EPA/600/A-94/198
STUDIES ON THE MICROBIAL
ECOLOGY OF POLYCYCLIC
AROMATIC HYDROCARBON
BIODEGRADATION
/. C. Mueller, S. E. Lantz, R. Devereux,
). D. Berg, and P. H. Pritchard
ABSTRACT
Soils with known history of exposure to polycyclic aromatic hydrocarbons
(PAHs) were collected from Norway, Germany, and the United Slates
and screened for the presence of PAH-degrading bacteria. Purified PAH-
degrading isolates were characterized by fatty acid profile analysis
(GC-FAME),substrate utilization patterns (Biolog™ assays), 16S rRNA
sequence comparisons, and total DNArDNA hybridizations. Microbial
respirometry and chemical analyses also were performed to define the
PAH-biodegradation potential of these soils. These studies showed that
all soils contaminated with PAI Is harbored competent PAH-degrading
bacteria that are biochemically similar and phylogenetically related.
I lowever, bioremediation strategies relying exclusively on indigenous
PAI 1 degraders should be closely evaluated for the ability to achieve
site-specific cleanup standards in a timely manner.
INTRODUCTION
The use of specially selected microorganisms to enhance bioremediation efforts
has proved effective in a number of applications, especially when combined with
bioreactor systems (Mueller ct al. 1993, Pritchard 1992). In our studies, the success-
ful use of such isolates to remediate soil and water contaminated with organic
wood preservatives (e.g., creosote, and pentachlorophenol [PCI'D has resulted
in the opportunity to employ these technologies at similarly contaminated sites
throughout the world.
Prior to distribution of these bioremediation strategies, concerns regarding the
import of nonindigenous microorganisms needed to be addressed. Toward litis
end, we embarked on a program to ascertain whether (1) microorganisms similar
to those used in our bioremediation strategy could be found in soils far removed
from each other geographically; (2) previous exposure of soil microorganisms to
PAH mixtures affected their PAH-degrading abilities; and (3) introduction of
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Muslter et al, 219
socially selected inoculant strains would offer any advantages, in terms of oper-
ating fx-rfoi mance, to bioremediation systems employing indigenous mici obiota.
MATERIALS AND METHODS
Acquisition of Soil Samples
Eight samples of soil with a history of exposure to PAII mixtures, such as
creosote or diesel fuel, were recovered from a variety of locations. Six of these
samples were obtained from creosote-contaminated sites in Norway (long-term
exposure), one was sent from a diesel-contaminaled site in Germany (recent spill),
and one was recovered from an abandoned wood-preserving facility (American
Creosote Works [ ACW|) in northwest Florida, USA (long-term exposure). Addi-
tionally, two soils with no known history of exposure to such chemicals were
recovered from agricultural farmland in south-central Illinois, USA.
Soil Analyses
Soil texture, moisture, nutritional status (NH«-N, NO,-N, available phos-
phorus, total phosphorus), water-holding capacity, and pH were determined in
accordance with standard methods for soil analyses (Pageetal. 1982). Analytical
methods for extraction and quantitative determination of pcntachlorophcnol and
41 creosote constituents by gas chromatography (CO are described elsewhere
(Mueller et al. 1989, 1991).
For microbiological analysis, triplicate 1.0-g samples (wet weight) were
placed in 9.0-mL volumes of sterile phosphate buffer (25 mM KH2P04, 25 mM
K2HP04, pi 1 7.1) and shaken vigorously for 15 min (350 rpin). Soil suspensions
were allowed to settle for 1 min before serial dilution in the same buffer. Total
heterotrophic plate counts were performed with each soil sample using a standard
heterotrophic plate count medium (Luria-Bertani agar; Maniatis et al. 1982) and
standard microbiological methods (duplicate samples plated in replicate) (Page
etal. 1982). Phenanthrene (PI Hi)- and fluoranlhene (FLA)~degrading bacteria were
enumerated using an overlay technique (Kiyohara et al. 1981). These values were
recorded as the number of colony-forming units (CFUs) on carbon-substrate-free
mineral salts agar that cleared the hydrocarbon substrate after 12 to 14 days of
incubation at 2S°C.
