Hexadecane mineralization and denitrification in two diesel fuel-contaminated soils

FEMS Microbiology Ecology 32 (2000) 17^23

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Hexadecane mineralization and denitri¢cation in two diesel fuel-contaminated soils Re¨al Roy *, Charles W. Greer Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montre¨al, Que. H4P 2R2, Canada Received 4 September 1999 ; received in revised form 15 December 1999 ; accepted 15 December 1999

Abstract The effect of nitrate, ammonium and urea on the mineralization of [14 C]hexadecane (C16 H34 ) and on denitrification was evaluated in two soils contaminated with diesel fuel. In soil A, addition of N fertilizers did not stimulate or inhibit background hexadecane mineralization (4.3 mg C16 H34 kg31 day31 ). In soil B, only NaNO3 stimulated hexadecane mineralization (0.91 mg C16 H34 kg31 day31 ) compared to soil not supplemented with any nitrogen nutrient (0.17 mg C16 H34 kg31 day31 ). Hexadecane mineralization was not stimulated in this soil by NH4 NO3 (0.13 mg C16 H34 kg31 day31 ), but the addition of NH4 Cl or urea suppressed hexadecane mineralization (0.015 mg C16 H34 kg31 day31 ). Addition of 2 kPa C2 H2 did not inhibit the mineralization process in either soil. Denitrification occurred in both soils studied when supplemented with NaNO3 and NH4 NO3 , but was not detected with other N sources. Denitrification started after a longer lag in soil A (10 days) than in soil B (4 days). In soil A microcosms supplemented with NaNO3 or NH4 NO3 , rates of denitrification were 20.6 and 13.6 mg 31 31 NO3 day31 , respectively, and in soil B, they were 18.5 and 12.5 mg NO3 day31 , respectively. We conclude that denitrification 3 kg 3 kg may lead to a substantial loss of nitrate, making it unavailable to the mineralizing bacterial population. Nitrous oxide was an important end-product accounting for 30^100% of total denitrification. These results indicate the need for preliminary treatability studies before implementing full-scale treatment processes incorporating commercial fertilizers. Crown copyright ß 2000 Published by Elsevier Science B.V. All rights reserved. Keywords : Soil; Bacterium ; Nitrate ; Ammonium; Hexadecane ; Denitri¢cation; Nitrous oxide

1. Introduction In the context of the biodegradation of organic pollutants, such as petroleum hydrocarbons, in terrestrial environments, addition of commercial fertilizers is a common practice to stimulate the soil indigenous micro£ora in degrading the target pollutants (biostimulation) [1,2]. In a study of the Exxon Valdez oil spill in Alaska, it was found that the most critical factor for successful bioremediation was the concentration of NH‡ 4 in the pore water following the application of an oleophilic fertilizer [3]. A commercial fertilizer typically contains three forms of N: urea, nitrate and ammonia. Although these nutrients may all be directly assimilated by various soil microorganisms, including those involved in the degradation of petroleum hydrocarbons, they may also be transformed by other members of the soil microbial community [4]. For instance, minerali-

* Corresponding author. Tel. : +1 (514) 496-6316; Fax: +1 (514) 496-6265; E-mail: [email protected]

zation of urea may lead to the production of NH‡ 4 which 3 3 in turn may be oxidized to NO2 and NO3 by nitrifying 3 bacteria [5]. Ultimately, NO3 2 and NO3 may be dissimilated to N2 by denitrifying bacteria under anoxic conditions [6]. This last process leads to a loss of N fertilizer from terrestrial ecosystems that may represent up to 1 kg N ha31 year31 in fertilized agricultural ¢elds [7^9]. In the context of bioremediation of contaminated soil, little is known about the fate of N fertilizer. Denitri¢cation may also be important in the context of mineralization of petroleum hydrocarbons under anoxic conditions [10]. A growing number of studies are showing that, under denitrifying conditions, several aromatic compounds, for instance toluene, xylene, phenols, cresols, and naphthalene may be degraded [11,6]. Only recently, Bregnard et al. [12] have shown the degradation of an aliphatic compound, pristane, under denitrifying conditions. In the ¢eld, anoxic microsites, in otherwise oxic soils, frequently occur in soil aggregates [13,14]. Under such conditions, in a diesel-contaminated soil, denitri¢cation may occur and contribute to the biodegradation of petroleum hydrocar-

