zOh DD ..... ’0’. l ON
.0:000.0:.000 05300.22. 44 ..:...C. I on

.’ ....... ’ ....... '. . . . . . >. l 0‘
.. .e..e.. .. 8
.. 1 cm
l 2.
r. on
I cm

I 8..

 

 

 

I. ....

"093999433 301. “lo

80

high co-oxidation capacities (more than 60% with transformation capacities >
than 90%) and the overall number of isolates obtained (55% TOM positives).
There was no effect of the primary carbon source on the transformation capacity
proﬁle for TOM positive strains, but differences can be seen when TOM
negative strains are analyzed. More than 50% 0f the TOM negative strains
grown on toluene showed less than 50% co-oxidation capacity when toluene

was the primary carbon source, whereas more than 50% of TOM negative

strains co-oxidized TCE when grown on phenol.

Discussion

We analyzed population levels and the diversity of dominant phenol and
toluene degrading bacteria isolated at different times from an aquifer that has
been treated with different CAH’s, phenol and toluene by using molecular and
conventional microbiological techniques. Microbial population density is an
important determinat of biodegradation rates (Simkins and Alexander, 1984).
We used standard MPN techniques (Alexander, 1982) to quantify numbers of
specific phenol and toluene degraders and respective TCE co-oxidizers. For the
evaluation of the number of toluene and phenol degraders, the concentration of
substrate used was critical. Our results showed that populations were probably
underestimated by the previous methods used. High concentration of substrates
May also select resistant populations that have little signiﬁcant ecological

il'hportance since this condition is usually found in contaminated sites where

81

biodegradation is occurring. We have previously shown that toluene
concentration was also critical for enrichments and isolation of pure cultures of
toluene-degrading denitriﬁers (Fries et al., 1994). Phenol and toluene degraders
were found to be 9.5% and 0.7%, respectively, of total heterotrophic bacteria.
Phenol has been the substrate used at this site for two previous feeding
regimens Which may have enriched phenol degraders in the site.

The majority of the isolates can grow on both phenol and toluene as the
primary carbon source although phenol degradation was a trait found in 90% of
the isolates. A larger percentage of phenol grown strains were more effective in
cooxidizing TCE than toluene grown strains. This suggests that phenol may be
better than toluene for ﬁeld application.

Most of the dominant strains isolated from the glass beads were also
isolated from the water samples suggesting that they are good surface
colonizers. Very few strains were isolated from water samples exclusively.
Species identiﬁcation by FAME analysis recognized only 25% of the isolates at
an index above 0.5, and these isolates were placed in 11 different genera.
Forty-eight percent of the isolates could not be identiﬁed by FAME analysis. This
is similar to what is found with other groups of environmental isolates, namely

that the database inadequately reﬂects environmental strains.

The most surprising feature of this study was the dominance of the toluene

Ortho-monoxygenase gene in these aromatic degraders. Hybridization signals

ft‘Om Southern blots showed that the toluene ortho-hydroxylase is the dominant

82

toluene degrading pathway. The variation in RFLP patterns obtained, signal
intensity and restriction size of the bands suggests divergence of sequences for
this gene in the populations, although it may have had a common ancestral
origin. The lack of hybridization of many strains to any of the ﬁve probes tested
suggests unknown pathways or enzymes. Indeed, a new gene sequence for
toluene degradation that is biochemically similar to the dioxygenase pathway of
Pseudomonas putida F1 has been isolated (Gerben J. Zylstra, personal
communication). Following toluene injection into the aquifer, ortho-cresol was
detected as an intermediate (Hopkins and McCarty, 1995) providing additional
evidence for dominance of the ortho-hydroxylase pathway among the organisms
present and effective in this ﬁeld site.

Although separate information on TCE degradation rates for the ﬁve well
known toluene degraders that harbor different genes is available in the literature
(Winter et al., 1989; Wackett and Gibson, 1988, Folsom et al, 1990), direct
comparison among organisms is difﬁcult because of signiﬁcant differences
among methods used. This is the ﬁrst study that compared these strains under
the same conditions. The efﬁciency of TCE cooxidation was related to the gene
sequence present on the organisms. The same observations has been made for
methane monooxygenase where the soluble form of that enzyme is more
effective in TCE degradation (Oldenhius et al, 1989; Tsien et al., 1989; Alvarez-

Cohen et al, 1992). We found some cross hybridization between the clones for

the toluene ortho-hydroxylase gene and the toluene meta-hydroxylase, and the

83

strains harboring those sequences, Burkholderia cepacia G-4 and Burkholderia
pickettii PKO1. were the most efﬁcient TCE cooxidizers. TCE transformation
capacity varied widely between the Moffett Field strains and ranged from
approximately 1 to 100%. The diversity in the transformation capacity proﬁle
also indicates that a single population was not dominant at this site. A possible
explanation for this fact is that populations may respond in different ways to the
interactions between the natural substrate and other chemicals serving as
substrates for the oxygenases systems. This was also shown by Folsom et al.
(1990) in experiments with equal concentrations of phenol and TCE. A decrease
of about 50% in the rate of phenol degradation was observed, suggesting a
competitive mechanism, and possibly toxic effects, between the natural
substrate and the fortuitous substrate, TCE.

In the present study we have shown the diversity of multiple phenol and
toluene degrading strains from glass bead bioﬁlms and planktonic communities.
The majority of these isolates harbor pathways similar to the toluene ortho-
hydroxylase gene, but the lack of signal to all known genes for more than half of
the isolates suggests that more diversity exist at the gene sequence level of

toluene oxidation than what is currently known.

84

Acknowledgments
We thank Helen Corlew-Neuman for conducting the FAME analysis. This work

was supported by EPA , Grant N° CR 822029-01-0 and the NSF Center for

Microbial Ecology, Grant N° BIR 9120006.

List of References

Alexander, M. 1982. Most probable number method for microbial populations, p.
815-820. In A L. Page (ed.), Methods of soil analysis. Part 2. Chemical and
microbiological properties. American Society of agronomy, Madison, WI,
USA

Alvarez-Cohen, L. , P. L. McCarty, E. Boulygina, R. 8. Hanson, G. A.
Brusseau, and H. C. Tsien. 1992. Characterization of a methane urilizing
bacterium from a bacterial consortium that rapidly degrades
trichloroethylene and chloroform. Appl. Environ. Microbiol. 58:1886-1893.

Alvarez-Cohen, L. and P. L. McCarty. 1991a. Product toxicity and cometabolic
competitive inhibition modeling of chloroform and trichloroethylene by
methanotrophic resting cells. Appl. Environ. Microbiol. 57:1031-1037.

Alvarez-Cohen, L. and P. L. McCarty. 1991b. Effects of toxicity, aeration, and
reductant supply on trichloroethylene transformations by a mixed
methanotrophic culture. Appl. Environ. Microbiol. 57:228-235.

Arciero, D. T. T. Vanelli, M.Logan, and A. B. Hooper. 1989. Degradation of
trichloroethylene by the ammonia-oxidizing bacterium Nitrosomonas
europaea. Biochem. Biophys. Res. Commun. 159:640-643.

Dabrock, B., J. Riedel, J. Bertram, and G. Gottschalk. 1992.
lsopropylbenzene (cumene) a new substrate for the isolation of
trichloroethylene-degrading bacteria. Arch. Microbiol. 15829-13.

de Bruijn, F. J. 1992. Use of repetitive (repetitive extragenic palindromic and
enterobacterial repetitive intergeneric consensus) sequences and the
polymerase chain reaction to ﬁngerprint the genomes of Rhizobium meliloli
isolates and other soil bateria. Appl. Environ. Microbiol. 58:2180-2187.

Elmerich, C. ,B. L. Dreyfus, and J. P. Aubert. 1982. Genetic analyses of
nitrogen ﬁxation in a tropical fast-growing Rhizobium. EMBO J. 1:499-503.

Ensingn, S. A., M. R. Hyman, and D. J. Harp. 1992. Cometabolic degradation
of chlorinated alkenes by alkene monooxygenase in a propylene-grown
Xantobacter strain. Appl. Environ. Microbiol. 58:3038-3046.

Ewers, J., D. Freier-Schroder, and H. J. Knackmuss. 1990. Selection of
trichloroethene(‘l' CE) degrading bacteria that resist inactivation by TCE.
Arch. Microbiol. 154:410-413.

Fogel, M. M., A. Taddeo, and S. Fogel. 1986.8iodegradation of chlorinated
ethenes by a methane-utilizing mixed culture. Appl. Environ. Microbiol.
51:720-724.

Folsom, B. R., P. J. Chapman and P. H. Pritchard. 1990. Phenol and
trichloroethylene degradation by Pseudomonas cepacia G4: kinetics and
interactions between substrates. Appl. Environ. Microbiol. 56:1279-1285.

Fries, M. R., J. Zhou, J. Ghee-Sanford, and J. M. Tiedje. 1994. Isolation,
characterization, and distribution of denitrifying toluene degraders from a
variety of habitats. Appl. Environ. Microbiol. 60:2802-2810.

Harayama, 8., M. Rekik, M. Wubbolts, K. Rose, R. A. Leppik, and K. N.
Timmis. Characterization of ﬁve genes in the upper-pathway operon of tol
plasmid pWWO from Pseudomonas putida and identiﬁcation of the gene
products. J. Bacteriol. 171:5048-5055.

Harker, A. R. and Y. Kim. 1990. Trichloroethylene degradation by two
independent aromatic independent pathways in Alcaligenes eutrophus
JMP134. Appl. Environ. Microbiol. 56:1 179-1181.

Henry, S. H. and D. Grbic-Galic. 1991. Inﬂuence of endogenous and
exogenous electron donors and trichloroethylene oxidation toxicity on
trichloroethylene oxidation by methanotrophic cultures from a groundwater
aquifer. Appl. Environ. Microbiol. 57:236-244.

Henry, S. H. and D. Grbic-Galic. 1990. Effect of mineral media on
trichloroethylene oxidation by aquifer metanotrophs. Microb. Ecol. 20: 151-
169.

Holben, W. E., J. K. Jansson, B. K. Chelm, and J. M. Tiedje. 1988. DNA probe
method for the detection of speciﬁc microorganisms in the soil bacterial
community. Appl. Environ. Microbiol. 54:703-711.

Hopkins, G. D., L. Semprini, and P. L. McCarty. 1993. Microcosm and In situ
ﬁeld studies of enhanced biotransformation of trichloroethylene by phenol-
using microorganisms. Appl. Environ. Microbiol. 59:2277-2285.

Hopkins, G. D., and P. L. McCarty. 1995. Field evaluation of ln-Situ aerobic
cometabolism of trichloroethylene and three dichloroethylene isomers using
phenol and toluene as the primary substrates. Environ. Sci. Technol.
(submitted for publication)

Johnson, G. R., and R. H. Olsen. 1993. Characterization of clones from
Pseudomonas sp. JS150 encoding substituted phenol and benzene
metabolic pathways analogous to Pseudomonas strains PKO1 and G4,
abstr. K-82, p.274. Abstr. 93th Annu. Meet. Am. Soc. Microbiol. 1993.

Krieg, N. R. 1981. Enrichment and isolation, p. 112-142. In P. Gerhardt, R. G.
E. Murray, R. N. Costilow, E. W. Nester, W. A Wood, N. R. Krieg, and G. 8.
Phillips. (ed.), Manual of methods for general bacteriology. American
Society for Microbiology, Washington, DC.

Little, C. D., A. V. Palumbo, S. E. Herbes, M. E. Lidstrom, R.L. Tyndall, and
P. J. Gilmer. 1988. Trichloroethylene biodegradation by a methane-
oxidizing bacterium. Appl. Environ. Microbiol. 54:951-956.

Malalchowsky, K. J., T. J. Phelps, A. B. Teboli, D. E. Minnikin and D. C.
Mite. 1994. Aerobic mineralization of trichloroethylene, vinyl chloride, and
aromatic compounds by Rhodococcus species. Appl. Environ. Microbiol.
60:542-548.

Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a
laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Nelson, M. J. K.,S. O. Montgomery, and P. H. Pritchard. 1988.

Trichloroethyleene metabolism by microorganisms that degrade aromatic
compounds. Appl. Environ. Microbiol. 54:604-606.

Nelson, M. J. K.,S. 0. Montgomery, E. J. O’Neil and P. H. Pritchard. 1987.
Biodegradation of trichloroethylene and involvement of an aromatic
biodegradative pathway. Appl. Environ. Microbiol. 53:949-954.

Nelson, M. J. K.,S. O. Montgomery, E. J. O’Neil and P. H. Pritchard. 1986.
Aerobic metabolism of trichloroethylene by a bacterial isolate. Appl.
Environ. Microbiol. 522383-384.

Oldenhuis, R., R. L. J. M. Vink, D. B. Jansen, and B. Witholt. 1989.
Degradation of chlorinated aliphatic hydrocarbons by Methylosynus

87

trichosporium OB3b expressing soluble methane monooxygenase. Appl.
Environ. Microbiol. 55:2819-2826.

Olsen R. H., J. J. Kukor , and B. Kaphammer. 1994. A novel toluene-3-
manaoxygenase from Pseudomonas pickettii PKO1. J. Bacteriol. 176:3749-
3756.

Owens, J. D., and R. M. Keddie. 1969. The nitrogen nutrition of soil and
herbage coryneform bacteria. J. Appl. Bacterial. 32:388-347.

Rasche, M. E., M. R. Hayman, and D. J. Harper. 1991. Factors limiting
aliphatic chlorocarbon degradation by nitrossomonas europaea: cometabolic
inactivation of ammonia monooxygenase and substrate speciﬁcity. Appl.
Environ. Microbiol. 57:2986-2994.

Roberts, P. V., G. D. Hopkins, D. M. Mackay and L. Semprini. 1990. Aﬁeld
evaluation of in-situ biodegradation of chlorinated ethenes: Part 1,
Methodology and field site characterization. Ground water 28:591-604.

Sasser, M. 1990. Technical Note#102: Tracking a strain using the Microbial
Identiﬁcation System. MIS Inc., North Newark, Del.

Shields, M. S. (1994) Personal communication.

Simkins, S., and M. Alexander. 1984. Models for mineralization kinetics with
the variables of substarte concentration and population density. Appl.
Environ. Microbiol. 47: 1299-1 306.

Smibert, R. M., and N. R. Krieg. 1981. General characterization, p.409-443. In
P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A Wood, N.
R. Krieg, and G. 8. Phillips. (ed.), Manual of methods for general
bacteriology. American Society for Microbiology, Washington, DC.

Tsien, H. C., G. A. Brusseau, R. S. Hanson and L. P. Wackett. 1989.
Biodegradation of trichloroethylene by methylosinus OB3b. Appl. Environ.
Microbial. 55:3155-3161.

Vogel, T. M. and P. McCarty. 1985. Biodegradation of tetrachloroethylene ta
trichloroethylene, dichlaraethylene, vinyl chloride , and carbon dioxide under
methanogenic conditions. Appl. Environ. Microbiol. 49: 1 080-1 083.

Wackett, L.P., and S. R. Householder. 1989. Toxicity of trichloroethylene to
Pseudomonas putida F1 mediated by toluene dioxygenase. Appl. Environ.
Microbiol.55:2723-2725.

Wackett, L.P., D. T. Gibson. 1988. Degradation of trichloroethylene by toluene
dioxygenase in whole cell studies with Pseudomonas putida F 1. Appl.
Environ. Microbiol.54z1703-1708.

Wilson, J. T. and B. H. Wilson. 1985. Biotransformations of trichloroethylene
in soil. Appl. Environ. Microbiol. 37:242-243.

Winter, R. B., K. Yen and B. D. Ensley. 1989. Efﬁcient degradation of
trichloroethylene by a recombinant Escherichia coli. Bio/1' echnalagy 7:282-
285.

Yen, K.-M, M. R. Karl, L. M. Blatt, M. J. Simon, R. 8. Winter, P. R. Fausset, H.
8. Lu, A. A. Harcourt, and K. K. Chen. 1991. Cloning and characterization
of a Pseudomonas mendocina KR1 gene cluster encoding toluene-4-
monooxygenase. J. Bacterial. 173:5315-5327.

Zylstra, G. J. 1995. Personal communication
Zylstra, G. J. and D. T. Gibson. 1989. Toluene degradation by Pseudomonas

putida F1: Nucleotide sequence of the todC1CZBADE genes and their
expression in Escherichia coli. J. Biol. Chem. 264:14940-14946.

Appendix A

Table A1. Total number of heterotrophs, toluene, phenol and TCE

degraders recovered using different concentrations of

substrates, and present on three different samples from

 

 

 

 

 

 

 

 

 

the Moffett Field aquifer.
Sample Concertmion (ugIml) MPN (cells/ml of slurry)
Tol. Phe. TCE Primerysubetrie TCE co-oxidizers
cell n° 5% 01' cell n° $96 Cl
GI!“ 50 - - 7.011103 2111103, 23:104
beads 50 - 1 1.1x1a‘ 3.3x1a3, 3.sx1a‘ 7.01:103 2.1):103, 231:10‘
5 - - 4.91:1 05 10:10", 1 0:10"
5 - 0-5 3.51005 1.11110“, 1.2110“5
- - - 4.3::107 . 1.31:1 a7, 10:10“
- 50 - 2:11:105 7.31:10‘. 7.911105
- 50 1 1.31110:5 3.91:10‘, 4.311105 7.91:10‘ 2.41:10‘2611105
- 5 - 50:108 10:10“, 1.871107
- 5 0-5 7.91005 20:105. 20:106
0.22m 50 - - 3.31003 1.01:10’,1.1x10‘
ﬁlter 50 - 1 7.911103 20:103. 20:10‘ 79:103 20:103. 20:10‘
5 - - 20:105 7.31:10‘. 70:10"
5 - 0-5 201105 7.31:10‘, 70:10”
- - - 20:10“ 7.311107, 70:10"
- 50 - 3.51005 1.111105. 1.2:1a6
- 50 1 4.2005 1.5100”, 10:10“ 5.41:105 1.6):105,1.8x106
- 5 - 3.41:10‘3 1.0110“, 1.811107
- 5 0-5 3.51:10es 1.11110“, 1.21107
600011 50 - - 3.3x10‘ 1.0:10‘. 1.111105
ﬁt" 50 - 1 3.31110‘ 1.0:10‘, 1.111105 2.0:10‘ 7.311103, 7.91:10‘
5 - - 3.51005 1.11110“. 1.2:10'3
5 - 0-5 1.31:108 3.91:105, 4.311106
- - - 2.0110“ 7.31:1 07, 70:10"
- 50 - 3.311107 1.01107, 1.1100"
- 50 1 2411106 70:105. 7.91:106 3.5::105 1.111105. 1.21106
- 5 - 2:11:106 7.311105, 7.9.0106
- 5 0-5 20:10“ 7.31:1 a“, 70:10"

 

Table A. 1. (continued)

S—2 5
5
S-3 5
5
S-4 5
5

5
5

9181

5
5

0.5

0.5

0.5

0.5

0.5

0.5

' CI. conﬁdence interval

b

3.0002 .0000
0000’. 8.000
0.000 .3000
1.5100 .1000
1.5100 .1000
7.3100 .7000
21100 .2000
1.1100 .1200
1.1100 .1200
1.1x10 .1200
7.000 .7000

~100au~auuA
OGG‘I‘OOGAU

4.01002

1.11:10‘

29x10

3.510 0

1.11002. 1.0003

0.00 0‘. 3000‘

0.00 03. 900 03

1.11:10‘.1.200‘s

91

Appendix B

Table 8.1. Original data on all isolates from Moffett Field. Isolates are arranged
according to common REP-PCR groups. source of isolate. and use of

toluene and phenol are also shown.

