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Biodegradation 12: 201–207, 2001.
© 2001 Kluwer Academic Publishers. Printed in the Netherlands. 201
Repeated inoculation as a strategy for the remediation of low
concentrations of phenanthrene in soil
Egbert Schwartz∗& Kate M. Scow
Graduate Group in Ecology and Department of Land, Air and Water Resources, One Shields Ave. University of
California at Davis, Davis, CA 95616-8627, USA (∗author for correspondence: 151 Hilgard Hall, University of
California, Berkeley CA 94720-3110, USA
Accepted 14 February 2001
Key words: bioaugmentation, bioavailability, bioremediation, phenanthrene degradation, sorption
Abstract
Phenanthrene, a polycyclic aromatic hydrocarbon, becomes increasingly unavailable to microorganisms for degrad-
ation as it ages in soil. Consequently, many bioaugmentation efforts to remediate polycyclic aromatic hydrocarbons
in soil have failed. We studied the effect of repeatedly inoculating a soil with a phenanthrene-degrading Arthrobac-
ter sp. on the mineralization kinetics of low concentrations of phenanthrene. After the first inoculation, the initial
mineralization rate of 50 ng/g phenanthrene declined in a biphasic exponentialpattern. By three hundred hours after
inoculation, there was no difference in mineralization rates between the inoculated and uninoculated treatments
even though a large fraction of the phenanthrene had not yet been mineralized. A second and third inoculation sig-
nificantly increased the mineralization rate, suggesting that, though the mineralization rate declined, phenanthrene
remained bioavailable. Restirring the soil, without inoculation, did not produce similar increases in mineralization
rates, suggesting absence of contact between cells and phenanthrene on a larger spatial scale (>mm) is not the cause
of the mineralization decline. Bacteria inoculated into soil 280 hours before the phenanthrene was added could not
maintain phenanthrene degradation activity. We suggest sorption lowered bioavailability of phenanthrene below an
induction threshold concentration for metabolic activity of phenanthrene-degrading bacteria.
Introduction
Polycyclic aromatic hydrocarbons(PAH’s) are suspec-
ted toxins that accumulate in soils and sediments due
to their insolubility in water and lack of volatility (Pig-
natello & Xing 1996; Scow & Johnson 1997). In a
process termed aging, sorbed PAH’s become increas-
ingly resistant to extraction and degradation (Kelsey
et al. 1997; Schwartz & Scow 1999). PAH’s may mi-
grate into rigidly structured forms of organic matter,
soot, or in between the clay bi-layer from which diffu-
sion into the soil solution is extremely slow (Luthy et
al. 1997). Exposure from contaminated ground water
requires that phenanthrene partitions from resistantly
sorbed into bioavailable compartments. Presently, risk
associated with PAH exposure is based upon the total
concentration in soil and not just the bioavailable frac-
tion in soil even though the majority of aged PAH’s are
resistantly sorbed (Kelsey & Alexander 1997).
Remediation of PAH’s in soil is difficult because
only low concentrations of pollutant are bioavailable
despite the presence of extensive contamination in the
sorbed phase (Hughes et al. 1997). It is unclear if aged
PAH’s are permanently sorbed in soil or if they de-
sorb very slowly, thereby replenishing the fraction of
available PAH’s. If the latter is the case, activity of
pollutant degrading-organisms on low available sub-
strate concentrations must persist for extended periods
of time.
In one remediation approach, termed bioaugment-
ation, soil is inoculated with pollutant-degrading or-
ganisms. This strategy often fails, not because the
inoculum is unable to degrade phenanthrene in soil,
but because the pollutants are not available. One ap-
proach in bioaugmentation relies on a single inocula-
202
tion to establish the inoculum in the soil community.
However, successful persistence of bacterial strains
through inoculation into non-sterile soils is rare (Acea
et al. 1988). The consequences of a successful inva-
sion on other ecosystem processes also remain poorly
understood. A second approach to bioaugmentation is
to require degradation activity by the inoculum only
briefly. Temporary activity of an inoculum is much
easier to achieve and abates concerns for unintended
bioaugmentation consequences, but may remediate
only a small fraction of pollution. Repeated inocula-
tion may promote degradation of more contamination,
including aged pollution that is slowly desorbing. In
this paper we investigate the feasibility of repeatedly
inoculating a non-sterile soil with a phenanthrene-
degrading bacterium in order to promote the degrad-
ation of low concentrations of phenanthrene in soil.
