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Effects of sludge-amendment on mineralization of pyrene and microorganisms in sludge and soil
Chemosphere 45 (2001) 625±634
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Eects of sludge-amendment on mineralization of pyrene and microorganisms in sludge and soil Charlotte Klinge
a,1
, Bo Gejlsbjerg a, Flemming Ekelund b, Torben Madsen
a,*
a
b
DHI ± Water and Environment, Agern All e 11, DK 2970 Hùrsholm, Denmark Department of Terrestrial Ecology, Zoological Institute, University of Copenhagen, Universitetsparken 15, DK 2100 Copenhagen é, Denmark Received 12 May 2000; accepted 30 November 2000
Abstract Hydrophobic contaminants sorb to sludge in wastewater treatment plants and enter the soil environment when the sludge is applied to agricultural ®elds. The mineralization of pyrene was examined in soil, in sludge mixed homogeneously into soil, and in sludge±soil systems containing a lump of sludge. Sludge-amendment enhanced the mineralization of pyrene in the soil compared to soil without sludge, and the most extensive mineralization was observed when the sludge was kept in a lump. The number of protozoa, heterotrophic bacteria and pyrene-mineralizing bacteria was much higher in the sludge compared to the soil. The amendment of sludge did not aect the number of protozoa and bacteria in the surrounding soil, which indicated that organic contaminants in the sludge had a little eect on the number of protozoa and bacteria in the surrounding soil Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: Biodegradation; Sludge-amended soil; Bacteria; Protozoa; Pyrene
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants in sewage sludge originating from, e.g. gasoline, coal tar and motor oil, and they are led to wastewater treatment plants primarily with domestic sewage, via run-o from roads and atmospheric deposition from combustion of fuels. A previous monitoring of organic contaminants in sludge showed that the sum of the concentrations of 18 speci®c PAHs in sludge was in the range between 0 and 27 mg/kg on dry weight (d.w.) basis (Tùrslùv et al., 1997). Unsubstituted high molecular weight PAHs like, e.g. pyrene are resistant to
*
Corresponding author. Tel.: +45-451-69200; fax: +45-45169292. E-mail address: [email protected] (T. Madsen). 1 Present address: Novo Nordisk A/S, Laurentsvej 59, bygn. 8K, DK 2880 Bagasvñrd, Denmark.
bacterial attack under strictly anaerobic conditions and are in general not considered toxic to soil organisms (Pothuluri and Cerniglia, 1994). Pyrene sorbs strongly to organic matter (log Kow of 5.18, Edwards et al., 1991; estimated log Koc of 4.55, Abdul et al., 1987) and thus, its bioavailability is limited in organic rich systems like sludge-amended soils. When applied to agricultural ®elds, most of the dewatered sludge is distributed in lumps which disintegrate over time. The presence of essentially anaerobic sludge lumps in the ®eld implies that anaerobic±aerobic gradients will establish in the soil. Wastewater sludge contains a high number of microorganisms including prokaryotic bacteria and eukaryotic protozoa. The bacteria present in sludge may enhance the biodegradation of toxic chemicals in sludge-amended soil, while, on the other hand, protozoa graze on bacteria. The dynamics within the microbial population in soil may be aected either by direct contact with the sludge and the sludge-bound contaminants or by diusion of contaminants into the
0045-6535/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 0 ) 0 0 6 0 2 - 0
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surrounding soil. The aim of this study was to examine the mineralization of a hydrophobic contaminant, pyrene, in sludge-amended soil and to study the eects of sludge-amendment on the numbers of protozoa, total heterotrophic bacteria, and pyrene-degrading bacteria. 2. Materials and methods 2.1. Chemicals Radiolabelled: [4,5,9,10-14 C]pyrene (32.3 mCi/mmol and 58.7 mCi/mmol; radiochemical purity >98%) was purchased from Sigma Chemical (St. Louis, MO, USA). Non-labelled pyrene of analytical purity was purchased from Sigma Chemical (St. Louis, MO, USA). 2.2. Preparation of pyrene±soil and pyrene±sludge complexes The soil used in the experiments was a coarse sandy soil collected from the upper 20 cm of an ecologically cultivated agricultural ®eld in Jyndevad, Denmark. The soil has the following characteristics: Coarse sand, 76.8%; ®ne sand, 12.2%; silt, 4.1%; clay, 3.9%; organic matter, 3.0%, organic carbon, 1.7%; pH 6.0 (Hansen, 1976) and a maximum water holding capacity (WHC) of 40% (w/w) (determined in this study). Dewatered activated sludge was collected from a municipal wastewater treatment plant in Lundtofte, Denmark. The approximate dry matter content of the sludge was 28% (w/w). The sludge was stored at 4°C until use. Before use, the concentration of pyrene in the sludge was determined by gas chromatography±mass spectrometry (GC±MS) as previously described (Madsen and Kristensen, 1997). [14 C]Pyrene or non-labelled pyrene was added to sludge or soil from stock solutions in acetone. Thereafter the solvent was allowed to evaporate for approx. 30 min while ¯ushing with N2 and pyrene was homogeneously mixed into the sludge or the soil. The resulting pyrene± sludge or pyrene±soil complexes were incubated for 24 h at 4°C in the presence of an N2 atmosphere to allow the pyrene to sorb to the sludge or soil and minimize biodegradation during this period.
sludge±soil mixture (d.w.) corresponding to approx. 580,000 dpm per bottle. Parallel 124-ml bottles with soil was prepared by adding 40 g of soil (d.w.) spiked with 40 lg of [14 C]pyrene per kg soil (d.w.) to each bottle. For both systems, Milli-Q water was added to 70% of the WHC. A vial containing 2 ml of 0.5 N KOH was placed inside the bottles to trap 14 CO2 , and the mineralization of the radiolabelled pyrene was followed by liquid scintillation counting (Madsen and Kristensen, 1997). The bottles were left open for approx. 10 min at each replacement of KOH to restore the oxygen content of the headspace. Incubation of the test bottles was static at 15°C in darkness. The experiment was carried out in four replicates and had a duration of 222 days. Experiment B: In order to mimic the sludge-lumps that are present in sludge-amended soil, a lump of 3 g (d.w.) of the pyrene±sludge complex (approx. 1.0 mg [14 C]pyrene/kg d.w.) was placed in a stainless steel net (1mm mesh; 4 4 1:4 cm3 ). The net containing the sludge-complex was surrounded by a 2-cm layer of soil (total: 360 g soil d.w.) in 610-ml glass jars (Fig. 1). Another set of 610-ml glass jars contained the same amounts of [14 C]pyrene-amended sludge and soil but in this case, the pyrene±sludge complex was mixed homogeneously with the soil. The resulting concentration in the homogeneous mixture was thus 8.3 lg of [14 C]pyrene per kg sludge±soil mixture (d.w.). The sludge±soil ratio was 1:120 for both the inhomogeneous and the homogeneous sludge±soil system. Milli-Q water was added to 55% of the maximum WHC and the incubation took place at 15°C in the dark. The headspace volume was 260 ml and there was approximately 1,070,000 dpm [14 C]pyrene in each glass jar. A vial containing 4 ml of 0.5 N KOH was placed
2.3. Sludge±soil mineralization experiments Experiment A: The mineralization of [14 C]pyrene was examined in sludge±soil mixtures and in soil without sludge. In this experiment, the sludge was mixed homogeneously into soil to obtain a sludge:soil ratio of 1:50 dry weight (d.w.). A pyrene±sludge complex (0.8 g d.w. sludge) containing approx. 2.0 mg [14 C]pyrene per kg sludge (d.w.) was mixed homogeneously with 40 g (d.w.) of soil in 124-ml glass bottles. The resulting concentration in the bottles was 40 lg of [14 C]pyrene per kg
Fig. 1. Inhomogeneous sludge±soil mixture (Experiment B). Schematic presentation of experimental set-up. A sludge lump was placed in a glass container and surrounded by soil. Zone 1 (z1) was the sludge lump (4.0 cm wide 4.0 cm deep 1.4 cm high). Zone 2 (z2) was de®ned as soil less than 3 mm from the sludge lump. Zone 3 (z3), the bulk soil, was de®ned as soil more than 15 mm from the sludge lump.