Soil Enrichment with PAHs
Soils were enriched for PAH-degrading microorganisms by adding 5.0 mL
of a 10% soil slurry (prepared in sterile mineral salts medium) to 250-mL Erlen-
tncyer flasks containing 45 mL of sterile mineral salts medium (Mueller et al.
1990). Either PHE or FLA previously had been added to each flask in sterile
acetone (evaporated) for a PAI I concentration of 500 mg/L. Soils were incubated
in the dark with shaking (150 rpm) at 30°C for 14 days. Following 14 days of
-------
220 Binreniedmlimi of Chlorinated and PAH Compounds
aerobic incubation and enrichment, cultures were diluted 1:5 (vol/vol) with fresh
mineral salts medium including PANsat 500mg/L. This transfer procedure was
repeated two more times for a total of four enrichments over a 10-week period.
Screening Enrichment Cultures for PAH Degraders
After the second and fourth enrichments, liquid samples were removed from
each vessel and screened for the presence of bacteria capable of using PI IR and
FLA as primary growth substrates. Once iiuli vidual colonies were single-colony
purified, they were transferred to utilizablo-carbon-free agar and complex agar,
then overlain with PHE or FLA as described by Kiyohara et al. (1981). Plates
were incubated for 14 days prior to scoring for individual colonies exhibiting
zones of clearing of the PAN substrate.
Colonies demonstrating PAI 1-clearing abilities were purified and transferred
to 125-mL F.rlenmeyer flasks containing 25 mL mineral silts broth plus 500 mg/L
1*1 IF; or FLA as the sole carbon and energy source. Cultures were incubated for
5 to 7 days with shaking (150 rpm) at 30°C. Bacterial growth at the expense of
the PAH substrates was measured by visual assessment of turbidity and by moni-
toring changes in absorbance at 550 nm. As a control, growth in carbon-free
mineral salts broth also was monitored.
Microbiological Characterizations
Once the PAH-degrading ability of purified cultures had been validated,
cultures were characterized by CC-FAMEand the Biolog™ Microplate System™
(Microbe Inotech Laboratories, Inc., St. Louis, Missouri). The taxonomic relation-
ships among these strains was analyzed by evaluating similarity measures from
GC-FAME and substrate utilization patterns with principal component analysis
(Jacobs 1990).
Phylogenetic relationships were determined by 16S ribosomal RNA sequence
comparisons. Universal primers and the polymerase chain reaction were used
to amplify 16S rRNA genes (Weisburg et al. 1991) from select PAH-degrading
bacterial strains CRE7, CRE11, CRE12, Psmdomonas paucimobilis strain EPA505,
and the type strain of Pseudomotws paucimobilis ATCC 29837. Reverse transcriptase
sequencing with the rRNA template as the primer was used to generate 16S rRNA
sequence information for select PAH-degrading strains (Lane et al. 1985). Total
DNA:DNA hybridizations were performed between select PAI I-degraders to
define homology (Amann et al. 1992).
Manometric Respirometry Studies
Into 14 flasks (250-mL Erlenmeyer flasks), each containing 10 mL of a 33%
soil slurry, was added a predetermined level of inorganic nutrients. Duplicate
flasks of each slurry were amended with 500 mg/L naphthalene (NAI I), PI-IE,
or FLA; readily utilizable carbon (250 mg/L glucose + 250 mg/L glycerol); or
5(X) mg/L specification creosote no. 450 (American Wood-Preserver's Association).
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Mueller et ai
221
Two flasks received no supplemental carbon (to discern the effect of nutrient
amendment and aeration), and two more flasks served as killed cell controls
(acidified to pi 1 2.0 with 1 N MCI plus 3.7% formaldehyde) for each soil tested.
Microbial respirometi ic responses (rate of liberation of CO? and the simul-
taneous consumption of 02 were determined at 8-hr intervals over an 8-day
incubation period (23°C, 100 rpm shaker speed) with a MicroOxyrnax respirometer
(Columbus Instruments, Columbus, Ohio). At the end of each incubation period,
slurries from nutrient-amended only, creosote-amended, and killed-cell (control)
treatments were extracted and analyzed for the presence of creosote constituents
as previously described (Mueller et al. 1991). These values were compared with
those determined at lime zero for each soil.
