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Stimulation of Diesel Fuel Biodegradation by IndigenousNitrogen Fixing Bacterial Consortia
M.F. Piehler,1 J.G. Swistak,1 J.L. Pinckney,2 H.W. Paerl1
1 The University of North Carolina at Chapel Hill, Institute of Marine Sciences,
Morehead City, NC 28557, USA2 Texas A&M University, Department of Oceanography, College Station, TX 77843, USA
Received: 29 December 1998; Accepted: 6 April 1999
A B S T R A C T
Successful stimulation of N2 fixation and petroleum hydrocarbon degradation in indigenous mi-
crobial consortia may decrease exogenous N requirements and reduce environmental impacts of
bioremediation following petroleum pollution. This study explored the biodegradation of petro-
leum pollution by indigenous N2 fixing marine microbial consortia. Particulate organic carbon
(POC) in the form of ground, sterile corn-slash (post-harvest leaves and stems) was added to diesel
fuel amended coastal water samples to stimulate biodegradation of petroleum hydrocarbons by
native microorganisms capable of supplying a portion of their own N. It was hypothesized that
addition of POC to petroleum amended water samples from N-limited coastal waters would
promote the growth of N2 fixing consortia and enhance biodegradation of petroleum. Manipulative
experiments were conducted using samples from coastal waters (marinas and less polluted control
site) to determine the effects of POC amendment on biodegradation of petroleum pollution by
native microbial consortia. Structure and function of the microbial consortia were determined by
measurement of N2 fixation (acetylene reduction), hydrocarbon biodegradation (14C hexadecane
mineralization), bacterial biomass (AODC), number of hydrocarbon degrading bacteria (MPN),
and bacterial productivity (3H-thymidine incorporation). Throughout this study there was a con-
sistent enhancement of petroleum hydrocarbon degradation in response to the addition of POC.
Stimulation of diesel fuel biodegradation following the addition of POC was likely attributable to
increases in bacterial N2 fixation, diesel fuel bioavailability, bacterial biomass, and metabolic ac-
tivity. Toxicity of the bulk phase water did not appear to be a factor affecting biodegradation of
diesel fuel following POC addition. These results indicate that the addition of POC to diesel-fuel-
polluted systems stimulated indigenous N2 fixing microbial consortia to degrade petroleum hydro-
carbons.
Correspondence to: M.F. Piehler, Fax: (252) 726-2426; E-mail:
mpiehler@email.unc.edu
MICROBIALECOLOGY
Microb Ecol (1999) 38:69–78
DOI: 10.1007/s002489900157
© 1999 Springer-Verlag New York Inc.
Introduction
Coastal development in the United States has been increas-
ing at a rapid rate [25]. Petroleum pollution in waterways,
ports, and marinas is an unfortunate consequence of inten-
sive human use of the marine environment in these regions.
Enclosed areas such as harbors, bays, and marinas have the
highest frequency of moderate-sized spills and are the site of
activities responsible for the bulk of chronic petroleum pol-
lution [17]. Diesel fuel is widely used in marine engines and
accounts for 45% of the total volume of petroleum pollution
introduced to US waterways [30]. Diesel fuel has been found
to have significant detrimental effects on the marine micro-
bial community [28], but has also been shown to be biode-
graded by indigenous marine microorganisms [11]. Under-
standing the microbially mediated fate of this common and
growing source of coastal pollution is critical to develop-
ment of effective and environmentally benign bioremedial
techniques.
Biodegradation of petroleum hydrocarbons in marine en-
vironments can be limited by many factors, including nu-
trient availability (usually N), bioavailability of the pollutant,
bacterial biomass (both total and hydrocarbon degraders),
and toxicity of the pollutant on microorganisms degrading
the pollutants [16]. Bioremedial methods designed for use in
coastal environments attempt to maximize biodegradation
while minimizing perturbations of ecosystem structure and
function. Commonly applied bioremedial methods (e.g.,
mechanical removal and fertilizer addition) may lead to fur-
ther ecological damage in sensitive environments [10]. This
study explored the potential role of the microbial commu-
nity in the biodegradation of petroleum pollution by indig-
enous N2 fixing marine microbial consortia. We also sought
to explore potential bioremedial methods with minimal en-
vironmental impacts on coastal diesel fuel pollution for areas
in which N has been found to be the primary nutrient lim-
iting biodegradation of petroleum [24].