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bons. In this case, the addition of a commercial fertilizer containing nitrate may also act as a source of electron acceptors for indigenous denitrifying diesel-degrading bacteria. The objective of the present study was to evaluate the e¡ect of various nitrogen fertilizers (urea, NH4 Cl, NaNO3 and NH4 NO3 ) on the mineralization of hexadecane (C16 H34 ) in aerobic microcosms of two di¡erent soils with similar levels of C10 -C50 contamination. Denitri¢cation which may be responsible for loss of N fertilizer in soil was also evaluated during the mineralization process using the acetylene (C2 H2 ) blockage technique [15,16]. 2. Materials and methods 2.1. Soils The characteristics of the two soils used for these studies are presented in Table 1. Standard procedures were used for the determination of total petroleum hydrocarbon (C10 -C50 ) [17], total carbon [18], total Kjeldahl nitrogen [19] and total phosphorus [20], which were performed either in our lab or by the Soil Testing Lab of McGill University. The water content was measured by an Electronic Moisture Analyzer MA30 (Sartorius, Goettingen, Germany). Both soils were sampled from diesel-contaminated areas. Soil A was from a military installation in Bagotville, Que., Canada, and soil B was from a gas station in Montreal, Que., Canada. Soils were stored at 4³C until used. Both soils had similar levels of petroleum hydrocarbon contamination (2000 and 3800 mg kg31 soil), and the total carbon and total mineral nitrogen contents were higher in soil A than in soil B. The C/N ratios, on a mass basis, were 32 and 81 in soils A and B, respectively. Total phosphorus and water contents were lower in soil A than in soil B. 2.2. Soil microcosms Experiments were performed in aerobic soil microcosms consisting of 10 g (wet weight) of soil in 100-ml glass serum bottles capped with te£on-lined rubber stoppers and sealed with aluminum crimps. Unlabeled hexadecane (50 mg ml31 ), dissolved in hexane, was added as 20-Wl aliquots to all soil microcosms to a ¢nal concentration of 100 mg kg31 soil. Sterile stock solutions of NaNO3 (1 M), NH4 Cl (1 M), NH4 NO3 (0.5 M) and urea (0.5 M) were added separately in 400-Wl aliquots to soil micro-

cosms and no change in soil pH greater than 0.3 units occurred following the additions. These N supplements were equivalent to 560 mg N kg31 wet soil, which is in the upper range for fertilization studies, but typical for denitri¢cation assessments [21,22]. In control £asks, 400 Wl sterile distilled water was added instead of nitrogenous salts solutions. For each treatment, triplicate £asks were prepared. Moreover, two sets of microcosms were prepared: one set for the determination of [14 C]hexadecane mineralization and the other for the determination of denitri¢cation rates using the acetylene blockage technique [15]. All microcosms were incubated statically in the light at 25³C. Sterile microcosms were prepared as previously described for the control. Soil microcosms were sterilized for 1 h on two consecutive days. Hexadecane and C2 H2 (2 kPa) were added to these microcosms. 2.3. Hexadecane mineralization The mineralization experiments followed a modi¢ed technique from Whyte et al. [23]. A 10-ml test tube containing 0.5 M KOH (1 ml) was placed inside the soil microcosms (CO2 trap). A mixture of [1-14 C]hexadecane (speci¢c activity 2.2 mCi mmol31 or 0.081 GBq mmol31 ) (Sigma^Aldrich, Mississauga, Ont., Canada) with unlabeled hexadecane (50 mg ml31 in hexane) was added to each soil microcosm as 20-Wl aliquots. The amount of radioactivity added to soil microcosms was equivalent to 1948.5 Bq with a total concentration of hexadecane of 462 Wmol kg31 (104 parts per million (ppm)). The KOH was replaced periodically and its radioactivity content was determined by liquid scintillation spectrometry (Tri-carb 2100TR, Packard Instruments, Meriden, CT, USA) [24]. Rates of mineralization were computed as the cumulative fraction of [14 C]CO2 produced from the initial [14 C]hexadecane added. We also prepared a series of KOH trap-microcosms with C2 H2 (2 kPa) to evaluate the e¡ect of this alkyne on hexadecane mineralization. 2.4. Denitri¢cation and respiration rates Denitri¢cation was measured by determination of N2 O production in the presence of C2 H2 (2 kPa) in soil microcosms without KOH traps [15,18]. Acetylene is an inhibitor of the N2 O reductase of denitrifying bacteria that prevents N2 O reduction to N2 [25,26]. Respiration was measured by accumulation of CO2 in the headspace of £asks. Gas samples (0.5 ml) were withdrawn from the headspace of the soil microcosms and analyzed by gas