RESULTS AND DISCUSSION
Soil Analyses
The results of the physicochemical analyses used to characterize the soils
are summarized in Table 1, In general, all measured soil parameters were within
a range conducive to biological activity. However, contaminated soils had rather
low levels of available nitrogen and, to a certain extent, available phosphorus
(Bray PI).
Microbiological analysis of soils prior to enrichment with PAHs showed that
all soils harbored culturable heterotrophic bacteria (Table 2). With the exception
of the ACW 47 site, all soils with a history of exposure to PA Us also had a discern-
ible number of PHE-degraders, and, with the exception of soils UN 1 and ACW 47,
a relatively high number of FLA-degraders. Conversely, the "control soils"
(SIU ARC, SIU BRC), with no known history of exposure to PAIls, hail no
detectable I'l Hi- or FLA-degraders.
The presence of PCP at a relatively high concentration (>250 nig PCI'/kg soil
dry wt) in soil collected from the abandoned American Creosote Works site
(sample ACW 47), in combination with creosote at an average concentration
>500 mg creosote PAHs/kg soil dry wt), may represent the reason for the low
initial numbers of PIIE- and FLA-degraders (i.e., toxicity). In the case of the UN 1
soil sample, the only soil being recently impacted by diescl fuel (accidental
highway spill) and not long-term creosote exposure (<1 month exposure at the
lime of sampling), the high number of PHE-degraders along with a rather low
number of FLA-degraders may be the result of low-level exposure to PHE
(e.g., diesel vapors) or adaptation to structurally related compounds (Bauer &
Gipone 1988).
PAIl Enrichment Studies
Using PHE as a growth substrate, all soils except SIU ARC and SIU BRC pro-
duced turbid cultures within the first week of the first enrichment. All subsequent
transfers produced turbid cultures within the first 2 to 3 days of incubation.-
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TABLE 1. Description and characterization of soils used for the isolation of PAH-degraders.
Origin/Location
of Soil
Exposure
History
Classification
Nutrient Analysis (mg/kg soil dry wt)
NH4-N NOj-N Bray PI Bray P2
pH
Field
Capacity
(% wgt)
Norwegian Soils
-
Rade 1
Creosote
Loamy sand
2
10
0
28
6.4
7.1
Rade 2
Creosote
Sand
4
6
6
13
5.9
7.4
Lillestram 1
Creosote
Sand
10
6
10
19
6.2
16.0
Lillestram 2
Creosote
Loam
3
5
16
28
7.2
37.0
Drammen
Creosote
Sand
1
6
12
25
7.4
8.1
Hommelvik
Creosote
Loamy sand
9
31
3
23
7.2
10.7
German Soil
UN 1
Mixed PAHs
Sand
1
0
82
87
7.4
23.9
American Soils
ACW47
Creosote /PCP
Sand
3
1
20
20
6.7
14.9
SIU ARC
None known
Silt loam
20
13
34
47
6.8
29.2
SIU BRC
None known
Silt loam
0
9
35
48
6.4
20.3
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Mueller el at.
223
TABLE 2. Enumeration of total aerobic, culturable heterotrophic, phenanlhrene-,
and fluoranlhene-degrading bacteria in soils.
Total
Phenanlhrene
Fluoranthene
1 Ieterotrophs!M
Degraders'''
Degraders'"
Soil'"
log Cl'U/mL slurry"1'
Rade 1
6.47
4.87
5.82
Rade 2
6.00
4.44
5.85
Lillestrom 1
7.57
3.93
5.26
Lillestrom 2
7.63
3.65
3.74
Drammen
6.18
4.13
5.16
Hommelvik
6.98
4.04
3.85
UN 1
6.43
5.49
<3.00
ACVV 47
6.60
<3.00
<3.00
SIU ARC
7.00
<2.00
<2.00
SIU ORC
7.31
<2,00
<2.00
(a) See Tabic 1 for description of soils.
(b) Tofal helerotrophs cm I.uria-Bertani agar after 5 days incubation at 28°C.