 

 

a b a c c
isolate REP Source of isolate Toluene Phenol
MF-S E-FSOT 10-4 0(10-5) 100 100

100 100

109 E-cso'r 10-4-(10-5) 100 100
MF-7 E-FSOT 10-4b(10-5) 100 0.0
100 0.0

12 E-cso'r 10-«(10-5) 100 0.0

9 E-CSOT 10-4a(10-5) 100 0.0
MF-11 E-FSOT 104-(106) 0.0 100
100' 100

2 E-GB5OT 1050(10-5) 100 100
3 E-GBSOT 10-Sa(10-6) 100 100
4 E-GBSOT 1050(10-5) 100 100
MF-13 E-CSOT 1040(10-6) 100 100
100 100

MF-15 E-cso'r 10-4c(10-7) 100 100
100 100

20 E-cso'r 1040(10-6) 100 100
21 E-CSOT 10-«(10-8) 100 100
6 E-FSOT 1040(10-7) 100 100
NIP-18 5-00501' 10-5.(10-6) 00 100
99 100

17a E-GBSOTT 1050005) 80 100
103 E-csor 10-«(10-6) 70 100
55 E-GBSOTT 1050(10-6) so 100
NIP-19 E-FSOT 10-4c(10-6) 100 100
100 100

14 E-csoT10-«(10-6) 100 100
10 E-FSOT 10-«(10-6) 100 100
176 E-CSOT10-4a(10-6) 100 100
177 E-GBSOTT10-Sa(10-6) 100 100

1 E—GBSOTT 10-5a(10-6) 100 100
25 E-FSOT 1040(10-6) 100 100
184 E-FSOT 10-4a(1o-6) 100 100
45 0085017 10-5a(10-6) 100 100
49 5.0507 10-4a(10-6) 100 100
50 E-GBSO'IT 10-5a(10-8) 100 100
172 E—CSOT10-40(10-6) 100 100

 

Table 8.1. (continued)

92

 

NIP-23 8 E-GBSOP 1055(10-6) 0.0 100
0.0 100
27 E-GBSOP 10.5.(10-7) 0.0 100
28 E-FSOP 1O-50(10-6) 0.0 100
29 E-FSOP 10-5a(10-6) 0.0 100
30 5:509 10-50(10-6) 0.0 100
31 E-FSOP 10-5a(10-6) 0.0 100
32 E-CSOP 10-7a(10-6) 0.0 100
33 E-CSOP 10-7a(10-6) 0.0 100
34 E-CSOP 10-7a(10-7)MD 0.0 100
MF-24 9 5.00509 10-5a(10-4) 0.0 0.0
0.0 100 *
38 1500501: 10-5b(10-5) 0.0 100
1115-39 10 E-FSOPT 10-6b(10-6) 50 100
40 100
41 E-FSOPT 10-6b(10-6) 30 100
MF-52 11 5430507 10-5a(10-4) 0.0 100
. - 40 100
MF-53 12 000500 1050(10-4) 100 100
100 100
MF-54 13 E-FSOT 1040(10-5) 100 100
100 100
MF-58 14 5.00509 1050(10-7) 100 100
100 100
60 00509 10-7a(10-6) 100 100
61 E-CSOP 10-7a(10-6) 100 100
123 E-CSO‘IT10-5a(10-6) 100 100
NIP-59 15 1506501: 1055(10-5) 100 100
100 100
MF-62 16 E-050P 10—7a(10-5) 100 100
100 100
MF-63 17 E-GBSOPT 104511005) 100 100
100 100
64 15005091" 10511004) 100 100
65 54305097 1055(10-5) 100 100
37 E-GBSOPT 10-5b(10-6) 100 100
42 E-C§OPT 1o-ea(10-6) MD 100 100
43 E-CSOPT 10-6a(10-6)MD 100 100
44 E-FSOPT 10-6b(10-6) 100 100
35 54335097 10-5b(10-5) 100 100
36 54395097 1055(10-5) 100 100
69 E-FSOPT 10-6b(10-6) 100 100
70 E—CSOPT 10-6a(10-6) 100 100
71 e-csopr 10-6a(10-6) 100 100
72 E-CSOPT 10-6a(10-6) 100 100
73 E-CSOPT 10-6a(10-6) 100 100
74 E-CSOPT 10-6a(10-6) 100 100
75 E-csoPT 10-6a(10—6) 100 100
76 E-C50PT 10-6a(10-6) 100 100
77 E-FSOPT 10—6b(10-6) 100 100
70 15.55097 10—6b(10-6) 100 100
79 E-FSOPT 10-6b(10-61 100 100

 

Table 8.1. lcontinued)

93

 

NIP-66 18 E-FSOPT 10-6b(10-6) 40 100
10 100

40 15-55091 10-6b(10-6) 20 100
MF-so 19 E-FSOT 10-4.(10-6) 100 100
100 100

101 E-FSOPT 1065(10-6) 100 100
MF-82 (3) E-CSOT 1040(10-5) 100 100
0.0 100

35 E—FSOT 10-«(10-6) 80 100
MF-84 20 5430501“ 1055(10-5) 100 100
100 100

NIP-84w 21 E-GBSOT 1055(10-5) 100 100
100 100

MF-88 22 E-FSOT 10-«(10-6) 100 100
100 100

167 E-FSOT 104.0045) 100 100
MF-89 23 E-FSOT 10-«(10-5) 0.0 100
0.0 100

MF-91 24 0095011 1050(10-5) 0.0 100
0.0 100

117 E-FSOTT 10-«(10-4) 0.0 100
130 E-F50P 1055(10-5) 0.0 100
MF—92 25 5-09501' 1055(10-5) 100 100
100 100

90 E-CSOP 107-(105) 100 100
MF-107 26 54305017 1055(10-5) 100 100
100 100

112 E-cesmT10-5-(10-6) 100 100
MF-108 27 5035011 1055(10-5) 0.0 100
0.0 100

109 0005071055004) 0.0 100
110 E-casorr 1055(10-4) 0.0 100
MF-115 (3) 5.005077 10-6a(10-5) 0.0 100
0.0 100

116 15005017 10-50(10-6) 0.0 100
105 150507 10-4a(10-6) 0.0 100
140 E-CSOPT 10-6a(10-6) 0.0 100
51 5.00507 10-5a(10-6) 0.0 100
150 E-CSOPT 10-6a(10-6) 0.0 100
151 E-CSOPT 1065(10-6) 0.0 100
152 E-CSOPT 10-6a(10-6) 0.0 100
153 E-csoPT 10-6a(10-6) 0.0 100
154 E-CSOPT 10-6a(10-6) 0.0 100
155 E-CSOPT 10-Ga(10-6) 0.0 100
156 E-csoPT 10-6a(10-6) 0.0 100
157 E-050PT 106.006) 0.0 100
158 E-csoPT 1060(10—6) 0.0 100
159 E-CSOPT 10-8:(10-6) 0.0 100
MF-118 28 E-F501'l' 1045(10-5) 100 65
100 30

NIP-119 29 5.55011 10-«(10-5) 0.0 100
0.0 100

 

Table 8.1. (continued)

94

 

MF-121 30 E-CSOTI' 10-5b(10-6) 100 100
100 100

120 005017 10-5b(10-6) 100 100
NIP-128 31 1500509 10-5a(10-6) 100 100
100 100

126 E-GBSOP 10—5a(10-6) 100 100
122 E-GBSOP 1055(105) 100 100
124 503509 10-5a(10-5) 100 100
125 E-GBSOP 10-5a(10-6) 100 100
MF-141 32 E-csoPT 10-60(10-7) 100 100
100 100

137 E-CSOPT 10-60(10-6) 100 100
139 55501911054105) 100 100
MF—163 33 E-GBSOT 1050(10-5) 100 100
100 100

160 E-GBSOT 105-(104) 100 100
MF-188 34 E—FSOT 1045(10-5) 100 100
100 100

170 E-C5OT10-«(10-6) 100 100
171 E-C5OT 1040(10-5) 100 100
173 E-CSOT10-4a(10-5) 100 100
174 E-C50T 10-4a(10-6) 100 100
MF-175r 35 E-CSOT 1045(10-7) 100 100
100 100

127 5433509 10-5-(10-6) 100 100
129 E-GBSOP 10-Sa(10-6) 100 100
131 E-FSOP 10-6a(10-5) 100 100
132 E-FSOP 10610106) 100 100
133 E-FSOP 10-6a(10-4) 100 100
134 E—FSOPT10-6a(10-6) 100 100
133 E-F50PT 10-6a(10-6) 100 100
96 E-GBSOPT 10—5a(10-5) 100 100
97 54335097 1055(10-5) 100 100
102 E-FSOPT 10—6a(10-6) 100 100
56 15005097 10-5a(10-5) 100 100
47 E-GBSOPT 10-5a(10-5) 100 100
142 5035091 10-5a(10-6) 100 100
143 E-GBSOPT 10-5a(10-7) 100 100
144 E-GBSOPT 10-5a(10-5) 100 100
145 E-GBSOPT 10-5a(10-6) 100 100
146 54335097 10-5a(10-4) 100 100
147 E—GBSOPT 1055(10-7) 100 100
148 E-GBSOPT 10-5a(10-5) 100 100
149 E-GBSOPT 10-50(10-6) 100 100
MF-179 36 15095017 10-Sa(10-6) 100 100
100 100

NIP-180 37 E-GBSOTI’ 10-5a(10-6) 100 100
100 100

NIP-181 33 54335017 10-50(10-6) 20 100
. 10 100

MF-182 39 0005011 10-50(10—6) 100 100
100 100

94 E-FSOPT 10-60(10-6) 100 100
95 E-FSOP 10-6a(10-5) 100 100

 

Table 8.1. (continued)

 

MF-183 (37) E-GBSOTT 10-5:(10-6) 100 100
100 100
MF-185 4O E-CSOTT 10-5a(10-7) 0.0 100
0.0 100
188 E-CSOTT 10-51I(10-6) 0.0 100
190 E-CSOTT 10-Sa(10-6) 0.0 100
191 E-CSOTT 10-Sa(10-7) 0.0 100
165 E-FSOP 10-6a(10-5) 0.0 100
166 E-FSOP 10-6:(10-6) 0.0 100
MF-189 41 E—GBSOT 1O-50(1O-4) 100 100
100 100
No growth on toluene or phenol
16,17,161 ,93,162 E-GBSOT 10-50 0.0 0.0
111,114,113 E-GBSOTT 10-Sa 0.0 0.0
57.26. E-GBSOP 10-50 0.0 0.0
67.98.100 E-GBSOPT 10-50 0.0 0.0
8.87.86.164 E-FSOT 10-4a 0.0 0.0
46 E-FSOTT 10-40 0.0 0.0
68.99 E-FSOPT 10-60 0.0 0.0
22,81,83,104.106 E-CSOT 10-40 0.0 0.0
48 E-CSOTT 10-Sa 0.0 0.0
186,187 E-CSOP 10-7a 0.0 0.0
136,135,192 E-C5OPT 10-Ga 0.0 0.0
MF-l-l-2-1 42 D-1O-20 0.0 100
0.0 100
2 D-10-20 0.0 100
3 D—10-Za 0.0 100
MF-l-l-2-4 43 D-10-2c 0.0 100'
0.0 100
6 04 O-Za 0.0 100
7 D-10-Za 0.0 100
8 D-10-2c 0.0 100
10 D—10-Za 0.0 100
1 1 D-10-2c 0.0 100
MF-H-2-9 44 D-10-2c 0.0 100
0.0 100
24 D—1O—20 0.0 100
MF-H—2-12 45 D—1 O-Za 0.0 100
0.0 100
18 D-10-Za 0.0 100
19 D-1 O-Za 0.0 100
20 D—10-Za 0.0 100
MF-l-l-2-13 ' 46 D-10-Za 0.0 100
- 0.0 100
16 D-10-21I 0.0 100
17 0-1 O-Za 0.0 100
26 D-10-20 0.0 100
No growth on toluene or phenol
22,23,255 D—10—3a 0.0 0.0

 

Table 8.1. (continued)

96

 

MF-301 (7) E-T-10-4b (10-6)MD 100 100
100 100
302 E-T-10-4b (10-6) 100 100
303 E-T-10-4b (10-6) 100 100
305 E-TT-10-5b (10-6) 100 100
306 E-TT-10-5b (10-6) 100 100
316 E-P-10-5b (10-5) 100 100
318 E-PT-10-5a (10-5) 100 100
321 E-PT-10-5a (10-5) 100 100
322 E-PT-10-5a (10-5) 100 100
323 E-PT-10-Sa (10-5) 100 100
324 E-PT-10-5a (10-4) 100 100
332 E-PT-10-Sa (10-5) 100 100
333 E-PT-10-5a (10-5) 100 100
338 E-PT-1 O-Sa (10-5) 100 100
339 E—PT-10-56 (10-5) 100 100
343 EFT-1060 (10-5) 100 100
MF-304 47 E-TT-10-50(10-5) 0.0 0.0
0.0 100
317 E-‘lT-10-50(10-4) 0.0 100
MF-340 48 E-P-10-50(10-5) 100 0.0
100 0.0
341 E-P-10-5a(10-5) 100 0.0
MF-342 49 E-P-10-5a(10-6) 0.0 0.0
100' 100*
No growth on toluene or phenol
307,308,327 E-T-10-50 0.0
309.310,311,319,320.334 E-TT-lO-Sa 0.0
312.313,314.315.325,326 E-P-10-Sa 0.0
328,329.330,331.334.335,336.337 E-PT-10-Sa 0.0
MF-H-3-3 (7.8.9) (33) D-10-7a MD 100 100
100 100
Mf-H-3-5 (7) D-10-5a 100 100
100 100
No growth on toluene or phenol
MF-H-3-1 D-10-6a
MF-H-3-2 0.10-55
MF-H—3-4 D-10-60
MF-H-3-6 D-10-5a
NIP-401 (7) E-T-10-51-(10-6) MD 100 100
100 100
402 E-T-10-50(10-6) MD 100 100
403 E-P-10-53(10-5) 100 100
404 E-P-10-5a(10-5) 100 100
406 E-T-10-50(10-5) 100 100
407 E-T-10-5a(10-5) 100 100
408 E-TT-10-5a(10-5) 100 100
409 E-1T-10-50(10-5) 100 100
423 E-Tr-10-5b(10-5) 100 100
424 E-TT-10-5b(10-5) 100 100
425 E-TT-10-5b(10—5) 100 100

 

Table 8.1. (continued)

97

 

MF-405 50 159-1055005) 0.0 100
0.0 100

443 554055005) 0.0 100
447 sp.-1055005) 0.0 100
MF-411 51 157-1055005) 0.0 97
0.0 100

410 E—T-10-5a(10—5) 0.0 100
MF-412 (7) 5-7-105500-6) 100 100
100 100

426 5-7-10-51100-5) 100 100
MF-413 52 E-TT-10-50(10—7)MD 100 100
100 100

427 E-TT-10-Sa(10-7) 100 100
423 E-TT-10-50(10—7) 100 100
429 arr-1050007) 100 100
MF-414 53 err-105005) 100 100
100 100

105-415 54 541-105-005) MD 40 100
100 100

430 157-1055005) 100 100
431 5421055005) 100 100
432 E-PT-10-Sa(10-6) 100 100
435 E-PT-10-5a(10-6) 100 100
446 E-P-10-Sa(10-5) 100 100
NIP-416 (24) 1549-1055005) 0.0 0.0
0.0 100

455 E-P-105o005) 0.0 100
MF-419 55 155-1055005) 0.0 0.0
100 0.0

417 157-1055004) 100 0.0
436 E-1T-10-5500-5) 100 0.0
437 5-PT-105a00-4) 100 0.0
438 E-P-10-5a(10-5) 100 0.0
456 155-105.004) 100 0.0
MF-421 (35) 157-1055005) 100 100
100 100

41s ram-105.004) 100 100
422 E-PT-10-5a00-5) 100 100
433 54121055005) 100 100
419 5.7-1 0-5a(10-6) 100 100
420 E-T-10-Sa(10-5) 100 100
440 E-P-105a00-4) 100 100
434 5010-55005) 100 100
461 E-T'r-10-5a00-6) 100 100
MF-441 (17) 1551-1055005) 100 100
100 100

442 15-15-105.005) 100 100
457 513-10-5000-4) 100 100
453 E-PT-10-5a(10-5) 100 100
459 155-1055005) 100 100
444 5540-50004) 100 100
MF-445 56 E-1T-10-5500-6) 100 0.0
100 0.0

 

Table 8.1. (continued)

 

MF-448 57 E-PT-10-Sa(10-5) 0.0 100
100 100
MF-449 58 E-T-10-5b(10-4) 100 100
100 100
460 E-T-10-5b(10-4) 100 100
MF-450 59 E-T-10-5b(10-5) 0.0 100
100 100
452 E-T-10-5b(10-5) 100 100
MF-451 60 E—T-10-5b(10-5) 100 100
100 100
MF-453 (28) E-T-10-53(10-5) 0.0 100
0.0 100
463 E-T-10-50(10-5) 0.0 100
MF-454 61 E-T-10-5b(10-5) 0.0 100
0.0 100
MF-462 62 E-P-10-50(10-6) 0.0 100
0.0 100
MF-464 (7) E-PT-10-50(10-6) 100 100
100 100
No growth on Toluene or Phenol
439,465 E-T-10-5a
446,447,455 E-Tr-10-5a
456 E-P-10-Sa
466,467,468 E-PT-10-Sa
MF-H-4-1 (63) D-10-83 MD 0.0 100
MF-I-l-4-5 63 D-10-7a MD 0.0 100
0.0 100
MF-H-4-2 (24) D—10-7a MD 0.0 100
0.0 100
MF-H-4-4 (35) D-10-63 MD 100 100
100 100
MF-H-4-6 (1 7) D-10-6a 100 100
100 100
No growth on toluene or phenol
MF—H—4-3 64 D-10-7a 0.0 30
0.0 0.0

 

a
Isolates were obtained after dilution series of original samples (D) or from MPN Positive tube at

extiction (E) from the same samples amended with phenol (P) or toluene (T) at 50 ppm, with (PT

or TT) or without TCE. Original dilutions and MPN positive tubes at extinction were diluted and

plated on: M-RZA (a), toluene vapors (b), phenol vapors. The ﬁrst number indicates the dilution

at which the isolate was obtained from the natural sample and the second indicates the level of

the isolate in the enriched MPN tube (e.g. 10-7=107). Isolates were puriﬁed by three tranfers on

M-RZA medium. tested for their ability to degrade Toluene or phenol. Sources of inoculum for
the ﬁrst sample from the aquifer: GB=Glass beads. F=ﬁlter (water from the aquifer ﬁltered

through a 0.2 pm ﬁlter). C= cotton ﬁlter (bacteria collected in a cotton ﬁlter exposed to the ﬂux of
water from the aquifer). Samples 2. 3. and 4 consisted of glass beads only. Samples 2. 3. and 4

MPN tubes had 25 ppm phenol or toluene with or without TCE.

Isolates numbered as 001-199 (sample 1) were obtained before the treatments with phenol and
toluene were applied to the ﬁeld for the year of 1993.
Isolates numbered as 201-299 (sample 2) were obtained after the phenol treatment and 1,1-

DCE + TCE.

Isolates numbered as 301-399 (sample 3) were obtained after the phenol treatment + TCE.
Isolates numbered as 401-499 (sample 4) were obtained after the toluene treatment + TCE.

99

t’REP-PCR grouping of isolates. Positive degraders were grouped by REP PCR analysis (plates
were grouped by similar colony morphologies). Isolates in parentheses represent REP groups
identiﬁed as identical to a previous observed pattern.

C% of toluene removed from the headspace of a vial or phenol disapearance from the medium
when compared to a noninoculated control and measured by GC/HPLC analysis. respectively. at
15 days of incubation; or (*) second evaluation at 30 days.

Chapter III

Microbial succession during a ﬁeld evaluation of phenol and toluene as the
primary substrates for TCE co-oxidation.

Introduction

Trichloroethylene (T CE) is one of the most common pollutants of
groundwater throughout the United States. Anaerobic degradation of TCE and
tetrachloroethylene (PCE) in the laboratory has been reported (Bower and
McCarty, 1983; Vogel et al, 1987; Vogel and McCarty, 1985). but these
transformations are often incomplete and can result in the accumulation of
equally harmful lesser chlorinated metabolites, c-DCE. t-DCE, 1.1-DOE and vinyl
chloride (Vogel et al.. 1987). In 1985. VVrlson and Wilson demonstrated aerobic
degradation of TCE in soil enriched with natural gas. Later, several aromatic
compounds were also shown to induce TCE degradation (Nelson et al.. 1987;
Wackett asnd Gibson, 1988; Viﬁnter et al.. 1989). These aromatic oxygenases
are responsible for the cometabolic transformation of this compounds.

Bioremediation remains potentially the most cost effective cleanup
technology for chlorinated aliphatic hydrocarbons (CAH’s). Field experiments
have been carried out at the Moffett Federal Airﬁeld, Mountain View. California,
to test the effectiveness of several primary carbon and energy sources for

stimulating the bioremediation of CAH’s. Methane was an effective substrate for

100

101

the transformation of t-DCE and vinyl chloride, but not for TCE and c-DCE
(Roberts et al.. 1990; Semprini et al., 1990; Semprini and McCarty, 1991;
Semprini et al 1991). Phenol was subsequently found to be effective for TCE
and c-DCE removal (Hopkins et al., 1993a; Hopkins et al., 1993b). The
successful ﬁeld demonstration of phenol stimulating TCE removal prompted
further investigations to optimize its use and to compare its effectivenes for the
removal of TCE and its lesser chlorinated products, c-DCE, t-DCE, 1.1-DCE, and
vinyl chloride (Hopkins and McCarty. 1995).

Many studies have been carried out in the laboratory on selected culturable
isolates responsible for biodegradation of aromatic compounds and their TCE
co-metabolic potential, but few studies have focused on a more comprehensive
understanding of the community selected by such cometabolic treatments and
the dynamics of that community in response to treatments.