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inside each jar to absorb 14 CO2 , which was quanti®ed as described above. The experiment was carried out in triplicates and had a duration of 80 days. The oxygen concentrations in zones 1±3 (Fig. 1) and in the homogeneous mixtures, were measured at the end of the experiment by use of an oxygen microelectrode (outer diameter, 150 lm; Unisense, Arhus, Denmark) connected to a picoamperemetre (PA2000, Unisense, Arhus, Denmark). A two-point calibration of the electrode was performed in water amended with sodiumdithionite (anaerobic) and water in equilibrium with the atmosphere at 15°C. The measurement in picoampere was then calculated as percentage O2 saturation. Oxygen concentrations were measured at 1 mm steps from the surface of the soil (or sludge lump) by using a micromanipulator. In the experiment with the sludge lump, the soil overlaying the sludge lump was removed before measurements. The oxygen concentrations were measured in triplicates for each zone. The aerobic part of the pro®le was de®ned by an O2 concentration > 1% of atmospheric saturation. At this O2 concentration the average condition in the system is aerobic, whereas aerobic respiration and denitri®cation compete as electron acceptors at lower O2 concentrations (Wiesmann, 1994). After oxygen measurements, the amounts of 14 C in zones 1±3 were quanti®ed: On the basis of the O2 measurements, the outer 2.5 mm of the sludge lump was assumed to constitute the aerobic surface layer. The sludge lump (zone 1) was carefully separated from the soil and divided into two parts (the aerobic surface layer and the anaerobic center). The outer approx. 1.8 mm of the 2.5 mm surface layer was removed and this part was used to determinate the 14 C in the aerobic surface layer. The central part of the sludge (approx. 3.0 mm from the surface to the center of the lump) was used to determine the 14 C in the anaerobic center. The two parts were dried separately at 105°C for 24 h and homogenized before the 14 C was quanti®ed by combustion (600°C, 4 min) of 0.1g subsamples in excess of oxygen. Internal standards were used to correct for color quenching, and controls directly spiked with [14 C]pyrene were used to correct for the eciency of the procedure. 2.4. Calculations The maximum biodegradation rates were estimated by linear regression as percentage of the added 14 C mineralized per day and as lg pyrene per day for each replicate during the period in which the fastest rate was observed (n P 3 and r2 P 0.95). For calculation of the rate in lg pyrene, the background pyrene concentration in sludge was added. The total mineralization at the end of the experiments was calculated as the accumulated 14 CO2 production in percent of the added [14 C]pyrene. For statistics, the Student's t-test for unrelated samples was used.
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2.5. Bacteria and protozoa Glass jars containing sludge distributed either inhomogeneously (Fig. 1) or homogeneously in soil were set up as described for Experiment B with the sole deviation that non-labelled pyrene was used. The controls included soil and sludge without addition of pyrene and acetone, soil and sludge with addition of acetone which was allowed to evaporate, and soil without sludge. All series were prepared in triplicates and incubated at 15°C in darkness. Glass jars were harvested after 0, 6, 15 and 29 days for the inhomogeneous mixture and after 3 and 43 days for the homogeneous mixture. From the harvested jars, 5.0 g wet weight of the material was collected from each of the de®ned zones in the inhomogeneous mixtures and randomly from the homogeneous mixture by pooling samples taken with a glass tube (inner diameter 3 mm). The pooled samples from each zone was diluted in 100 ml of a mineral medium (Ridgway et al., 1990), which was supplemented with 1.3 g Fe-EDTA/l instead of Fe(NH4 2 SO4 2 , and shaken for 30 min. This suspension was diluted and used for enumeration of bacteria and protozoa. The number of protozoa was determined by the most probable number (MPN) method (Darbyshire et al., 1974). By use of this method, an estimate of the total number of protozoa, both the active and encysted, is obtained. Enumeration of protozoa was carried out at 0, 4, 17 and 29 days for the inhomogeneous mixture and after 3 and 43 days for the homogeneous mixture. After shaking for 30 min, the sample was diluted in Ne's amoeba saline medium (NaCl 12 g/l, KH2 PO4 , 13.6 g/l, Na2 HPO4 ; 2H2 O 17.8 g/l, CaCl2 ; 2H2 O, 0.4 g/l, MgSO4 ; 7H2 O, 0.4 g/l) with 0.1 g of Tryptic Soy Broth/l (Difco Laboratories, Detroit, MI, USA). The dierent dilutions were added to microtitre plates (Costar, No 3598, 8 12 wells) and the plates were incubated in darkness at 15°C. Protozoa were counted after one and four weeks by use of a light microscope with 200 magni®cation. The MPN was determined as described by Rùnn et al. (1995). Agar plates were prepared from the mineral medium (Ridgway et al., 1990) by adding 1.5% Bitec agar, 1.0 % tryptone and 0.5% yeast extract (Difco Laboratories, Detroit, MI, USA). Aliquots of inoculum (0.1 ml) from 103 to 107 dilutions of sludge or soil samples were distributed onto the agar plates. The plates were incubated at 20°C and colony forming units (CFU) were enumerated after 6 days. To quantify the number of bacteria capable of using pyrene as the sole source of carbon and energy, agar plates (Noble agar, Difco Laboratories, Detroit, MI, USA) without tryptone and yeast extract were prepared. Aliquots of the inoculum were distributed onto the agar plates as described above. Then 5% (w/v) pyrene in hexane:acetone mixture (1:1 v/v) was sprayed onto the agar plates resulting in white crystal-
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line pyrene layer at the surface (Kiyohara et al., 1982; Heitkamp and Cerniglia, 1988). After incubation at 28°C for up to 14 weeks, colonies surrounded by a transparent zone were regarded as pyrene-degrading bacteria. 3. Results 3.1. Sludge±soil mineralization experiments The chemical analysis of the intact dewatered sludge sample showed that the concentration of contaminant pyrene was 0.2 mg/kg (d.w.) Also other PAHs were found in the sludge, e.g. benzo (b + j + k)¯uoranthene (0.44 mg/kg), ¯uoranthene (0.13 mg/kg), and benzo(a)pyrene (0.10 mg/kg). The mineralization of [14 C]pyrene in soil and in sludge±soil mixtures (Experiment A) showed that the addition of sludge to soil markedly enhanced the mineralization of pyrene (Fig. 2). The experiment was stopped after 222 days, where the mineralization of [14 C]pyrene corresponded to approx. 30% of the 14 C added in the sludge±soil mixture and 12% of the 14 C added in the soil. The mineralization rate in the soil was relatively constant over the entire experimental period. Pyrene-mineralization increased when the sludge was present in a lump compared to the homogeneous sludge±soil mixture (Experiment B, Fig. 3). The maximum rates and the extent of mineralization after 80 days in the two experiments are shown in Table 1. The presence of sludge clearly enhanced the mineralization of pyrene compared to the mineralization in soil. The maximum pyrene mineralization rate (in % per day) in the 1:50 sludge±soil mixture was not signi®cantly dierent from the rate in the 1:120 mixture (Table 1) P > 0:05; n 3; 4:
Fig. 3. Mineralization of pyrene in sludge±soil mixtures (Experiment B). 14 C-labelled pyrene was added to sludge that either was placed as a lump in a glass container and surrounded by soil (see also Fig. 1) or mixed homogeneously with the soil. Vertical bars indicate standard deviations of three replicates. Where invisible, deviations are within the symbols.
Oxygen pro®les were measured in the homogeneous sludge±soil mixture and in each of the three zones (zones 1±3) in the inhomogeneous mixture in (Experiment B, Fig. 4). Aerobic conditions (> 80% of atmospheric saturation) were present in the homogeneous sludge±soil mixture (data not shown). The O2 concentrations were almost stable 6 mm from the surface and below in the homogeneous mixture and in zones 2 and 3 in the inhomogeneous mixtures. In zone 1, the oxygen pro®le showed increasing oxygen concentrations from the center towards the lower surface of the sludge lump. This pro®le was identical to the pro®le from the upper surface to the center shown in Fig. 4. In the inhomogeneous mixture the soil was aerobic (zone 2, less than 3 mm from the lump; and zone 3, more than 15 mm from the lump). The sludge lump (zone 1) was dominated by anaerobic conditions at a depth of approx. 2.5 mm below the surface (O2 concentration < 1% of atmospheric saturation). This indicates that aerobic conditions dominated in the outer 2.5 mm surface layer at the end of the experiment, which corresponded to approx. 51% of the sludge lump. 3.2. Recovery of
Fig. 2. Mineralization of pyrene in soil and in homogeneous sludge±soil mixture (Experiment A). 14 C-labelled pyrene was added to either sludge mixed homogeneously with soil at a ratio of 1:50 or to soil without sludge. Vertical bars of standard deviations of four replicates are within the symbols.
14
C
Recovery of the residual radiolabelled pyrene was determined by combustion of sludge and soil subsamples at the end of Experiment B. Controls showed that by drying samples at 105°C, approx. 10% of the added 14 C was lost from sludge or soil. Therefore, all residual values were calculated from an initial 14 C activity, which was normalized to 90% of the added amount. Combustion of 0.1-g subsamples from the surface layer and the center of the lump yielded approx. 3422 (577) dpm and 21,676 (373) dpm, respectively. Assuming that approx. 51% of the lump was aerobic, the residual 14 C
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Table 1 Maximum mineralization rates in Experiments A and Ba Experiment
Sludge±soil mixture
Conc. of pyrene (mg/kg d.w.)
Mineralized after 80 days (% of added 14 C)
Max. rate (% of added
A A B B
Soil only 1:50 0.120 Sludge lumpb
0.04 0.04 0.0083 1.00
5.2 (0.03)A 19.8 (0.03)B 16.9 (0.17)C 23.6 (0.36)D
0.06 0.37 0.48 0.90
14
C/day)
(0.01)A (0.08)B (0.04)B (0.14)C
Max. rate (lg pyrene/daykg d.w.) 0.024 (0.004)A 0.22 (0.048)B 0.031 (0.003)A 9.04 (1.40)C
a
Values in parentheses are standard deviations of four replicates (Experiment A) and three replicates (Experiment B). Values followed by dierent letters are signi®cantly dierent P < 0:05. Background pyrene concentration in sludge was 0.2 mg/kg d.w. b Average value calculated on the basis of the dry weight of the sludge lump, as pyrene did not diuse to the surrounding soil.