(r) f'AI t-degraders based on the number of colonies to clear PAH substrates on minimal
medium after 14 days incubation af 30°C.
(d) CPU = colony forming units.
Simultaneously, all undissolved PI Us crystals were removed, and rapid changes
in medium coloration were observed.
Using FLA as an enriching substrate, the observed growth responses were
very similar lo those recorded in the presence of PI IE. I lore, fluoranthene biodeg-
radalion was apparent within the first 10 days of enrichment for all soils except
SIU ARC, SIU BRC, UN 1, and Hommefvik. Growth (as determined by visually
apparent increases in turbidity, change in medium coloration, and disappearance
of undissolved FLA crystals) with inocula from Momrnel vik and UN 1 soil became
evident within 21 days of enrichment. Following 40 days of incubation, biodegra-
dation or solubilization of PI IE and FLA in liquid medium inoculated with micro-
organisms recovered from the SIU ARC and SIU BRC soils was not observed.
The enrichment culture conditions used in these studies (e.g., excess inorganic
nutrients, elevated temperatures, mixing, and aqueous solutions saturated with
PAHs) are substantially different than expected environmental conditions.
However, they closely resemble conditions associated with btoreactor operations
that are widely used in the bioremediation industry (Berg et al., this volume;
Mueller et al. 1993). Thus, while the relatively low number of PAI1 degraders
from unexposed sites may not fully reflect the potential for long-term adaptation,
the fact that non-PAl l-history soils did not readily yield PI IE or FLA degraders
suggests that, in the event of recent contamination, bioremediation of PAHs on
a short-term basis may require the use of inoculants.
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224
HioremeJmlkm nf Chlorinated and PAIi GmipmftiJj
Respirometric Analyses
Hie respiratory activity of indigenous microflora from all soils with a worded
history of exjiosure to I'AlIs was stimulated upon the addition of nil organic carlum
sources, as well as with the addition of inorganic nutrients alone. Using soil from
the f lommclvik site as a typical example, increased respiratory responses were
observed upon the addition of individual PA1 ls(NAH, PI IE, or FLA) and creosote
(Figure 1, top and middle panels). These responses were shown to be above and
beyond that expected from the conversion of resident carbon, which includes creo-
sote PA I Is, tine to the addition of inorganic nutrients and aeration. I lence, these
chemicals represented ulilizable carbon sources to the indigenous microdot,!. Con-
versely, both soils with no known exposure to PAI ls(SIU soils)showed very limited
response to the addition of PAI Is or inorganic nutrients, but the indigenous soil
microflora rapidly mineralized added glucose and glycerol (Figure 1, bottom panel).
In all cases, no activity was observed in the poisoned systems. Compared with
analytical chemistry data, increases in respiratory activities generally were associ-
ated with accelerated biodegradalion of monitored creosote constituents (data not
shown).
Microbial Ecology and Bacterial Taxonomy
All soils with a history of PAI 1 exposure, except ACVV 47, harbored PI IF- and
FLA-degrading bacteria of various genera. Of the many isolates recovered, the
degradative abilities of 13 PI IF-degraders, 14 FLA-degraders, and 1 pentachloro-
phenol degrader (Resnick & Chapman 1990), isolated from myriad contaminated
sites, were positively verified (Table 3, data not shown). Iliese strains were
processed for characterization and identification based on GC-FAMFand BiologIM
assays ( Table 4).
Because the established databases for both GC-FAME and Biolog™ assays
arc focused predominantly on the identification of pathogenic microorganisms
and those of clinical importance, many of the identifications made have low
similarity coefficients (Table 4). Hence, most of the identifications were considered
to be suggestive rather than conclusive. Principal component analyses using
GC-FAME profiles of each of the PAH-degrading bacteria showed that many
microorganisms isolated from US. soils were closely related to microorganisms
recovered from other soils in the United States and Europe (Figure 2).