In this work we studied microbial population succession as a result of
injection into the aquifer of different CAH’s. In addition. we evaluated the
inﬂuence of a shift in carbon source from phenol to toluene on aquifer microbial
community structure. We evaluated community composition by both classical
growth dependent microbiological procedures, as well as by analysis of 16S
rRNA patterns derived from DNA extracted from the entire community. The
dominant populations selected were rather stable over treatments and quite

resilient following introduction of a toxic CAH.

102

Material and Methods

Field sample site. The experimental site is located at the Moffett Federal Air
Field (formerly the Moffett Naval Air Station) Mountain View, California. A
complete description of the site is provided in chapter 2.

Field treatments and sampling. The ﬁeld experiment was conducted by Gary
Hopkins of Stanford University. The experiments analyzed in this chapter are
the same as described for 1993 in chapter 2 but the emphasis here is on the
sucession of populations responding to the four treatments. A summary of the
sequence of chemicals injected into the aquifer is given in Table 2.1. of chapter
2. Brieﬂy. sample 1 represented the microbial population present in the aquifer
following phenol and TCE injections the previous ﬁeld seasons and after 8
months of inoperation of the ﬁeld test site. Sample 2. was exposed to phenol,
TCE and 1.1-DOE, sample 3 was exposed to phenol and TCE-and sample 4 was
exposed to toluene and TCE. For samples 2, 3 and 4, new sterilized glass bead
bag were placed in the sampling well on the same day new substrate addition
began and remained in the ﬁeld for 20-26 days before removal for analysis.
Since new. uncolonized samples were provided before each treatment the
results reﬂect only colonization (growth) due to treatments and not survival by
indigenous ﬂora which would have been the case if aquifer solids were sampled.
Analysis of microbial biomass and extraction of total microbial community
DNA. To collect the biomass from the glass beads or ﬁlters, these samples were

shaken with sterile saline solution, pH 7, in sterile 250 ml Nalgene plastic

103

bottles. The supernatant was transferred to a clean, sterile bottle, followed by
centrifugation. The pellet was retained and the supernatant discarded. At least
eight extractions were performed in this manner from each sample. The ﬁnal
pellet was resuspended in saline to yield a biomass concentration that
corresponded to 10 9 glass beads/ml of saline. This turbid resuspended
solution was used for MPN determinations. direct isolation of microbial
populations and total microbial community DNA extraction (chapter 2). DNA was
extracted according to the direct lysis protocol developed by Holben et al.
(1933)

For the MPN determinations and isolation of dominant populations the basic
experimental protocol presented in Figure 2.2. (chapter 2) was followed. The
estimated population density and the 95% conﬁdence intervals were calculated
by standard methods (Alexander,1982). The dilution level from which each
isolate was obtained was recorded to determine which strains repeated
overtime. and their approximate population levels.

Species diversity index. Species richness (d) was calculated using the
expression of Pielou (1975): d= S-1 I log N. in which S=number of species and
N= number of individuals. We used identical REP patterns as a surrogate for
bacterial “species”. While this does not reﬂect true species. it does reﬂect
patterns of diversity in the community.

FAME - The isolates analyzed for cellular fatty acids (FAME) were precultured

on M-R2A and inoculated into Erlenmeyer ﬂasks containing 0.3% (wlv) tryptic

104

soy broth (T88) (Krieg. 1981). Cells were harvested by centrifugation. The cell
pellet was placed into a screw-capped test tube and stored at -20°C until
analysis. Saponiﬁcation. methylation and extraction were performed using the
procedure described in the MIDI manual (Sasser, 1990). Gas chromatography
data was compared to a fatty acid identification library (Microbial Identiﬁcation
Systems, Inc., Newark. Del.) for possible isolate identiﬁcation. Cluster analysis
and dendograms of fatty acid proﬁles were generated using NTSYS software
(Applied 8iostatistics, lnc., Setauket. N.Y.). An Euclidian similarity matrix was
computed from non-standardized data and distances between strains were
calculated using unpaired group mean averages(UPGMA). Goodness of ﬁt
between the Euclidian matrix and resulting tree was compared using the
cophonetic correlation coefﬁcient (Romesburg, 1984). For the construction of
the dendogram. we determined the fatty acid composition of the following
standard strains grown on the same media as the isolates: Hydrogenophaga
pseudoﬂava (MIDI database version 3.8). Arthrobacter sp. strain 53 (Haack et
al.. 1994). Pseudomonas putida F1, Pseudomonas putida PaW1. Pseudomonas
mendocina KR. Burkholderia cepacia G-4 and Burkholderia pickettii PK01 (ref.
Zhou). The FAME proﬁles of the Azoarcus strains were determined in this study.
Molecular methods. Repetitive extragenic palindromic REP- PCR patterns
were obtained from cells using Rep-1 and Rep-2 primers and the polymerase
chain reaction (de Bruijn, 1992). Ampliﬁcation was performed using a Model

9600 Perkin-Elmer Cetus Thermocycler. Products (10 pl) were separated by

105

electrophoresis in 1.5% agarose gels and stained with ethidium bromide.
Ampliﬁcation was primarily done using individual colonies grown on M-RZA, but
for isolates with poor or no ampliﬁcation, DNA was extracted from cells and used
as the template for PCR amplification. For isolates with very close patterns, the
same stock of primers and reagents were used in the analysis.

The biomass extracted and resuspended from glass beads and ﬁlters from
samples 1. 2, 3 and 4 was used for the community DNA extraction. The pellet
was resuspended in phosphate buffer and DNA was extracted according to the
direct lysis protocol developed by Holben et al. (1988). Extracted DNA was
subjected to ampliﬁcation of the ribosomal RNA genetic loci by the polymerase
chain reaction (PCR). Universal primers were used to amplify an approximately
1,500 base pairs fragment using a forward primer which corresponds to
nucleotide positions 8-27 (5’-AGAG'ITI’GATCCTGGCTCAG-3’; primer A) of
Escherichia coli 16S rRNA and a reverse primer corresponding to the
complement of positions1541 to 1518 (5’-AAGGAGGTGATCCAGCCGCA—3’;
primer H) (Ulrike et al.. 1989). All primers were synthesized with an Applied
8iosystem DNA synthesizer at the Macromolecular Structure and Sequencing
facility at Michigan State University. In general, ampliﬁcation was done in 100
pl total reaction volume containing 100 ng of total community extracted DNA or
DNA from pure cultures of different isolates as template. 1 0M of each primer,
250 (M of each deoxynucleoside triphosphate. 10 ul of 10X Taq buffer, and 2.5

Units of Taq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT). PCR was

106

performed in an automated thermal cycler(9600 Perkin-Elmer Cetus. Nonrvalk.
CT) with an initial denaturation [95°C,130 s]. followed by 30 cycles of
denaturation [92°C, 70 s]. annealing [55°C, 30 s]. and extension [72°C, 130 s],
and a single ﬁnal extension [72°C, 6 min]. An aliquot of 10 ul was run in 0.7%
agarose gel to evaluate the quality of the ampliﬁed fragment. In general
ampliﬁcation yielded greater than 5 ug of PCR product. Ampliﬁed DNA from
community (25)") or bacterial isolates (15 pl) was digested according to
manufacturers speciﬁcations for at least 3 h with restriction endonucleases
(Haelll. HpaIl and Sau3A; Boehringer Mannheim. Indianapolis, IN). The 16S
rDNA restriction fragments were then concentrated to a lesser volume by
Iyophilization for 10 min and then electrophoresed on 4% (wlv) NuSieve 3:1
agarose gel (FMC, Indianapolis. IN) in 0.5x TAE [Tris acetate-EDTA] at 7
volts/cm. and stained in a 0.5 ug/ml ethidium bromide solution. The resulting
fragmentation patterns were then used as the fingerprint for the identiﬁcation of
bacterial or community genomes.

Analytical methods - Toluene and TCE were measured by gas
chromatography equipped with a flame ionization detector(GC/FID), a DB-624
capillary column (J&W Scientiﬁc. Folson, CA) and a headspace sampler. The
autosampler vials were equilibrated at 30°C, the column at 90°C and injector and
detector at 200°C. He was the carrier gas. Phenol was analyzed by reverse-
phase HPLC with a Hibar RT C13 column (E. Merck), with a ﬂow rate of 1.5

ml/min of 66:33:01 Hzo-CH3CN-H3PD4 and a W detector set to 218 nm.

107

Results

Succession determined from studies of isolates. No signiﬁcant differences
in the relative abundance of total heterotrophs, phenol degraders. toluene
degraders, phenol catalyzed TCE co-oxidizers and toluene catalyzed TCE co-
oxidizers were observed when TCE. c-DCE or t-DCE were injected into the
aquifer along with phenol or with toluene (Figure 3.1.a.). In contrast, when 1.1-
DCE was injected along with phenol and TCE (sample 2), a drastic reduction in
all populations was observed (Figure 3.1.3.). Once 1.1-DOE was removed
(sample 3) populations levels increased and reached densities similar to those
at the beginning of the experiment. While 1.1-DOE had an effect on population
densities, the primary carbon source did not since the addition of phenol (sample
3) or toluene (sample 4) produced the same population densities. It is
noteworthy that the populations of toluene degraders rose markedly after
feeding with phenol (sample 3) and that phenol degraders were at equal density
to toluene degraders after feeding with toluene (sample 4).

Species diversity indices were used to express changes in relative
diversity among successional stages. Due to technical difﬁculties in speciating a
large number of bacterial isolates we used REP band patterns to reflect the
effects of CAH’s and primary substrates on species richness. Species richness
was greatly reduced by 1.1-DOE and remained low during the subsequent
phenol feeding (sample 3) but increased to original levels following the toluene

feeding (Figure 3.1.0). A taxonomic evaluation of the isolates showed that

108

Figure 3.1. (A) Populations of total heterotrophs, phenol and toluene degraders
and TCE co-oxidizers obtained from glass beads sampled following each of the
four indicated treatments, I, II, III, IV. For the initial sample, the populations of
toluene and phenol degraders were obtained with 50 ppm carbon substrate.
Populations for samples II. III. and IV were obtained with 25 ppm of carbon
substrate. (8) Species richness index calculated as suggested by Pielou (1975)
considering isolates from glass beads with different REP sequences as different

”species”.

109

 

 

 

 

 

 

 

 

A B s/ - m
z
/ ..
/.
/. 1
/.
/ O
.. .. %
.. I m
. m
2 _. -
mm... x. w...
3.1 \\ ..
00me \x
0 . ...
mmm \.\
2+2 1. - o
_ _ _ _ _ _ _ _ _ _ _ _ _
m 8 6 .4 2 0 mm H mm m 8 .6 4 2

Autumn nee—a “_o m \ «£2593
..anac 3:202. no no;

Aooocoacoa 0mm.
0005.0: 00.0009.

Toluene
4- TCE

Phenol

Phenol
+ TCE

+ TCE

4- 1,1 DCE

IV

III

II

110

disturbance by 1.1-DCE led to the survival of relatively few populations
compared with unperturbed communities. and that these surviving populations
were predominantly gram positives (Figure 3.4. and chapter 2).

We also investigated the effect of carbon sources and CAH’s on the
composition of the aquifer bacterial communities by tracking changes in speciﬁc
populations using REP-PCR. Isolates from successive sampling that had similar
REP sequences were identiﬁed and run side-by-side on agarose gels to conﬁrm
their similarities (Figure 3.2). Strains that were found at more than one sampling
time and from several samples or media were judged to be more dominant in the
ﬁeld. The distribution of the REP group members isolated was inﬂuenced by the
ﬁeld treatments. Isolates represented by REP groups MF-19. MF-63, MF-89.
MF-108, MF-163 and MF-175 were among the most commonly isolated and
dominant populations in the initial sample (Figure 3.3.). However, after the
phenol and 1.1-DOE treatment very few isolates were obtained, and those found
(sample 2) had REP sequences not seen before. This suggests that one of
these compounds was detrimental to the initial bacterial populations dominating
the aquifer. Once 1.1-DCE was removed. but phenol was still being applied. two
isolates with REP patterns resembling groups MF-19 and MF-163 were again
detected as the most commonly isolated populations (sample 3). When the
phenol injection was switched to toluene, isolates representing REP groups MF-
19. MF-63. MF-89, MF-108 and MF-175 (sample 4) were again isolated at a high

frequency. These isolates accounted for almost all of the isolates recovered in

111'

Figure 3.2. REP-PCR fingerprint patterns of Moffett Field isolates generated by
using chromosomal DNA. Isolates that were observed to repeat overtime were
runned side-by-side for pattern comparisons. Lanes 1. 15. 16 and 28 are size

markers, with the base pairs indicated on the left.

H2

ﬁn-
153
..w

» l
22] Li
‘ ii

i!

ll

1 »
p.

5.8,
see

1%

a
.23

we
~ I"

4 “O

 

 

113

 

 

 

       
   

  
  

     
 
  

 

12

mmmmm 1
MMMMM Egggégéggmm
Meg; mm
.5 555555555510
222222222
0 m
- m h
222222222222mm
MW
................................... .. m
5555555555 t
222222222
_ _ _ _ _ _ _ _ _ q A _
41I. mm 9 8 7 6 5 4 3 2 4| 0

03:5: 3:303 .o no.—

Field sample (time of sample)

Figure 3.3. REP genotypes “persistent during the course of

the experiment. The population levels for each

isolate is estimated using the most dilute tube

from which the strain was isolated.

114

the initial sample indicating a succession back to the original microbial
community in this aquifer.

Comparison of the microbial groups from different aquifer samples. FAME
analysis was done on all isolates with different REP patterns from different
samples. FAME was then used as an independent method to evaluate
differences among isolates including some with the same REP pattern but
isolated from different samples. 80th FAME and REP data could then be used
to evaluate which isolate types were more frequently isolated over the
treatments. Initial populations present in the ﬁeld were very diverse based on
the number of isolates with different REP patterns (chapter 2) and on the number
of fatty acid methyl ester (FAME) groups obtained. The FAME analysis grouped
the isolates with different REP patterns from the four samples into 30 distinct
groups of strains at Euclidian distances of less than 20 (Figure 3.4). MIDI
literature suggests that the Euclidian distance scale permits a preliminary
indication of me relatedness of entries at the genus, species. and subspecies
levels. which are respectively, 25. 10. and 6 Euclidian distances (Sasser, 1990).
In our cluster analysis we included fatty acid data of well characterized strains
that are known to harbor ﬁve different pathways for toluene degradation. None
of the isolates clustered (< 20) with these isolates: Pseudomonas putida F1.
Pseudomonas putida PaW1, Pseudomonas mendocina KR or Burkholderia
cepacia G-4, or with a common soil isolate Arthrobacter sp. One isolate (MP-13)

clustered at an Euclidian distance of less than 10 with Burkholderia pickettii

115

Figure 3.4. Dendogram based on the fatty acid profiles of the isolates. FAME

groups I through XXX were defined at a Euclidian distance of 20. (r xy: 0.86).

(Isolates from: sample I, 0 II 9 III. ando IV).

Euclidian distance

116

 

Marcus

MF-f 07

mucus
Amorous
Marcus

Iﬂﬂﬂilﬂ'll’ﬂﬂiﬂ

{£53 “m

=3

toluiirycus TOL-4

tolulitycus Til-17

tolulirycus Tel-19
sp. 85b2

ﬂl

toluli usTd-15

tolulirycus Td-t
tolulitycus Tel-2
tolulr‘rycus T64

Hydmgsnophsgs postman

MF-304
MF-413
MF—414
MF-ss
MF-421
HRH-+4
MF=-17s
MF—s
MF-OO
DIP-183
MF-t 05
MF-141
MF-445
MF-Oz
MF-tt
MF-tsz
MF-t 70
MIMI-#5
MF—f!
Asosrcus
MF-301
M11404
Ins-so
MF-401
MF-fo
MF-412
MF-H-3-5
MF=-13

lﬂﬂﬂﬂlﬂﬂﬂﬂﬂﬂl 00'

"ﬁlm

WW

Burkholderia pickettii PK01
Pseudomonas putida F1
Pseudomonas putida PAWt

MF=-419

5°
Burkholderia cepacia G-4
Pseudomonas mendocina KR

tar-13
"F4 9.
sen-2.4
um:
"F405
145-100
115-340
um
MF-M
Its-11.2.1
rap-32
um
urea-4.2
DIP-416
Hm
MF-411
06-02-12
MF-u
um
“F4 91

I'IIHﬂ°ﬂl'ﬂ°l°ﬂﬂ

Anhmbsctsr sp. strain 53

117

PKO1. w However, many isolates and a known control, Azoarcus indigens V832T
a nitrogen ﬁxing organism (Reinhold-Hurek et al., 1993), also clustered in this
group at an Euclidian distance of less than 20. We also included in this cluster
analysis, the fatty acid composition of the recently identiﬁed new genus of
toluene-degrading denitriﬁers Azoarcus tolulyticus, since many of the isolates
from Moffett Field resembled this genus based on FAME analysis, toluene
degradation and denitrification properties (chapter 4). In our earlier work (Fries
et al.,1994). we isolated several toluene-degrading denitriﬁers from different
geographical regions of the world. Those isolates when analyzed by FAME
were reported as Hydrogenophaga pseudoﬂava (MIDI system Version 3.8).
Cluster analysis of these isolates and the inclusion of the FAME proﬁle for
Hydrogenophaga pseudoﬂava and Azoarcus sp. 85b2 (another nitrogen ﬁxing
isolate). conﬁrmed that the new isolates clustered with H. pseudoﬂava (Figure
3.4.). Gram positive isolates clustered in 12 different groups( I, II, III, IV, V. VI.
XVII. XXIV, XXVI, XXVIII. XXIX, and XXX). Most of the gram positive isolates
were retrieved in speciﬁc samples. However, group III gram positives was very
common in the aquifer since isolates belonging to this group were obtained from

samples 1, 3 . and 4 (Figure 3.3).

Community composition determined by ARDRA. We used ARDRA of
community DNA to evaluate (i) how similar the glass bead bioﬁlm community

was to the planktonic community, (ii) whether the community selected by phenol

118

was different from the one selected by toluene and (iii) whether the dominant
strains determined from the isolation studies also appeared to be the dominant
organisms by DNA analysis. The relatively low number of bands generated for
aquifer community DNA suggests that such communities are low in diversity or
that the diversity resides in a number of closely related organisms with the same
band patterns. The microbial community proﬁle obtained from glass beads.
representing the attached community was very similar to the profiles obtained for
the microbial community proﬁles obtained for ﬁltered water samples and
representing the planktonic community (Figure 3.5. A,8 and C).

The results obtained with the RFLP proﬁle from the original community and
subsequent communities obtained under different carbon sources strongly
supports the hypothesis that the structure of the aquifer microbial community did
not change under the treatments being compared (Figure 3.5). The number of
unique bands per treatment out of the total restriction bands seen using three
different restriction enzymes were 2115 for the initial sample. 1I15 after phenol
treatment and 1/15 after toluene treatment (T able 3.1). These numbers show
considerable stability in the community over treatments.

All but one of the most dominant isolated strains showed a distinctive
restriction fragment pattern for the ampliﬁed rDNA when digested with Hpall
(Figure 3.5.A). The exception was MF-19 and MF-11 which showed similar
patterns. Digestion with Hae III (Fig. 3.5.8). and Sau3A (Fig. 3.5.C)

distinguished between these isolates. The patterns observed for all dominant

119

Figure 3.5. ARDRA analyses of aquifer community DNA and of pure cultures
dominant in the aquifer. The PCR amplified 16$ rRNA gene product was
digested with Hpall (A). Hae Ill (8), and SEIBA (C), electrophoresed on a
0.5XTAE (WN) NuSieve (FMC) agarose, and stained with ethidium bromide.
The lanes show PCR products from aquifer glass beads (AQ. GB) of sample i,
cells retained on the 0.22 pm filter (Filter 1) and cotton fiber filtered water (Filter
2) from sample I, glass beads from sample ill (phenol amendment). glass beads

from sample IV (toluene amendment) and the indicated pure cultures.

120

 

IZI

 

122

 

123

Table 3.1. Number of unique ARDRA bands for the indicated sample
relative to the total bands in that sample. The template DNA

was extracted from glass beads sampled after three field

 

 

 

 

 

treatments.
Enzyme Initial sample' Phenol4-TCEb Toluene+ TCE°
H86 I" 2/9 . 1/9 1/9
Hpa II 0/4 0/4 0/4
830 3A 0/2 0/2 0/2
Total 2/15 1/15 1/15

 

‘Beginning of the experiment ( 8 months of field inoperation)
t’after phenol-TCE injection (20-26 days exposure)
cafter toluene—TCE injection (20-26 days exposure).