Table 1 Summary of the characteristics of the soils studied Soil

C10 -C50 (mg kg soil31 )

C (mg kg soil31 )

N (mg kg soil31 )

P (mg kg soil31 )

C/N

H2 O content (%)

A B

2 000 3 800

44 000 24 300

1 388 300

63 930

32 81

54 75

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chromatography (GC). The concentration of each gas in the headspace was calculated based on a standard curve and using the gas law to calculate the amount (mol) of each gas. Since the soils were not slurried, we considered that the dissolved fraction of the various gases would be marginal compared with the headspace. 2.5. Analytical methods Gases (N2 O, CO2 ) were measured by GC either on a HP6890 GC (Hewlett^Packard, Palo Alto, CA, USA) with a thermal conductivity detector (TCD) or on a SRI 8610C gas chromatograph (SRI, Torrance, CA, USA) with a TCD, a £ame ionization detector (FID) and an electron capture detector (ECD) in parallel. The HP6890 was con¢gured in the following manner: the TCD was set at 225³C and the oven at 60³C; a 2 mU3.1 mm stainless steel column packed with HaysepQ (Supelco, Bellefonte, PA, USA) ; helium (ultra high purity grade, Prodair, Montreal, Que., Canada) was the carrier gas £owing at 40 ml min31 . The detection limit of the TCD for N2 O was 800 ppmv and the response was linear up to 16 kPa. The SRI 8610C GC had the following con¢guration: the TCD was set at 100³C, the FID at 150³C and the ECD at 250³C; the oven was set at 60³C, and each detector was connected to a separate column (2 mU3.1 mm stainless steel packed with Porapak Q (Supelco)). Helium (ultra high purity grade, Prodair) was used as carrier gas with £ow rates of 23 ml min31 for the TCD, 20 ml min31 for the FID and 30 ml min31 for the ECD. The detection limits were : N2 O, 8 ppmv (ECD) or 200 ppmv (TCD); CH4 , 20 ppmv (FID) or 800 ppmv (TCD); and CO2 , 300 ppmv (TCD). The TCD response was linear for N2 O, CH4 , CO2 (up to 20 kPa), the FID response was linear for CH4 up to 1.5 kPa, and the ECD response was linear for N2 O up to 300 ppmv. For gas determinations, 0.5 ml of the gas samples was injected into the GC system with simultaneous integration of peaks using PeakSimpleII software (SRI). Gas standards were injected at the beginning and at the end of each day of analysis. A gas standard which contained 8033 ppmv of each of the following gases : CH4 , CO2 , N2 O and C2 H2 , was prepared at the beginning of each day of analysis. Calibrated gas standards (990 ppmv N2 O in N2 ;