For example, strain CRE7 (PHE-degrader isolated from the ACVV site at
Pensacoja, Florida, and used commercially in a bioremediation process) was
found to be closely related to PI IE-degrading strains N2P5 and N2P6(Radesoil,
Norway); strains N3P2 and N3P3 (Lillestrom soil, Norway); and strains PJC2288,
2289, and 2295 (PAH degraders also from the Pensacola site). Likewise, FLA-
degrading bacteria very similar to strain EPA505 were recovered from Germany
(strains G IF1 and G1F2) and geographically separate sites in the United States
(strains PJC 2286,2287). Gram-positive "mycobacterial" strains PJC 2282, PJC 2283,
and FDA PYR-1 were found to be very closely related to each other, but, as
expected, distinctly different from Gram-negative bacterial isolates.
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Mueller et al.
225
?oo -
soluble nufnenis
ofucose/qlycerol
phpnanlhreno
?00 -
400
0
1 6
32
S
1 t 2 120
80
4
1 44
.
O —
S o
w «
3
v a
o
a v>
° ^
O *
400
700 -
o
a o
P «
¦200-
32
64
80
96
400
200 -
#¦#
-200 -
-400
0
40
96
112 128 144
1 6
32
64
80
Incubation Time (Hr»)
IlGURtI1. Res pirn lory response of indigenous microorganisms to organic and
inorganic amendments. Respiratory activity of soil microorganisms present
in Homtnelvik soil contaminated with creosote (top and middle panels),
and SIU ARC farmland soil with no known history of exposure to PAI Is
(bottom panel).
Phylogenctic studies using 16S rRNA sequence comparisons showed that two
PAH-degrading strains, isolated from creosote-contaminated sites in the USA
(strains CRE7 and CRE11), were related to Psewivmoitas aeruginosa. The 16S rRN A
sequences between the PAH-degradcrsand P. aeruginosa were 92 to 93% similar.
Correlations developed between 16S rRNA sequence similarity and %DN A relaled-
noss (Amann et al. 1992, Devereux et al. 1990) suggest that, at 92% 16S rRNA
-------
1
2
3
4
3
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Bacteria isolated from PAH-contaminated sites for their ability to degrade phenanthrene or fluoranthene.
Culture Number
Source/ Relerence
Enrichment Substrate
Soil of Origin
EPA505
Mueller et al. 1990
Fluoranthene
Pensacola, Honda
PJC 2282
U.S, EPA, GBERL
Fluoranthene/pyrene
Pensacola, Florida
PJC 2283
U.S. EPA, GBERL
Fluoranthene/pyrene
Live Oaks, Honda
PJC 2285
US. EPA, GBERL
Fluoranthene
live Oaks site, Horida
PJC 2286
U.S. EPA, GBERL
Fluoranthene
Live Oaks site, Horida
PJC 2287
U.S. EPA, GBERL
Fluoranthene
Pensacola, Horida
PJC 2288
U.S. EPA, GBERL
Phenanthrene
Pensacola, Horida
PJC 2289
U.S. EPA, GBERL
Phenanthrene
Live Oaks, Horida
PJC 2295
U.S. EPA, GBERL
Phenanthrene
Pensacola, Horida
CRE7
Mueller et al. 1989
Phenanthrene
Pensacola, Horida
CRE11
Mueller et al. 1989
Phenanthrene
Pensacola, Honda
CRE12
Mueller et al. 1989
Phenanthrene
Pensacola, Horida
AK Phen6
Mueller et al. 1992
Phenanthrene
Prince William Sound, Alaska
NIFt
This study
Fluoranthene
Rade, Norway
N2P5
This study
Phenanthrene
Rade, Norway
N2P6
This study
Phenanthrene
Rade, Norway
N3P2
This study
Phenanthrene
Lillestrom, Norway
N3P3
This study
Phenanthrene
Lillestrom, Norway
N3F1
This study
Fluoranthene
Lillestrom, Norway
N3F2
This study
Fluoranthene
Lillestrom, Norway
N4F4
This study
Fluoranthene
Lillestrom, Norway
N5F4
This study
Fluoranthene
Drammen, Norway
N6F4
This study
Fluoranthene
Hommelvik, Norway
Gin
This study
Fluoranthene
Germany
C1F2
This study
Fluoranthene
Germany
G1P1
This study
Phenanthrene
Germany
G2P2
This study
Phenanthrene
Germany
FDA PYK-1
Heitkamp & Cerniglia 1988
Pyrene
Port Aransas, Texas
SR3
Resnick & Champman 1990
Pemachlorophenol
northwest Horida
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Mueller el 2)
I'JC 2289 Pffutionutnns pseudtmiallei (0.177)
I'JC 2295 Pfcuihmumns ce/wicm (0.059)
CRH7 I'seudnnmms ce/mcia (0.399)
CRF.lt Pscmitmitmas acrstghuKtt (0.778!