 

124

aquifer isolates could also be discerned in the community DNA for the three
samples under study (Table 3.2.). This ﬁnding indicates that the dominant
community proﬁles can be accounted for by the dominant isolates suggesting
that these isolates are dominant members of the community. Only one band. of
approximately 460 to 517 base pairs (Figure 3.5.8) was present in all community
samples but could not be accounted for by the bands from culturable isolates.
The correspondence in dominance determined by isolation with dominance
determined from extracted community DNA suggests that non-culturable forms

are not signiﬁcant in this study.

Discussion

We evaluated succession of microbial populations in an aquifer field site in
order to understand how different CAH’s and two primary carbon sources
affected the dynamics of that natural community. Similar population densities
for total heterotrophs, toluene and phenol degraders, as well as TCE co-
oxidizers. were observed when the aquifer was injected with TCE, c-DCE or t-
DCE and with phenol or toluene as the primary carbon sources. However. all
population sizes were drastically reduced when 1.1-DCE was injected into the
aquifer. TCE, which is known to have a toxic effect on cultures of TCE co-
oxidizers (Wackett and Gibson, 1988; Wackett and Householder, 1989; Zylstra
et al.,1989; VVrnter et al.. 1989) did not cause a measured effect on populations

detected. In this study. the presence of TCE did not affect ﬁeld population levels

 

125

Table 3.2. Number of common ARDRA bands obtained from pure cultures
thought to be the most dominant members of the community
compared to the ARDRA bands obtained from DNA extracted
from glass beads of samples i. ll, III and IV. The number of

bands seen in community DNA but not seen in isolates tested

is shown in parentheses.

 

 

 

 

 

 

 

 

Enzyme GI | | s

MELI MEl! MEQ MEIZE MElﬂﬁ
Has III 68(4) 09(4) 710(2) cats) 50(3)
Hpa ll 214(6) 2MB) 314(2) 20(2) 214(1)
Sau 3A 22(4) 29(4) 2012) auto) 012(2)
Total 1005114) 1015(1) 1211516) 121155) ”15(6)
Enzyme e

MELt 0E1! 0E6: 015115 MEttﬂ
Hae Ill 543(3) 60(3) 60(3) 50(3) 519(3)
Hpa ii 215(3) 36(2) 315(0) 95(1) 05(2)
830 3A 212(0) 21210) 2010) 20(0) 02(2)
Total 1011616) 1111515) "116(3) 1016(4) 6116(7)
Enzyme G c

MEu MEI! MEQ MEIR MElzlzﬂ
H86 III til9(3) 01913) 50(3) 519(3) 50(3)
Hpa II 25(3) 95(2) 315(0) 315(1) 36(2)
Sau 3A 212(0) 21210) 212101 2720) 012(2)
Tota| 10/16(6) 11l18(5) 11/16(3) 1006(4) 3716(7)

 

 

'Beginning of the experiment ( 8 months of field inoperation)
l’after phenol-TCE injection ( 20-26 days exposure)
°after toluene-TCE injection (20-26 days exposure).

126

but 1.1-DCE seems to have caused extreme toxicity effects for many microbial
populations.

The ﬁrst signs of environmental stress usually occur at the population level,
affecting especially sensitive species. If there is sufﬁcient redundancy, other
species may ﬁll the functional niche occupied by the sensitive species (Odum,
1985; 1990; Schindler. 1990). Indeed, we observed a drastic reduction in
species diversity. as measured by patterns of REP richness during the injection
of 1,1-DCE. The populations isolated after this ﬁeld treatment were
predominantly gram positive bacteria and were only able to degrade phenol.
These survivors may have a unique resistance to 1,1-DCE. From a
bioremediation point of view. this fact must be considered carefully, since these
surviving populations may not be efﬁcient for the cometabolic transformation of
the desired CAH. None of the isolates obtained after injection of 1,1-DCE
(sample 2) carried the toluene ortho-monooxygenase gene despite the fact that
this was the most common gene observed in isolates from sample 1 (chapter 2).
One explanation is that toxicity of 1.1-DOE was directly related to this enzyme
system, since the 1.1-DOE injection prevented isolates with homology to this
gene from colonizing the glass beads.

Some populations were observed to repeat overtime as well as following the
injection of the toxic 1.1-DOE. Population levels in sample 3. returned to original
levels in sample 1. but not in species richness. Toluene addition helped return

the original richness to the community. The same populations found after

 

127

phenol addition occured after toluene addition. This observation can be
explained by the fact that most phenol degraders isolated from the site are also
toluene degraders (chapter 2). Therefore, we conclude that some populations
are better competitors for the primary carbon sources. but at the same time, very
sensitive to 1.1-DOE.

8y cluster analysis of the cellular fatty acids isolates representing many
different groups of degraders (30) were obtained. This indicates that a
taxonomically very diverse community was selected in this aquifer. This
includes twelve distinct gram positive groups and a group that, based on fatty
acid methyl esters. resembles Azoarcus sp. (chapter 4). From all four
characterized communities these results indicate that the initial (community 1)
and the ﬁnal (community 4) were very similar, but the community obtained from
sample 2 (community 2) was very different from the others and of low diversity.
These results corroborate the other evidence found for toxicity and selection of
particular populations by 1.1-DOE.

ARDRA analysis provided an alternative method that does not rely on
culturing to examine the structure of microbial communities by describing 16s
rRNA ﬁngerprint patterns of bacterial assemblages. In this study. ARDRA
analysis suggested similar community structures when phenol and toluene were
the primary carbon sources, consistent with the interpretation from analysis of
the dominant isolates. The majority of bands observed in ARDRA from

community DNA were found in ARDRA for the most common isolates.

128

suggesting that we did isolate the major populations from the site and that
unculturable populations were not important in this study.

In this study we examined microbial structure of an aquifer communities by
standard MPN techniques, isolation of the most dominant populations and
differentiation of these populations by REP-PCR analysis, and restriction
endonuclease analysis of ampliﬁed 16$ rRNA gene fragments from pure
cultures and total community DNA A “Species” richness index showed a
marked effect on population diversity when 1.1-DOE was injected into the
aquifer. 80th the structure and biochemical ability for primary carbon source
oxidation of the aquifer community were altered due to this CAH. We also found
a rebound of microbial populations to the original community structure once 1.1-
DCE was removed. This study also showed that toluene selected the same
population that had been selected on phenol suggesting that these two carbon

sources were rather equivalent in their biochemical potential.

Acknowledgments
This work was supported by EPA , Grant N° CR 822029-01-0 and the NSF

Center for Microbial Ecology, Grant N° BIR 9120006.

129

List of References

Alexander, M. 1982. Most probable number method for microbial populations. p.
815-820. In A L. Page (ed.), Methods of soil analysis. Part 2. Chemical and
microbiological properties. American Society of agronomy, Madison, Wi.
USA

Bower, E. J., and P. L. McCarty. 1983. Transformation of 1 and 2-carbon
halogenated aliphatic compounds under methanogenic conditions. Appl.
Environ. Microbiol. 45:1286-1294.

de Bruijn, F. J. 1992. Use of repetitive (repetitive extragenic palindromic and
enterobacterial repetitive intergeneric consensus) sequences and the
polymerase chain reaction to ﬁngerprint the genomes of Rhizobium meliloti
isolates and other soil bateria. Appl. Environ. Microbiol. 58:2180—2187.

 

Fries, M. R., J. Zhou, J. Chas-Sanford, and J. M. Tiedje. 1994. Isolation.
characterization, and distribution of denitrifying toluene degraders from a
variety of habitats. Appl. Environ. Microbiol. 60:2802-2810.

Haack, S. K., H. Garchow, D. A. Odelson, L. J. Fomey, and M. J. Klug. 1994.
Accuracy. reproducibility, and interpretation of fatty acid methyl ester profiles
of model bacterial communities. Appl. Environ. Microbiol. 60:2483-2493.

Holben, W. E., J. K. Jansson, 8. k. Chelm, and J. M. Tiedje. 1988. DNA probe
method for the detection of speciﬁc microorganisms in the soil bacterial
community. Appl. Environ. Microbiol. 54:703-711.

Hopkins, G. D., J. Munakata, L. Semprini, and P. L. McCarty. 1993a.
Trichloroethylene concentration effects on pilot ﬁeld-scale in-situ groudwater
bioremediation by phenol-oxidizing microorganisms. Environ. Scie. and
Technol. 27:2542-2547.

Hopkins, G. D., L. Semprini, and P. L. McCarty. 1993b. Microcosm and In situ
ﬁeld studies of enhanced biotransformation of trichloroethylene by phenol-
utilizing microrganisms. Appl. Environ. Microbiol. 59:2277-2285.

Hopkins, G. D., and P. L. McCarty. 1995. Field evaluation of ln-Situ aerobic
cometabolism of trichloroethylene and three dichloroethylene isomers using
phenol and toluene as the primary substrates. Environ. Scie. and Technol.
(submitted for publication)

Krieg, N. R. 1981. Enrichment and isolation. p. 112-142. In P. Gerhardt, R. G.
E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood. N. R. Krieg. and G. 8.

130

Phillips. (ed.), Manual of methods for general bacteriology. American
Society for Microbiology, Washington. DC.

Nelson, M. J. K.,S. O. Montgomery, E. J. O’Neil and P. H. Pritchard. 1987.
Biodegradation of trichloroethylene and involvement of an aromatic
biodegradative pathway. Appl. Environ. Microbiol. 53:949-954.

Odum, E. P. 1985. Trends expected in stressed eco-system. 8ioscience
35:419-422.

Odum, E. P. 1990. Field experimental tests of eco-system-ievel hypotheses.
Trends Ecol. Evol. 5:204-205.

Pielou, E. C. 1975. Ecological diversity. John Wiley and Sons, New York.

Reinhold-Hurek, 8., T. Hurek, M. Giliis, 8. Hosts, M. Vancannet, K.
Kersters, and J. Ley. 1993. Azoarcus gen. nov.. nitrogen-ﬁxing
proteobacteria associated with roots of Kallar grass (Lepfochioa fusca (L.)
Kunth). and description of two species. Azoarcus indigens sp. nov. and
Azoarcus communis sp. nov. Intern. Journal of System. Bacteriol. 43:574-
584.

Roberts, P. V., G. D. Hopkins, D. M. Mackay and L. Semprini. 1990. Aﬁeld
evaluation of in-situ biodegradation of chlorinated ethenes: Part 1.
Methodology and ﬁeld site characterization. Ground water. 28:591-604.

Romesburg, H. C. 1984. Cluster analysis for researchers. Lifetime Learning
Publications. Belmont, Calif.

Sasser, M. 1990. Technical Note#102: Tracking a strain using the Microbial
identiﬁcation System. MIS Inc., North Newark,Del.

Schindler, D. W. 1990. Experimental perturbations of whole lakes as tests of
hypotheses concerning ecosystem structure and function. Oikos 57:25-41.

Semprini, L., and P. L. McCarty. 1991. Comparison between model
simulations and ﬁeld results for in-situ biorestoration of chlorinated
aliphatics. Part 1. 8iostimulation of methanotrophic bacteria. Ground
Water 29:365-374.

Semprini, L. , G. D. Hopkins, P. V. Roberts, D. Grbic-Galic, and P. L.
McCarty. 1991. A ﬁeld evaluation of in-situ biodegradation of chlorinated
ethenes. Part 3. Studies of competitive inhibition. Ground Water 29:239-
250.

 

131

Semprini, L. , P. V. Roberts, G. D. Hopkins, and P. L. McCarty. 1990. A
ﬁeld evaluation of in-situ biodegradation of chlorinated ethenes . Part 2.
Results of biostimulation and biotransformation experiments. Ground Water
28:715-727.

Ulrike, E., T. Rogall, H.8locker, M. Emde, and E. C. Bottger. 1989. Isolation
and direct complete nucleotide determination of entire genes.
Characterization of a gene coding for 168 ribossomal RNA Nucleic Acids
Res. 17:7843-7853.

Vogel, T. M. and P. McCarty. 1985. Biodegradation of tetrachloroethylene to
trichloroethylene, dichloroethylene, vinyl chloride , and carbon dioxide under
methanogenic conditions. Appl. Environ. Microbiol. 49: 1 080-1 083.

Vogel, T. M., C. S. Criddle, and P. McCarty. 1987. Transformation of
halogenated aliphatic compounds. Environ. Sci. Technol. 21:722-736.

 

Wackett, L.P., and D. T. Gibson. 1988. Degradation of trichloroethylene by
toluene dioxygenase in whole cell studies with Pseudomonas putida F 1.
Appl. Environ. Microbiol.54:1703-1708.

Wackett, L.P., and S. R. Householder. 1989. Toxicity of trichloroethylene to
Pseudomonas putida F1 mediated by toluene dioxygenase. Appl. Environ.
Microbiol.55:2723-2725.

Winter, R. B., K. Yen and B. D. Ensley. 1989. Efﬁcient degradation of
trichloroethylene by a recombinant Escherichia coli. Bio/Technology 7:282-
285.

Wilson, J. T. and B. H. Wilson. 1985. Biotransformations of trichloroethylene
in soil. Appl. Environ. Microbiol. 37:242-243.

Zylstra, G. J., L. P. Wackett and D. T. Gibson. 1989. Trichloroethylene
degradation by Escherichia coli containing the cloned Pseudomonas putida
F1 toluene dioxygenase genes. Appl. Environ. Microbiol. 55:3162-3166.

Chapter IV

Natural selection of denitriﬁers at a ﬁeld site amended with phenol and
toluene for TCE co-oxidation.

Introduction

Denitriﬁcation is the dissimilatory reduction of nitrate or nitrite to nitrogen
gases. A variety of bacteria occupying diverse habitats including soil. water,
foods. marine environment. and digestive tract have been reported as denitriﬁers
(Zumft. 1991). Their numbers in relation to total culturable populations in soil
and water habitats can vary from 0.01 to 9% and are in this range for all soil and
water habitats studied (T iedje. 1988).

Four enzymes are required to convert nitrate to nitrogen gas in respiratory
denitriﬁcation. Most of the knowledge about the enzyme systems is from gram
negative bacteria. The ﬁrst enzyme. nitrate reductase is membrane bound with
its active site facing the cytoplasm (Southamer, 1988). Nitrite reductase is
located in the periplasmic space. There are two major types of dissimilatory
nitrite reductases: those containing cytochrome cd, (cdy-dNirs) and those
containing copper (Cu-dNirs) in the active sites ( Shapleigh and Payne, 1985;
Hochstein and Tomlinson. 1988; Coyne et al., 1989). The major product of
nitrite reduction by nihite reductase is nitric oxide. which is subsequently

reduced to nitrous oxide by nitric oxide reductase. Nitric oxide reductases

132

 

133

puriﬁed so far are membrane bound and contain a b and c cytochrome (Heiss et

al.. 1989; Carr and Ferguson, 1990; Demastia et al.. 1991). In some bacteria

like P. aureofaciens and P. chlororaphis, N20 formation is the last step of
denitriﬁcation (Firestone et al.. 1979). In most denitriﬁers. however. N20 is

reduced to dinitrogen gas by a Cu—containing nitrous oxide reductase located in
the periplasmic space (Hochstein and Tomlinson. 1988; Stouthamer. 1988).
Biodegradation of aromatic compounds under aerobic conditions is well
known; oxygen is utilized for ring activation and cleavage as well as serves as
the electron acceptor for the complete oxidation of these compounds (Gibson
and Subramanian, 1984). The availability of oxygen. due to its low solubility in
water and inefﬁcient transport through saturated porous matrices such as soil
and sediments. is usually the rate limiting parameter for aromatic compound
biodegradation. It’s only been in recent years that anaerobic degradation of
these compounds has been conclusively established. The degradation of
toluene and phenol has been reported by pure cultures under denitrifying
conditions (Bakker, 1977; Dolﬁng et al., 1990; Schocher et al., 1991; Evans et
al., 1992; Chee-Sanford et al., 1992; Fries et al.. 1994). methanogenic
conditions (VVrlson et al., 1986; Grbic-Galic and Vogel. 1987), sulfate reducing
conditions (Edwards et al.. 1993; Rabus et al.. 1993) and ferric iron reducing
conditions (Lovley et al., 1989; Lovley and Lonergam, 1990). We have
developed techniques for the isolation of populations of anaerobic phenol and

toluene-degrading denitrifers where low (8.9. 5 ppm) concentrations of the

134

aromatic compound was an important factor to success (Fries et al., 1994).
Also. the stimulatory effects of nitrate addition on biorestoration of a fuel
contaminated site has been reported (Hutchins, 1991; Hutchins et al., 1991a;
Hutchins et al.. 1991b). Therefore. the ability to degrade aromatic compounds
under anaerobic conditions is now well documented.

An aquifer at the Moffett Federal Air Field, Mountain Wow. California. has
been under test with phenol and toluene as the primary carbon and energy
sources for stimulating the cometabolic remediation of several CAH’s (Hopkins
et al.. 1993; chapter 2). The cometabolic transformation of CAH’s is dependent
on the expression of oxygenases by microbial populations that use the aromatic
compound for growth. However, the limiting 02 , below 0.2 mg/L (Roberts et al.,
1990) and high nitrate (25 mg/L) in the ground water (Hopkins et al., 1993) may
favor the development of denitrifers. In the present study. we investigated
whether the high levels of nitrate and low 02 in the groundwater favored the
selection of denitriﬁers foilovving the injection of aromatic compounds.
F urthermore. if aromatic compounds are injected for the purpose of inducing the
production of oxygenases. it must be ensured that the levels of oxygen are
maintained since a different set of enzymes are induced under denitrifying
conditions for the degradation of these substrates (Altenschmidt and Fuchs,
1992; Evans et al.. 1992; Seyfried et al.. 1994; Ghee-Sanford et al., 1995). We
also conﬁrmed that anoxic conditions resulted in no TCE co-oxidation by isolates

able to degrade toluene under denitrifying conditions.

 

 

135

Material and Methods
Field sample site. The experimental site is located at the Moffett Federal Air

Field (formerly the Moffett Naval Air Station) Mountain \ﬁew. California.
Chemical analysis of water from the sampling site revealed the following
concentrations: phosphate less than 0.5 mglL, nitrate, 25 mglL. sulfate. 700
mgIL (Hopkins et al.. 1993). A complete description of the site is provided
elsewhere (chapter 2).

Microbial strains. Isolation procedures and some characteristics of the strains
from the Moffett Field used in this work are provided in chapter 2 and chapter 3.

Standard strains, Azoarcus tolulyticus Td-isolates have been described (Fries et.
al.. 1994), Pseudomonas pickettii PK01 was kindly provided by Dr. Ron Olsen
and Azoarcus sp.SSb2 and Azoarcus indigens V832T was kindly provided by Dr.
Barbara Reinhold-Hurek.

Most probable number of heterotrophs and denitrifiers. Two samples were
used to compare population levels of denitrifiers and heterotrophs from the site.
Sample 1 consisted of ﬁltered water from the SSEGB well (Figure 2.1.. chapter
2) using a 0.22 um ﬁlter. and sample 2 was a core sample from aquifer material
collected during the construction of a new (third) experimental leg at the ﬁeld site
in 1994, on a location adjacent to the old wells but which had no previous
exposure to the water ﬂow from previous ﬁeld experiments. To collect the

biomass from the ﬁlter. this sample was shaken with sterile saline solution, pH

 

136

7, in sterile 250 ml Nalgene plastic bottle. To collect the biomass from the
sand—gravel, 300 g of this sample was shaken with sterile saline solution. pH 7,
in a sterile 250 ml Nalgene plastic bottle. The supernatant was collected and
resuspended to a visual turbidity similar to the one obtained for the ﬁlter. The
slurry obtained from the filter and the sand core sample were used for MPN
determinations.

The estimated population density and the 95% conﬁdence intervals were
calculated by standard methods (Alexander,1982). Resuspended material
collected from ﬁlters and subsurface material were serially diluted and 1 ml of
each dilution was transferred to sterile 20 ml vials after which 9 ml of the specific
medium was added. Two media were used for MPN determination of denitriﬁer
population density: nutrient broth amended with 5 mM N0; (T iedje, 1982) and a
denitriﬁcation medium. The denitriﬁcation medium consisted of the salt mixture
(SM) (chapter 2) and the following carbon sources (final concentration per liter):
Proteose peptone, 0.5 g; yeast extract. 0.5 g; casamino acids. 0.5 g;

potassium acetate, 1 g; sodium succinate. 1 g. The tubes were sealed with
Teﬂon-lined stoppers and incubated without shaking. Denitriﬁcation was
evaluated by observing gas bubble formation in inverted Durham tubes (N2). by
measuring the presence of NO and N20 in the dilution tube headspace by gas

chromatography and by measuring the concentrations-of nitrate and nitrite in the
medium supernatant by HPLC analysis. A vial was assumed to be positive for

denitriﬁcation when bubbles were detected in Durham tubes. or when we

 

137

detected NO in the vial headspace. The total heterotrophic population in both
samples was determined by the MPN method also using two media: the
standard TSB medium (Krieg. 1981 ) and liquid M-RZA (chapter 2) medium. The
results were scored by analyzing visible turbidity. Five tubes were inoculated
from each serial dilution. The incubation temperature throughout this work was
30°C.