19

1010 ppmv CH4 and 978 ppmv C2 H4 in N2 ) (Prodair) were also used. 2.6. Statistical analysis Rates of hexadecane mineralization were computed by linear regression of the amount of hexadecane mineralized over time using Excel 5. The time period used for the regression was di¡erent for each soil. For soil A, data from day 3 to day 6 showing maximum mineralization were used while for soil B, data from day 4 to day 32 were used. The equality of these rates (slopes) was tested for each soil independently by using the method described in Sokal and Rohlf [27]. For both soils, the rates (slopes) between the various treatments were not all equal. Rates computed for each treatment were then compared using the TP method described in [27] because the sampling times were all the same for a given soil. 3. Results 3.1. Hexadecane mineralization Mineralization of hexadecane in soil A began immediately after the addition of substrate (Fig. 1.I). Mineralization approached a maximum of ca. 60% within 30 days of incubation. Addition of the various N fertilizers had no e¡ect on the mineralization of hexadecane by the indigenous microbial population in this soil. Rates of hexadecane mineralization (days 3^6) varied from 6.7 to 9.4 mg C16 H34 kg31 day31 (Table 2). Addition of C2 H2 (2 kPa) did not inhibit the mineralization of hexadecane in this soil (Fig. 1.II and Table 2). Mineralization of hexadecane in soil B (Fig. 1.III) occurred after a brief lag and at a slower rate than in soil A (Fig. 1.I). In the absence of any N supplement, the rate of hexadecane mineralization (days 4^32) was 0.17 þ 0.01 mg C16 H34 kg31 day31 (Table 2). Addition of NaNO3 increased the rate of hexadecane mineralization while NH4 Cl reduced it when compared to the soil with no N supplement (Table 2). Addition of urea to this soil also decreased the rate of hexadecane mineralization to the same extent as did NH4 Cl. Supplementing the soil with

Table 2 Maximum rates of hexadecane mineralization in two soils with similar levels of C10 -C50 contamination and with various N sources Soil

Water

NH4 Cl

NaNO3

NH4 NO3

Urea

A

3C2 H2 8.8 (0.4)a;b +C2 H2 8.1 (0.5)a;b 3C2 H2 0.17 (0.01)B;C +C2 H2 0.20 (0.02)B;C

9.4 (1.0)a 7.9 (0.8)a;b 0.015 (0.001)C 0.011 (0.001)C

9.3 (0.3)a 8.1 (0.2)a;b 0.91 (0.01)A 0.85 (0.03)A

8.9 (0.4)a;b 8.4 (0.4)a;b 0.13 (0.02)B;C 0.28 (0.07)B

7.2 (0.5)a;b 6.7 (1.0)a;b 0.02 (0.00)C 0.01 (0.00)C

B

Sterile 0.00 (0.00)b 0.00 (0.00)C

Rates are expressed in mg C16 H34 kg31 day31 . Each value represents the regression coe¤cient with its S.E.M. in parentheses. Values with di¡erent letters indicate signi¢cant di¡erences (P = 0.05) between treatments for a given soil based on unplanned comparisons among a set of regression coe¤cients using the TP method [27]. Lower case and upper case indicate independent testing of soil A and soil B.

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Fig. 1. Mineralization of [14 C]hexadecane (100 ppm) in soil A (left) and in soil B (right) in the absence (I, III) and in the presence (II, IV) of C2 H2 (2 kPa). Each data point is the average of triplicate £asks þ 1 S.E.M.

NH4 NO3 did not have a signi¢cant e¡ect on the hexadecane mineralization rate (0.13 þ 0.02 mg C16 H34 kg31 day31 ) when compared to the unsupplemented soil. The addition of C2 H2 (2 kPa) to the headspace of microcosms with soil B did not inhibit hexadecane mineralization (Fig. 1.IV). Regardless of the N supplement, rates of hexadecane mineralization were independent of the presence of C2 H2 (Table 2). 3.2. Denitri¢cation and loss of N fertilizer In the presence of C2 H2 , the accumulation of N2 O was detected after a lag of 10 days in microcosms with soil A when supplemented with NaNO3 or NH4 NO3 , and to a much lesser extent when NH4 Cl or urea were added (Fig. 2.I). Rates of denitri¢cation were higher with NH4 NO3 (1.7 þ 0.2 Wmol N2 O £ask31 day31 ) as the N supplement. Considering the quantity of NO3 3 added initially, it was possible to estimate the amount of N supplement that was lost through denitri¢cation in this soil. It was found that when NH4 NO3 was used, loss of N reached 35% of the initial NO3 3 concentration after 30 days of incubation, while it reached only 10% within the same incubation period when NaNO3 was added as supplement.