[14 C]pyrene after 80 days corresponded to 23.6% of the added 14 C (Table 1). The total 14 C-recovery in the sludge lump corresponded, therefore, to only 59% of the added radioactivity. Additional control trials revealed that the poor 14 C recovery was mainly caused by loss of 14 C during combustion of dried sludge and soil subsamples (data not shown). A low residual value of 14 C-concentration was observed in zone 2 < 0:6% of the initial concentration in the adjacent zone 1), while no 14 C was found in zone 3, con®rming that only a small amount of the 14 C added to the sludge entered the surrounding soil. Fig. 4. Oxygen saturation measured in the sludge lump and the surrounding soil (inhomogeneously sludge±soil mixture, Experiment B). ± O ± The sludge lump (z1); ± ± Soil less than 3 mm from the lump (z2); ± ± Soil more than 15 mm from the lump (z3). The pro®le was measured from the upper surface of each zone and vertically downwards every 1.0 mm. The pro®le for zone 2 was measured just next to the sludge lump and downwards. Horizontal bars indicate standard deviations of measurements of three replicates. Where invisible, deviations are within the symbols.
which was recovered in the aerobic surface layer and in the anaerobic center represented 5% and 30% of the added 14 C, respectively. The total mineralization of
3.3. Protozoa and bacteria Only few changes in the numbers of protozoa and bacteria were observed during the experiment (Figs. 5 and 6). The results from the enumeration of the protozoa and bacteria show that at all sampling times (except day 4) there were signi®cantly more microorganisms in the sludge compared to the soil P < 0:05; n 3. Only at day 4, the protozoa were more numerous in soil (zone 2) than in sludge P < 0:05; n 3 (Fig. 5). Between the ®rst two samplings the number of protozoa and bacteria increased in the soil (zones 2 and 3; P < 0:05; n 3). The two last samplings showed an
Fig. 5. Total number of protozoa in the sludge lump (z1), soil less than 3 mm from the sludge lump (z2) and soil more than 15 mm from the sludge lump (z3), Experiment B. Columns having one or more letters in common in the superscript are not signi®cantly dierent P > 0:05. Vertical bars indicate standard deviations of three replicates.
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Fig. 6. Total number of bacteria in the sludge lump (z1), soil less than 3 mm from the sludge lump (z2) and soil more than 15 mm from the sludge lump (z3), Experiment B. Columns having one or more letters in common in the superscript are not signi®cantly dierent P > 0:05. Vertical bars indicate standard deviations of three replicates.
increase in the number of protozoa and the number of bacteria had increased in the last sample in the sludge P < 0:05; n 3). With the exception of the number of bacteria at day 5, there was no signi®cant dierence P > 0:05 between numbers of protozoa or bacteria in zones 2 and 3 which indicates that the sludge did not aect the microorganisms in the surrounding soil (Figs. 5 and 6). The number of bacteria decreased between days 6 and 29 in both zones 2 and 3 (P < 0:05; n 3), while no decrease in the number of protozoa in zones 2 and 3 was observed. The average numbers of protozoa and bacteria in homogeneous mixtures were 5:4 104 ( 2:7 104 ) MPN/g (d.w.) and 3:5 107 ( 2:3 107 ) CFU/g (d.w.) mixture, respectively (data not shown). No signi®cant change in these numbers was seen by comparison of samples collected at days 3 and 43. The number of pyrene-degrading bacteria in the sludge was 1:1 106 (7:2 105 ) CFU/g (d.w.), while these constituted only 1:4 104 (1:5 104 ) CFU/g (d.w.) in the soil (detailed data not shown). 4. Discussion 4.1. Sludge±soil mineralization experiments We found that the mineralization of pyrene in an agricultural soil was highly enhanced when the soil was amended with sludge. This could be caused by several factors aecting the biodegradation potential and the bioavailability of pyrene in soil and sludge. The biodegradation potential is primarily determined by the biomass of speci®c degraders and the redox conditions, while the bioavailabilty is determined by complex in-
teractions between the concentration of substrate and the partitioning into dissolved and sorbed fractions and diusion of sorbed substrate in organic matter over time. Pyrene mineralization in soil without sludge-amendment occurred at a lower and more constant rate, than when sludge was added (Fig. 2, Table 1). Sludge consists of a high amount of organic carbon (approx. 26% organic C, dry weight basis, Tùrslùv et al., 1997), and the amount of organic carbon determines the amount of pyrene sorbed. Therefore, the concentration of pyrene in the pore-water was approx. 30% higher in the soil (66 ng/l) compared to the sludge-amended soil (51 ng/l) in Experiment A (based on log Koc (pyrene) 4.55, Kd Koc foc ; foc soil 0:017 and foc (sludge) 0.26). These concentrations are probably well below the expected Km value for degradation of pyrene (Alexander, 1994; Schwartz and Scow, 1999) and therefore, based solely on kinetic considerations, the mineralization rate should be slightly higher in the soil. As the rate, on contrary, was much lower in soil, we suggest that this was primarily caused by a higher number of active pyrene-mineralizing bacteria in the sludge±soil system. This was supported by the counting of pyrene-degrading bacteria in Experiment B, where the number of speci®c pyrene-degraders in the sludge was two orders of magnitude higher than in the soil. The mineralization of pyrene may also have been enhanced because the soil bacteria were stimulated by nutrients in the sludge. Earlier studies have shown enhanced mineralization when soil bacteria were supplied with carbon. Reilley et al. (1996) showed enhanced degradation of pyrene when excess carbon was added to a rhizophere and concluded that an increased bacterial population was the reason,
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whereas Wischmann and Steinhart (1997) observed that compost enhanced PAH degradation in a contaminated soil. The mineralization of pyrene was markedly higher when the pyrene-spiked sludge was applied in a lump compared to the mineralization in a homogeneous mixture, although the conditions in the center of the lump were anaerobic, while the homogeneously mixed system was aerobic (Fig. 4, Table 1). Normally, pyrene is only mineralized under aerobic conditions (e.g. Pothuluri and Cerniglia, 1994; Hurst et al., 1996), although evidence exists that document the mineralization of three- and four-ringed PAHs under anaerobic, denitrifying conditions in pure culture experiments (Coates et al., 1997) and under anaerobic sulphate-reducing conditions in petroleum-contaminated habor sediment (McNally and Mihelcic, 1998). However, we do not regard the anaerobic mineralization of pyrene as a quantitatively important process in sludge or soil as earlier work (Gejlsbjerg et al., in press) showed that no significant pyrene mineralization could be observed in anaerobic soil±sludge mixtures. Therefore, we assume that the mineralization observed in this work occurred mainly in the outer aerobic layer of the sludge lump. The combustion of sludge subsamples showed that only approx. 5% of the added 14 C was present in the aerobic part of the sludge after ended incubation. The recovery of only approx. 30% of the added 14 C in the center of the lump was probably a result of a poor eciency of the sludge combustion procedure which implies that most of the missing 14 C would be related to the anaerobic part of the sludge lump. On basis of the 14 CO2 data and the 14 C recovered in the aerobic part of the sludge, a maximum value of approx. 30% of the added 14 C could be related to degradation of pyrene (mineralized or biomass incorporated) in the aerobic part of the sludge, which constituted approx. 51% of the sludge lump at the end of the experiment. This must be regarded as the maximum aerobic volume during the experiment, as the oxygen penetration into the sludge is expected to increase over time as a result of a decrease in organic matter and water content. The diusion of pyrene from the anaerobic to the aerobic zone within the sludge lump during the experiment was probably of no importance. We observed that less than 0.6% of the added 14 C was found in zone 2 at the end of the experiments and based on the highest possible diusion it is estimated that less than 2% of pyrene initially present in the anaerobic zone could have diused to the aerobic zone during the experiment. Thus, extensive mineralization of pyrene occured in the aerobic part of the sludge lump and the associated aerobic mineralization rate in the sludge was much higher than the mineralization rate in the mixed sludge±soil system. This dierence could partly have been caused by the higher concentration of pyrene in the pore water in the sludge lump compared to the homogeneous mixture