Materials and methods
Mineralization experiments
Forbes soil (3.96% organic carbon, pH 5.6, cation ex-
change capacity 14.0 meq/100g, 47% sand, 38% silt,
15% clay, 0.333 bar moisture = 40.3%, further de-
scribed in (Scow et al. 1994)) was passed through a
2mmsieveandstoredina4◦C cold room. One week
prior to the experiments, the soil was brought up to a
moisture content of 0.222 bar or 26.3% and stored at
room temperature. A mixture of 14 C-labeled (Sigma
Chemical Co., St Louis MO, >98% purity, specific
activity of 59 mCi/mmol) and non-isotopically labeled
phenanthrene was added to 20 grams dry weight soil
in 100 µl of methylene chloride. Phenanthrene was
mixed into the soil with a spatula by hand for 2
minutes. The soil was incubated at 28 ◦Cinanair-
tight pint mason jar with a trap containing 1 ml of
0.5 N NaOH. Three replicate samples were prepared
for each experimental treatment. The base was peri-
odically sampled, and its radioactivity was measured
in a liquid scintillation counter (Beckman Instruments,
Inc., Fullerton, CA). The mason jar was opened dur-
ing sampling to ensure sufficient oxygen remained
available over the course of the incubation.
Arthrobacter, strain RP17
Strain RP17 was isolated by Mark Fuller from an
organic soil in the Sacramento River Delta through en-
richment culturing on phenanthrene. Strain RP17 can
use phenanthrene as its sole carbon and energy source
and mineralize it to CO2. Partial sequencing of the 16S
ribosomal gene (GenBank accession # AY005127),
as well as phospholipid fatty acid analysis, showed
that strain RP17 is an Arthrobacter sp. Its closest
known relative, based on 16S rDNA comparison, is
Arthrobacter polychromogenes.
Preparation of inoculum
Cells of Arthrobacter strain RP17 were cultivated on
50 ml mineral media (3.47 g/L KH2PO4,4.27g/L
K2HPO4,1.23g/L(NH
4)2SO4, 0.46 g/L MgSO4, 17.6
mg/L CaCl2, 1 mg/L FeSO4,3mg/LH
3BO3,2mg/L
CoCl2,1mg/LZnSO
4,0.3mg/LMnCl
2,0.3mg/L
Na2MoO4,0.2mg/LNiCl
2, 0.1 mg/l CuCl2, 250ng/L
starch, 250 ng/L peptone, 250 ng/L yeast extract) con-
taining phenanthrene as the major carbon source. The
cells were harvested through centrifugation at 8,000
rpm for 10 minutes and were subsequently washed
three times as follows. The cells were resuspended
in 40 ml of phospho-saline buffer (PSB) (50mM po-
tassium phosphates, 0.85% NaCl, pH 6.9) and cent-
rifuged at 1,000 rpm for 5 minutes. Solids, such as
phenanthrene crystals, pelleted at that point, but cells
remained in suspension. The supernatant was trans-
ferred to a new centrifuge tube and spun, again, at
8,000 rpm for 10 minutes. After the final wash, the
cells were resuspended in 2 ml of PSB. Dilutions were
made from this suspension using PSB. The cell dens-
ities of the inocula were measured through dilution
plating on 1/10 strength (3 g/L) tryptic soy agar plates.
Population density experiment
The indigenous microbial community of Forbes soil
does not degrade phenanthrene rapidly, but the min-
eralization rate can be increased substantially through
inoculation with strain RP17. Cells were added to For-
bes soil at densities of: 4.3 ×107,2.83×106, and 2.67
×105cfu/g dry soil. Bacteria were inoculated into soil
in the mason jars in 100 µl volumes of PSB dilutions
and stirred with a spatula by hand for 2 minutes to
promote even distribution within the soil. Initial inocu-
lation occurred 24 hours after the phenanthrene had
been added to soil.
Phenanthrene mineralization potential experiment
To test whether the inoculum retained the ability to
mineralize phenanthrene in the absence of phenan-
threne, 2.83 ×106cfu/g dry soil cells were added to
203
soil 280 hours before phenanthrene was added. Bac-
teria and phenanthrene were added to soil as described
above.