Biodegradation of petroleum hydrocarbons by N2 fixing
microbial consortia has been described before. In N-limited
sandy soils, Toccalino and co-workers [29] found elevated
rates of hydrocarbon biodegradation correlated with in-
creased N2 fixation. C loading from petroleum pollution in
N-limited aquatic systems may also select for N2 fixing het-
erotrophic bacteria [9]. The addition of POC in the form of
corn slash (post-harvest leaves and stems) to coastal water
samples increases N2 fixation by native heterotrophic bacte-
ria by providing labile carbon and a surface to which the
bacteria could attach [6]. The bacteria attached to corn-slash
particles were instrumental in forming reduced anoxic mi-
crozones which may have further facilitated O2 sensitive N2
fixation. Additionally, enhanced biodegradation of diesel
fuel has been observed following the addition of POC to
seawater samples [24].
Within microbial consortia, N2 fixation and petroleum
hydrocarbon degradation were stimulated to increase bio-
degradation of petroleum hydrocarbons by microorganisms
capable of supplying a portion of their own N. This may
result in a significantly reduced need for exogenous N as
fertilizer, and decreased detrimental environmental impacts
from bioremediation. POC additions to diesel fuel amended
coastal water samples were tested to assess effects on micro-
bial N2 fixation, diesel fuel bioavailability, bacterial biomass,
and bacterial metabolic activity. The influence of these fac-
tors on biodegradation of petroleum hydrocarbons associ-
ated with particle surfaces was investigated.
MethodsSampling Sites
Experiments were conducted on water samples collected from
Morehead City Yacht Basin (MCYB) and Bogue Sound (BS) (Fig.
1), situated within the Newport River Estuarine System (NRES),
North Carolina. NRES averages 1 m depth at mean low tide with an
average flushing time through the estuary to Beaufort Inlet of ap-
proximately 6 d [14]. The system is N-limited with respect to
primary productivity [14]. Sampling in Bogue Sound was con-
ducted from the pier at the University of North Carolina’s Institute
of Marine Sciences (IMS). There were no docked or moored boats
at this sampling location, although a public boat ramp is located
approximately 100 m east of the IMS pier. Bogue Sound is a full
salinity tidal sound with sandy beaches in the sample area [14].
MCYB is a medium capacity marina (slips for 60 docked boats)
located in Calico Creek. MCYB is also a full-salinity tidal system
and is surrounded by salt marsh.
Biodegradation and N2 Fixation
Petroleum hydrocarbon degradation/N2 fixation experiments were
conducted in Pyrex flasks, incubated at ambient temperature on a
shaker table, and included parallel samples for petroleum hydro-
carbon degradation and N2 fixation (nitrogenase activity) measure-
ments. Nitrogenase activity (NA) and petroleum hydrocarbon deg-
radation measurements were made 2, 4, 8, 16, and 32 days after
initiation of incubations. Diesel fuel (Amoco Oil Company) was
added at concentrations approximating a spill (1% v/v) [20] to
water samples from Morehead City Yacht Basin. Post-harvest corn
plants were obtained from Open Grounds Farm (Carteret County,
NC), ground in a Wiley mill, and autoclaved for use in experi-
ments. Corn slash particles were added at a concentration of 1.67 g
70 M.F. Piehler et al.
L−1 and were approximately 1000 µm in diameter. The POC con-
centration added was based on N2 fixation maxima obtained by
POC addition [6] and estimates of the POC additions possible
without inducing anoxia. Experiments were conducted in Novem-
ber 1995 and August 1996 and treatments included diesel fuel and
POC and control (diesel fuel only).
Biodegradation Biodegradation was estimated by measuring 14C-
hexadecane mineralization to 14CO2 [3]. 250 ml screw-top bottles
were equipped with center well collectors filled with 2 N KOH and
folded strips of cotton paper to trap 14CO2 generated by biodeg-
radation. Then, 100 ml of sample water collected from either BS or
MCYB was added to bottles with 1 ml diesel fuel spiked with 14C
hexadecane (Amersham Inc.) (0.045 µCi/sample). Well collectors
were sampled at regular intervals over a 4 week period and paper
strips collected from the wells were placed in 7 ml scintillation vials
with 5 ml CytoScint scintillation cocktail (ICN Inc.) and counted in
a Beckman LS5000TD liquid scintillation counter. Abiotic controls
(HgCl2 poisoned—1 mM final concentration) were run to account
for nonbiological generation of 14CO2 or trapping of volatilized14C-hexadecane. The measurement of 14CO2 generated was used to
calculate relative rates of biodegradation.
