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MICROBIAL ECOLOGY Microb Ecol (1999) 38:69–78 DOI: 10.1007/s002489900157 © 1999 Springer-Verlag New York Inc.

Stimulation of Diesel Fuel Biodegradation by Indigenous Nitrogen 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, USA 2 Texas A&M University, Department of Oceanography, College Station, TX 77843, USA Received: 29 December 1998; Accepted: 6 April 1999

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B S T R A C T

Successful stimulation of N2 fixation and petroleum hydrocarbon degradation in indigenous microbial consortia may decrease exogenous N requirements and reduce environmental impacts of bioremediation following petroleum pollution. This study explored the biodegradation of petroleum 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 consistent 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 activity. 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-fuelpolluted systems stimulated indigenous N2 fixing microbial consortia to degrade petroleum hydrocarbons.

Correspondence to: M.F. Piehler, Fax: (252) 726-2426; E-mail: [email protected]

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Introduction Coastal development in the United States has been increasing at a rapid rate [25]. Petroleum pollution in waterways, ports, and marinas is an unfortunate consequence of intensive 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 pollution [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 microbial community [28], but has also been shown to be biodegraded by indigenous marine microorganisms [11]. Understanding the microbially mediated fate of this common and growing source of coastal pollution is critical to development of effective and environmentally benign bioremedial techniques. Biodegradation of petroleum hydrocarbons in marine environments can be limited by many factors, including nutrient 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 further ecological damage in sensitive environments [10]. This study explored the potential role of the microbial community in the biodegradation of petroleum pollution by indigenous N2 fixing marine microbial consortia. We also sought to explore potential bioremedial methods with minimal environmental impacts on coastal diesel fuel pollution for areas in which N has been found to be the primary nutrient limiting 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 increased N2 fixation. C loading from petroleum pollution in N-limited aquatic systems may also select for N2 fixing heterotrophic 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 bacteria by providing labile carbon and a surface to which the bacteria could attach [6]. The bacteria attached to corn-slash

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particles were instrumental in forming reduced anoxic microzones 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 biodegradation 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 microbial N2 fixation, diesel fuel bioavailability, bacterial biomass, and bacterial metabolic activity. The influence of these factors on biodegradation of petroleum hydrocarbons associated with particle surfaces was investigated.

Methods Sampling 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 approximately 6 d [14]. The system is N-limited with respect to primary productivity [14]. Sampling in Bogue Sound was conducted 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 hydrocarbon degradation and N2 fixation (nitrogenase activity) measurements. Nitrogenase activity (NA) and petroleum hydrocarbon degradation 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 experiments. Corn slash particles were added at a concentration of 1.67 g

Biodegradation of Diesel Fuel by Nitrogen Fixing Consortia

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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).

L−1 and were approximately 1000 µm in diameter. The POC concentration 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 November 1995 and August 1996 and treatments included diesel fuel and POC and control (diesel fuel only). Biodegradation Biodegradation was estimated by measuring 14Chexadecane 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 biodegradation. 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 volatilized 14 C-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. Following 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 nitrogen as the carrier was used to separate the gases. Rates were expressed in terms of ethylene generated per unit time.

Bacterial Community Structure and Function Bacterial productivity, total bacterial counts, and number of hydrocarbon degrading bacteria were measured using experimental mesocosms (72 L). Additionally, microscopic analyses were performed 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 uptake 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 duplicate microcentrifuge tubes containing 100 µl cold 100% trichloroacetic acid (TCA), and refrigerated. Following 60 min of incubation in a water bath, triplicate 1.8 ml aliquots were again removed from vials and placed in microcentrifuge tubes containing

72 100 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 activity of 3H-thymidine added to samples. In order to remove unincorporated 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 replaced 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 determined. 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 dilutions 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 experiments. Microscopy Microscopic analyses of the POC particles were performed 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 oxygen tension. Observations were performed following the September 1995 mesocosm experiment. Tetrazolium salt additions of 2,3,5-triphenyl-3tetrazolium 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 formation (low oxygen tension zones) [23]. Samples were fixed in absolute ethanol for examination using scanning electron microscopy 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 14

C-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 14Chexadecane (100K dpm/sample) spiked diesel fuel were added to 100 ml water samples in 250 ml serum vials. Bottles were incubated

M.F. Piehler et al. 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 following 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 concentrated by evaporation under a stream of N2 gas. 14C in the condensed 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 toxicity 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 Microtox 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 multiple range test p < 0.05 (BMRT) [19].

Results Microscopy TTC amended samples showed a consistent pattern of anoxic zones on or associated with POC particles. Ten slides were prepared and examined and a representative micrograph 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 particles were extensively colonized by bacteria of various morphologies (Fig. 3).