Repeated inoculation of soil microcosms
Selected microcosms were reinoculated twice more,
306 and 497 hours after the first inoculation, with 4.3
×107cfu/g dry soil and 4.1 ×107cfu/g dry soil
respectively. As in the other experiments, the cells
were delivered in 100 µl of phospho saline buffer and
mixed into the soil for 2 minutes by hand with a spat-
ula. To test if mixing in inoculating soils repeatedly
affected phenanthrene degradation kinetics, 100 µlof
PSB without cells was stirred into soil with a spatula
by hand for 2 minutes. Remixing occurred 306 and
again 497 hours after the inoculum was added to soil.
Immediately prior to restirring, the phenanthrene min-
eralization rate of inoculated treatments was similar to
uninoculated control soils.
Effect of phenanthrene concentration on
mineralization kinetics
The relationship between the concentration of phenan-
threne added to soil and the fraction mineralized was
investigated by adding three different concentrations,
50 ng/g, 5 µg/g, and 500 µg/g, of phenanthrene to soil
and inoculating it with 5.55 ×105cfu/g soil 24 hours
later. The cells and phenanthrene were added to soil as
described above.
Measurement of fraction of phenanthrene that
evolved as CO2in mineral media
The fraction of 20 ng/ml phenanthrene that evolved
as CO2was determined in 50 ml of mineral media
inoculated with 5.83 ×108cfu of RP17. The ex-
periment was performed twice, and both times three
replicate samples were prepared for each experimental
treatment. Mineralization curves were fit with first or-
der, single exponential equations using Kaleidagraph
(Synergy Software, Reading, PA). Cells from a 10-ml
aliquot were pelleted through centrifugation at 8,000
rpm for 10 minutes and subsequently washed three
times with PSB. The pellet was resuspended in one
ml of methanol and mixed with 5 ml of scintillation
cocktail. The radioactivity was measured in a liquid
scintillation counter. Radioactivity present in the su-
pernatant as well as the wash solution was measured.
These results were used to calculate the ratio between
the radioactivity stored in cells and the sum of the
radioactivity stored in cells and that evolved as CO2.
Results
The indigenous population in Forbes soil mineralized
50 ng/g phenanthrene very slowly (Figure 1). After
280 hours, only 1.8% of the phenanthrene was miner-
alized. By 306 hours after Forbes soil was inoculated
with 4.3 ×107,2.83×106,or2.67×105cfu strain
RP17/g dry soil, 19.6, 4.7, and 2.0% of the added
phenanthrene had mineralized respectively. Phenan-
threne mineralization in microcosms to which 4.3 ×
107or 2.83 ×106cfu/g dry soil were added were
fit well with a double exponential decay curve. Min-
eralization kinetics in the uninoculated soil and soil
inoculated with 2.67 ×105cfu/g dry soil were best
fit with a zero order equation. Mineralization rates in
all treatments diverged most approximately 26 hours
after inoculation, at which time the mineralization rate
in soil inoculated with the highest cell density was
41 times greater than in uninoculated soil. After 279
hours, degradation rates were similar in all treatments:
3.8 ×10−4%/g hr, 3.8 ×10−4%/g hr, 5.2 ×10−4
%/g hr, and 1.3 ×10−3%/g hr for the uninoculated,
2.7 ×105,2.8×106, and 4.3 ×107cfu/g treatments,
respectively.
The second inoculation resulted in an immediate
increase in the mineralization rates in all treatments
(Figure 1). Within 32 hours after reinoculation, min-
eralization rates increased 7.1, 13.7, and 13.8 times in
soils initially inoculated with 4.3 ×107,2.83×106,
and 2.67 ×105cfu/g dry soil, respectively. The third
inoculation, at 497 hours, increased the mineralization
rate, 2.7, 2.6, and 3.0 times in microcosms initially
inoculated with 4.3 ×107,2.83×106,and 2.67 ×105
cfu/g dry soil, respectively. The maximum mineraliza-
tion rate occurred 27 hours after the third inoculation,
similar to what was observed after the 1st and 2nd in-
oculations. Whereas only 7.3% of phenanthrene was
mineralized in microcosms initially inoculated with
2.83 ×106cfu/g soil, 25.6% more was mineralized
when the soil was inoculated two additional times.