N2 Fixation Nitrogenase (the enzyme responsible for N2 fixation)
activity (NA) was measured using the acetylene reduction assay
[27]. Samples, 50 ml, were incubated in 72 ml serum vials for the
same period as the biodegradation samples. During the final 4 h of
the incubation, 5 ml of CaC2 generated acetylene was injected
through the flanged stoppers into the inverted serum vials. Follow-
ing the 4 h incubation, 2 ml headspace gas samples were taken and
placed in evacuated 2 ml autosampler vials for analysis by a Shi-
madzu GC-9A using flame ionization detection (FID) to determine
the amount of ethylene generated. Sample- and acetylene-only
blanks were run to account for generation of ethylene from sources
other than acetylene reduction by microorganisms. A 2 m stainless
steel Poropak T filled column held at 80°C with high-purity nitro-
gen as the carrier was used to separate the gases. Rates were ex-
pressed in terms of ethylene generated per unit time.
Bacterial Community Structure and Function
Bacterial productivity, total bacterial counts, and number of hy-
drocarbon degrading bacteria were measured using experimental
mesocosms (72 L). Additionally, microscopic analyses were per-
formed on samples from mesocosm experiments. Mesocosms were
filled with Bogue Sound water and incubated in outdoor ponds at
IMS. Subsamples for rate measurements were taken at 1, 2, and 4
weeks for mesocosms. Diesel fuel additions ranged from 0.01 to
0.60% v/v. Mesocosm experiments were conducted in August 1995,
September 1995, November 1995, and February 1996. Treatments
to water samples were POC (corn-slash and diesel fuel) and control
(diesel fuel only).
Bacterial Productivity Bacterial productivity was measured by up-
take of 3H-thymidine into cellular macromolecules [7, 18, 26].
Vials were spiked with 20 µl 3H-thymidine (64 Ci mmol−1, ICN
Inc.), and 1.8 ml from each vial was immediately removed to du-
plicate microcentrifuge tubes containing 100 µl cold 100% trichlo-
roacetic acid (TCA), and refrigerated. Following 60 min of incu-
bation in a water bath, triplicate 1.8 ml aliquots were again re-
moved from vials and placed in microcentrifuge tubes containing
Fig. 1. Map showing location of the sampling sites near Morehead City, North Carolina, USA. Sites included Bogue Sound (BS) and
Morehead City Yacht Basin (MCYB).
Biodegradation of Diesel Fuel by Nitrogen Fixing Consortia 71
100 ml of 100% TCA, to a final concentration of 5% v/v, and
refrigerated. During incubation, an additional 100 µl was removed
from each sample vial and placed in scintillation vials containing 5
ml Ecolume (ICN) scintillation cocktail to determine the total ac-
tivity of 3H-thymidine added to samples. In order to remove un-
incorporated TCA-soluble 3H-thymidine, the refrigerated samples
were rinsed two times with 5% v/v TCA [26]. Cold samples were
microcentrifuged for 10 min at 14,000 rpm to concentrate a pellet
of biomass in the bottom of the tubes. Using a blunt-tipped steel
needle, the supernatant was aspirated with weak vacuum and re-
placed with 1 ml cold 5% v/v TCA. This rinse was repeated a
second time, and the second 5% v/v TCA rinse replaced with 1 ml
Ecolume scintillation cocktail. Activity of samples was determined
using a Beckman LS 5000TD liquid scintillation counter. Data are
presented from the September 1995 mesocosm experiment.
Microbial Community Structure Prior to every experiment and also
following the mesocosm experiments, total bacterial community
biomass and number of hydrocarbon degrading bacteria were de-
termined. Acridine orange direct counts (AODC) were used to
assess total bacterial biomass [13]. Samples were sonicated for 30 s
in ice to remove bacteria from particles and fields were counted
until at least a total of 100 cells were encountered. The number of
hydrocarbon degraders was estimated using a modified five-tube
most probable number (MPN) technique [3]. Serial decimal dilu-
tions were made into mineral media in 5 ml capped test tubes with
diesel fuel as the sole carbon source. Turbidity was used as the
positive indicator of growth and standard MPN tables [1] were
used to estimate hydrocarbon degrading bacteria per unit volume
water sample. Data are presented from all four mesocosm experi-
ments.