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(a rough estimate based on the relation between Koc ; Kd and foc that were described above gives 108 and 12 ng/l for the sludge lump and the homogeneous mixture, respectively). The higher concentration of pyrene in the sludge lump probably caused a growth in the number of pyrene-mineralizing, which exceeded the growth of these bacteria in the homogeneous sludge±soil mixture. A growth of pyrene-mineralizing bacteria in the sludge lump was indicated by the initial exponential phase in the inhomogeneous mixture in Fig. 3. The concentration of pyrene in the homogeneous mixture may have been lower than the threshold value for bacterial growth (Alexander, 1994). Also the concentration of sorbed pyrene was much higher in the sludge lump. Mineralization of sorbed pyrene may be possible as indicated by Guerin and Boyd (1992) who showed that two bacterial strain were able to use sorbed napthalene. In addition, microbiological mediated desorption (Stucki and Alexander, 1987), and solubilisation of sorbed pyrene by biosurfactants (Willumsen and Karlson, 1996) or by surfactants present in the sludge (Madsen and Kristensen, 1997) may also have increased the bioavailable concentration of pyrene in the sludge. The higher concentration of pyrene present in the sludge lump may also have caused an increased availability of pyrene compared to the homogeneous mixture. Chung and Alexander (1999) and White and Pignatello (1999) showed that an increasing concentration of pyrene and phenanthrene in soil increased the bioavailable fraction of the two PAHs. In our experiments, the mineralization of pyrene reached a stable plateau of approx. 20±30% of the added radioactivity (Figs. 2 and 3). In Experiment B, the plateau was reached at the highest level in the inhomogeneous mixture. Mineralization of PAHs frequently show a biphasic pattern, where the ®rst, rapid mineralization is caused by desorption of PAH partitioned into a less condensed part of the soil organic matter (rubbery phase). The following, slower mineralization phase is caused by the slower desorption from the condensed interior regions in soil organic matter (glassy phase) (Schwartz and Scow, 1999). White et al. (1999) showed that the mineralization rate for phenanthrene in soil was determined by the desorption rate, which decreased over time, and Guthrie and Pfaender (1998) found that the pyrene desorption rate was reduced after a period with biological activity. Although little is known concerning the desorption over time from sludge, it is possible that sequestering and aging processes are of minor importance for the availability of chemicals in sludge compared to soil. The pyrene sorbed to the sludge may be released over time as a result of degradation of the sludge matrix instead of being sequestered into the soil mineral and organic fractions. It has been shown that the decrease in desorption of organic contaminants in soil was mainly due
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to sequestering of the organic contaminants into condensed (glassy) regions of the soil humus. It was also found that the desorption was higher for young than for older humus (Huang and Weber, 1997), and it is likely that sludge could be regarded as young organic material with little or no condensed regions. This implies that the major part of the sorbed contaminants in sludge may be associated with material that allow a relatively high desorption. 4.2. Protozoa and bacteria The rationale behind the de®nition of three zones in the experiment was to investigate the eects of addition of sludge and/or pyrene on the microorganisms in and in close proximity to the sludge. The numbers of protozoa and bacteria (CFU) found in soil and sludge are in accordance with previous studies of soil and organic rich ``hotspots'' (Christensen et al., 1992; Ekelund and Rùnn, 1994; Humphreys and Banks, 1995; Carmichael and Pfaender, 1997; Griths et al., 1998). The addition of pyrene to the sludge and soil did not aect the numbers of protozoa and bacteria (data not shown). We attribute the increase in protozoan numbers in the sludge on days 17 and 29 to an increase in the number of food bacteria, which may have been caused by an increase in the aerobic volume of the sludge lump during the experiment. The samples taken from the sludge lump (zone 1) contained both the aerobic and anaerobic sludge volume and, therefore, greater dierences may have occurred in the aerobic volume of the sludge. The higher concentration of protozoa in the sludge lump compared to the homogeneous mixture and the soil may have resulted in a higher turnover of bacterial biomass in the sludge lump and thereby higher re-mineralization of 14 C incorporated into bacteria. However, probably only a minor part of the pyrene degraded is incorporated into the biomass, so this eect cannot account for all of the increase in mineralization in the sludge lump. Bouchez et al. (1997) showed that for two bacterial strains degrading pyrene, the incorporation into the biomass was approx. 14% of the amount of pyrene mineralized. The initial stimulation of microorganisms in the soil was probably due to the addition of moisture to the airdried soil. The turnover times for bacteria and many small protozoa are only a few hours (Heitkamp and Cerniglia, 1988; Ekelund and Rùnn, 1994; Ekelund, 1996), and hence, the observed numbers of microorganisms might have been dierent, if the samples had been taken at other days. The numbers of bacteria and protozoa in the zones outside the sludge lump were not aected by the sludge indicating limited mobility of the organic compounds in the sludge. This observation is in agreement with the results reported by Rùnn et al. (1999) who found that the stimulating eect of dead root material on protozoa
and bacteria was insigni®cant, a few mm away from the roots. There were also no observable eects on the protozoa and bacteria in our homogeneous sludge±soil mixtures. This indicates that the sludge did not aect the total number of microorganisms in the surrounding soil. The number of pyrene-degrading bacteria was much higher in the sludge compared to the soil. The standard deviation on the number of pyrene-degrading bacteria in the soil was large, but their occurrence was indicated by the mineralization of pyrene in soil without sludge amendment (Fig. 2). Although not detected by the methods used in our experiment, it is possible that the pyrene-mineralizing bacterial population in the sludge had a lower survival when the sludge was mixed homogeneously with the soil due to a shift in the environment or due to the dilution of the pyrene to concentration that did not sustain bacterial growth.
5. Conclusions The following conclusions may be derived from the present study: 1. The dewatered activated sludge which was used in this study had a high potential for mineralization of pyrene and the presence of a high number of pyrene-degrading bacteria in the sludge suggests that these microorganisms were responsible for mineralization of pyrene in the sludge-amended soil. 2. When the dewatered sludge was distributed in lumps, a rapid mineralization of pyrene occurred at the surface of the lumps, and the total amount mineralized was higher than in a homogeneous mixture of sludge and soil. 3. The numbers of protozoa and bacteria were much higher in the sludge compared to the surrounding soil. The numbers of protozoa and bacteria in the soil were not aected by the sludge.
Acknowledgements This study was ®nanced by The Danish Environmental Research Programme, Center for Sustainable Land Use, and Management of Contaminants, Carbon and Nitrogen. Flemming Ekelund was supported by a grant from the Danish Strategic Environmental Research Initiative 1998-2001 (BIOPRO).
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C. Klinge et al. / Chemosphere 45 (2001) 625±634 Alexander, M., 1994. Biodegradation and Bioremediation. Academic press, London. Bouchez, M., Blanchet, D., Besnainou, B., Leveau, J.Y., Vandecasteele, J.P., 1997. Kinetic studies of biodegradation of insoluble compounds by continuous determination of oxygen consumption. J. Appl. Microbiol. 82, 310±316. Carmichael, L.M., Pfaender, F.K., 1997. The eect of inorganic and organic supplements on the microbial degradation of phenanthrene and pyrene in soils. Biodegradation 8, 1±13. Christensen, S., Griths, B., Ekelund, F., Rùnn, R., 1992. Huge increase in bacterivores on freshly killed barley roots. FEMS Microbiol. Ecol. 86, 303±310. Chung, N., Alexander, M., 1999. Eect of concentration on sequestration and bioavailability of two polycyclic aromatic hydrocarbons. Environ. Sci. Technol. 33, 3605±3608. Coates, J.D., Woodward, J., Allen, J., Philip, P., Loveley, D.R., 1997. Anaerobic degradation of polycyclic aromatic hydrocarbons and alkanes in petroleum-contaminated marine harbor sediments. Appl. Environ. Microbiol. 63, 3589±3593. Darbyshire, J.F., Wheatley, R.E., Greaves, M.P., Inkson, R.H.E., 1974. A rapid method for estimating bacterial and protozoan populations in soil. Rev. Ecol. Biol. Sol. 11, 465± 475. Edwards, D.A., Luthy, R.G., Liu, Z., 1991. Solubilization of polycyclic aromatic hydrocarbons in micellar nonionic surfactant solutions. Environ. Sci.Technol. 25, 127±142. Ekelund, F., 1996. Growth kinetics of ®ve common heterotrophic soil ¯agellates. Eur. J. Soil Biol. 32, 15±24. Ekelund, F., Rùnn, R., 1994. Notes on protozoa in agricultural soil, with emphasis on heterotrophic ¯agellates and naked amoebae and their ecology. FEMS Microbiol. Rev. 15, 321± 353. Gejlsbjerg, B., Klinge, C., Madsen, T. Mineralization of organic contaminants in sludge±soil mixtures. Environ. Toxicol. Chem., 20(4), in press. Griths, B.S., Wheatley, R.E., Olesen, T., Henriksen, K., Ekelund, F., Ronn, R., 1998. Dynamics of nematodes and protozoa following the experimental addition of cattle or pig slurry to soil. Soil. Biol. Biochem. 30, 1379±1387. Guerin, W.F., Boyd, S.A., 1992. Dierential bioavailability of soil-sorbed naphthalene to two bacterial species. Appl. Environ. Microbiol. 58, 1142±1152. Guthrie, E.A., Pfaender, F.A., 1998. Reduced pyrene bioavailability in microbially active soils. Environ. Sci. Technol. 32, 501±508. Hansen, L., 1976. Soil types at the Danish state experimental stations (In Danish: Jordtyper ved Statens Forsùgsstationer). Tidsskrift for Planteavl 80, 742±758. Heitkamp, M.A., Cerniglia, C.E., 1988. Mineralization of polycyclic aromatic hydrocarbons by a bacterium isolated from sediment below an oil ®eld. Appl. Environ. Microbiol. 54, 1612±1614. Huang, W., Weber, W.J., 1997. A distributed reactivity model for sorption by soils and sediments: 10 relationships between desorption, hysteresis, and the chemical characteristics of organic domains. Environ. Sci. Technol. 31, 2562± 2569. Humphreys, P.N, Banks, C.J., 1995. The use of sequencing batch activated sludge reactors to determine nitrogen