CKH12 nrrwg»mw (9.366)
AK PI IKN6 No ID ft'scudominuis snccharijihiln (NA)
N1F1 Xanlhamtmas malhiphilia (0.35)
N21'S i'^euihrnumm rqwrw (0.294)
N2I'6 Pfeiiilommis ce/ncii (0.114)
NJH Psciidtmumas ce/ncia (0.379)
Mira Psettilommuis aciitihi!a (0.11)
GII'2 Ai ineltthtcler oilnmcrtku-s (0.4(1)
FDA PYR-1 Mycolmcterium /moforluitum (0.059)
SR3 Pscudtmutnaf Mcrlwrtithila (0.603)
No ID [insufficient growth 36 h|
Ci'.p'<(("; 13 (0,65)
Cinyiielxicterium imrmbilix (0.59)
No ID Ipoor growthl
(a) Closest speaes identification listed in brackets [ I when no identification is made. Underscore
indicates a match to the clinical database of Microbe Inotcch Laboratories, St. Louis, MO.
sequence similarity and ca 50% DNA relatedness, the PAH-degrading strains
are related to P. aeruginosa M the gcnus/s|xx'ics level where greater than 20% DNA
relatedness indicates a genus-level relationship.
Likewise, results of DNA:DNA hybridization studies showed that strains
isolated from Norwegian and U.S. sitesalso were somewhat related at the genus
level. For example, the U.S. isolate strain CRE7 demonstrated 56% and 36%
DNA:DNA homology with the Norwegian isolate N2P5 and the German isolate
G11'2, respectively, all l>eing isolated for their ability to utilize PI IE as a sole carbon
source. These relationships follow theGC-FAME principal component analyses
presented in Figure 2. Further, the U.S. strain EPA505 demonstrated 24 and 35%
DN A:DN A homology with the ATCC type strain of Pscudomonas )nucitnobilis and
the Norwegian strain N2P5, respectively.
-------
228
Rioremediatum of Chlorinated mid PA! I ComfminJt
2288, 2289,
2295
N2P6, CRE7,
N2P5
40 _
N3P2
30
N3FI
CRE12
20 _
GIP2
CRE I
N4F4
N3F2
FDA PYR-!
2282, 2283
228 7
AK phen6, 2286
GIF2 EPA505
2285
-20
-40
-20
20
0
40
Principal Component 2
FIGURE 2. Principal component analysis with GC-FAME data from PAH-
degrading bacteria isolated from geographically distant PAH-contaminated
sites.
CONCLUSIONS
All soils with a history of PAH exposure yielded microbial populations compe-
tent for the degradation of thePAHsphenanthreneand fluoranthene. According
to GC-FAME, Biolog™, 16S rRNA sequence similarity, and DNA:DNA homology,
many of these PAH-degrading microorganisms appeared to be closely related
phenotypicaffy and phylogenetically to similar typesof organisms isolated from
soils at geographically distant sites in the United States. The technique employed
to enrich PAH-degrading bacteria thus appeared to select similar types of micro-
organisms that are indigenous to contaminated soils at each site. Admittedly,
this does not necessarily include all types of microorganisms that may play a
role in the biodegradation/bioremcdiation of PAHs (e.g., fungi).
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Mueller el at.