Microscopy Microscopic analyses of the POC particles were per-
formed to describe the structure of the attached bacterial commu-
nity, to determine the relationship of the diesel fuel and POC, and
to examine microscale heterogeneity of oxygen tension. Observa-
tions were performed following the September 1995 mesocosm
experiment. Tetrazolium salt additions of 2,3,5-triphenyl-3-
tetrazolium chloride (TTC, 0.01% wt/v) were made to samples to
identify areas of low oxygen tension [23]. TTC amended samples
were examined using dark field microscopy (Nikon Labophot-2,
400× total magnification) to detect areas of formazan crystal for-
mation (low oxygen tension zones) [23]. Samples were fixed in
absolute ethanol for examination using scanning electron micros-
copy to assess the magnitude and structure of the attached bacterial
community associated with POC particles. Dark field microscopy
was also utilized to determine the relationship of the diesel fuel to
the particles.
Fate of Diesel Fuel
14C-hexadecane was used as a tracer to assess the fate of diesel fuel
in a seawater sample with corn-slash (POC) amendments in a set of
experiments conducted in September 1996. Corn-slash and 14C-
hexadecane (100K dpm/sample) spiked diesel fuel were added to
100 ml water samples in 250 ml serum vials. Bottles were incubated
outside on a shaker table for 48 h with 2 N KOH filled center well
collectors [3]. Fate of the compound was assessed using the fol-
lowing protocol. Filter paper from the center well collectors was
collected and counted in a Beckman LS5000TD liquid scintillation
counter (Cytoscint cocktail was used throughout, ICN, Inc.). This
was the “mineralized” fraction. Samples were then filtered through
glass fiber filters (Whatman GFF). The filtrate contained both the
“separate” and “soluble” fractions (their combined magnitude was
determined by difference). Filters were then extracted three times
with 5 ml hexane. The hexane extract was collected and concen-
trated by evaporation under a stream of N2 gas. 14C in the con-
densed hexane extract was counted using a liquid scintillation
counter and constituted the “reversibly sorbed” fraction. Finally,
the filters and particles which constituted the “sorbed” fraction
were counted by a liquid scintillation counter. Biotic and abiotic
treatments were run to separate physical and biological effects of
POC addition on bioavailability of diesel fuel.
Toxicity Experiments
Water samples (30 ml) were incubated in 72 ml serum vials for 48
h with the following treatments: control (water sample only), diesel
fuel, diesel fuel + POC, and abiotic diesel fuel + POC, in triplicate.
Twenty-ml water subsamples were taken following the incubation
period from just below the immiscible petroleum layer with a glass
Pasteur pipette. The Microtox assay [8] was used to compare tox-
icity from the bulk phase of samples incubated with and without
POC. Five-minute acute toxicity tests were performed on a range of
sample volumes from 20 to 200 µl. Data were analyzed using Mi-
crotox software and EC50 calculations were made. Data presented
were obtained during September 1996.
Data Analysis
SPSS statistical software was used for all analyses (SPSS Inc.). Data
were analyzed using a one-way ANOVA with treatment as the main
factor and rate measures as the response variable. A-posteriori
comparisons of means were performed using the Bonferroni mul-
tiple range test p < 0.05 (BMRT) [19].
Results
Microscopy
TTC amended samples showed a consistent pattern of an-
oxic zones on or associated with POC particles. Ten slides
were prepared and examined and a representative micro-
graph from the dark field TTC addition experiment is shown
with anoxic microzones on a corn particle (Fig. 2). Scanning
electron microscopy consistently revealed that the corn par-
ticles were extensively colonized by bacteria of various mor-
phologies (Fig. 3).
72 M.F. Piehler et al.
Bacterial Community Changes
Changes in bacterial productivity, bacterial biomass, and
number of hydrocarbon degraders following POC addition
to diesel fuel amended samples were examined. POC addi-
tion was found to significantly increase total bacterial bio-
mass in diesel fuel amended samples (ANOVA, p < 0.05)
(Fig. 4). The magnitude of enhancement was very similar at
each concentration, and POC addition increased both total
bacterial biomass and the number of hydrocarbon degraders
at every concentration tested (100–6000 µl L−1). The num-
ber of hydrocarbon degraders increased significantly follow-
ing POC addition to samples with diesel fuel at every con-
centration tested except 100 µl L−1 (Fig. 5, 95% C.I.). The
maximum increase in number of hydrocarbon degraders
observed occurred at 300 µl L−1 and there was a decrease in
enhancement at each higher concentration of diesel fuel
tested. Bacterial productivity, measured as 3H-thymidine in-
corporation, was significantly higher in samples with POC
and diesel fuel compared to additions of diesel fuel alone
(ANOVA, p < 0.05) (Fig. 6). Maximum productivity rates in
the POC and diesel fuel treatments occurred at the 1 and 2
week sample times.