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balances and optimum periods of pre-areation denitri®cation. Environ. Technol. 16, 549±558. Hurst, C.J., Sims, R.C., Sims, J.L., Sorensen, D.L., McLean, J.E., Huling, S., 1996. Polycyclic aromatic hydrocarbon biodegradation as a function of oxygen tension in contaminated soil. J. Haz. Mat. 51, 193±208. Kiyohara, H., Nagao, K., Yana, K., 1982. Rapid screen for bacteria degrading water-insoluble, solid hydrocarbons on agar plates. Appl. Environ. Microbiol. 43, 454±457. Madsen, T., Kristensen, P., 1997. Eects of bacterial inoculation and nonionic surfactants on degradation of polycyclic aromatic hydrocarbons in soil. Environ. Toxicol.Chem. 16, 631±637. McNally, D.L., Mihelcic, J.R., Luecking, D.R., 1998. Biodegradation of three- and four-ring polyaromatic hydrocarbons under aerobic and denitrifying conditions. Environ. Sci.Technol. 32, 2633±2639. Pothuluri, J.V., Cerniglia, C.E., 1994. Microbial metabolism of polycyclic aromatic hydrocarbons. In: Chaudhry, G.R. (Ed)., Biological Degradation and Bioremediation of Toxic Chemicals. Chapman & Hall, London, pp. 95±125. Reilley, K.A., Banks, M.K., Schwab, A.P., 1996. Organic chemicals in the environment. Dissipation of polycyclic aromatic hydrocarbons in the rhizosphere.. J. Environ. Qual. 25, 212±219. Ridgway, H.F., Safarik, J., Phipps, D., Carl, P., Clark, D., 1990. Identi®cation and catabolic activity of well-derived gasoline-degrading bacteria from a contaminated aquifer. Appl. Environ. Microbiol. 56, 3565±3575. Rùnn, R., Ekelund, F., Christensen, S., 1995. Optimizing soil extract and broth media for MPN-enumeration of naked amoebae and heterotrophic ¯agelates in soil. Pedobiologia 39, 10±19. Rùnn, R., Griths, B., Ekelund, F., Christensen, S., 1999. Spatial distribution and successional pattern of microbial activity and microfaunal populations on decomposing barley roots. J. Appl. Ecol. 33, 662±672. Schwartz, E., Scow, K.M., 1999. Using biodegradation kinetics to measure availability of aged phenanthrene to bacteria inoculated into soil. Environ. Toxicol. Chem. 18, 1742± 1746. Stucki, G., Alexander, M., 1987. Role of dissolution rate and solubility in biodegradation of aromatic compounds. Appl. Environ. Microbiol. 53, 292±297. Tùrslùv, J., Samsùe-Petersen, L., Rasmussen, J.O, Kristensen, P., 1997. Use of waste products in agriculture. Environmental Project No. 366, Danish Environmental Protection Agency (EPA), Copenhagen, Denmark. White, J.C., Hunter, M., Nam, K., Pignatello, J.J., Alexander, M., 1999. Correlation between biological and physical availabilities of phenanthrene in soils and soil humin in aging experiments. Environ. Toxicol. Chem. 18, 1720± 1727. White, J.C., Pignatello, J.J., 1999. In¯uence of bisolute competition on the desorption kinetics of polycyclic aromatic hydrocarbons in soil. Environ. Sci. Technol. 33, 4292±4298. Wiesmann, U., 1994. Biological nitrogen removal from qastewater. Adv. Biochem. Eng./Biotechnol. 51, 113±154. Willumsen, P.A., Karlson, U., 1996. Screening of bacteria, isolated from PAH-contaminated soils, for production of
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biosurfactants and bioemulsi®ers. Biodegradation 7, 415± 423. Wischmann, H., Steinhart, H., 1997. The formation of PAH oxidation products in soil and soil/compost mixtures. Chemosphere 35, 1681±1698.
Bo Gejlsbjerg, M.Sc. from the University of Copenhagen, 1997. Research in biodegradation and ecotoxicity of organic contaminants.
Charlotte Klinge, M.Sc. in ecotoxicology from the University of Copenhagen, 1999.
Torben Madsen, Ph.D. in environmental microbiology from the University of Copenhagen 1991.
Flemming Ekelund, Ph.D. Research includes the taxonomy, ecology and ecotoxicology of soil protozoa.
www.elsevier.com/locate/chemosphere
Eects of sludge-amendment on mineralization of pyrene and microorganisms in sludge and soil Charlotte Klinge
a,1
, Bo Gejlsbjerg a, Flemming Ekelund b, Torben Madsen
a,*
a
b
DHI ± Water and Environment, Agern All e 11, DK 2970 Hùrsholm, Denmark Department of Terrestrial Ecology, Zoological Institute, University of Copenhagen, Universitetsparken 15, DK 2100 Copenhagen é, Denmark Received 12 May 2000; accepted 30 November 2000
Abstract Hydrophobic contaminants sorb to sludge in wastewater treatment plants and enter the soil environment when the sludge is applied to agricultural ®elds. The mineralization of pyrene was examined in soil, in sludge mixed homogeneously into soil, and in sludge±soil systems containing a lump of sludge. Sludge-amendment enhanced the mineralization of pyrene in the soil compared to soil without sludge, and the most extensive mineralization was observed when the sludge was kept in a lump. The number of protozoa, heterotrophic bacteria and pyrene-mineralizing bacteria was much higher in the sludge compared to the soil. The amendment of sludge did not aect the number of protozoa and bacteria in the surrounding soil, which indicated that organic contaminants in the sludge had a little eect on the number of protozoa and bacteria in the surrounding soil Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: Biodegradation; Sludge-amended soil; Bacteria; Protozoa; Pyrene
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants in sewage sludge originating from, e.g. gasoline, coal tar and motor oil, and they are led to wastewater treatment plants primarily with domestic sewage, via run-o from roads and atmospheric deposition from combustion of fuels. A previous monitoring of organic contaminants in sludge showed that the sum of the concentrations of 18 speci®c PAHs in sludge was in the range between 0 and 27 mg/kg on dry weight (d.w.) basis (Tùrslùv et al., 1997). Unsubstituted high molecular weight PAHs like, e.g. pyrene are resistant to
*
Corresponding author. Tel.: +45-451-69200; fax: +45-45169292. E-mail address: [email protected] (T. Madsen). 1 Present address: Novo Nordisk A/S, Laurentsvej 59, bygn. 8K, DK 2880 Bagasvñrd, Denmark.
bacterial attack under strictly anaerobic conditions and are in general not considered toxic to soil organisms (Pothuluri and Cerniglia, 1994). Pyrene sorbs strongly to organic matter (log Kow of 5.18, Edwards et al., 1991; estimated log Koc of 4.55, Abdul et al., 1987) and thus, its bioavailability is limited in organic rich systems like sludge-amended soils. When applied to agricultural ®elds, most of the dewatered sludge is distributed in lumps which disintegrate over time. The presence of essentially anaerobic sludge lumps in the ®eld implies that anaerobic±aerobic gradients will establish in the soil. Wastewater sludge contains a high number of microorganisms including prokaryotic bacteria and eukaryotic protozoa. The bacteria present in sludge may enhance the biodegradation of toxic chemicals in sludge-amended soil, while, on the other hand, protozoa graze on bacteria. The dynamics within the microbial population in soil may be aected either by direct contact with the sludge and the sludge-bound contaminants or by diusion of contaminants into the
0045-6535/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 0 ) 0 0 6 0 2 - 0
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surrounding soil. The aim of this study was to examine the mineralization of a hydrophobic contaminant, pyrene, in sludge-amended soil and to study the eects of sludge-amendment on the numbers of protozoa, total heterotrophic bacteria, and pyrene-degrading bacteria. 2. Materials and methods 2.1. Chemicals Radiolabelled: [4,5,9,10-14 C]pyrene (32.3 mCi/mmol and 58.7 mCi/mmol; radiochemical purity >98%) was purchased from Sigma Chemical (St. Louis, MO, USA). Non-labelled pyrene of analytical purity was purchased from Sigma Chemical (St. Louis, MO, USA). 2.2. Preparation of pyrene±soil and pyrene±sludge complexes The soil used in the experiments was a coarse sandy soil collected from the upper 20 cm of an ecologically cultivated agricultural ®eld in Jyndevad, Denmark. The soil has the following characteristics: Coarse sand, 76.8%; ®ne sand, 12.2%; silt, 4.1%; clay, 3.9%; organic matter, 3.0%, organic carbon, 1.7%; pH 6.0 (Hansen, 1976) and a maximum water holding capacity (WHC) of 40% (w/w) (determined in this study). Dewatered activated sludge was collected from a municipal wastewater treatment plant in Lundtofte, Denmark. The approximate dry matter content of the sludge was 28% (w/w). The sludge was stored at 4°C until use. Before use, the concentration of pyrene in the sludge was determined by gas chromatography±mass spectrometry (GC±MS) as previously described (Madsen and Kristensen, 1997). [14 C]Pyrene or non-labelled pyrene was added to sludge or soil from stock solutions in acetone. Thereafter the solvent was allowed to evaporate for approx. 30 min while ¯ushing with N2 and pyrene was homogeneously mixed into the sludge or the soil. The resulting pyrene± sludge or pyrene±soil complexes were incubated for 24 h at 4°C in the presence of an N2 atmosphere to allow the pyrene to sorb to the sludge or soil and minimize biodegradation during this period.