229
Given thai PA I I-degraders appeared to be indigenous to geographically
diverse PAI (-contaminated soils, then if thestinuitalory effect of controlled milri-
alion, mixing, and aeration on the activity of the indigenous microflora results
in acceptable rates anil extents of biodegradation of targeted chemicals, then, on
a silo-specific basis, it may be (x)ssible to rely solely on the activity of such micro
organisms to facilitate site remediation (see Berg el al., this volume). Despite the
phylogenetic similarities among these organisms, however, their catabulic abilities
would seem to be the most important consideration from a bioremedialion perspec-
tive. Thus, in theevent that indigenous microorganisms do not perform favorably,
then utilization of nonindigenous microbes in optimized bioremedialion systems
could be advantageous for cost-efficient, effective biorcmediation. Based on the
resu Its of these* studies, theexport/importof the nonindigenous bacteria used in
these studies to augment bioremedialion efforts would not seem to represent the
introduction of exotic biota, and thus would pose no discernible ecological risk.
ACKNOWLEDGMENTS
We thank Myke'lle I Icrlsgaard and Barbara Artlet (Technical Resources, Inc.,
Gulf Breeze, Florida), Sheree Enfinger (U.S. Environmental Protection Agency,
Environmental Research Laboratory, Gulf Breeze, Florida), and Stephanie Willis
(University of New Hampshire) for technical assistance; Brian Klubek (SIU-
Carbondalc, Illinois) for soil analyses; Bruce Hemming (Microbe Inolech
Laboratories, St. Louis, Missouri) for help in the interpretation of GC-FAME and
Biolog™ results; and Peter Chapman (U.S. EPA, ERL, Gulf Breeze, Florida) and
Carl Cerniglia (U.S. Food and Drug Administration, National Center for
Toxicological Research, Jefferson, Arkansas) for donating PAI l-ilegrading strains
for comparative analyses. Soil from Germany was provided by Wolfgang Fabig
(Umwellshulz Nord, Germany).
Financial support for these studies was provided by the Norwegian State
Railway (NSB) and the U.S. EPA (Gulf Breeze). Iliese studies were performed
-------
230 Hioreniedmtiim of Chlorinated and I'AH Compounds
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TECHNICAL REPORT DATA
{Please read Instructions on the reverse before completing)
1. REPORT NO. 2,
EPA/600/A-94/198
3. REC
k> TITLE ANO SUBTITLE
¥ STUDIES ON THE MICROBIAL ECOLOGY OF PAH
BIODEGRADATION
5. REPORT OATE
6. PERFORMING ORGANIZATION CODE
7. AUTHORISI 1 ] 2
J.G. Mueller , S, E. Lantz , R. Devereux ,
J.D, Berg , and P.H, Pritchard
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME ANO ADDRESS
^ S P B Technologies, Inc., Gulf Breeze, FL;
Aquateam, Oslo, Norway
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. ENVIRONMENTAL PROTECTION AGENCY
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
GULF BREEZE, FLORIDA 32561-5299
13. TYPE OF REPORT ANO PERIOD CQV6REO
14. SPONSORING AGENCY CODE
15. SlJPPLPMf NTAHY NOTES
: Bioremediation of Chlorinated and Polycyclic Aromatic Hydmr^r-bo" Conoound-
Robert E, Hinchee, et. al. fnd.1 r r,f>wic PnhlishPr^, Boca Haton, FL. p. 218-230. 1994
16. ABSTRACT
Soils with known history of exposure to polycyclic aromatic
hydrocarbons (PAHs) were collected from Norway, Germany and the
United States and screened for the presence of PAH-deqradinq
bacteria. Purified PAH-degrading isolates were characterized by
fatty acid profile analysis (GC-FAME), substrate utilization
assays), 16S rRNA sequence comparisons, and total
DNA:DNA hybridizations. Microbial respirometry and chemical
analyses were also performed to define the PAH-biodeqradation
potential of these soils. These studies showed that all soils
contaminated with PAHs developed competent PAH-degrading bacteria
hat are biochemically similar and phylogenetically related
However, bioremediation strategies relying exclusively on
indigenous PAH degraders should be closely evaluated for the
ability to achieve site-specific clean up standards in a timelv
manner. 1
17. KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
b. IDENTIFIERS/OPEN ENDEO TERMS
c. COSati Field/Croup
18. DISTRIBUTION STATEMENT
19. SECURITY CLASS (Tha Report)
21. NO. OF PACES
12
20. SECURITY CLASS (This pages
22. PRICE
EPA Form 2220-1 (R«». 4-77) previous coit-on .5 obsolete.
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