Diesel Fuel Degradation and N2 Fixation
Average mean temperature, total bacterial counts, and num-
ber of hydrocarbon degrading bacteria are shown for each
experiment in Table 1. POC addition elevated N2 fixation
significantly above the control in each experiment (ANOVA,
p < 0.05) (Fig. 7a,b). The pattern of biostimulation of N2
fixation was similar in each of the experiments from August
and November, shown in Fig. 7a and 7b, respectively. Ni-
trogenase activity was relatively low after 2 days, peaked at
Fig. 2. Dark-field photomicrograph of a corn particle incubated
with tetrazolium salts. Dark areas are sites of formazan crystal
formation and indicate reduced microzones.
Fig. 3. Scanning electron micrographs of a corn particle incu-
bated with diesel fuel in seawater. Numerous attached bacteria of
varied morphology are apparent.
Biodegradation of Diesel Fuel by Nitrogen Fixing Consortia 73
4–8 days, and declined through days 8–32. In the August
experiment (Fig. 7a) the amount of N fixed through the
duration of the experiment was determined by estimating
the area under the curve and was found to be approximately
2.6 mg N. The November experiment (Fig. 7b) showed N2
fixation rates of a lower magnitude than the August experi-
ment, resulting in the fixation of less N (∼0.13 mg N).
Diesel fuel degradation was elevated above the control
from the start of the August experiment, lower through days
8–16, and increased to maximum at 32 days (Fig. 7a). In
November the rates of diesel fuel degradation were low
through 8 days, rose quickly to their peak at 16 days, and
remained elevated (∼350% of control) at 32 days (Fig. 7b).
POC addition elevated diesel fuel degradation significantly
above the control in each case (ANOVA, p < 0.05). In the
August experiment approximately 9% of the 14C-hexadecane
added was mineralized and collected in the 14CO2 traps, and
in the November experiment approximately 5% of the label
added was mineralized and collected.
Fate of Diesel Fuel Following POC Addition
The fate of 14C-hexadecane in abiotic and biotic microcosms
after 48h is shown in Fig. 8. No difference was found in the
partitioning of 14C-hexadecane in biotic and abiotic micro-
cosms (ANOVA, p > 0.05). Additionally, the abiotic and
biotic mineralized and sorbed fractions were not statistically
distinguishable, despite the apparent differences (BMRT).
The majority of the labeled hexadecane was found to be
“reversibly sorbed” with the POC (Fig. 8). The separate/
soluble fraction was the next largest in magnitude, followed
by sorbed, and the mineralized fraction was the smallest.
Toxicity
There was no detectable toxicity in any of the volumes of any
of the treatments tested using the Microtox assay [8]. EC50s
could not be calculated because there was no concentration
dependent reduction in luminescence.
Discussion
The central goal of this study was to explore the feasibility of
stimulating a naturally occurring N2 fixing microbial con-
sortium to degrade petroleum pollution in coastal waters. In
an effort to understand the overall effect of POC addition on
the bacterial community, changes in bacterial biomass,
number of hydrocarbon degraders, and bacterial productiv-
ity were documented following the addition of POC. Bacte-
rial biomass increased following POC addition to diesel fuel
amended samples at every concentration tested. The increase
Fig. 4. Bacterial counts (AODC) with diesel fuel and POC and
diesel fuel alone amendments to mesocosm samples from Bogue
Sound. Diesel fuel was added over a range of concentrations and
two data points were taken at the highest concentration tested
(6000 µl L−1).
Fig. 5. Number of hydrocarbon degrading bacteria (MPN) with
diesel fuel and POC and diesel fuel alone amendments to meso-
cosm samples from Bogue Sound. Asterisks indicate concentrations
at which the 95% confidence intervals for the number of hydro-
carbon degrading bacteria in the two treatments did not overlap.
