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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