Production of N2 O in the presence of C2 H2 was observed in soil B, only when supplemented with NaNO3 or NH4 NO3 but not when this soil was supplemented with other N sources (Fig. 2.III), and the initial time lag before the initiation of denitri¢cation was shorter (4 days). In soil B, denitri¢cation occurred concurrently with hexadecane mineralization, as indicated by their highly significant (P 9 0.01) correlation (r2 = 0.868) [27,28], whereas in soil A, hexadecane mineralization was completed before denitri¢cation had started. Between 24% (NaNO3 ) and 34% (NH4 NO3 ) of the N supplement was lost through denitri¢cation after 32 days incubation. In the absence of C2 H2 , N2 O accumulated in microcosms of soil A (Fig. 2.II). N2 O was emitted from the 16th day of incubation and was especially signi¢cant when NaNO3 (0.34 þ 0.09 Wmol N2 O £ask31 day31 ) or NH4 NO3 (0.47 þ 0.06 Wmol N2 O £ask31 day31 ) were used as N supplements. The molar ratio of N2 O/N2 in this soil was 0.3. The N2 O production rates represent a loss of 9% (NH4 NO3 ) or 3% (NaNO3 ) of the initial NO3 3 supplement after 30 days of incubation. Accumulation of N2 O as a free intermediate in the microcosm headspace was even more signi¢cant in microcosms from soil B (Fig. 2.IV). N2 O was emitted from

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Fig. 2. Denitri¢cation in soil A (left) and in soil B (right) supplemented with hexadecane (100 ppm) in the absence (I, III) and in the presence (II, IV) of C2 H2 (2 kPa). Each data point is the average of triplicate £asks þ 1 S.E.M.

day 4 and thereafter increased continuously in the headspace. N2 O production rates were similar following the addition of NaNO3 or NH4 NO3 (1.1 þ 0.0 Wmol £ask31 day31 ). No N2 O production was observed when other N supplements were added to this soil. The N2 O/N2 molar ratios were 1.0 or 0.7 when NH4 NO3 or NaNO3 were used as N supplements, respectively. Of the initial NO3 3 applied, the amount lost as N2 O after 32 days of incubation was 33% (NH4 NO3 ) or 15% (NaNO3 ). 4. Discussion The C/N ratio is often used as a criterion to decide if N fertilizer would be required for e¡ective bioremediation of a contaminated soil. This ratio is a useful indicator of the N limitation of a system, yet it is not su¤cient to predict the outcome of the bioremediation of a site following application of N fertilizers. For instance, in this study, we investigated two soils with similar petroleum hydrocarbon contamination levels. A lower C/N ratio in soil A (32) than in soil B (81) indicated that the former soil was less N-limited than the latter. Accordingly, mineralization of hexadecane in soil A was 51 times faster than in soil B without nitrate addition. Addition of N fertilizer did not stimulate or inhibit the mineralization of hexadecane in

soil A. These results suggest that hexadecane mineralization in soil A was not N-limited, but it was P-limited if the C/P ratio (698) is considered in this soil. In soil B, addition of NH4 NO3 , NH4 Cl or urea as N sources did not stimulate hexadecane mineralization, unlike other reports generally showing stimulation of petroleum biodegradation following addition of NH4 NO3 or other ammonium salts [1]. Even more surprising, addition of NH4 Cl or urea inhibited hexadecane mineralization. A number of reasons may explain this observation : a pH shift [29] following nitri¢cation, nitrite accumulation from nitri¢cation [30] or inhibition of petroleum-degrad3 ing bacteria by NH‡ 4 or Cl ions [31,32]. The exact mechanism is not known. In this soil, the C/N ratio poorly predicted the e¡ect of N supplementation on hexadecane degradation. These results suggest that the addition of N fertilizers, although improving the C/N ratio, may not necessarily improve the biodegradation of pollutants in contaminated soils. In contrast to the other fertilizers, the addition of NaNO3 to soil B stimulated hexadecane mineralization. Although it was almost ten times lower than in soil A. Stimulation of hexadecane mineralization by NaNO3 in soil B may be explained by: (1) the use of NO3 3 as a N source by bacteria involved in hexadecane mineralization, or (2) as an electron acceptor, in the absence of O2 , by