Two data points are shown at the highest concentration tested
(6000 µl L−1), both of which were significantly different.
74 M.F. Piehler et al.
in biomass was likely due to stimulation of bacterial growth
in the presence of increased surface area for attachment,
some labile carbon from the corn, and an increase in the
bioavailability of the petroleum hydrocarbons for bacterial
degradation following POC addition.
The effect of POC addition on the number of hydrocar-
bon degraders was dependent on the concentration of diesel
fuel. At a 100 µl L−1 diesel fuel addition there were no more
hydrocarbon degraders in the POC treatment than in the
diesel-fuel-only control. With 300, 2000, and 6000 µl L−1
diesel fuel added, POC addition increased hydrocarbon de-
grader abundance significantly above control levels. The
maximum observed increase in hydrocarbon degraders fol-
lowing POC addition was at a concentration of 300 µl L−1
diesel fuel. Number of hydrocarbon degraders was inversely
proportional to the quantity of diesel fuel added. The overall
trend of increased hydrocarbon degrader abundance follow-
ing addition of POC was attributed to POC providing a
surface for bacterial attachment in close proximity to petro-
leum hydrocarbons. This may have increased bioaccessibility
of diesel fuel either by direct access to sorbed diesel fuel or
by increasing the mass transfer of diesel fuel by reducing the
diffusion distance [31].
Elevation of levels of bacterial productivity, bacterial bio-
mass, and hydrocarbon degraders by the addition of POC
enhanced the potential for petroleum hydrocarbon degrada-
tion. Researchers have found varied responses of bacterial
productivity to petroleum pollution [4]. Enhancement of
bacterial productivity following POC addition in these ex-
periments was thought to result, in part, from an increase in
number and proportion of attached bacteria. Attached bac-
teria have been observed to be more metabolically active
than free-living bacteria [22, 15]. Also, the bacterial metabo-
lism of labile organic carbon from the corn particles may
have contributed to increased bacterial productivity.
The addition of POC to diesel fuel amended coastal water
Fig. 6. Bacterial productivity (3H-thymidine uptake) with diesel
fuel (2000 µl L−1) and POC and diesel fuel alone amendments to
mesocosm samples from Bogue Sound incubated over a period of
four weeks. Error bars are one standard deviation.
Table 1. Bacterial community measurements and average mean
temperature for the petroleum hydrocarbon degradation and N2
fixation experiments
Date Cells ml−1Hydrocarbon
degraders ml−1Average mean T
(°C)
November 1995 1.48 × 106 0.6(0.2, 1.5) 15.5August 1996 2.7 × 106 1.7(0.6, 4.4) 24.6
Cell counts are mean values obtained using AODC. Number of hydrocar-bon degraders was determined by MPN and presented as mean value withthe 95% confidence interval in parentheses.
Fig. 7. Diesel fuel biodegradation (14C hexadecane mineraliza-
tion) and N2 fixation (acetylene reduction) in MCYB samples
through 32 day incubations in 250 ml flasks. Data in (A) are from
August 1996 and data in (B) are from November 1995. Data are
presented as percentage of control (diesel fuel only) and error bars
are one standard deviation.
Biodegradation of Diesel Fuel by Nitrogen Fixing Consortia 75
samples increased rates of both petroleum hydrocarbon
mineralization and NA significantly above rates observed in
controls. Other researchers have found petroleum hydrocar-
bon biodegradation supported by N from N2 fixation [29].
Enhanced NA may have increased microbial utilization of
petroleum hydrocarbons. Petroleum hydrocarbons may
have been utilized either as energy to support N2 fixation or
through increased cellular metabolism utilizing this “new” N
source. Results from the August experiment indicated the
latter as a more likely explanation. In this experiment, diesel
fuel degradation was elevated through the first 4 days of the
experiment and then decreased to near the level of the con-
trol, possibly because of nutrient (likely N) depletion. Bio-
degradation rates were then constant through 15 days and
increased to a peak at 32 days. This maximum in diesel fuel
degradation was likely due to the microbial remineralization
of N fixed during the peak between days 4 and 10, which was
considerable (∼2.6 mg N fixed through the experiment). N2
fixation was probably not sustained at maximal levels be-
cause of either elevated fixed N concentrations or localized
depletion of labile organic carbon, both of which are known
to inhibit NA [22].