Restirring the soil, inoculated with 2.83 ×106
cells, 306 and 496 hours after inoculation resulted
in a small, but significant increase in the phenan-
threne mineralization rate (p <0.015) (Figure 2). The
mineralization rate increased 38% after the first restir-
ring and an additional 25% after the second restirring.
Approximately 5.1% and 6.5% of the initially added
204
Figure 1. Phenanthrene mineralization rate (top) and cumulative
mineralization (bottom) in soils inoculated with strain RP17 mul-
tiple times; 1st inoculation: 4.30 ×107cfu/g, 2nd and 3rd inocu-
lations: 4.10 ×107cfu/g (), 1st inoculation: 2.83 ×106cfu/g,
2nd and 3rd inoculations: 4.10 ×107cfu/g (), 1st inoculation:
2.83 ×106cfu/g, no further inoculations (), 1st inoculation: 2.67
×105cfu/g, 2nd and 3rd inoculations: 4.10 ×107cfu/g (), no
inoculations (). Arrows show times of 2nd and 3rd inoculations.
phenanthrene was mineralized by the first and second
restirrings, respectively.
Arthrobacter strain RP17 cells did not maintain
their full potential to mineralize phenanthrene 300
hours after being inoculated in Forbes soil (Figure 3).
Twenty six hours after the phenanthrene was added,
the mineralization rate in the soil to which 300 hours
before 2.83 ×106cfu/g cells were added was 0.00047
%/g hr, whereas in soil where 2.83 ×106cfu/g cells
were added 24 hours after the phenanthrene was ad-
ded, the phenanthrene was mineralized at 0.001638
%/g hr.
Approximately 56.4% of the 500 µg/g phenan-
threne added to Forbes soil and inoculated with 5.55
×105cfu/g RP17 was mineralized 425 hours after in-
oculation, whereas only 31.1% and 3.4% mineralized
in microcosms containing 5 µg/g and 50 ng/g phenan-
threne, respectively (Figure 4). The kinetics of miner-
alization of the three concentrations also differed. The
Figure 2. Phenanthrene mineralization rate of soil mixed 306 and
496 hours after 2.83 ×106cells RP17/g soil were added. Arrows
show times soils were mixed again.
Figure 3. Phenanthrene mineralization rate of cells added to soil
280 hours before phenanthrene spike () in comparison to the min-
eralization rate of soil to which cells were added 24 hours after
phenanthrene was added (), uninoculated soil ().
mineralization rate in the 50 ng/g treatment remained
relatively constant and was fit best by a zero order
equation. The mineralization rate in soil spiked with
5µg phenanthrene/g soil declined biphasically. The
mineralization kinetics in the microcosms containing
500 µg/g phenanthrene showed a brief lag phase with
a mineralization maximum not occurring until 200
hours after the cells were added to the soil and was
subsequently followed by an exponential decline in
the mineralization rate (Figure 5). Using a quantitat-
ive polymerase chain reaction method, Schwartz et al.
(2000) showed that the lag phase was associated with
growth of RP17.
205
Figure 4. Cumulative phenanthrene mineralization in soil inocu-
lated with 5.55 ×105cfu/g Arthrobacter, strain RP17 and amended
with phenanthrene: 500 µg/g phenanthrene (), 5 µg/g phenan-
threne (), 50 ng/g phenanthrene ().
Figure 5. Mineralization rate of 20 ng/ml phenanthrene in mineral
media inoculated with 5.83 ×108cfu Arthrobacter, strain RP17.
Experiment 1(), experiment 2 ().
Approximately 70% of 20 ng phenanthrene/ml
mineral media added to a pure culture of RP17 cells
evolved as CO2,while the rest was stored in the cell’s
biomass. The degradation constant equaled 0.407/h in
the first experiment and .371/h in the second experi-
ment (Figure 5). The degradation kinetics of 20 ng/ml
phenanthrene were fit well with a single exponential
equation, as was expected with low concentrations of
phenanthrene.
Discussion
In Forbes soil, the linear sorption isotherm for phen-
anthrene indicated that only 0.3% of the phenanthrene
was present in the aqueous phase (Johnson 1997). Pre-
sumably, bacteria can only access pollutants dissolved
in the aqueous phase for degradation (Ogram et al.