sludge±soil mixture (d.w.) corresponding to approx. 580,000 dpm per bottle. Parallel 124-ml bottles with soil was prepared by adding 40 g of soil (d.w.) spiked with 40 lg of [14 C]pyrene per kg soil (d.w.) to each bottle. For both systems, Milli-Q water was added to 70% of the WHC. A vial containing 2 ml of 0.5 N KOH was placed inside the bottles to trap 14 CO2 , and the mineralization of the radiolabelled pyrene was followed by liquid scintillation counting (Madsen and Kristensen, 1997). The bottles were left open for approx. 10 min at each replacement of KOH to restore the oxygen content of the headspace. Incubation of the test bottles was static at 15°C in darkness. The experiment was carried out in four replicates and had a duration of 222 days. Experiment B: In order to mimic the sludge-lumps that are present in sludge-amended soil, a lump of 3 g (d.w.) of the pyrene±sludge complex (approx. 1.0 mg [14 C]pyrene/kg d.w.) was placed in a stainless steel net (1mm mesh; 4 4 1:4 cm3 ). The net containing the sludge-complex was surrounded by a 2-cm layer of soil (total: 360 g soil d.w.) in 610-ml glass jars (Fig. 1). Another set of 610-ml glass jars contained the same amounts of [14 C]pyrene-amended sludge and soil but in this case, the pyrene±sludge complex was mixed homogeneously with the soil. The resulting concentration in the homogeneous mixture was thus 8.3 lg of [14 C]pyrene per kg sludge±soil mixture (d.w.). The sludge±soil ratio was 1:120 for both the inhomogeneous and the homogeneous sludge±soil system. Milli-Q water was added to 55% of the maximum WHC and the incubation took place at 15°C in the dark. The headspace volume was 260 ml and there was approximately 1,070,000 dpm [14 C]pyrene in each glass jar. A vial containing 4 ml of 0.5 N KOH was placed
2.3. Sludge±soil mineralization experiments Experiment A: The mineralization of [14 C]pyrene was examined in sludge±soil mixtures and in soil without sludge. In this experiment, the sludge was mixed homogeneously into soil to obtain a sludge:soil ratio of 1:50 dry weight (d.w.). A pyrene±sludge complex (0.8 g d.w. sludge) containing approx. 2.0 mg [14 C]pyrene per kg sludge (d.w.) was mixed homogeneously with 40 g (d.w.) of soil in 124-ml glass bottles. The resulting concentration in the bottles was 40 lg of [14 C]pyrene per kg
Fig. 1. Inhomogeneous sludge±soil mixture (Experiment B). Schematic presentation of experimental set-up. A sludge lump was placed in a glass container and surrounded by soil. Zone 1 (z1) was the sludge lump (4.0 cm wide 4.0 cm deep 1.4 cm high). Zone 2 (z2) was de®ned as soil less than 3 mm from the sludge lump. Zone 3 (z3), the bulk soil, was de®ned as soil more than 15 mm from the sludge lump.
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inside each jar to absorb 14 CO2 , which was quanti®ed as described above. The experiment was carried out in triplicates and had a duration of 80 days. The oxygen concentrations in zones 1±3 (Fig. 1) and in the homogeneous mixtures, were measured at the end of the experiment by use of an oxygen microelectrode (outer diameter, 150 lm; Unisense, Arhus, Denmark) connected to a picoamperemetre (PA2000, Unisense, Arhus, Denmark). A two-point calibration of the electrode was performed in water amended with sodiumdithionite (anaerobic) and water in equilibrium with the atmosphere at 15°C. The measurement in picoampere was then calculated as percentage O2 saturation. Oxygen concentrations were measured at 1 mm steps from the surface of the soil (or sludge lump) by using a micromanipulator. In the experiment with the sludge lump, the soil overlaying the sludge lump was removed before measurements. The oxygen concentrations were measured in triplicates for each zone. The aerobic part of the pro®le was de®ned by an O2 concentration > 1% of atmospheric saturation. At this O2 concentration the average condition in the system is aerobic, whereas aerobic respiration and denitri®cation compete as electron acceptors at lower O2 concentrations (Wiesmann, 1994). After oxygen measurements, the amounts of 14 C in zones 1±3 were quanti®ed: On the basis of the O2 measurements, the outer 2.5 mm of the sludge lump was assumed to constitute the aerobic surface layer. The sludge lump (zone 1) was carefully separated from the soil and divided into two parts (the aerobic surface layer and the anaerobic center). The outer approx. 1.8 mm of the 2.5 mm surface layer was removed and this part was used to determinate the 14 C in the aerobic surface layer. The central part of the sludge (approx. 3.0 mm from the surface to the center of the lump) was used to determine the 14 C in the anaerobic center. The two parts were dried separately at 105°C for 24 h and homogenized before the 14 C was quanti®ed by combustion (600°C, 4 min) of 0.1g subsamples in excess of oxygen. Internal standards were used to correct for color quenching, and controls directly spiked with [14 C]pyrene were used to correct for the eciency of the procedure. 2.4. Calculations The maximum biodegradation rates were estimated by linear regression as percentage of the added 14 C mineralized per day and as lg pyrene per day for each replicate during the period in which the fastest rate was observed (n P 3 and r2 P 0.95). For calculation of the rate in lg pyrene, the background pyrene concentration in sludge was added. The total mineralization at the end of the experiments was calculated as the accumulated 14 CO2 production in percent of the added [14 C]pyrene. For statistics, the Student's t-test for unrelated samples was used.
627
2.5. Bacteria and protozoa Glass jars containing sludge distributed either inhomogeneously (Fig. 1) or homogeneously in soil were set up as described for Experiment B with the sole deviation that non-labelled pyrene was used. The controls included soil and sludge without addition of pyrene and acetone, soil and sludge with addition of acetone which was allowed to evaporate, and soil without sludge. All series were prepared in triplicates and incubated at 15°C in darkness. Glass jars were harvested after 0, 6, 15 and 29 days for the inhomogeneous mixture and after 3 and 43 days for the homogeneous mixture. From the harvested jars, 5.0 g wet weight of the material was collected from each of the de®ned zones in the inhomogeneous mixtures and randomly from the homogeneous mixture by pooling samples taken with a glass tube (inner diameter 3 mm). The pooled samples from each zone was diluted in 100 ml of a mineral medium (Ridgway et al., 1990), which was supplemented with 1.3 g Fe-EDTA/l instead of Fe(NH4 2 SO4 2 , and shaken for 30 min. This suspension was diluted and used for enumeration of bacteria and protozoa. The number of protozoa was determined by the most probable number (MPN) method (Darbyshire et al., 1974). By use of this method, an estimate of the total number of protozoa, both the active and encysted, is obtained. Enumeration of protozoa was carried out at 0, 4, 17 and 29 days for the inhomogeneous mixture and after 3 and 43 days for the homogeneous mixture. After shaking for 30 min, the sample was diluted in Ne's amoeba saline medium (NaCl 12 g/l, KH2 PO4 , 13.6 g/l, Na2 HPO4 ; 2H2 O 17.8 g/l, CaCl2 ; 2H2 O, 0.4 g/l, MgSO4 ; 7H2 O, 0.4 g/l) with 0.1 g of Tryptic Soy Broth/l (Difco Laboratories, Detroit, MI, USA). The dierent dilutions were added to microtitre plates (Costar, No 3598, 8 12 wells) and the plates were incubated in darkness at 15°C. Protozoa were counted after one and four weeks by use of a light microscope with 200 magni®cation. The MPN was determined as described by Rùnn et al. (1995). Agar plates were prepared from the mineral medium (Ridgway et al., 1990) by adding 1.5% Bitec agar, 1.0 % tryptone and 0.5% yeast extract (Difco Laboratories, Detroit, MI, USA). Aliquots of inoculum (0.1 ml) from 103 to 107 dilutions of sludge or soil samples were distributed onto the agar plates. The plates were incubated at 20°C and colony forming units (CFU) were enumerated after 6 days. To quantify the number of bacteria capable of using pyrene as the sole source of carbon and energy, agar plates (Noble agar, Difco Laboratories, Detroit, MI, USA) without tryptone and yeast extract were prepared. Aliquots of the inoculum were distributed onto the agar plates as described above. Then 5% (w/v) pyrene in hexane:acetone mixture (1:1 v/v) was sprayed onto the agar plates resulting in white crystal-
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line pyrene layer at the surface (Kiyohara et al., 1982; Heitkamp and Cerniglia, 1988). After incubation at 28°C for up to 14 weeks, colonies surrounded by a transparent zone were regarded as pyrene-degrading bacteria. 3. Results 3.1. Sludge±soil mineralization experiments The chemical analysis of the intact dewatered sludge sample showed that the concentration of contaminant pyrene was 0.2 mg/kg (d.w.) Also other PAHs were found in the sludge, e.g. benzo (b + j + k)¯uoranthene (0.44 mg/kg), ¯uoranthene (0.13 mg/kg), and benzo(a)pyrene (0.10 mg/kg). The mineralization of [14 C]pyrene in soil and in sludge±soil mixtures (Experiment A) showed that the addition of sludge to soil markedly enhanced the mineralization of pyrene (Fig. 2). The experiment was stopped after 222 days, where the mineralization of [14 C]pyrene corresponded to approx. 30% of the 14 C added in the sludge±soil mixture and 12% of the 14 C added in the soil. The mineralization rate in the soil was relatively constant over the entire experimental period. Pyrene-mineralization increased when the sludge was present in a lump compared to the homogeneous sludge±soil mixture (Experiment B, Fig. 3). The maximum rates and the extent of mineralization after 80 days in the two experiments are shown in Table 1. The presence of sludge clearly enhanced the mineralization of pyrene compared to the mineralization in soil. The maximum pyrene mineralization rate (in % per day) in the 1:50 sludge±soil mixture was not signi®cantly dierent from the rate in the 1:120 mixture (Table 1) P > 0:05; n 3; 4:
Fig. 3. Mineralization of pyrene in sludge±soil mixtures (Experiment B). 14 C-labelled pyrene was added to sludge that either was placed as a lump in a glass container and surrounded by soil (see also Fig. 1) or mixed homogeneously with the soil. Vertical bars indicate standard deviations of three replicates. Where invisible, deviations are within the symbols.