Biodegradation of Diesel Fuel by Nitrogen Fixing Consortia

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.

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 addition was found to significantly increase total bacterial biomass 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 number of hydrocarbon degraders increased significantly following POC addition to samples with diesel fuel at every concentration 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

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Fig. 3. Scanning electron micrographs of a corn particle incubated with diesel fuel in seawater. Numerous attached bacteria of varied morphology are apparent.

tested. Bacterial productivity, measured as 3H-thymidine incorporation, 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 number 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. Nitrogenase activity was relatively low after 2 days, peaked at

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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).

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 experiment, 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 microcosms (ANOVA, p > 0.05). Additionally, the abiotic and biotic mineralized and sorbed fractions were not statistically

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Fig. 5. Number of hydrocarbon degrading bacteria (MPN) with diesel fuel and POC and diesel fuel alone amendments to mesocosm samples from Bogue Sound. Asterisks indicate concentrations at which the 95% confidence intervals for the number of hydrocarbon 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.

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 consortium 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 productivity were documented following the addition of POC. Bacterial biomass increased following POC addition to diesel fuel amended samples at every concentration tested. The increase

Biodegradation of Diesel Fuel by Nitrogen Fixing Consortia

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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.

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 hydrocarbon 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 degrader abundance significantly above control levels. The maximum observed increase in hydrocarbon degraders following 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 following addition of POC was attributed to POC providing a

Table 1. Bacterial community measurements and average mean temperature for the petroleum hydrocarbon degradation and N2 fixation experiments Date

Cells ml−1

Hydrocarbon degraders ml−1

Average mean T (°C)

November 1995 August 1996

1.48 × 106 2.7 × 106

0.6(0.2, 1.5) 1.7(0.6, 4.4)

15.5 24.6

Cell counts are mean values obtained using AODC. Number of hydrocarbon degraders was determined by MPN and presented as mean value with the 95% confidence interval in parentheses.

Fig. 7. Diesel fuel biodegradation (14C hexadecane mineralization) 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.

surface for bacterial attachment in close proximity to petroleum 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 biomass, and hydrocarbon degraders by the addition of POC enhanced the potential for petroleum hydrocarbon degradation. Researchers have found varied responses of bacterial productivity to petroleum pollution [4]. Enhancement of bacterial productivity following POC addition in these experiments was thought to result, in part, from an increase in number and proportion of attached bacteria. Attached bacteria have been observed to be more metabolically active than free-living bacteria [22, 15]. Also, the bacterial metabolism 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

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Fig. 8. Fate of diesel fuel determined by using 14C-hexadecane as a tracer. Abiotic and biotic models are shown.

samples increased rates of both petroleum hydrocarbon mineralization and NA significantly above rates observed in controls. Other researchers have found petroleum hydrocarbon 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 control, possibly because of nutrient (likely N) depletion. Biodegradation 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

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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 because 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 enhancement very similar to those observed in the August experiment. 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 mineralized 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 increased diesel fuel biodegradation. Analysis of the fate of diesel fuel following POC addition revealed a significant change in short-term fate and a probable increase in bioavailability. Two days after the addition of POC to biotic microcosms, 0.13% of the labeled hexadecane 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. “Reversibly 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 bioavailable 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 experiments. 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 petroleum pollutants [5]. However, Microtox assays did not detect any toxicity in any of the water samples analyzed.

Biodegradation of Diesel Fuel by Nitrogen Fixing Consortia

Reduced toxicity following POC addition to polluted systems 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 performed 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 oxygen 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 requirements (N2 fixation is inhibited by oxygen and hydrocarbon degradation occurs most effectively at high levels of oxygen). Scanning electron microscopy revealed particles to be heavily colonized by bacteria of various sizes and morphologies. This was anticipated because of the elevated bacterial counts following POC addition. Dark field microscopy also showed the relationship of the diesel fuel to the particles. 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 enhancement of petroleum hydrocarbon degradation in response to the addition of POC. In the August and November N2 fixation/biodegradation experiments, 9% and 5%, respectively, of the labeled hexadecane added was mineralized to 14CO2. Stimulation of diesel fuel biodegradation following the addition of POC was likely due to increased microbial N2 fixation providing supplies of available N, diesel fuel bioavailability, bacterial biomass, and metabolic activity. Toxicity of the bulk water phase 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 the growth of indigenous N2 fixing microbial consortia to degrade petroleum hydrocarbons.

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We thank L. Kelly for technical assistance. This research was supported by EPA cooperative agreement #821946-01-0.

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