The November experiment showed patterns of enhance-
ment very similar to those observed in the August experi-
ment. The magnitude of enhancement of NA was much
lower, however, and the estimated total amount of N fixed
through the experiment was about 80% less than in the
August experiment. Despite this fact, the magnitude of diesel
fuel degradation and the total amount of diesel fuel miner-
alized in the POC treatment was similar to the levels seen in
the August experiment. This suggests that either a small
amount of fixed N was necessary to enhance diesel fuel
degradation or that other factors were contributing to in-
creased diesel fuel biodegradation.
Analysis of the fate of diesel fuel following POC addition
revealed a significant change in short-term fate and a prob-
able increase in bioavailability. Two days after the addition
of POC to biotic microcosms, 0.13% of the labeled hexa-
decane had been mineralized, 3.44% was sorbed, and 22.3%
was in an immiscible separate phase or in the water phase
(soluble). Nearly three-quarters of the 14C-hexadecane tracer
was found to be “reversibly sorbed” to the particles. “Re-
versibly sorbed” tracer was defined as that which remained
with the particles following filtration and was removed by
hexane extraction. Using fate and bioavailability of organic
pollutants in sediments as an analogue [12], it was assumed
that the reversibly sorbed fraction would likely be bioavail-
able and the sorbed fraction would not. The addition of POC
may have increased bioavailability of diesel fuel above the
level with no POC by increasing the oil/water interface and
attracting diesel fuel to the particles colonized by bacteria.
In addition to the biotic fate experiments described
above, parallel abiotic trials were conducted to determine the
importance of biotic processes in determining the fate of
diesel fuel. The fate partitioning of hexadecane was found to
be statistically indistinguishable in the biotic and abiotic ex-
periments. As anticipated, the biotic mineralized and sorbed
values were higher, but the difference was not statistically
significant because of the short incubation time (2 days).
The action of POC on fate and, in turn, bioavailability was
found to occur independent of biotic processes.
Toxicity has been found to limit biodegradation of pe-
troleum pollutants [5]. However, Microtox assays did not
detect any toxicity in any of the water samples analyzed.
Fig. 8. Fate of diesel fuel determined by using 14C-hexadecane as
a tracer. Abiotic and biotic models are shown.
76 M.F. Piehler et al.
Reduced toxicity following POC addition to polluted sys-
tems has been observed to enhance biodegradation [2]. POC
addition had no effect on toxicity in these experiments. The
lack of measurable toxicity in any treatments tested indicates
there was no increase in toxicity resulting from elevated
biodegradation of diesel fuel, which has been indicated as a
concern in other studies [32].
Microscopic analyses of the POC particles were per-
formed to describe the structure of the attached bacterial
community, to determine the relationship of the diesel fuel
and POC, and to examine microscale heterogeneity of oxy-
gen tension. Well-developed oxygen depleted microzones
were detected on corn particles. It was hypothesized that
microscale oxygen tension variability allowed N2 fixing and
petroleum hydrocarbon degrading microbes to function in
close proximity despite their disparate environmental re-
quirements (N2 fixation is inhibited by oxygen and hydro-
carbon degradation occurs most effectively at high levels of
oxygen). Scanning electron microscopy revealed particles to
be heavily colonized by bacteria of various sizes and mor-
phologies. This was anticipated because of the elevated bac-
terial counts following POC addition. Dark field microscopy
also showed the relationship of the diesel fuel to the par-
ticles. Diesel fuel was found to be in contact with many of
the particles examined and bacteria were usually apparent on
both the particle and the diesel fuel droplet.
Throughout this study there was a consistent enhance-
ment of petroleum hydrocarbon degradation in response to
the addition of POC. In the August and November N2 fixa-
tion/biodegradation experiments, 9% and 5%, respectively,
of the labeled hexadecane added was mineralized to 14CO2.
Stimulation of diesel fuel biodegradation following the ad-
dition of POC was likely due to increased microbial N2
fixation providing supplies of available N, diesel fuel bio-
availability, bacterial biomass, and metabolic activity. Tox-
icity of the bulk water phase did not appear to be a factor
affecting biodegradation of diesel fuel following POC addi-
tion. These results indicate that the addition of POC to diesel
fuel polluted systems stimulated the growth of indigenous
N2 fixing microbial consortia to degrade petroleum hydro-
carbons.
Acknowledgments
We thank L. Kelly for technical assistance. This research was
supported by EPA cooperative agreement #821946-01-0.
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