Oxygen pro®les were measured in the homogeneous sludge±soil mixture and in each of the three zones (zones 1±3) in the inhomogeneous mixture in (Experiment B, Fig. 4). Aerobic conditions (> 80% of atmospheric saturation) were present in the homogeneous sludge±soil mixture (data not shown). The O2 concentrations were almost stable 6 mm from the surface and below in the homogeneous mixture and in zones 2 and 3 in the inhomogeneous mixtures. In zone 1, the oxygen pro®le showed increasing oxygen concentrations from the center towards the lower surface of the sludge lump. This pro®le was identical to the pro®le from the upper surface to the center shown in Fig. 4. In the inhomogeneous mixture the soil was aerobic (zone 2, less than 3 mm from the lump; and zone 3, more than 15 mm from the lump). The sludge lump (zone 1) was dominated by anaerobic conditions at a depth of approx. 2.5 mm below the surface (O2 concentration < 1% of atmospheric saturation). This indicates that aerobic conditions dominated in the outer 2.5 mm surface layer at the end of the experiment, which corresponded to approx. 51% of the sludge lump. 3.2. Recovery of
Fig. 2. Mineralization of pyrene in soil and in homogeneous sludge±soil mixture (Experiment A). 14 C-labelled pyrene was added to either sludge mixed homogeneously with soil at a ratio of 1:50 or to soil without sludge. Vertical bars of standard deviations of four replicates are within the symbols.
14
C
Recovery of the residual radiolabelled pyrene was determined by combustion of sludge and soil subsamples at the end of Experiment B. Controls showed that by drying samples at 105°C, approx. 10% of the added 14 C was lost from sludge or soil. Therefore, all residual values were calculated from an initial 14 C activity, which was normalized to 90% of the added amount. Combustion of 0.1-g subsamples from the surface layer and the center of the lump yielded approx. 3422 (577) dpm and 21,676 (373) dpm, respectively. Assuming that approx. 51% of the lump was aerobic, the residual 14 C
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Table 1 Maximum mineralization rates in Experiments A and Ba Experiment
Sludge±soil mixture
Conc. of pyrene (mg/kg d.w.)
Mineralized after 80 days (% of added 14 C)
Max. rate (% of added
A A B B
Soil only 1:50 0.120 Sludge lumpb
0.04 0.04 0.0083 1.00
5.2 (0.03)A 19.8 (0.03)B 16.9 (0.17)C 23.6 (0.36)D
0.06 0.37 0.48 0.90
14
C/day)
(0.01)A (0.08)B (0.04)B (0.14)C
Max. rate (lg pyrene/daykg d.w.) 0.024 (0.004)A 0.22 (0.048)B 0.031 (0.003)A 9.04 (1.40)C
a
Values in parentheses are standard deviations of four replicates (Experiment A) and three replicates (Experiment B). Values followed by dierent letters are signi®cantly dierent P < 0:05. Background pyrene concentration in sludge was 0.2 mg/kg d.w. b Average value calculated on the basis of the dry weight of the sludge lump, as pyrene did not diuse to the surrounding soil.
[14 C]pyrene after 80 days corresponded to 23.6% of the added 14 C (Table 1). The total 14 C-recovery in the sludge lump corresponded, therefore, to only 59% of the added radioactivity. Additional control trials revealed that the poor 14 C recovery was mainly caused by loss of 14 C during combustion of dried sludge and soil subsamples (data not shown). A low residual value of 14 C-concentration was observed in zone 2 < 0:6% of the initial concentration in the adjacent zone 1), while no 14 C was found in zone 3, con®rming that only a small amount of the 14 C added to the sludge entered the surrounding soil. Fig. 4. Oxygen saturation measured in the sludge lump and the surrounding soil (inhomogeneously sludge±soil mixture, Experiment B). ± O ± The sludge lump (z1); ± ± Soil less than 3 mm from the lump (z2); ± ± Soil more than 15 mm from the lump (z3). The pro®le was measured from the upper surface of each zone and vertically downwards every 1.0 mm. The pro®le for zone 2 was measured just next to the sludge lump and downwards. Horizontal bars indicate standard deviations of measurements of three replicates. Where invisible, deviations are within the symbols.
which was recovered in the aerobic surface layer and in the anaerobic center represented 5% and 30% of the added 14 C, respectively. The total mineralization of
3.3. Protozoa and bacteria Only few changes in the numbers of protozoa and bacteria were observed during the experiment (Figs. 5 and 6). The results from the enumeration of the protozoa and bacteria show that at all sampling times (except day 4) there were signi®cantly more microorganisms in the sludge compared to the soil P < 0:05; n 3. Only at day 4, the protozoa were more numerous in soil (zone 2) than in sludge P < 0:05; n 3 (Fig. 5). Between the ®rst two samplings the number of protozoa and bacteria increased in the soil (zones 2 and 3; P < 0:05; n 3). The two last samplings showed an
Fig. 5. Total number of protozoa in the sludge lump (z1), soil less than 3 mm from the sludge lump (z2) and soil more than 15 mm from the sludge lump (z3), Experiment B. Columns having one or more letters in common in the superscript are not signi®cantly dierent P > 0:05. Vertical bars indicate standard deviations of three replicates.
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Fig. 6. Total number of bacteria in the sludge lump (z1), soil less than 3 mm from the sludge lump (z2) and soil more than 15 mm from the sludge lump (z3), Experiment B. Columns having one or more letters in common in the superscript are not signi®cantly dierent P > 0:05. Vertical bars indicate standard deviations of three replicates.
increase in the number of protozoa and the number of bacteria had increased in the last sample in the sludge P < 0:05; n 3). With the exception of the number of bacteria at day 5, there was no signi®cant dierence P > 0:05 between numbers of protozoa or bacteria in zones 2 and 3 which indicates that the sludge did not aect the microorganisms in the surrounding soil (Figs. 5 and 6). The number of bacteria decreased between days 6 and 29 in both zones 2 and 3 (P < 0:05; n 3), while no decrease in the number of protozoa in zones 2 and 3 was observed. The average numbers of protozoa and bacteria in homogeneous mixtures were 5:4 104 ( 2:7 104 ) MPN/g (d.w.) and 3:5 107 ( 2:3 107 ) CFU/g (d.w.) mixture, respectively (data not shown). No signi®cant change in these numbers was seen by comparison of samples collected at days 3 and 43. The number of pyrene-degrading bacteria in the sludge was 1:1 106 (7:2 105 ) CFU/g (d.w.), while these constituted only 1:4 104 (1:5 104 ) CFU/g (d.w.) in the soil (detailed data not shown). 4. Discussion 4.1. Sludge±soil mineralization experiments We found that the mineralization of pyrene in an agricultural soil was highly enhanced when the soil was amended with sludge. This could be caused by several factors aecting the biodegradation potential and the bioavailability of pyrene in soil and sludge. The biodegradation potential is primarily determined by the biomass of speci®c degraders and the redox conditions, while the bioavailabilty is determined by complex in-
teractions between the concentration of substrate and the partitioning into dissolved and sorbed fractions and diusion of sorbed substrate in organic matter over time. Pyrene mineralization in soil without sludge-amendment occurred at a lower and more constant rate, than when sludge was added (Fig. 2, Table 1). Sludge consists of a high amount of organic carbon (approx. 26% organic C, dry weight basis, Tùrslùv et al., 1997), and the amount of organic carbon determines the amount of pyrene sorbed. Therefore, the concentration of pyrene in the pore-water was approx. 30% higher in the soil (66 ng/l) compared to the sludge-amended soil (51 ng/l) in Experiment A (based on log Koc (pyrene) 4.55, Kd Koc foc ; foc soil 0:017 and foc (sludge) 0.26). These concentrations are probably well below the expected Km value for degradation of pyrene (Alexander, 1994; Schwartz and Scow, 1999) and therefore, based solely on kinetic considerations, the mineralization rate should be slightly higher in the soil. As the rate, on contrary, was much lower in soil, we suggest that this was primarily caused by a higher number of active pyrene-mineralizing bacteria in the sludge±soil system. This was supported by the counting of pyrene-degrading bacteria in Experiment B, where the number of speci®c pyrene-degraders in the sludge was two orders of magnitude higher than in the soil. The mineralization of pyrene may also have been enhanced because the soil bacteria were stimulated by nutrients in the sludge. Earlier studies have shown enhanced mineralization when soil bacteria were supplied with carbon. Reilley et al. (1996) showed enhanced degradation of pyrene when excess carbon was added to a rhizophere and concluded that an increased bacterial population was the reason,