FAME . Protocols for FAME analyzes and construction of a dendogram have
been described in chapter two. For the construction of the dendogram, the fatty
acid composition of Hydrogenophaga pseudoﬂava was obtained from MIDI
database version 3.8.

Isolate characterization. The denitriﬁcation ability of individual isolates was
evaluated by observing gas bubble formation in inverted Durham tubes, by
detecting the presence of NO and/or N20 in the vial headspace by gas
chromatography and by measuring the concentration of nitrate and nitrite in the
medium by HPLC analysis. A 10% inoculum grown aerobically on the
denitriﬁcation media was used for the denitriﬁcation test. To test the capabilities
of the isolates to grow on phenol or toluene under denitrifying conditions. a

heavy inoculum from M-R2A plates. of each isolate was transferred to sterile
20 ml vials after which 5 ml of anaerobic BS + N03' medium plus 25 ppm
phenol or toluene was added (Fries et al.. 1994). The media with the aromatic

substrates were added in an anaerobic chamber ﬁlled with nitrogen gas and 3%

hydrogen. The vials were sealed with sterile butyl rubber Teﬂon-lined septa and

138

incubated for at least 2 weeks before phenol or toluene disappearance was
evaluated. Controls from the same batch of medium but without cells were
incubated at the same time as a reference to determine the amount of phenol or
toluene removed. Degradation activity was deﬁned as at least 95% loss of
substrate in the headspace as measured by GC analysis for toluene or in the
culture’s supernatant for phenol.

Trichloroethylene co-oxidation under aerobic versus anaerobic
denitrifying conditions. Toluene-degrading denitriﬁers were grown under

anaerobic conditions in BS + NO3’ + 50 ppm of toluene (chapter 2). Inocula (1/2
ml) were transferred to 20 ml sterile auto sampler vials and 4.5 ml of 88 + N03'

+ 50 ppm of toluene and 0.8 ppm TCE was added to the vials and the vials were
sealed with Teﬂon-lined stoppers. For evaluating anaerobic TCE co-oxidation
vials were prepared similarly except that the medium was added in the anaerobic
chamber. Degradation was determined by substrate disappearance in the
headspace after 15 days of incubation as measured by GC analysis, when
compared to non-inoculated controls.

Molecular methods. The gene probes used in this study. i.e. the heme-type
nitrite reductase. from Pseudomonas stutzen' JM 300. the toluene-ortho-
monooxygenase from Pseudomonas sp. JS150 and Pseudomonas cepacia G-4
along with the protocols for hybridization are described in chapter 2 and 5.
Genomic DNA was obtained by standard methods (Elmerich et al.,1982) from

pure cultures of isolates grown on M-R2A broth under aerobic conditions.

139

Restriction endonuclease digestion of DNA was performed according to
manufacturer’s speciﬁcations. Digested DNA was size fractioned by
electrophoresis in 0.7% agarose gels and transferred to nitrocellulose
(polyester-supported BAS 68380; Schleicher & Schuell, Keene. NH.) as
previously described (Fries et al.. 1994).

Analytical methods. Toluene and TCE were measured by gas chromatography
equipped with a ﬂame ionization detector (GCIFID), a DB—624 capillary column
(J&W Scientiﬁc, Folson, CA) and a headspace sampler. The autosampler vials

were equilibrated at 30°C, the column at 90°C and injector and detector at

200°C. He was the carrier gas. N03] and NOz' concentrations were determined

in culture supernatant by HPLC analysis using a Partisil 10 SAX column
(Whatman. Clifton, NJ.). W detection at 210 nm. and 50 mM phosphate (pH 3.0)
as eluant. N20 and NO were measured by GC/ECD. using a Poropak Q column
at 55°C. 300°C detector temperature and 95% argon-5% methane as carrier
gas. Phenol was analyzed by reverse-phase HPLC with a Hibar RT C13 column
(E. Merck), with a ﬂow rate of 1.5 ml/min of 66:33:01 HZO-CH3CN-H3PO4 and a

W detector set to 218 nm.

 

140
Results
Heterotrophic and denitrifying population densities. The populations of
cultivatable heterotrophs and denitriﬁers were compared for two samples from
the aquifer using two different media to estimate each group. Similar

heterotrophic populations (7.9x105- 2.4x106 cells/ml of slurry) were observed.

irrespective of the medium. for the core sample from an untreated area from the
site, and the ﬁltered water sample from the aquifer that had received carbon
additions for the two previous ﬁeld seasons (T able 4.1). The proportion of the
total heterotrophic population comprised of denitriﬁers was also similar for the
two habitas with 4.9% and 8.1% capable of denitriﬁcation in the water (treated)

and sand (untreated) samples, respectively.

Denitrlficatlon products in MPN tubes. During the MPN evaluation of
denitriﬁer populations some of the MPN tubes at the low and high dilutions
showed no turbidity for the water sample yet some nitrate was consumed. This
occured in both nutrient broth and denitriﬁcation medium. Headspace analysis
of these tubes (40 % of the total positive MPN tubes) revealed the presence of

NO (nitric oxide) (Table 4.2.). None of the tubes where NO was detected
produced N2 gas. but all accumulated some N20.
We obtained 348 numerically dominant isolates from the Moffett Field

experimental site. of which 273 were positive for toluene and/or phenol

degradation. REP-PCR analysis revealed that the 273 isolates procuced 63

 

141

Table 4.1. Number of denitriﬁers and heterotrophs observed in a sample
that is on the path of carbon ﬂow (ﬁlter) compared with a
sample from the aquifer (sand) that has not been subjected to
treatments at the Moffett Field site.

 

 

 

 

 

 

Sample Medium' Cell number 95% CI”
(x 10‘) (x 10‘)

Filter CM 7.9 2.4, 26
NB 2.4 0.7. 7.9

T83 79 24, 260

M-RZA 130 39, 430

Sand DM 13 3.9, 43
NB 17 52, 56

T38 130 39, 430

M-RZA 1 240 73, 790

 

a
DM (denitriﬁcation media) and NB (nutrient broth) amended with 5 mM nitrate

were used to evaluate total numbers of denitriﬁers. TSB (T ryptic soy broth) and
M-R2A (modiﬁed R2A) minus nitrate were used to evaluate the total number of
heterotrophs.

95% conﬁdence interval.

142

Table 4.2. An example of diversity of denitriﬁcation products in MPN
tubes from a Moffett Field ﬁltered water sample.

 

MPN Turbidity Denitriﬁmtion products
tube

 

 

N05 N02' NO N20

 

 

 

10"
10'1
10‘1
10‘1
10'1
10‘2
10*2
10‘2
10‘2
10'2
10°
104
10°
10°
10'3
10“
10‘
104
10“
10*
10‘
10‘
10*5
10*5
1015
1015 - 3.9 - - -
10‘ - 4.0 - - -
10‘ - 4.0 - - -
10‘ - 4.5 - - -
10‘3 - 4.4 - - -

.L
on
l+r+t
r+l+r

N
0
_L
b
...
...

1+++++++++++++++1++1+1++
I r t
h- O .
o _s

r+++rr+++1 1

1+++Il+++1 1

1
5“ I
o
I I
I I
I I
IIIru-slun++nuu+++++++u++t+l++3

 

143

different REP-groups (chapter 2). Forty-six percent of the isolates are
denitriﬁers with nitrogen gas as the end product. 5% produced nitrous oxide. one
isolate produced nitric oxide. 25% reduced nitrate to nitrite. Twenty-two percent
of the isolates did not reduced nitrate in denitriﬁcation media (Table 4.3. and
Figure 4.1). Three of the most dominant and most commonly isolated strains
from sample 1, MF-175. MF-19. MF-163 were reisolated again in samples 3. and
4, on heterotrophic (non-selective) medium, MF-H-4-4. MF-H-3—5, MF-H-3-3.
respectively. All of these isolates are denitriﬁers. When probed for the type of

nitrite reductase gene . 41% of the denitriﬁer isolates (Table 4.3. and Figure 4.1)

hybridized to the heme cd1 nitrite reductase gene. probe from Pseudomonas

stutzeri JM300. This type of nitrite reductase was shown by Coyne et al. (1989)

to be the most frequent in strains collections from nature.

Characteristics of an unusual NO-producing denitrifier, MF-18. The results
obtained in the MPN tubes, were unusual in respect to the appearance of nitric
oxide in some tubes. These results can be potentially explained by one of
isolates. strain MF-18. This strain was independently isolated four times from
glass beads and cotton ﬁlter samples in medium containing toluene and
presence or absence of TCE. This strain produced large quantities of NO when
given nitrate or nitrite under aerobic and anaerobic conditions (Table 4.4.). This
NO concentration could be sufficient to prevent turbidity from developing in MPN

tubes or pure culture. The isolate did not grow with nitrous oxide as an electron

 

144~

Table 4.3. Characteristic denitriﬁcation products of Moffett Field isolates,
hybridization to the heme-type nitrite reductase gene probe. and
capability of toluene or phenol degradation under denitrifying
conditions.

 

Isolate Denitriﬁcation Hybridization Phenol Toluene

b c c
producta to cd1-dNir degradation degradation

 

 

MF-S N02- -
MF-7 +
MF—1 1 4-
MF-13 N20
MF—15 +
MF-18 NO
MF-19 +
MF—23 +
MF-24 -
MF-39 N02 - -

MF-52
MF-53
MFe54
MF-58
MF-59
MF-62
MF—63
MF-66
MF-BO
MF-84
MF-84w
MF-88
MF-89
MF—91
MF-92

MF-107
MF-108
MF-118
MF-1 19
MF-121

MF—128
MF-141
MF-163
MF-168
MF-175
MF-179

MF-180
MF-181

auraat+++raa+l114-{:134-4-4-

++++§'++++§.+II++++++++++I
r++r a
l t
r

|++
l
r

5,5

 

 

Table 4.3. (continued)

 

MF-1 82 N02- - ' -

MF185 + + - -

MF-189 N0; - - -

MF-H-2-1 N0; nd nd nd
MF-H-2—4 + nd nd nd
MF-H-2-9 - nd nd nd
MF-H-2-12 - nd nd nd
MF-H-2-1 3 N02. nd nd "d
MF-304 + nd nd nd
MF-340 No; nd nd nd
MF-342 N0; nd nd nd
MF-405 - nd nd nd
MF-41 1 - nd nd nd
MF-413 + nd nd nd
MF-414 N02— nd nd nd
MF-415 - nd nd nd
MF-419 + nd nd nd
MF-445 — nd nd nd
MF-448 N20 110 nd nd
MF-449 N0; nd nd nd
MF-451 N02. Dd 11d nd
MF-454 - nd nd nd
MF-462 No; nd nd nd
MF-H-4-5 - nd nd "d
B. cepacia G-4 NO," - - -

P. stutzeri JMSOO + + - -

B. pickettii PK01 + - - -

 

a -
Observed denitrification products were N2 (+) (as gas In Durham tubes). N02 by HPLC. and NO
and N20 by GCIECD detector.

0
Results from Southern blot hybridizations using the heme type nitrite reductase gene probe.

C
Growth of the isolates under anaerobic denitrifying conditions using phenol or toluene as the
only carbon and energy source.

146

Figure 4.1. Distribution among the 63 REP-PCR groups of the (a) characteristic

denitrification products, (b) capability to degrade phenol or toluene

under denitrifying conditions and (c) homology to the heme type
nitrite reductase gene probe.

 

 

148

 

2003030... «I + «25 xon 920 E 00:. :25 0
258.5% 52 a
.82 .2 2E N.» new .002 .2 2E Qm 0.0.5 ucozoaceocoo Ex... 0

 

 

 

 

 

 

52 52 52 52 - .82
oz 52 52 oz - .82 oxen 95.0
52 52 52 oz - o~z 0.1.2
05 52 on... .00 + .82
mu «.0 20 no. + .82
- - - - + No ._<
em 552 woe we. - .002
... ..v on. 3 - -aoz
- 1 - - - ococ coo.<
N02 .002 sz 02
92.5 €53
anos. 33300: 353.2 5 300.05 ..200800 300300:
$023.0 ocm

 

00.2.0385 2.0.0:...

.09.: £520 cos? 2-0.2 20.0.0 5 3030.0 cozooEono 2.2.0.0228 .v.v 030...

149

acceptor. The isolate did appear to begin growth as a denitrifier under argon
since N-anions were consumed and N gases were produced. but turbidity did
not developed. Growth could have been arrested early by the high
concentration of NO. To test this hypothesis we tried to grow this isolate in a
glove box (97% nitrogen and 3 % hydrogen) with nitrate or nitrite as an electron
acceptor and a cotton plug on the tube so that any nitric oxide (3 very insoluble
gas) could be exchanged into the headspace of the glove box No apparent
turbidity was observed (Table 4.4.). Growth was observed in tubes without
nitrite or nitrate in an aerobic headspace . and no NO was detected under this

conditions.

Strains that degrade toluene and phenol under denitrifying conditions. We
evaluated the ability of the isolates obtained from sample 1 (chapter 2)
representing different REP-PCR groups to degrade toluene or phenol under
anaerobic (denitrifying conditions). Five isolates (8%) were able to degrade
toluene and two of those isolates also degraded phenol using nitrate as an
electron acceptor (Table 4.3. and Figure 4.1.). All isolates were also aerobic
toluene degraders and three showed a distinct band of homology in Southern
blots when hybridized to the toluene ortho-hydroxylase of Pseudomonas cepacia
G-4. The efﬁciencies for TCE removal under aerobic conditions varied from
30% to 95% among the isolates. Under anaerobic denitrifying conditions. no
TCE was removed from the vial headspace of any of the isolates although all

toluene was consumed (Table 4.5.). This result was expected since it is

150

Table 4.5. Degradation of toluene under aerobic and anaerobic-
denitrifying conditions and percent removal of TCE by
toluene-degrading denitriﬁer isolates from Moffett Field‘.

 

 

 

 

 

 

Isolate TOMb Aerobicc Anaerobicc
probe
Toluene TCE Toluene TCE

MF-7 - 1 00 3O 1 00 O
MF-58 + 1 00 95 1 00 O
MF-66 + 50 50 1 00 0
MF-1 O7 - 1 00 4O 1 00 O
MF-1 1 8 + 100 40 100 O

 

a
The inoculum was grown under denitrifying conditions (50 ppm toluene) and. a 10 % inoculum
was transferred to a newvial before addition of test medium.

b
Southem blot hybridization to the toluene ortho monooxygenase gene (TOM) from
Pseudomonas sp. JS 150 and from Burkholderia cepacia G-4..

c
% Toluene and TCE removal from the headspace vials after two weeks of incubation (initial
concentrations were 50 ppm toluene and 0.8 ppm TCE).

 

 

151

generally accepted that oxygenases are necessary for TCE co-oxidation; this is
additional evidence for this fact.

The ﬁve new toluene-degrading denitriﬁer isolates appeared to be closely
related to each other and to the members of the new species Azoarcus
tolulyticus. The probable identiﬁcation is based on the fact that these isolates
share the following key features with the described Azoarcus tolulyticus strains
(Fries et al.. 1994; Zhou et al.. 1995): characteristic growth and colony
morphology on M-R2A medium, the same type and proportion of cellular fatty
acids , the same gram stain. catalase reaction. and characteristic cell
morphology when growing on M-RZA plates. We used the fatty acid data for the
new isolates and from the previously isolated Azoarcus tolulyticus as well as
some other Azoarcus sp., in a cluster analysis. All isolates clustered at an
Euclidian distance of less than 20 suggesting that they belong to the same

genus (Figure 4.2.).

Discussion
We investigated if selection for denitrifying populations occurred in an
aquifer that has high natural levels of nitrate and low levels of oxygen, by
evaluating population numbers. characterizing denitriﬁcation products of isolates
from the site and using molecular methods for identiﬁcation of denitrifying genes.
We used two different media for this comparison and found that 49-81% of the

total viable population was composed of denitriﬁers. This proportion is on the

 

I. —

152

 

28552..

2.0.00. 0208030 2.0.0:! 80.. 058.0 000.003. 002200.20 =2» 5.3 .0532
use. 50o: 52. 0255.. 869855522 52. 98 052. .o 20.05 .290 a... 650..

5958:2355

§§§_

5.36.35
38 .9 30.83.
as. 460535
222103.53
05.235335
3232-3 .33
3.355.355

«2.32.3303
B—ﬁ

5.262133
2...:

.5

8.!

00:88—- Sax-oam

 

o N.

 

T

 

 

 

Hi
We

 

 

 

 

 

n.
._

 

 

153

high end of the range for populations of denitrifiers present in common soils
(Tiedje, 1988 ). The similarities in denitriﬁer numbers and their proportion of the
total heterotrophic population for a sample collected outside the ﬂow path of
injected toluene or phenol and from a water sample in the ﬂow path indicates
that the injected carbon sources were not responsible for this selection.
However, the high nitrate low oxygen condition may have been sufficient to
select a high proportion of denitriﬁers in both samples regardless of whether
extra carbon was added.

More than half of the isolates obtained from the site (chapter 2) are true
denitriﬁers with either nitrogen gas or nitrous oxide as end products. The
number of nitrite accumulators was half the number of denitriﬁers. This is in
contrast to results obtained by Gamble et al. (1977), when evaluating samples
from world soils, who found a greater number for nitrite accumulators than
denitriﬁers. Only 22% of the isolates presented no reaction to nitrate when
tested for this property. Three of the most commonly isolated and most
dominant strains are also denitriﬁers. Based on these several observations, we
believe that the denitriﬁcation property helped enriched the denitrifying
populations at this site. Murray et al. (1992) has shown that denitriﬁcation
efﬁciency by an isolate was responsible for the competitive advantages for
growth under denitrifying conditions when compared with an inefﬁcient

denitriﬁer.

154

One isolate, MF-18. produced NO when growing on medium supplemented
with nitrate or nitrite and in tubes with an aerobic headspace. NO was also
produced by this strain when inoculated into an anaerobic medium, but no
increase in turbidity was observed. We suspected that the organism was
producing enough NO to be toxic and prevent its further growth. Hence, we tried
to grow this strain in a glove box in a non-sealed tube, thus allowing the toxic
NO to diffuse from the tube. However, no growth was observed under this
condition. We are not certain that the experimental protocol was appropriate for
NO removal or that the production of NO by the cell still damaged the cell before
it could escape. NO production by strains like MF-18 still offer the best
explanation why some MPN tubes at low dilution had no turbidity, but high
headspace NO and only partial N03' and NO; removal.

In natural communities we could postulate that other species may consume
this compound and in this way the isolate would maintain its niche by preventing
growth of other sensitive species. We consider this isolate to be an unusual
denitriﬁer based on the lack of visible growth under denitrifying conditions. This,
however, might be explained by NO toxicity. This is the first report of the
isolation of a denitriﬁer that produces NO in high amounts apparently preventing
its own growth. One explanation for the NO accumulation is that the nitrite
reductase is much more rapid than the nitric oxide reductase. Another
possibility is that other enzymatic mechanisms could produce NO in the absence

of a removal enzyme. Nitric oxide reductase cytochrome P450nor is one novel

155

mechanism for nitrate reduction that has been found in fungi, e.g. Fusarium
oxysporum (Shoun and Tanimoto, 1991).

We also found that 8% of the isolates harbor the unique property of
anaerobic growth on monoaromatic hydrocarbons, using nitrate as their electron
acceptor. This ﬁeld site was amended with phenol for two field seasons prior to
our sampling, and this may have enriched for anaerobic phenol (and toluene)
degraders. Monoaromatic hydrocarbons are common contaminants of ground
water, and also, biogenic sources of aromatic compounds have been described
for bacteria and fungi. Hydroxylated toluene, toluene and benzene has been
reported to be produced by aerobic and anaerobic populations in culture media,
and by natural anoxic hypolimnia of stratiﬁed lakes and anoxic river sediments
(Martin and Haider, 1969; Juttner and Henatsch, 1986; Juttner, 1988,
1990,1991; Fisher and Juttner, 1994). The fact that aromatic compounds are
toxic even at concentrations of 5-10 ppm, (Fries et al., 1994; chapter two) and
that many aromatic compounds are synthesized by microbial populations at
aerobic and anaerobic conditions led us to speculate that the ability of
populations to use these resources from an habitat, provides these organisms
with greater versatility anaerobic micro-niches. A natural source for toluene in
nature helps to explain the wide distribution and high population levels of
anaerobic toluene degraders in nature. All these isolates resemble Azoarcus
tolulytr‘cus (Fries et al., 1994; Zhou et al., 1995) from tests done so far; further

comparisons are under way to conﬁrm their identity.