1985), though there are isolated reports of bacteria
able to degrade sorbed chemicals (Guerin & Boyd
1992). In previous studies the availability of phen-
anthrene to RP17 declined in a biphasic curve, as
phenanthrene aged in soil (Schwartz & Scow 1999).
The first, rapid decline was completed within 150
hours after the phenanthrene was added to soil and
was followed by a much slower decline in bioavailab-
ility. Thirty two hours after phenanthrene was added
to soil, 2.85 times as much phenanthrene was avail-
able to RP17 than 611 hours after phenanthrene was
added. Thus, RP17 can only access a small fraction
of the phenanthrene at one time, and this fraction
increasingly becomes smaller as phenanthrene ages
in soil. Therefore, low bioavailability of phenan-
threne could be the primary reason for recalcitrance
of phenanthrene in soil and failure of bioaugmentation
attempts.
One obvious explanation of the observation that
degradation rates in all treatments became similar 300
hours after inoculation (Figure 1) was that the majority
of phenanthrene had sorbed and that, therefore, the
availability of phenanthrene, and not the population
density of phenanthrene-degrading organisms, was de-
termining the degradation rate. If this interpretation
were correct, one would expect that adding cells a
second or third time would not increase the mineraliz-
ation rate, because any remaining phenanthrene would
not be bioavailable. We observed, on the contrary, that
re-inoculation improved the mineralization rate (Fig-
ure 1), indicating that bioavailability alone did not
explain the decline in mineralization rate. Some of the
phenanthrene must have remained bioavailable, but
was not mineralized, because reinoculation increased
the mineralization rate (Figure 1).
It is unlikely that the source of the 14CO2evolution
after the 2nd and 3rd inoculation was the biomass that
had previously grown up on 14C-phenanthrene. The
amount of 14CO2evolved from soil after the second
inoculation is too great to be derived solely from bio-
mass grown up on phenanthrene. For instance, at the
time of the second inoculation (306 hours after the
initial inoculation), five percent of phenanthrene had
mineralized in soil inoculated with 2.83 ×106cfu/g.
In a pure culture study (Figure 5) in mineral media
with phenanthrene as a sole carbon source, 71% of
added phenanthrene evolved as CO2, while the rest
was stored in biomass. Using the yield measured in
pure culture, 1.45% of the radioactivity was stored
206
in biomass before the 2nd inoculation in the soil ex-
periment. After the second inoculation, 15.5% of the
initially added phenanthrene evolved as CO2(Figure
1). Furthermore, it is unlikely the turnover of RP17 in
soil was fast enough to account for any of the increases
in mineralization rates observed in this study. Mem-
bers of the genus Arthrobacter, which have a coccoid
dormant cell morphology, can survive extensive times
in a dormant state. Cells of Arthrobacter crystallopoi-
etes, for instance, remained 100% viable after having
been starved in phosphate buffer for 30 days (Boylen
& Ensign 1970). Quantitative polymerase chain re-
action measurements of the population dynamics of
RP17 cells grown up in Forbes soil, amended with 500
ppm phenanthrene, showed the population remained
stable, even though most of the phenanthrene had been
mineralized (Schwartz et al. 2000). The turnover of
microbial biomass in soil is often measured on the or-
der of months and not hours. For instance, in a silty
clay loam spiked with 500 µg/g 14C-glucose, active
soil microorganisms had a half life of 60 days (Wu et
al. 1993).
It is unlikely mineralization increased upon re-
peated inoculations, because newly added bacteria
were placed in the vicinity of phenanthrene that pre-
viously was spatially isolated from original inocu-
lations. Soil is so spatially complex that it can be
envisioned as containing a large number of isolated
compartments, each of which must be filled with a
phenanthrene-degrading bacterium to achieve remedi-
ation. This scenario suggests that the mineralization
rate declined because the inoculum, though remaining
active, had exhausted the phenanthrene in its imme-
diate vicinity. Remixing the soil should allow sorbed
bacteria of one compartment to come into contact with
available phenanthrene in another compartment. How-
ever, restirring the soil led to only a slight increase in
the phenanthrene mineralization rate (Figure 2). Also,
a low density inoculum, of 5.55 ×105cfu/g soil,
mineralized a large fraction (56%) of high concen-
trations (500 ppm) of phenanthrene while it mineral-
ized very little (<4%) of low concentrations (50 ppb)
phenanthrene (Figure 4). Spatial heterogeneity should
impede the mineralization of high concentrations of
phenanthrene as much as low concentrations.