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whereas Wischmann and Steinhart (1997) observed that compost enhanced PAH degradation in a contaminated soil. The mineralization of pyrene was markedly higher when the pyrene-spiked sludge was applied in a lump compared to the mineralization in a homogeneous mixture, although the conditions in the center of the lump were anaerobic, while the homogeneously mixed system was aerobic (Fig. 4, Table 1). Normally, pyrene is only mineralized under aerobic conditions (e.g. Pothuluri and Cerniglia, 1994; Hurst et al., 1996), although evidence exists that document the mineralization of three- and four-ringed PAHs under anaerobic, denitrifying conditions in pure culture experiments (Coates et al., 1997) and under anaerobic sulphate-reducing conditions in petroleum-contaminated habor sediment (McNally and Mihelcic, 1998). However, we do not regard the anaerobic mineralization of pyrene as a quantitatively important process in sludge or soil as earlier work (Gejlsbjerg et al., in press) showed that no significant pyrene mineralization could be observed in anaerobic soil±sludge mixtures. Therefore, we assume that the mineralization observed in this work occurred mainly in the outer aerobic layer of the sludge lump. The combustion of sludge subsamples showed that only approx. 5% of the added 14 C was present in the aerobic part of the sludge after ended incubation. The recovery of only approx. 30% of the added 14 C in the center of the lump was probably a result of a poor eciency of the sludge combustion procedure which implies that most of the missing 14 C would be related to the anaerobic part of the sludge lump. On basis of the 14 CO2 data and the 14 C recovered in the aerobic part of the sludge, a maximum value of approx. 30% of the added 14 C could be related to degradation of pyrene (mineralized or biomass incorporated) in the aerobic part of the sludge, which constituted approx. 51% of the sludge lump at the end of the experiment. This must be regarded as the maximum aerobic volume during the experiment, as the oxygen penetration into the sludge is expected to increase over time as a result of a decrease in organic matter and water content. The diusion of pyrene from the anaerobic to the aerobic zone within the sludge lump during the experiment was probably of no importance. We observed that less than 0.6% of the added 14 C was found in zone 2 at the end of the experiments and based on the highest possible diusion it is estimated that less than 2% of pyrene initially present in the anaerobic zone could have diused to the aerobic zone during the experiment. Thus, extensive mineralization of pyrene occured in the aerobic part of the sludge lump and the associated aerobic mineralization rate in the sludge was much higher than the mineralization rate in the mixed sludge±soil system. This dierence could partly have been caused by the higher concentration of pyrene in the pore water in the sludge lump compared to the homogeneous mixture
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(a rough estimate based on the relation between Koc ; Kd and foc that were described above gives 108 and 12 ng/l for the sludge lump and the homogeneous mixture, respectively). The higher concentration of pyrene in the sludge lump probably caused a growth in the number of pyrene-mineralizing, which exceeded the growth of these bacteria in the homogeneous sludge±soil mixture. A growth of pyrene-mineralizing bacteria in the sludge lump was indicated by the initial exponential phase in the inhomogeneous mixture in Fig. 3. The concentration of pyrene in the homogeneous mixture may have been lower than the threshold value for bacterial growth (Alexander, 1994). Also the concentration of sorbed pyrene was much higher in the sludge lump. Mineralization of sorbed pyrene may be possible as indicated by Guerin and Boyd (1992) who showed that two bacterial strain were able to use sorbed napthalene. In addition, microbiological mediated desorption (Stucki and Alexander, 1987), and solubilisation of sorbed pyrene by biosurfactants (Willumsen and Karlson, 1996) or by surfactants present in the sludge (Madsen and Kristensen, 1997) may also have increased the bioavailable concentration of pyrene in the sludge. The higher concentration of pyrene present in the sludge lump may also have caused an increased availability of pyrene compared to the homogeneous mixture. Chung and Alexander (1999) and White and Pignatello (1999) showed that an increasing concentration of pyrene and phenanthrene in soil increased the bioavailable fraction of the two PAHs. In our experiments, the mineralization of pyrene reached a stable plateau of approx. 20±30% of the added radioactivity (Figs. 2 and 3). In Experiment B, the plateau was reached at the highest level in the inhomogeneous mixture. Mineralization of PAHs frequently show a biphasic pattern, where the ®rst, rapid mineralization is caused by desorption of PAH partitioned into a less condensed part of the soil organic matter (rubbery phase). The following, slower mineralization phase is caused by the slower desorption from the condensed interior regions in soil organic matter (glassy phase) (Schwartz and Scow, 1999). White et al. (1999) showed that the mineralization rate for phenanthrene in soil was determined by the desorption rate, which decreased over time, and Guthrie and Pfaender (1998) found that the pyrene desorption rate was reduced after a period with biological activity. Although little is known concerning the desorption over time from sludge, it is possible that sequestering and aging processes are of minor importance for the availability of chemicals in sludge compared to soil. The pyrene sorbed to the sludge may be released over time as a result of degradation of the sludge matrix instead of being sequestered into the soil mineral and organic fractions. It has been shown that the decrease in desorption of organic contaminants in soil was mainly due
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to sequestering of the organic contaminants into condensed (glassy) regions of the soil humus. It was also found that the desorption was higher for young than for older humus (Huang and Weber, 1997), and it is likely that sludge could be regarded as young organic material with little or no condensed regions. This implies that the major part of the sorbed contaminants in sludge may be associated with material that allow a relatively high desorption. 4.2. Protozoa and bacteria The rationale behind the de®nition of three zones in the experiment was to investigate the eects of addition of sludge and/or pyrene on the microorganisms in and in close proximity to the sludge. The numbers of protozoa and bacteria (CFU) found in soil and sludge are in accordance with previous studies of soil and organic rich ``hotspots'' (Christensen et al., 1992; Ekelund and Rùnn, 1994; Humphreys and Banks, 1995; Carmichael and Pfaender, 1997; Griths et al., 1998). The addition of pyrene to the sludge and soil did not aect the numbers of protozoa and bacteria (data not shown). We attribute the increase in protozoan numbers in the sludge on days 17 and 29 to an increase in the number of food bacteria, which may have been caused by an increase in the aerobic volume of the sludge lump during the experiment. The samples taken from the sludge lump (zone 1) contained both the aerobic and anaerobic sludge volume and, therefore, greater dierences may have occurred in the aerobic volume of the sludge. The higher concentration of protozoa in the sludge lump compared to the homogeneous mixture and the soil may have resulted in a higher turnover of bacterial biomass in the sludge lump and thereby higher re-mineralization of 14 C incorporated into bacteria. However, probably only a minor part of the pyrene degraded is incorporated into the biomass, so this eect cannot account for all of the increase in mineralization in the sludge lump. Bouchez et al. (1997) showed that for two bacterial strains degrading pyrene, the incorporation into the biomass was approx. 14% of the amount of pyrene mineralized. The initial stimulation of microorganisms in the soil was probably due to the addition of moisture to the airdried soil. The turnover times for bacteria and many small protozoa are only a few hours (Heitkamp and Cerniglia, 1988; Ekelund and Rùnn, 1994; Ekelund, 1996), and hence, the observed numbers of microorganisms might have been dierent, if the samples had been taken at other days. The numbers of bacteria and protozoa in the zones outside the sludge lump were not aected by the sludge indicating limited mobility of the organic compounds in the sludge. This observation is in agreement with the results reported by Rùnn et al. (1999) who found that the stimulating eect of dead root material on protozoa
and bacteria was insigni®cant, a few mm away from the roots. There were also no observable eects on the protozoa and bacteria in our homogeneous sludge±soil mixtures. This indicates that the sludge did not aect the total number of microorganisms in the surrounding soil. The number of pyrene-degrading bacteria was much higher in the sludge compared to the soil. The standard deviation on the number of pyrene-degrading bacteria in the soil was large, but their occurrence was indicated by the mineralization of pyrene in soil without sludge amendment (Fig. 2). Although not detected by the methods used in our experiment, it is possible that the pyrene-mineralizing bacterial population in the sludge had a lower survival when the sludge was mixed homogeneously with the soil due to a shift in the environment or due to the dilution of the pyrene to concentration that did not sustain bacterial growth.
5. Conclusions The following conclusions may be derived from the present study: 1. The dewatered activated sludge which was used in this study had a high potential for mineralization of pyrene and the presence of a high number of pyrene-degrading bacteria in the sludge suggests that these microorganisms were responsible for mineralization of pyrene in the sludge-amended soil. 2. When the dewatered sludge was distributed in lumps, a rapid mineralization of pyrene occurred at the surface of the lumps, and the total amount mineralized was higher than in a homogeneous mixture of sludge and soil. 3. The numbers of protozoa and bacteria were much higher in the sludge compared to the surrounding soil. The numbers of protozoa and bacteria in the soil were not aected by the sludge.
Acknowledgements This study was ®nanced by The Danish Environmental Research Programme, Center for Sustainable Land Use, and Management of Contaminants, Carbon and Nitrogen. Flemming Ekelund was supported by a grant from the Danish Strategic Environmental Research Initiative 1998-2001 (BIOPRO).
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Bo Gejlsbjerg, M.Sc. from the University of Copenhagen, 1997. Research in biodegradation and ecotoxicity of organic contaminants.
Charlotte Klinge, M.Sc. in ecotoxicology from the University of Copenhagen, 1999.
Torben Madsen, Ph.D. in environmental microbiology from the University of Copenhagen 1991.
Flemming Ekelund, Ph.D. Research includes the taxonomy, ecology and ecotoxicology of soil protozoa.