156

TCE is a solvent that is postulated to be co-oxidized by many oxygenase
enzyme systems. We compared the aerobic versus denitrifying growth of
toluene-degraders and the effects of TCE levels on the isolates capable of
growth under these two oxic conditions. We demonstrated that anaerobic
pathways are not effective on TCE co-metabolism. This provides additional
evidence of the requirement for 02 and oxygenases for TCE co-metabolism.

The presence on this site of aromatic-degrading denitriﬁers is an important
consideration for bioremediation technologies. Populations of TCE degraders
may still be enriched under anaerobic conditions so that once any 0: is added

the TCE oxidation rate may proceed at a higher rate.

Acknowledgments

We thank Gary Hopkins for collecting the samples from the aquifer during the
ﬁeld experiment. This work was supported by EPA , Grant N° CR 822029-01-0

and the NSF Center for Microbial Ecology, Grant N° BIR 9120006.

List of References

Alexander, M. 1982. Most probable number method for microbial populations, p.
815-820. In A L. Page (ed.), Methods of soil analysis. Part 2. Chemical and
microbiological properties. American Society of agronomy, Madison, Vlﬁ,
USA

157

Altenschmidt, U. and G. Fuchs. 1992. Anaerobic toluene oxidation to
benzylalcohol and benzaldehyde in a denitrifying Pseudomonas strain. J.
Bacteriol. 174:4860-4862

Bakker, G. 1977. Anaerobic degradation of aromatic compounds in the
presence of nitrate. FEMS Letters 12103-108.

Carr, G. J. and S. J. Ferguson. 1990. The nitric oxide reductase of
Paracoccus denitriﬁcans. Biochem. J. 269:423-430.

Chee-Sanford, J., J. W. Frost, M. R. Fries, J. Zhou, and J. M. Tiedje. 1995.
Characteristics of a denitrifying toluene-degrading bacterium, Azoarcus
tolulitycus strain Tol-4, and evidence for its anaerobic toluene mineralization
pathway involving acetyl-CoA and cinnamoyl-CoA Submmited for
publication.

Ghee-Sanford, J., M. R. Fries, and J. M. Tiedje. 1992. Anaerobic degradation
of toluene under denitrifying conditions in bacterial isolate Tol-4, abst. Q-
233, p.374. Abstr. 92th Annu. Meet. Am. Soc. Microbiol. 1992.

Coyne, M. S. A. Arunakumari, B. A. B. A. Averil, and J. M.. Tiedje. 1989.

Immunological identiﬁcation and distribution of dissimilatory heme cd1 and
nonheme copper nitrite reductases in denitrifying bacteria. Appl. Environ.
Microbiol. 55:2924-2931.

Demastia, M., T. Turk, and T. C.. Hollocher. 1991. Nitric oxide reductase.
Purification from Paracoccus denitriﬁcans with use of a single collumn and
some characteristics. J. Biol. Chem. 266:10899-1 0905.

Dolfing, J., J. Zeyer, P. Blnder-Eicher, and R. P. Schwarzenbach. 1990.
Isolation and characterization of a bacterium that mineralizes toluene in the
absence of molecular oxygen. Arch. Microbiol. 154:336-341.

Edwards, E. A., L. E. Wills, D. Grbic-Galic, and M. Reinhard. 1993. Anaerobic
degradation of toluene and xylene under sulfate-reducing conditions. Appl.
Environ. Microbiol. 58:794-800.

Elmerich, C. ,B. L. Dreyfus, and J. P. Aubert. 1982. Genetic analyses of
nitrogen ﬁxation in a tropical fast-groMng Rhizobium. EMBO J. 1:499—503.

Evans, P. J., D. T. Mang, K. S. Kim, and L. Y. Young. 1991. Anaerobic
degradation of toluene by a denitrifying bacterium. Appl. Environ. Microbiol.
57:1139- 1145.

 

158

Evans, P. J.,W. Ling, B. Goldschmidt, E. R. Ritter, and L. Y. Young. 1992.
Metabolites formed during the anaerobic transformation of toluene and o-
xylene and their proposed relationship to the initial steps of toluene
mineralization. Appl. Environ. Microbiol. 58:496-501.

Firestone, M. K., R. B. Firestone and J. M. Tiedje. 1979. Nitric oxide as an
intermediate in denitrification: evidence from nitrogen-13 isotope exchange.

Biochem. Biophys. Res. Commun. 91:10-16.

Fischer, C. and F. Juttner. 1994. Isolation of a new toluene producing
Clostridium from anoxic sediments of a freshwater lake. Abstr. Annu. Meet.

Swiss Soc. Microbiol. 1994.

Fries, M. R., J.-Z Zhou, J. Chas-Sanford, and J. M. Tiedje. 1994. Isolation.
characterization, and distribution of denitrifying toluene degraders from a
variety of habitats. Appl. Environ. Microbiol. 60:2802-2810.

Gamble, T. N., M. R. Betlach, and J. M. Tiedje. 1977. Numerically
dominant denitrifying bacteria from world soils. Appl. Environ. Microbiol.
33:926-939.

Gibson, D. T., and V. Subramanian. 1984. Microbial degradation of aromatic
hydrocarbons, p. 181-252. In D. T. Gibson (ed.), Microbial degradation of
organic compounds. Marcel Dekker, Inc., New York.

Grbic-Galic, D., and T. M. Vogel. 1987. Transformation of toluene and benzene
by mixed methnogenic cultures. Appl. Environ. Microbiol. 53:254-260.

Heiss, B., K. Frunzke and W. Zumft. 1989. Formation of the N-N bond from
nitric oxide by menbrane-bound cytochrome be complex of nitrate-respiring
(denitrifying) Pseudomonas stutzeri. J. Bacteriol. 171:3288-3297.

Hochstein, L. I. and G. A. Tomlinson. 1988. The enzymes associated with
denitrification. Annu. Rev. Microbiol. 42:231-261.

Hopkins, G. D., L. Semprini, and P. L. McCarty. 1993. Microcosm and In situ
ﬁeld studies of enhanced biotransformation of trichloroethylene by phenol-
using microorganisms. Appl. Environ. Microbiol. 59:2277-2285.

Hutchins, S.R. 1991. Biodegradation of aromatic hydrocarbons by aquifer
microorganisms using oxygen, nitrate, or nitrous oxide as the terminal
electron aceptor. Appl. Environ. Microbiol. 57:2403-2407.

Hutchins, S.R., W. C. Downs, J. T. Wilson, G.B. Smith, D.A. Kovacs, D. D.
Fine, R. H. Douglass, and DJ. Hendrix. 1991. Effect of nitrate addition on

 

159

biorestoration of fuel-contaminated aquifer: Field demonstration.
Groundwater 29:68-76.

Hutchins, S.R., G.W. Sewell, D.A. Kovacs, and G.A. Smith. 1991a.
Biodegradation of aromatic hydrocarbons by aquifer microorganisms under
denitrifying conditions. Environ. Sci. Technol. 25:68-76.

Juttner, F. 1988. Benzene in the anoxic hypolimnion of a freshwater lake.
Natunlvissenschaften. 75: 1 51 -1 53.

Juttner, F. 1990. Distribution of toluene in tratiﬁed lakes and river dams of
southwest Germany. Verh. lntemat. Verein. Limnl. 24:279-281.

Juttner, F. 1991. Formation of toluene by microorganisms from anoxic
freshwater sediments. J. Anal. Chem. 339:785-787.

Juttner, F., and J. J. Henatsch. 1986. Anoxic hypolimnion is a signiﬁcant
source of biogenic toluene. Nature. 223:797-798.

Krieg, N. R. 1981. Enrichment and isolation, p. 112-142. In P. Gerhardt, R. G.
E. Murray, R. N. Costilow, E. W. Nester, W. A Wood, N. R. Krieg, and G. B.
Phillips. (ed.), Manual of methods for general bacteriology. American
Society for Microbiology, Washington, DC.

Lovley, D. R., M. J. Baedecker, D. J. Lonergan, I. M. Cozzarelli, E. J. P.
Phillips, and D. I. Segal. 1989. Oxidation of aromatic contaminants coupled
to microbial iron reduction. Nature 339:297—300.

Lovley,. R., and D. J. Lonergan. 1990. Anaerobic oxidation of toluene, phenol,
and p-cresol by the dissimilatory iron reducing bacterium GS-15. Appl.
Environ. Microbiol. 56:1858-1864.

Martin, J. P., and K. Haider. 1969. Phenolic polymers of Stachybotrys atra,
Stachybotrys chartarum and Epicoccum nigrum in relation to humic acid
formation. Soil Sci. 107:260-270.

Murray, R. E., L. L. Parsons, and M. S. Smith. 1992. Competition between
two isolates of denitrifying bacteria added to soil. Appl. Environ. Microbiol.
58:3890-3895.

Rabus, R., R. Nordhaus, W. Ludwig, and F. Widdel. 1993. Complete oxidation
of toluene under strictly anoxic conditions by a new sulfate-reducing
bacterium. Appl. Environ. Microbiol. 59:1444-1451.

160

Roberts, P. V., G. D. Hopkins, D. M. Mackay and L. Semprini. 1990. Aﬁeld
evaluation of in-situ biodegradation of chlorinated ethenes: Part 1,
Methodology and ﬁeld site characterization. Ground water. 282591-604.

Schocher, R. J., B. Seyfried, F. Vazquez and J. Zeyer. 1991. Anaerobic
degradation of toluene by pure cultures of denitrifying bacteria. Arch.
Microbiol. 157:7-12.

Seyfried, B., G. Glod, R. Schocher, A. Tschech, and J. Zeyer. 1994. Initial
reactions in the anaerobic oxidation of toluene and m-xylene by denitrifying
bacteria. Appl. Environ. Microbiol. 60:4047-4052.

Shapleigh, J. P., and W. J. Payne. 1985. Differentiation of c,d1 cytochrome
and copper nitrite reductase production in denitriﬁers. FEMS Microbiol. Lett.

26:275-279.

Shoun, H., and T. Tanimoto. 1991. Denitriﬁcation by the fungus Fusarium
oxysporum and involvement of cytochrome P-450 in the respiratory nitrite
reduction. J. Biol. Chem. 266:11078-11082.

Stouthamer. A. H. 1988. Dissimilatory reduction of oxidized nitrogen
compounds. p. 245-303. In: A J. B. Zehnder (ed), Biology of anaerobic
microorganisms. John \Mley & Sons, Inc., New York.

Tiedje. J. M. 1982. Denitriﬁcation, p. 1011-1026. In A L. Page (ed.), Methods
of soil analysis. Part 2. Chemical and microbiological properties. American
Society of agronomy, Madison, WI, USA

Tiedje. J. M. 1988. Ecology of denitriﬁcation and dissimilatory nitrate reduction
to ammonium, p. 179-244. In A Zehnder (ed.), Biology of anaerobic
microorganisms. Jonh \MIey& Sons, Inc., New York.

Wilson, B. H , G. B. Smith and J. F. Rees. 1986. Biotransformation of selected
alkylbenzenes and halogenated aliphatic hydrocarbons in methanogenic
aquifer material: a microcosm study. Environ. Sci. Technol. 20:997-1002.

Zhou, J.-Z., M. R. Fries, J. Chee-Sanford, and J. M. Tiedje. 1995.
Phylogenetic analyses of a new group of denitriﬁers capabla of anaerobic
growth on toluene and description of Azoarcus tolulyticus sp. nov. lntem.
Journal of System. Bacteriol. 45: ..... - ......

Zumft. W. G. 1991. The denitrifying prokariotes. p.554-582, In A Balows, et
al., (ed.), The prokaryotes, second edition. Springer-Verlag, Ina., New York.

 

Chapter V

Isolation, characterization, and distribution of denitrifying toluene
degraders from a variety of habitats

1B1

162

APPLIED AND ENVIRONMENTAL MICROBIOLOGY. Aug. 1994. p. 2802—2810
(rm-2240;94504.0(i+0
Copyright C 1994. American Society for MicrobIology

Vol. 60. No. 8

Isolation, Characterization, and Distribution Of Denitrifying
Toluene Degraders from a Variety Of Habitats

MARCOS R. FRIES.1 JIZHONG Zl-lOU.I JOANNE CHESE-SANFORD.2 AND JAMES M. TIEDJEL'?‘

Departments of Crop and Soil Sciences' and of lllt'crobiologv.2 Center for Microbial Ecology.
Michigan State University. East Lansing. Michigan 48824

Received l8 March 1994."Accepted 6 June 1994

Enrichments capable of toluene degradation under Oz-free denitrifying conditions were established with
diverse inocula including agricultural soils, compost, aquifer material, and contaminated soil samples from
dilerent geographic regions of the world. Successful enrichment was strongly dependent on the initial use of
relatively low toluene concentrations. typically 5 ppm. From the enrichments showing positive activity for
toluene degradation. 10 bacterial isolates were obtained. Fingerprints generated by PCR-amplified DNA. with
repetitive extragenic palindromic sequence primers, showed that eight of these isolates were dilereut. Under
aerobic conditions, all eight isolates degraded toluene, five degraded ethylbenzene, three consumed benzene,
and one degraded chlorobeneene. meta-Xylene was the only other substrate used anaerobically and was used
by only one isolate. All isolates were motile gram-negative rods, produced N, from denitrifiation. and did not
hydrolyze starch. All strains but one ﬁxed nitrogen as judged by ethylene production from acetylene, but only
four strains hybridized to the ruﬂlDK genes. All strains appeared to have heme nitrite reductase since their
DNA hybridized to the heme (nirS) but not to the Cu (nirU) genes. Five strains hybridized to a toluene
ortho-hydroxylase catabolic probe, and two of those also hybridized to a toluene meta-hydroxylase probe.
Partial sequences of the 165 rRNA genes of all isolates showed substantial similarity to 16$ rRNA sequences
of Azoarcus sp. Physiological, morphological, fatty acid, and 168 rRNA analyses indicated that these strains
were closely related to each other and that they belong to the genus Azoarcus. The activity and isolation of at
leastonetoluene-degradingdenitriﬁerfromthemajorityofthehabitattypesstndiedsnggestthatmicrobes

withthecapacitytogrowanaerobicallyontoluenearecommoninnature.

 

The monoaromatic hydrocarbons known as BTEX (ben-
zene, toluene. ethylbenzene, and xylenes) are one of the major
problems in environmental pollution. Their presence in
groundwater is a widespread problem because of the leakage
of underground petroleum storage tanks and spills at petro-
leum production wells, reﬁneries. pipelines, and distribution
terminals. Many governments have established cleanup stan-
dards for these chemicals in groundwater because of their
carcinogenic potential (4. 14).

Biodegradation of BTEX under aerobic conditions is well-
known; oxygen is utilized for ring activation and cleavage and
serves as the electron acceptor for the complete oxidation of
this compounds (15). The availability of oxygen. due to its low
solubility in water and its low rate of transport through
saturated porous matrices such as soil and sediments. is usually
the rate—limiting parameter for BTEX removal from contam-
inated sites. Therefore. BTEX biodegradation in the absence
Of Oxygen would be a very beneﬁcial remediation process. It
has only been in recent years that anaerobic degradation of
these compounds has been conclusively established. Of the
BTEX class of compounds. toluene seems to be the most easily
degraded under anaerobic conditions. The degradation of
toluene under denitrifying (3. 8. 11, 12, 21. 25. 36). methano-
genic ( 16. 40). sulfate-reducing (9. 32), and ferric iron-reducing
(27. 28) conditions has been reported. For bioremediation. the
most attractive electron acceptor is nitrate since it is water

soluble. not costly, and not seriously toxic and does not react
with other inorganic species present. such as ferric iron.

‘ Corresponding author. Mailing address: Center for Microbial
Ecology. Plant and Soil Science Building. Michigan State University.
East Lansing. MI 48824-1325. Fax: (517) 353-2917. Electronic mail
address; 21394jmt@msu.edu. '

However, very little is known about the organisms responsible
and how widely they are distributed in nature.

We report here on a new group of bacteria that grow on
toluene under denitrifying conditions and show that they
appear to be widely distributed in nature.

MATERIALS AND METHODS

Enrichments and isolations. Soils and sediments were col-
lected independently from various locations and handled by
procedures to prevent any cross-contamination. Samples (5 to
10 g) were incubated without shaking with 10 ml of basal salts
(BS) medium (31) amended with 5 mM KNO3 in sterile
centrifuge tubes. After 3 days of incubation, the enrichments
were centrifuged (1,300 X g for 10 min), the supernatant was
removed. and fresh sterile medium was added to the samples.
The samples were vortexed and reincubated. This protocol was
repeated one more time to deplete easily oxidizable carbon
from the samples that could potentially reduce the selection
for anaerobic toluene degraders. After the third incubation
period. the samples were centrifuged and the pellet was
resuspended in fresh medium; the large soil particles were then
allowed to settle. The tubes were transported to a Coy
anaerobic chamber, and 5 ml of the supernatant was trans-
ferred with a sterile syringe to a serum bottle containing 45 ml
of 88 medium containing 5 mM KNO3 and 5 ppm of toluene
that had been prepared by strict anaerobic protocol. Toluene
disappearance was evaluated by headspace analyses. Enrich-
ments positive for toluene degradation were spiked again with
toluene at a concentration of 5 ppm and then at 25 ppm. The
bottles were inverted and incubated. and the production of
bubbles on the surface of the Teﬂon-lined septa indicated
denitrification activity. Once bubble production ceased. typi-

2802

163

VOL. 60. 1994

cally after 3 days at this last stage of enrichment. three serial
transfers of a 10% inoculum were made per sample into fresh
medium containing 25 ppm of toluene. Anaerobic manipula-
tions throughout this work were done in the anaerobic cham-
ber and the resultant headspace of the bottles. and agar plates
was nominally 10% H3009? N2. The incubation temperature
throughout this work was 30°C.

These enriched samples were serially diluted and plated on
two different solid media: (i) BS medium plus 5 mM N03-
plus toluene vapors and (ii) modiﬁed RZA I M-RZA). based on
the original composition provided by Difco (Detroit. Mich.).
M-RZA had the following salt mixture composition per liter:
KHzPO,. 0.25 g: KZHPO.. 0.4 g: KNO3. 0.505 g1CaC12 - 2H20,
0.015 g; MgCl2 - 6HZO. 0.02 g: FeSO4 - 7HZO. 0.007 g: Nastu
0.005 g: NH.CI. 0.8 g: MnClz-4H20. 5 mg; H3803, 0.5 mg;
ZnClz. 0.5 mg; CoCl2 - 61:130. 0.5 mg; NiSO. - 6H20, 0.5 mg:
CuCl2 - 214120. 0.3 mg: and NaMoO2 - 21120. 0.01 mg. For solid
medium. 15 g of Bacto agar (Difco) was added. The pH was
adjusted to 7.0 before autoclaving. The carbon sources and
their concentrations. per liter. were yeast extract. 0.5 g: pep-
tone. 0.5 g: Casamino Acids. 05 g: dextrose. 05 g: soluble
starch. 0.5 g: and sodium pyruvate. 05 g. Glass petri dishes
were used for toluene vapor-based growth. and toluene was
added to a small vial inside an incubation jar. The amount of
toluene added to provide a ﬁnal concentration of 25 ppm was
calculated in the basis of the total volume of agar in the
incubation jar. After 1 week of incubation. sufﬁcient toluene to
provide 25 ppm was again added. The jar used in the toluene
vapor experiments was sealed with a Teﬂon-lined aluminum
sheet (Cole-Palmer. Chicago. III.) to prevent toluene absorp-
tion by the rubber sealer of the jar. The plates were incubated
under anaerobic and aerobic conditions.

For puriﬁcation of isolates. single colonies from different
plates and different dilutions were selected and puriﬁed at least
three times on M-RZA before further evaluation. Conﬁrma-
tion of toluene degradation under denitrifying conditions was
done by transferring a heavy inoculum of each isolate to sterile
20-tnl vials. recapping the vials with foam plugs. and transport-
ing the vials to an anaerobic chamber for headspace gas
exchange (at least 6 h). after which 10 ml of anaerobic BS
medium—NO,’-25 ppm of toluene was added. The vials were
sealed with sterile butyl rubber Teﬂon-lined septa and incu-
bated for at least 2 weeks before toluene disappearance was
evaluated. As a control, medium without nitrate containing
cells and toluene was incubated at the same time as the
medium with nitrate and was used as a reference to determine
nitrate-dependent toluene removal.