Population densities of RP17 cells measured with
a quantitative competitive polymerase chain reac-
tion method show the population was stable in soil
even after phenanthrene mineralization had ceased
(Schwartz et al. 2000). Therefore it is unlikely pred-
ation of RP17 by protozoa or viruses had an impact
on the degradation of phenanthrene. Addition of new
biomass to overcome a maintenance threshold that was
high due to predation is not a likely explanation for the
increase in mineralization rate upon reinoculation.
More likely, reinoculation cells were added with
induced metabolic pathways that overcame an in-
duction threshold. The concentration of bioavailable
phenanthrene may not have been high enough to
keep metabolic enzymes induced (Button 1985). Ex-
amples of substrate threshold concentrations include
aCorynebacterium species, which could mineralize a
large fraction of 100 ng/ml p-nitrophenol in sewage,
but not 26 ng/ml or 17 ng/ml (Zaidi et al. 1988).
A gram positive rod, bacteria strain T, was able to
mineralize only 1.2% of 0.56 µg/l toluene in mineral
media, but could mineralize 33.9% of 10 mg/L tolu-
ene (Roch & Alexander 1997). A greater fraction of
high concentrations of phenanthrene was mineralized
(Figure 4) because bioavailable phenanthrene con-
centrations were higher than the substrate threshold
level in soils with 500 µg/g phenanthrene and not
in soils spiked with 50 ng/g phenanthrene. Possibly,
the inoculum had reserves, such as glycogen or poly-
β-hydroxybutyrate, and once these were exhausted,
subsequent inoculations were required to mineralize
more phenanthrene.
Alternatively, Strain RP17 may have lost genes
encoding for phenanthrene metabolism after it was
inoculated into soil, and reinoculation provided fresh
genetic potential to degrade phenanthrene. To date we
have not been successful in isolating plasmids from
the strain and therefore we do not know if genes for
phenanthrene metabolism are encoded on a plasmid.
The strain does retain the ability to degrade phen-
anthrene after it has been grown in 10% tryptic soy
broth suggesting it does not lose its capacity to degrade
phenanthrene rapidly.
Unlike most polluted sites, the phenanthrene in
our studies was not aged extensively, and, therefore,
it may be difficult to relate our results directly to
environmental cleanup. However, it is possible that
substrate threshold concentrations also play an im-
portant role in degradation kinetics of aged PAH’s
because in these cases desorption rates and hence the
bioavailable concentrations are very low.
There are at least two major challenges to imple-
menting repeated inoculations of soils in remediation
efforts. The first is economic and concerns the cost
of repeatedly inoculating a site. The second is the po-
tential of stimulating predators, such as protozoa or
nematodes, through repeatedly adding inoculum, or
207
prey, to soil. In one study, the decline of bacterial
populations coincided with the increase of protozoan
populations (Acea et al. 1988). Eventually, repeated
inoculation may fail because predators consume the
inoculum before it can degrade much of the pollutant.
In summary, we showed it was possible to miner-
alize a large fraction of low concentrations of phenan-
threne in soil through repeated inoculation of the soil
with phenanthrene-degrading bacteria. More phenan-
threne was mineralized in soils that were inoculated
repeatedly than in soils that were only inoculated once
or that were not inoculated at all. We suggest that
the bioavailability of small concentrations of phen-
anthrene in soil is so low it is below the minimum
substrate threshold level and consequently that the
activity of the phenanthrene-degrading population can
not be maintained.
Acknowledgements
This work was supported by the NIEHS Superfund
Basic Research Program (P 42 ESO 4699), the Joint
Bioremediation Program of the Office of Biological
and Environmental Research at the U.S. Department
of Energy (96-NCERQA-10), the Kearney Founda-
tion of Soil Science, the Ecotoxicology Program of
the UC Toxic Substance Research and Education Pro-
gram, and the US EPA Center for Ecological Health
Research (R819658).
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