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denitrifying bacteria involved in the degradation of hexadecane. In order to evaluate the second possibility, we measured denitri¢cation by the C2 H2 blockage technique in soils A and B. Because C2 H2 may a¡ect monooxygenases [5] and possibly those involved in the degradation of alkanes [33] or more simply the growth of certain bacteria [34], it was important to assess the e¡ect of acetylene on hexadecane mineralization in soils A and B. The fact that acetylene had no signi¢cant e¡ect on the rate of hexadecane mineralization in soil A or B with no N supplement validated the use of the C2 H2 blockage technique to measure denitri¢cation [35,15]. A strong correlation between denitri¢cation and hexadecane mineralization in soil B supplemented with NaNO3 would suggest that denitrifying bacteria may have played a role in the mineralization of hexadecane in soil B. However, if denitri¢ers were involved in hexadecane mineralization, it may be expected that rates of hexadecane mineralization in soil B supplemented with NaNO3 would decrease in the presence of C2 H2 because under this condition, denitrifying bacteria are deprived of some energy generation by the blockage of nitrous oxide reductase. No signi¢cant decrease of hexadecane mineralization was observed in the presence of C2 H2 . Moreover, no signi¢cant increase of hexadecane mineralization was observed when NH4 NO3 was applied to soil B although denitri¢cation activity was similar to that observed with NaNO3 . Based on these considerations, it is more likely that assimilation rather than denitri¢cation of NO3 3 stimulated hexadecane mineralization in soil B when supplied with NaNO3 . Assessing denitri¢cation in soils A and B was also of interest because denitri¢ers may be possible competitors with petroleum-degrading bacteria that use NO3 3 as N source. Denitri¢ers may also be responsible for the local loss of N fertilizers through the coupling of denitri¢cation and nitri¢cation. In both tested soils, signi¢cant denitri¢cation occurred following supplementation with NaNO3 or NH4 NO3 . Denitri¢cation also occurred in soil A when treated with NH4 Cl or urea, but the denitri¢cation rates were very low compared to those with NaNO3 and NH4 NO3 and may be related to the presence of endogenous NO3 3 . In soil A, denitri¢cation started after hexadecane mineralization was almost complete, unlike in soil B where denitri¢cation started at the same time as hexadecane mineralization. Rates of denitri¢cation and loss of N fertilizer measured by the C2 H2 blockage technique were similar for both soils studied with either NH4 NO3 or NaNO3 . These results suggest that denitri¢cation may be responsible for a signi¢cant loss of added fertilizer. In summary, the application of N fertilizers may not always lead to increased biodegradation of petroleum hydrocarbons. A speci¢c N fertilizer (here NaNO3 3 with soil B) may be required to speci¢cally stimulate the mineralization of aliphatic petroleum hydrocarbons. The use of other N fertilizers such as NH‡ 4 or urea may lead to a suppression of the mineralization activity in some soils,

the mechanism of which is unknown. The use of the C/N ratio to determine the type and quantity of fertilizer to be added for soil bioremediation may not be su¤cient. The loss of N fertilizer through denitri¢cation may be signi¢cant (10^30% of initial NO3 3 input) and much of this loss may be via N2 O emissions (30^100%). Acknowledgements We thank Suzanne Labelle, Anca Mihoc, Chantale Beaulieu and Ste¨phane Deschamps for their technical assistance. We also thank Prof. Roger Knowles for providing a critical review of the manuscript.

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[16]

[17] [18]

[19]

[20]

[21]

[22]

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