Characterization of isolates. Cell size and shape were
observed by phase-contrast microscopy for cells grown anaer-
obically on toluene in liquid medium and for cells grown on
M-RZA agar. The latter were resuspended in water, and 5 ul
was added to a microscopic slide containing 5 pl of molten
0.4% agarose solution. mixed gently, and covered with a
coverslip. The medium for the nitrogen ﬁxation assay consisted
of BS free of nitrogen sources supplemented with ﬁlter-
sterilized solutions of (ﬁnal concentration per liter) biotin (0.1
mg). NazMoO. - 21420 (0.002 g). malic acid (5 g). and KOl-l
(4.5 g). adjusted to pH 7. Agarose (0.07%) was added to
provide for establishment of microaerobic conditions (7).
Inocula were grown on liquid M-RZA, and cells washed in
feline solution were inoculated into 20-ml serum vials contain-
mg 5 ml of medium. The vials were incubated for 48 h. and
mtrogenase activity was evaluated by the acetylene reduction
assay (1) after 6 and 24 h of incubation with acetylene.

Azospirillum brasr'liense sp7 was used as a positive control.

Denitriﬁcation was evaluated by observing gas bubble for-

DENITRIFYING TOLUENE DEGRADERS 2803

mation in inverted tubes and by measuring the presence of
N30 in the vial headspace by gas chromatography (GC). The
denitriﬁcation medium consisted of the salt mixture and the
following carbon sources (ﬁnal concentration per liter): Prote-
Ose peptone (0.5 g). yeast extract (0.5 g), Casamino Acids (0.5
g), potassium acetate ( l g). and sodium succinate (l g). Argon
was the headspace for the samples analyzed by GC.

Starch hydrolysis was performed by the standard method
(37) on M-RZA plates supplemented with 0.2% starch.

The isolates analyzed for cellular fatty acids were precul-
tured on M-R2A and streaked onto plates containing 0.3%
(wt/vol) tryptic soy broth solidiﬁed with 15 g of tryptic soy agar
(TSA) per liter (24). At least three plates of each isolate were
incubated for 72 to 96 h in order to obtain enough biomass for
the analysis. Cells were harvested from the plates by scraping
with a sterile loop. Saponiﬁcation. methylation. and extracﬁon
were performed by the procedure described previously (35).
Cluster analysis was carried out by using an in-house cluster
program and the MIDI software.

Detectable plasmids were screened for in isolates cultured
anaerobically on toluene-BS-NO,‘ medium. Cells were Iysed,
and plasmids were screened as described by Kado and Liu (23).

The anaerobic growth rate of the isolates in BS (containing
one-ﬁfth of the original EDTA concentrationi-S mM N033
50 ppm of toluene was determined by measuring the optical
density at 600 nm. The inoculum was grown under the same
conditions. Growth in TSA was evaluated every 12 h. and the
colony size was compared with that of M-RZA-grown cells.

To test for the isolates‘ capabilities to degrade benzene;
ethylbenzene: o-. m-, and p-xylenes: and chlorobenzene. inoc-
ula were grown on BS-NO,‘-toluene medium under both
denitrifying and aerobic conditions. Inocula (0.5 ml) were
transferred to 20-ml of sterile auto sampler vials. For aerobic
growth. 4.5 ml of BS-NOf—25 ppm of the aromatic substrate
was added to the vials, and the vials were sealed with Teﬂon-
lined stoppers. For anaerobic growth. the vials were prepared
similarly except that the media with the aromatic substrates
were added in the anaerobic chamber. Positive degradation
activity was deﬁned as at least 80% loss of substrate in the
headspace as measured by GC analysis. compared with that for
noninoculated controls.

Molecular methods. Repetitive extragenic palindromic
(REP)-PCR patterns were obtained from cells with Rep-1 and
Rep-2 primers by PCR (5). Ampliﬁcation was performed with
a model 9600 Perkin-Elmer Cetus thermocycler. Products (10
pl) were separated by electrophoresis in 1.5% agarose gels and
stained with ethidium bromide. Ampliﬁcation was primarily
done by using individual colonies grown on M-RZA. but for
isolates with poor or no ampliﬁcation. DNA extracted from
cells was used as the template for PCR ampliﬁcation. For
isolates with very similar patterns. the same stock of primers
and reagents were used in the analysis.

The gene probes used are described in Table 1. Escherichia
coli cultures carrying the plasmids with probes were grown for
plasmid ampliﬁcation in the presence of the appropriate
antibiotic. Plasmids were extracted by a standard protocol (29).
The probes were isolated as restriction fragments from their
respective vectors in 1% low-melting-point agarose. puriﬁed
with the Gene Clean kit (Bio 101. he. La Jolla. Calif). and
labelled with [a-32PldCTP (3.000 CilmM; Dupont. NEN Re-
search Products, Wilmington. Del.) by using a random hex-
amer priming kit from Boehringer Mannheim Biochemicals.
Labelled probes were separated from unincorporated nucle-
otides prior to use with a spun column (29). The probes were
used at approximately 10h cpmlml of hybridization fluid.

Genomic DNA was obtained by standard methods (10) from

2804 FRIES ET AL

164

Arm. ENVIRON. MICROBIOL.

TABLE 1. List of DNA probes used in this study

 

Laboratory source

 

n . .
(p333 9:12;) Gene encoded and probe sine Plasmud and refer ence“

Pseudomonas putida PaW1 Methyl monooxygenase (hydroxylase and NADH-ferredoxin pGSH2836 S. Harayama (18)
reductase). 2.35 kb (Semi-lindlll)

Pseudomonas putida F1 Toluene dioxygenase (large and small subunits of oxygenase. pDTG601 D. Gibson (44)
ferrodoxin. and part of reductase). 3.5 kb (EcoRl-Bglll)

Pseudomonas mendocina KR Toluene para-hydroxylase (monooxygenase and ferredoxin). pMY421 M. DeFlaun (42)
3.6 kb (EcoRl-EcoRl)

Pseudomonas pickettii PK01 Toluene meta-hydroxylase (a subunit of monooxygenase). pABl4AAva l R. Olsen (2. 30)
0.68 kb (Apal-Aval)

Pseudomonas sp. strain JS-150 Toluene ortho-hydroxylase. 2.2 ltb (EcoRV-Ht‘ndlll) pR020116 R. Olsen (22)"

Pseudomonas stutzeri JM300 Heme containing nitrite reductase. 0.7 kMDdel-Ddel) szGTh 2.4 J. Tiedje (38)

Pseudomonas sp. strain 179 Copper containing nitrite reductase. 1.9 kb (EcoRl-BamHl) pRTc1.9 J. Tiedje (41)

Pseudomonas stutzen’ ZoBell Nitrous oxide reductase. 1.2 kb (Pstl-Pstl) W. Zumft (39)

Micrococcus luteus 23$ rRNA. 0.47 kb (ECORI-Hindi") pAR17 K. Schleifer (33)

Rhizobium sp- Nitrogenase (rufHDK genes). 3.6 kb (Bglll-Xhol) pRSZ F. de Bruijn (10)

 

" Laboratories from which the clones were obtained from

" This strain was provided by J. C. Spain (17). The probe has strong hybridization to P. cepacia G-4 (22) (Fig. 3C) and likely reflects G-4-like sequences as well.

pure cultures of isolates and selected strains grown on M-RZA
broth under aerobic conditions. Restriction endonuclease di-
gestion of DNA was performed according to the manufactur-
er's speciﬁcations. Digested DNA was size fractioned by
electrophoresis in 0.7% agarose gels and transferred to nitro-
cellulose (polyester-supported BAS 68380; Schleicher &
Schuell, Keene. NH.) as previously described (29) with 20x
SSPE (1x SSPE is 0.18 M NaCl. 10 mM NaHzPO. [pH 7.04].
and 1 mM EDTA). The DNA on the ﬁlters was cross-linked by
UV light (Stratagene. La Jolla. Calif.). The solutions used for
DNA hybridization analysis have been described elsewhere
(19). The membranes were prehybridized for at least 24 h in
heat—sealed bags containing 100 jil of prehybridization ﬂuid
per cm2 of ﬁlter. Prehybridization ﬂuid contained 5x Den-
hardt solution. 5X SSPE. 50% formamide. and 200 p.g of
sonicated and denatured salmon sperm DNA per ml. The
hybridization solution was the same as that for prehybridiza-
tion but included 10% (wt/vol) dextran sulfate and was added
at 50 jil/cm2 of ﬁlter. The membranes were incubated for at
least 24 h. Two hybridization temperatures, 30°C (low strin-
gency) and 42°C (high stringency). were used. After hybridiza-
tion, the ﬁlters were washed once for 15 min with agitation at
30°C with 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M
sodium citrate)—-0.1% sodium dodecyl sulfate (SDS). For low-
stringency hybridization. a second wash for 30 min with 0.5x
SSC—0. l % SDS was performed. For high-stringency hybridiza-
tion. a second wash for 15 min with 0.5x SSC-0.1% SDS
followed by a third wash with 0.1 X SSC-0.1% SDS was
performed. This was followed by a ﬁnal wash at 55°C for 30
min with the last solution. After the washes. hybridization
signals were visualized by using the Betascope radioactive blot
analyzer (Betagen Corp.. Waltham. Mass.) or by autoradiog-
raphy with X-Omat AR ﬁlm (Kodak, Rochester. N.Y.) ex-
posed at -70°C with a Quanta Ill (Sigma, St. Louis. Mo.)
intensifying screen. Exposure times were 1 to 3 days. depend-
ing on the intensity of the radioactive signal. For reuse of the
same blot for another probe. the blots were stripped by
washing for 10 min in boiling water-0.1% SDS at least two
times. depending on the signal left on the blot after evaluation
with the Betasc0pe. A ﬁnal wash with 2x SSPE-0.1% SDS for
10 min completed the stripping protocol.

The 16S rRNA gene was ampliﬁed from genomic DNA by
PCR and cloned into a plasmid vector as described previously
(43). The 165 rRNA gene was sequenced from both directions
with an automated ﬂuorescence sequencer (model 373A;

Applied Biosystems. Foster City. Calif.) with the forward and
reverse primers. which span E. coli 16S rRNA gene positions
785 to 805 and 1115 to 1100. respectively. The DNA sequences
were compared with those in the Ribosomal Database Project
(26) and analyzed by using the programs in the Geneties
Computer Group software package (6) and in PHYLIP phy-
logeny inference package (13).

Analytical methods. Toluene: ethylbenzene: o-, m-. and
p-xylenes: benzene: and chlorobenzene concentrations were
measured with a GC equipped with an ﬂame ionization
detector. a DB-624 capillary column (J&W Scientiﬁc. Folson.
Calif), and a headspace sampler. The vials and bottles were
equilibrated at 30°C. the column was equilibrated at 90°C. and
the injector and detector were equilibrated at 200°C. He was
the carrier gas. Acetylene and ethylene were assayed by a
GCl-ﬁame ionization detection by using a DB-23 megabore
column (J&W Scientiﬁc) at 75°C. an injector at 160°C, and a
detector at 320°C with N2 as the gas carrier. N03” and N037
concentrations in culture supernatant were determined by
high-performance liquid chromatography (HPLC) analysis us-
ing a Partisil 10 SAX column (Whatman. Clifton. NJ.), UV
detection at 210 nm. and 50 mM phosphate (pH 3.0) as the
eluant. NO was measured by GC-electron capture detection
with a Poropak 0 column at 55°C. 300°C detector tempera-
ture. and 95% argon-5% methane as the carrier gas.

Nucleotide sequence accession numbers. The partial 16S
rRNA sequences of all new isolates have been placed in
Genbank under accession no. L 33687 to 1.33694.

RESULTS

Enrichments. Successful enrichments for denitrifying tolu-
ene degraders were obtained from about half of the samples
from both chemically contaminated and noncontaminated sites
(Table 2). Enrichments positive for toluene degradation were
obtained from widely separated and dissimilar habitats. Ten
isolates that were conﬁrmed as pure cultures and were able to
grow on toluene in the presence of nitrate and the complete
absence of oxygen were obtained (Table 2). The isolates also
came from a wide range of environments. Additional clones
that appeared to have toluene-degrading denitrifying activity
were obtained, but they were not studied further because it was
too difficult to conﬁrm purity, growth was too variable. or they
appeared to be identical or closely related to isolates already in
pure culture by REP-PCR analysis.

165

VOL. 60. 1994

TABLE 2 Source of inoculum. number of enrichments with
positive activity for toluene degradation under den n'ttrifying
condit loans and number ofisolates obtaIne do Ith

activity from each sample

 

Enrichment

 

Source of inoculum and (no positive “3::
descnpuon or sample total no (Slﬁin no ’
tested)
Noncontamtnat
Ca oon rainforcst sod 12 ll.
Michigan. muck soi 11‘. 2 (Td-ZO. Td-21)
Michigan. compou lec 1.1 1 (Td~15)
cum'alt soil 1,".7 l (Td-lb)
Hawaii Big Island forest soil 1‘5 INA
LKauaI sugar cane sot il 01
Siberia Koly‘ma Val ey permafrost 0.?
soil
Total 7/13 4
Contaminated with organic pollutants
razil: industrial waste 23 2 (T d—l7. Td-t9)
Rio Grande do Sul. Brazil; industrial 213 INA
sludge
OntarIo. Canada; pulp mill lagoon W [U
'ment
Bear lake. Michigan: aquifer. sand. U1 1 (GR-3)
petroleum (24 to 26-m deep)
Wexford. Michigan: aquifer. sand. 0!]
petroleum
Huntington Beach. California: 1!] 1 (Td-3)
manne. petroleum
Washingto ute 1‘2 2 (Td—l. Td-Z)
Six undescrtbed chemtcallv 116 IU
contammated soIls

Total 10!21 6

 

"1U. Isolation unsuccessful: INA. Isolation not attempted.

Successful enrichment and isolation were strongly depen-
dent on the use of relatively low toluene concentrations.
typically 5 ppm. This strategy was used because our early
isolation and enrichment attempts were unsuccessful after
extensive effort with 250 ppm and because we had noted a
considerable increase in the most probable number estimate of
aerobic toluene degraders when the t luene conce ceantr tion was
decreased from 250 to 50 ppm and again when reduced to 5

p ..Thus wer reasoned that toluennec toxicity could also be an
important factor for successful and isolation of
anaerobic toluene degraders. Once isolated. the cultures were
routinely cultured in E p of toluene and. once growing.
could be fed 50 ppm to obtain higher cell yields.

All isolates were obtained from plates of the M-RZA
medium incubated aerobically. Some isolates were obtained
from the anaerobic toluene vapors plus nitrate or M-R2A p us
nitrate medium. but by REP-PCR they were identical to the
ones isolated from the source on the aerobic medium. In
all cases. the denitrifying toluene degraders were pinpoint
colonies. Larger colonies were also picked. but these isolates
were either denitriﬁers or toluene degraders. Some of them
were capable of both functions but did not carry out both
under the same conditions. In some cases. it was very diﬂicult
to separate contaminating cells from the denitrifying toluene
degrader. Purity was based primarily on uniform and repeated
colony morphology after at least 2 weeks of incubation.

DENlTRlFYlNG TOLUENE DEGRADERS 2805

 

FIG. .REP-PCR ﬁngerprint patterns of toluene-degrader denitri-
ﬁer isolates generated by using chromosomal NA es 1 and 11
ize markers. with the base pairs indicated on the left.

Consistent REP-PCR patterns and consistent cell morphology

were used as con nﬁnnatory methods for uri

We also carried out parallel enrichments for denitrifying
benzene degraders. using the same enrichment conditions and
environmental samples as those used for the toluene enrich-

m.ents No activity could be conﬁrmed by measuring benzene
removal from any of the ennchme etsn

We used REP-PCR to screen for suﬂiciently different strains
for further study There were eight distinct proﬁles from the 10
conﬁrmed denitrifying toluene degraders (Fig. 1). One of the
isolates. GR- 3. hada REP -ttPCRpa em identical to that of our
previous isolate. Tol-4 (3). and was not studied further. OneS sect
of isolates with identical patterns came from he saern
(Michigan muck soil)a and may be siblings. therefore. only
isolate Td-Zl was studied further The other set of isolates with
identical patterns came fro Michigan agriculturala soil and a
compost pile; only the compost isolat d-l. studied
further since it came from a very diﬁerent environment.

Characteristics. The ability of the seven new isolates to
degrade related aromatic compounds under aerobic or anaer-
obic conditions Is very limited. except that all isolates can also

degrade toluene aerobically (Table 3). Several of the cultures
were initially nega aitve for aerobic toluene use: but after
repeated experiments and optimizing conditions. all were
shown to be capable of aerobic toluene consumption. albeit
some showed weak ability. Five strains couldu sethe alkylated
analog. ethylbenzene. aerobically, but none couldu use it anaer—
obically Three isolates dben nnze e, and one sed chloro—
benzene aerobically. meta- -Xylene was the only substrate used
anaerobically other than toluene. and it was used by only one
1501 a t.e The anaerobic pathway seems very speciﬁc for toluene.
and the aerobic substrate range is much more limited than it is
for the well-studied aerobic toluene degraders (Table 3).

166

 

 

 

 

2806 FRIES ET AL APPL ENVIRON. MICROBIOL
TABLE 3. Removal of diﬁerent substrates by toluenedegrading isolates and well-known aerobic strains in BS medium under
aerobic and anaerobic (denitrifying) conditions after 2 weeks of incubation“
Ethyl _
1mm: Benzene Toluene benzene o-Xylene an Xylene p-Xylene Chlorobenzene

Ac" Ana‘ Ae Ana Ae Ana Ae Ana Ac Ana Ae Ana Ac Aria
Tol-4 -— -— + 4» + — - - - — — — — —
Td-l — - + + + - - - — - - - — —
Td-2 - — : + — -> - - - - — - — —
Td-3 r. - z + - - - - — — — — — —
Td-15 - — + + + — — - — + — - — —
le7 + - + - + - - — — — - — -
Td-19 - -— z: - - — - - — - - — + -
Td-21 — — + «r + - — -— - — - — - —
Pseudomonas cepacia G4 + -— + — ~.- - + — — — + — + —
Pseudomonas mendocina KR + - + - + - + — + - —‘ - + —
Pseudomonas pickettii PK01 + - + — + - -:- — + — + — - —
Pseudomonas putida Fl -‘- — + — + — + - + - + - — -
Pseudomonas putida PaW1 + 4 — + - + -— -— — + -— — —

 

' Denotes more than 80% removal from headspace vial: -. negative activity. :.
” Ac. aerobic conditions.
' Ana. anaerobic (denitrifying) conditions

All seven isolates had similar major features but also showed
minor differences which conﬁrmed that they were not identical
strains (Table 4). All were gram-negative rods and, motile,
produced N2 from denitriﬁcation. and did not hydrolyze starch.
They did not grow well aerobically on complex media such as
TSA. Growth could begin to be seen on 1/10 strength TSA as
very sparse tiny colonies only after 48 h of incubation. M-R2A
is the best medium that we have found for growth on plates.
Colonies of l to 4 mm in diameter can be obtained after 36 to
72 h of incubation. N2 ﬁxation was shown by subsurface pellicle
formation in semisolid medium free of combined nitrogen, by
ethylene production from acetylene (all but isolate Td-15). and
by DNA from four of seven strains hybridized to the ruﬂ-IDK
genes (Table 4).

The major fatty acids for all strains studied and their
concentration ranges were cis:9 16:0 (42.3 to 61.9%). 16:0 (21.5
to 38.6%). 12:0 (4.4 to 12.9%). 3-OH-10:0 (1.7 to 7.7%), 14:0
(0.9 to 1.55%). cyclo 17:0 (0 to 4.11%), and 18:0 (less than
1%). Species identiﬁcation based on total fatty acids and
cluster analysis was n0t possible since the analysis gave a
similarity index of less than 0.4 by using the MIDI database.

activity often delayed. All substrates at 3 ppm concentration.

The morphology of all strains grown anaerobically on tolu-
ene was small rods, typically 1.4 to 2.1 pm in length (Fig. 2A).
When grown on M-RZA agar, however, all formed longer cells.
2.1 to 2.8 pm (Fig. 28). and some isolates (i.e.. Td-3. Td-15.
Td-17. and Td-19) had a tendency to form chains (Fig. 2C).

DNA isolated from the seven new isolates and ﬁve well-
studied aerobic toluene degraders was digested and hybridized
on Southern blots to various probes to determine which strains
carried similar sequences. Hybridization with a universal 23$
rRNA probe conﬁrmed that sufﬁcient DNA was in all lanes
and that all strains were different (Fig. 3A). The probe for the
denitrifying Cu-nitrite reductase gene (nirU) showed no hy-
bridization (data not shown). but the heme-nitrite reductase
probe (m'rS) hybridized to all strains. suggesting that they are
all denitriﬁers with the heme-type enzyme (Fig. 3B). The
nitrous oxide reductase probe (nosZ) hybridized to all strains
and in diﬁ'erent positions (data not shown). Probes for the ﬁrst
steps in all ﬁve aerobic toluene-degrading pathways were used.
but only the probe for the onho-hydroxylase (Pseudomonas sp.
strain 18150) (Fig. 3C) and the meta-hydroxylase (1’. pickettii
PK01) (Fig. 3D) genes showed hybridization. Five strains (all

TABLE 4. Summary of characteristics of the different denitrifying toluene degraders‘

 

Growth on

 

 

 

[some Gromh on M-RZA mo TSA Growth (11) D‘“""““"°“ N'"°8°“ “mm Starch Detectable
for 48 h ratc” hydrolysis plasmids
48 h 96 h N; N30 N0; Pellicle‘ Acetylene‘ Probe’
Tol-4 - : + 8—13 + + + + + + — -
Td-l + : -- 6—7 — + z + + + -— -
Td-2 + z - 5.7 + + + + + + -— -
Td-3 + - - 7-8 + + + + +‘ — - <—
r445 + - + 6—7 + + 4 + — — - —
Td-17 + - + 5—7 + + - + + — — -
Td-l9 + : - 5—7 - + + + +1 - - e
Td-Z] + — 4- 5.7 + + + + + + — —

 

" e. positive activtty: —. negative activity. 2. variable activity.

" Cells grown on BS-SO ppm of toluene under denitrifying conditions. Doubling time is expressed in hours.

" Observation of bubbles on the surface of the Teﬂon-lined septum.

‘ Formation of growth pellicle on nitrogen-free medium.

" Detection of ethylene from acetylene after at least 24 h of incubation.

’ Presence of distinct band on Southern blot hybridized with MIHDK probe.

" Ethylene production detectable only after 24 h of incubation with acetylene.

 

A ' Wm

Vot. 60.1994
———-,———7-— “f: -w—:- - -
-‘u 9 3 ‘f;. ‘. e~
o . >. . ,:
a u
o
\
\ ’ ‘
O

 

. . base-contrast photomiaographs of isolates Td-z (A).
Td-3 (B). and Td-I7 (C). Isolate Td-Z was grown on BS—toluene liquid
medium to late exponential phue. Isolates Td-S and Td-l7 were grown
on M-RZA solid medium for 48 h. Bars. 14 pm

167

DENIT'RIH’ING TOLUENE DEGRADERS 2807

but isolates Tcl- 3 and Td- 15) showed strong signals to the

ortho-hydroxylase pr The meta-h roxylase probe hybrid-
ized to DNA froml two strains that also showed hybridization to
the ortho- hydr rxvo se.probes but the Shybridizing bands were in
different positions 3for the two probe

The sequence of approximatelyR 280 Anucleotide bases. corre-

sponding to the E. coli 16$ rR gene sequence from
nucleotide 810 to 1090 was obtained for the seven isolates
The percent similarities among these isolates ranged from 97.8
to 100%. The partial sequences of these isolates showed strong
similarity to 16$ rRNA gene sequences of the nitrogen- -ﬁxing
genus Azoarcus (20). The similarities are Azoarcus sp. strain
Sb52 (90. 7 to 91.5%). Azoarcus sp. strai BH72 (92. 6 to
94.1%), andAzoaIc us (93. 4 to 94. 9%). The phyloge-
netic tree constructed by the maximum parsimony method
showed that all the toluene~denitrifying isolates form a phylo-
genetically coherent unit clustered withA .indigenr and Azoar-
cus sp. strain BH72 (Fig 4). Very similar tree topologies were
also obtained by distance matrix and maximum likehood
methods (data not shown).

DISCUSSION

Pure cultures of anaerobic toluene degraders have not been
easy to isolate. After a decade of effort by many capable
laboratories, seven isolates that use nitrate as an electron
acceptor (3. 8. 12. 36). one that uses Fe(IIl) (28). and one that
uses sulfate have been reported (32). This study yielded 10 new
isolates and additional active enrichments. e believe that the
most important reason for the improved succes rate of
enrichment and isolation was the strategy of avoiding toxicity
by never exposing the culture to more than 5 ppm of toluene
during the initial enrichment proce§. Also important were
exhausting the residual available carbon before adding tolu-
ene. isolating cells by aerobic growth on M-RZA. and being
sure to select the pinpoint colonies. This procedure. however.
may have selected for only a certain group of denitrifying
toluene degraders and may explain why we were unsuccessful
in obtaining isolates from some of the active enrichments.

The seven new isolates appear to be closely related to each
other and to be members of the genus Azoarcus. The identiﬁ-
cation is based on the fact that these isolates share the
following key features with the described Azoarcus strains (20.

): all 165 rRNA sequences fall within the cluster for this
genus. they ﬁx nitrogen. they have the same type andp propor-
tion of cellular fattv acids as do the described strains. and they
have similar morphology when gro own on complex medium.
They do have some phenotypic and ecologic differences from
the described Azoarcus strains. The new strains all denitrify
and grow poorly on TSA which are not characteristics of the
previously described strains (34). Also. ll of 12 previously
described strains were isolated from the roots of tropical

and are considered to be rhizosphere-associative nitro-
gen ﬁxers. None of our isolates came from plant rhizospheres.
Three came from environments in which plants had recently
grown (Michigan muck and agricultural soil). but four came
from soils contaminated with chemical wastes in industrial
areas. one from an aquifer 24 to 26 m underground and one
from a compost pile. Thus. the ecological niche of the new
isolates may be very diﬁerent from that described for the
previous isolat te.s

All our isolatesh are able to use toluene aerobically as well as
anaerobically, hwould not be expected if these are
independent traits. cPerhapsthe the common
steps reﬂect a crno on phylogenetic heritage. or were a result
of concurrent selection resulting from continued use of the

168

NFL. Emacs. MICRONOL

 

 
 

23130-

 
 

9416-

  
 
 

6557-

 

23l30-

 

5557,

 

 

 

 

 

 

 

 

 

 

 

 

 

 

of genome DNA from pure cultures of toluene degraders digested with 5:011] and hybridized With the
ase (C). and toluene nim-
pairs) are indicted on the left.

hybridization '
BS rRNA (A) heme nitrite reductase (B). toluene ortho-h
ridizations were done under high (A and B) and low (C and D) stringency conditions. Size markers (inbue

FIG. 3. Southern
following
Hyb

169

VOL. 60. I994

Alcaligenes eutrophus
Rubrivivax gelatinosus
Rmoocyclus purpureus
Thiobacillus thiooxidans
Spirillum volutans

Azoarcus sp. strain $5b2

Td—3

Td-19

Td-17

Til-21

 
  
  
  
 

  
  
   

 

Azoarcus indigens
Azoarcus sp. strain BH72
— Eacherlclia coi

FIG. 4. Phylogenetic position of the toluene-degrading denitriﬁer
(Td) isolates. This tree was constructed by using the programs
SEOBOOT. DNAPARS. and CONSENSE in PHYLIP 3.5 and rooted
by reference to E. coli”. The numbers under the nodes are the booutrap
conﬁdence estimates on the branches in 100 replicates. All other 165

rRNA gene sequences were obtained from the Ribosomal Database
Project (26).

 

same substrate under both aerobic and anaerobic conditions.
The phylogenetic heritage of the aerobic pathway at least is not
necessarily expected because the aerobic toluene pathway has
often been found on transmissible plasmids. The aerobic
substrate range. however. is far more limited than that found
for the well-studied aerobes. suggesting that the Azoarcus
aerobic pathway(s) has unique features.

Hybridization at high stringency of the subunit probe for the
toluene ortho-hydroxylase pathway to DNA from ﬁve of the
Azoarcus isolates suggests the presence of this gene in these
strains and hence that the ortho-hydroxylase pathway may be
responsible for aerobic toluene metabolism. Two of the strains
may also have the meta-hydroxylase pathway since they also
hybridized to this probe at different positions on the Southern
blot. Having three different toluene pathways. two aerobic and
at least one anaerobic. in one strain is perhaps unexpected.
Two of the strains had detectable plasmids. but the aerobic
toluene pathway probes did not hybridize to the plasmids.

This study indicates that denitrifying toluene degraders are
widely distributed in nature: and thus if nitrate were present or
added. toluene should be removed. This conclusion is based on
ﬁnding anaerobic toluene removal in such diverse and widely
distributed environments as a pristine rain forest in Cameroon,
industrial sites in two states of Brazil. a forest preserve in the
young geographically isolated island of Hawaii. a marine beach
in California. a deep sandy aquifer in Michigan. 3 wood pulp
treatment lagoon in Ontario. and a compost pile in Michigan.
The only different environment that did not yield an enrich-
ment was permafrost soils collected from a region adjacent to
the Arctic Ocean in eastern Siberia. Many samples did not
yield enrichments. however. This may be due to the difﬁculty of
successfully enriching these organisms or to the fact that such
organisms may not be present in every gram of nature. The
poor enrichment record for the undescribed contaminated
samples is likely due to general toxicity from the chemical

DENITRIFYING TOLUENE DEGRADERS 2809

contaminants since chemical odors were apparent in these
samples.

ACKNOWLEDGMENTS

We thank Helen Garchow for performing the FAME analysis and
Frans de Bruijn for the use of facilities for the nitrogen fixation assay.

This project was supported by NIEHS Superfund Research Pro-
gram. grant no. E5049“. and the facilities were provided to the
Center for Microbial Ecology by NSF grant no. BIR9120006. M.R.F.
acknowledges Fellowship support from CAPES. Brazil.

REFERENCES

l. Bergman. F. J. 1980. Measurement of nitrogen ﬁxation by direct
means. p. 65-110. In F. J. Bergersen (ed.). Methods for evaluating
biological nitrogen ﬁxation. John Wiley 6: Sons. Chichester.
United Kingdom.

7. Byrne. A. M.. J. J. Kukor. and R. ll. Olsen. 1993. Nucleotide
sequence analysis of the tbu operon encoding the toluene-3-
monooxygenase from Pseudomonas pickettii PK01. abstr. K31. p.
274. Abstr. 93th Gen. Meet. Am. Soc. Microbiol. 1993. American
Society for Microbiology. Washington. DC.

3. Choc-Sanford. J.. M. R. Fries. and J. M. Tiedk. 1992. Anaerobic
degradation of toluene under denitrifying conditions in bacterial
isolate Tol-4. abstr. 0-233. p. 374. Abstr. 92th Geri. Meet. Am.
Soc. Microbiol. 1992. American Society for Microbiology. Wash-
ington. DC.

4. Dean. 8. J. 1985. Recents ﬁndings on the genetic toxicology of
benzene. toluene. xylenes and phenols. Mutat. Res. 154:153-181.

5. dc Bruijn. I". J. 1992. Use of repetitive (repetitive extragenic
palindromic and enterobacterial repetitive intergeneric consensus)
sequences and the polymerase chain reaction to ﬁngerprint the
genomes of Rhizobium meldou‘ isolates and other soil bacteria.
Appl. Environ. Microbiol. 58:2180-2187.

6. Demennx. J- P. ﬂneberli. and 0. Smithles. 1984. A comprehen-
sive set of sequence analysis programs for the VAX. Nucleic Acids
Res. 12:387-395.

7. Doberelner. J.. and J. M. Day. 1976. Associative symbiosis in
tropical grasses: characterization of microorganisms and dinotro-
gen ﬁxing sites. p. 518—538. In E. W. Newton. and C. J. Nyman
(ed.). Proceedings of the lst International Symposium on Nitrogen
Fixation. Washington State University Press. Pullman. Wash.

8. Dolfing. J., J. Zeyer. P. Binder-Belicia and R. P. Sebwnnenbneb.
1990. Isolation and characterization of a bacterium that mineral-
izes toluene in the absence of molecular oxygen. Arch. Microbiol.
154536—341.

9. Edwards. E. A. L. E. Wills. M. Reinhard. and D. Grbié-Galié.
1992. Anaerobic degradation of toluene and xylene by aquifer
microorganisms under sulfate-reducing conditions. Appl. Environ.
Microbiol. 58:794—800.

10. Elmerich. C.. B. L Dreyfu. and J. P. Anbert. 1982. Genetic
analyses of nitrogen ﬁxation in a tropical fast-growing Rhizobium.
EMBO J. 1:499—503.

11. EvansP.J..W.Ling.B.Goldsehntldt.E.R.thter.nndLY.
Young. 1992. Metabolites formed during the anaerobic transfor-
mation of toluene and o-xylene and their proposed relationship to
the initial steps of toluene mineralization. Appl. Environ. Micro-
biol. 58:496-501.

12. Evans. P. J.. D. T. Mung. K. S. Kim. and I. Y. Yonng.1991.
Anaerobic degradation of toluene by a denitrifying bacterium.
Appl. Environ. Microbiol. 57:1139—1145.

13. Felaenstein. J. 1989. PHYLIP—phylogeny inference package (ver-
sion 3.2). Cladistics 5:164-166.

14. Fishbein. L 1985. An overview of environmental and toxicological
aspects of aromatic hydrocarbons. ll. Toluene. Sci. Total Environ.
42:267-288.

15. Gibson. D. T- and V. Subramanian. 1984. Microbial degradation
of aromatic hydrocarbons. p. 181-252. In D. T. Gibson (ed.),
Microbial degradation of organic compounds. Marcel Dekker.
Inc. New York.

16. GrbieoGallé. D.. and 'I'. M. Vogel. 1987. Transformation of toluene
and benzene by mixed methanogenic cultures. Appl. Environ.
Microbiol. 53:254-260.

2810

I7.

18.

24.

FRIES ET AL.

Haigler. B. E. C. A. Peﬂigrw. and J. C. Spain. 1992. Biodegra-
dation of mixtures of substituted benzenes by Pseudomonas sp.
strain JSISO. Appl. Envrron. Microbial. 582237-2244.
Hanyaina. S.. M. Rekili'. M. Wtibbolts. K. Rose. R. A. Leppllt. and
K. N. Timmis. Characterization of ﬁve genes in the upper-pathway
operon of TOL plasmid pWWO from Pseudomonas putida and
identiﬁcation of the gene products. J. Bacteriol. 171:50-18—5055.

. Holben. W. LJ. KJansson. B. KdeIILandJ. M. Tiedje. 1988.

DNA probe method for the detection of speciﬁc microorganisms
in the soil bacterial community. Appl. Environ. Microbiol. 54:703-
71 l.

. ﬂurek. T- S. Barman C. R. Woese. and B. Reinhold-Honk. 1993.

168 rRNA-targeted polymerase chain reaction and oligonucleo-
tide hybridization to screen for Azoarcus spp.. grass-assomated
dtazotrophs. Appl. Environ. Microbiol. 59:38l6-3824.

. Hutchins. S. IL. G. W. Sewell. D.A. Kovaes. and G. A. Smitb.1991.

Biodegradation of aromatic hydrocarbons by aquifer microorgan-
isms under denitnfying conditions. Envrran. Set. Technol. 25:68-
70.

.... Johnson. G. R.. and R. H. Olsen. 1993. Characrerization of clones

from Pseudomonas sp. JSISO encoding substituted phenol and
benzene metabolic pathways analogous to Pseudomonas strains
PK01 and G4. abstr. K-SZ. p. 274. Abstr. 93th Gen. Meet. Am.
Soc. Microbiol. 1993. American Society for Microbiology. Wash-
ington. D.C.

. Karla. C. L. and S.-‘I'. Do. 1981. Rapid procedure for detection

and isolation of large and small plasmids. J. Bacteriol. [45:1365-
1373.

Krieg. N. R. 1981. Enrichment and isolation. p. 112-142. In P.
Gerhardt. R. G. E. Murray. R. N. Costilow. E. W. Nester. W. A.
Wood. N. R. Krieg. and G. B. Phillips (ed.). Manual of methods
for general bacteriology. American Society for Microbiology.
Washington. DC.

. Kukor. J. J., and R. B. Olsen. 1990. Diversity of toluene degra-

dation following long term exposure to BTEX in situ. p. 405-421.
In D. Kamely. A. Chakrabarty. and G. S. Ornenn (ed.). Biatech-
nology and biodegradation. Gulf Publishing Co.. Houston. Tex.

.thqG.J.Olsen.B.LMaidaLM.J.McCamiR.

OverbeehTJ. Mocke.T.LMarsb.andC.R.Woese. 1993. The
Ribosomal Database Project. Nucleic Acids Res. 21(Suppl.):3021-
3023.

.Lovley,D.R..M.J.Bnedecker.D.J.lonetgnn.l.M.CunlellL

E. J. P. Phillips. and D. 1. Seal. 1989. Oxidation of aromatic
contaminants coupled to microbial iron reduction. Nature (Lon—
don) 339:297—300.

. Lovley. D. R. and D. J. Inner-gas. 1990. Anaerobic oxidation of 5

toluene. phenol. and p-cresol by the dissimilatory iron-reducing
organism. GS-IS. Appl. Environ. Microbiol. 56:1858—1864.

.. . Maniatis. T. E. F. Fritsch. and J. Sambrook. 1982. Molecular

30.

cloning: a laboratory manual. Cold Spring Harbor Laboratory.
Cold Spring Harbor. NY.

Olsen. R. H., J. J. Kukor. and B. J. Kaphammer. 1994. A novel
toluene-B-monooxygenase pathway cloned from Pseudomonas
pickettii PK01. J. Bacteriol. 176:3749-3756.

170

31.

35.

37.

39.

41.

APPL. ENVIRON. MICROBIOL.

Owens. J. D.. and R. M. Keddie. 1969. The nitrogen nutrition of
soil and herbage coryneform baCteria. J. Appl. Bacterial. 32:335—
347.

. Rabus. R.- R. NordbaIIS. W. LndWEg. and F. Widdel. 1993. Cam-

plete oxidation of toluene under strictly anoxic conditions by a new
sulfate-reducing bacterium. Appl. Envrron. Microbial. 59:1444—
1451.

. A. W. laidwig. and K.-H. Schleifer. 1988. DNA
probes with diﬂerent speciﬁcities from a cloned 23$ rRNA gene of
Micrococus (tum. J. Gen. Microbiol. [34:1197—1204.

. Reinhold-Hunk. B. T. Hunk. M. Gillis. B. Haste. M. Vancanneyt.

K. Reuters. and J. De Ley. I993. Azoarcus gen. nov.. nitrogen.
ﬁxing Proreobactena associated with roots of Kallar grass (Lepro-
chloa fusca (L) Kunth). and description of two species. Azoarcus
indigens sp. nov. and Azoarcus carnmunis sp. nov. Int. J. Syst.
Bacteriol. 43:574—584.

Sasser. M. 1990. Technical note 101: identiﬁcation of bacteria by
gas chromatography of cellular fatty acids. MIDI. Inc. North
Newark. Del.

.Seboeber,R.J..B.Seyfried.F.Vaaquez.andJ.Zeyer.l991.

Anaerobic degradation of toluene by pure cultures of denitrifying
bacteria. Arch. Microbiol. 157:7-12.

Snilbert. R. M.. and N. R. Krieg. 1981. General characterization. p.
409-443. In P. Gerhardt. R. G. E. Murray. R. N. Costilow. E. W.
Nester. W. A Wood. N. R. Krieg. and G. B. Phillips (ed.). Manual
of methods for general bacteriology. American Society for Micro-
biology. Washington. DC.

. Smith. G. B., and J. M. Tiedje. 1992. Isolation and characterization

of nitrite reductase gene and its use as a probe for denitrifying
bacteria. Appl. Environ. Microbiol. 58:376—384.

Viebroclt. A.. and W. G. Zumft. 1988. Molecular cloning. heterol-
ogous expression. and primary structure of the strucrural gene for
the copper enzyme nitrous oxide reductase from denitrifying
Pseudomonas sauzeri. J. Bacteriol. 170:4658—4668.

. Wilson. B. H., G. B. Smith. and J. F. Rees. 1986. Biotransforma-

tion of selected alkylbenzenes and halogenated aliphatic hydro-
carbons in methanogenic aquifer material: a microcosm study.
Environ. Sci. Technol. 20:997—1002.

Ye. R. W. M. R. Fries. S. G. Bezborodnikov. B. A. Averill. and
J. M. Tiedje. 1993. Characterization of the structural gene for a
copper containing nitrite reductase and its homology to other
denitrifiers. Appl. Environ. Microbiol. 59:250—254.

. Yell. K.-M.. M. R. Karl. L M. Blatt, M. J. Simon. R. 8. Winter.

P. R. Fausset. H. S. LII. A. A. Harcourt. and K. K. Chen. 1991.
Cloning and characterization of a Pseudomonas mendoema KR1
gene cluster encoding toluene-4-monooxygenase. J. Bacteriol.
173:5315—5327.

.ZbomJ.-LM.R.Fries.J.C.Chee—Sanford.andJ.M.Tiedje.

Unpublished data.

. Zylstra. G. J., and D. 1'. Gibson. 1989. Toluene degradation by

Pseudomonas putida F1: nucleotide sequence of the todCICZ
BADE genes and their expression in Escherichia coli. J. Biol.
Chem. 264:14940—14946.

nRIEs

nxWMMMMWWHj/lﬁﬂ' W

O