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Increased microbial catabolic activity in diesel contaminated soil following addition of earthworms (Dendrobaena veneta) and compost
Soil Biology & Biochemistry 40 (2008) 2970–2976
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Increased microbial catabolic activity in diesel contaminated soil following addition of earthworms (Dendrobaena veneta) and compost Zachary A. Hickman, Brian J. Reid* School of Environmental Sciences, University of East Anglia, Norwich, Norfolk NR4 7TJ, UK
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 March 2008 Received in revised form 12 June 2008 Accepted 13 August 2008 Available online 24 September 2008
This study sought to assess the influence of compost and earthworms (Dendrobaena veneta) upon the level of hydrocarbon catabolism in petroleum contaminated forecourt soil (extractable petroleum hydrocarbons (EPH) 10 þ 1.8 g kg1 and total 16 United States Environment Protection Agency (USEPA) polycyclic aromatic hydrocarbons (PAH) 1.62 0.5 g kg1). The catabolic activity of the indigenous microorganisms within uncombined materials (soil and compost) and within the combined treatments (soil plus compost; either with or without earthworms) was assessed by 14C-radiorespirometry (14Chexadecane, 14C-toluene and 14C-phenanthrene). Maximum levels of catabolic activity were observed (at the end of the incubation period; 84 d) for all three compounds in the combined contaminated soil, compost and earthworm mixtures. Significant (p < 0.05) enhancement factors (relative to the soil only control) in catabolic activity in the combined treatments (soil:compost (1:0.5)) of 3.6 times, 1.5 times and 3.5 times were observed for 14C-hexadecane, 14C-phenanthrene and 14C-toluene, respectively; with maximum levels of catabolic activity for these substrates being 68.6 1.7%, 37.9 5.3% and 85.9 1.3%. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Earthworm Hydrocarbon Catabolism Diesel Compost
1. Introduction Catabolically competent microorganisms are often present within soils contaminated with organic compounds. However, biodegradation may be limited on account of: low compound availability/accessibility, oxygen limitations, pH, nutrient limitations, suboptimal C:N:P ratio, temperature, inappropriate moisture conditions, toxic levels of contaminants (or co-contaminants), and/ or inadequate concentrations of terminal electron acceptors (Atlas, 1981; Alexander, 2000; Boopathy, 2000; Reid et al., 2000; Romantschuck et al., 2000; Semple et al., 2003). Thus, bioremediation methodologies often require the optimisation or enhancement of such environmental and biological conditions. It is widely recognised that earthworms can significantly and positively affect the soil environment in terms of soil organic matter dynamics and turnover, improved soil structure, improved soil fertility (Edwards and Bohlen, 1996; Lavelle et al., 2004; Kersante et al., 2006), breakdown of soil particles, substrate aeration and moisture retention and drainage (Edwards and Bohlen, 1996). Many of these actions and factors, such as the excretion of protein rich mucous, reworking and fragmentation of carbon and deposition of cast excreta are stimulators for soil microorganisms (Edwards and Bohlen, 1996; Brown and Doube, 2004). Thus, within
* Corresponding author. Tel.: þ44 1603 592357; fax: þ44 1603 591327. E-mail address: [email protected] (B.J. Reid). 0038-0717/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2008.08.016
the arena of bioremediation, the use of earthworms to improve soil conditions and to subsequently promote microbial numbers, diversity and activity should realise benefits for levels of catabolic activity and subsequent enhancement of organic contaminant biodegradation. Indeed, recent work has shown that earthworms, through their biological, chemical and physical actions upon soils, can assist in increased losses of PAHs (Contreras-Ramos et al., 2006), crude oils (Schaefer and Filser, 2007) and PCBs (Tharaken et al., 2006). A recent article (Hickman and Reid, 2008) provides a review of these manuscripts and the wider literature with respect to how earthworms might assist bioremediation. Beyond the isolated application of earthworms for the bioremediation of hydrocarbon contaminated soil, the co-application of both compost and earthworms is putatively advantageous. Whilst the compost provides additional microbial numbers and diversity, nutrients, pH buffering, and improved moisture retention (Semple et al., 2001), earthworm digestive actions result in greater soil particle surface areas, which could theoretically improve accessibility of bound or sequestered contaminants to degrader microorganisms (Verma and Pillai, 1991; Gevao et al., 2001). Significantly, the release of previously bound contaminants (Gevao et al., 2001), providing they are subsequently degraded, would facilitate attainment of lower bioremediation end points. This work relates the influence of treatment type (namely, contaminated soil only, compost only, and soil:compost mixtures (in ratios 1:0.5 and 1:2 wt/wt), in the presence and absence of earthworms) to observed levels of catabolic activity assessed by
Z.A. Hickman, B.J. Reid / Soil Biology & Biochemistry 40 (2008) 2970–2976 14 C-hydrocarbon respirometry. Ultimately, this work sought to establish if earthworms and compost, as individual additions and in combination with each other, enhanced hydrocarbon catabolic activity. Catabolic potential was assessed by 14C-radiorespirometry using three representative hydrocarbons, namely, 14C-hexadecane, 14 C-toluene and 14C-phenanthrene. These hydrocarbons are commonly found in petroleum impacted soils and have previously been used in other studies (Foght et al., 1990; Robinson et al., 1990; Geerdink et al., 1996; Prenafeta-Boldu et al., 2002; Reid et al., 2002; Ostberg et al., 2007).
2. Materials and methods Treatments included soil and compost mixtures at ratios of 1:0.5 and 1:2 (wt/wt), in addition to contaminated soil only and compost only control treatments. For 84 d, half of the treatments were incubated with earthworms (W) (ten earthworms per kg material), and half without earthworms (NW) (n ¼ 5 of each treatment). Samples were taken at 0 d and 84 d for 14C-respirometer assays. 2.1. Experimental set-up Mature compost (C:N ratio of 19:1; moisture content of 70% of maximum water holding capacity (WHC); pH of 7.2) was produced from shredded leaves, grass and other green material (50%) and stalks and woodier materials (50%) that underwent rapid composting utilising an enclosed force aerated system. The composted material continued to ‘mature’ outdoors for approximately 4 months prior to use. Aged petroleum contaminated soil (EPH 10 1.8 g kg1; total 16 USEPA PAH 1.62 þ 0.5 g kg1 (hereafter referred to as SPAH); sand 0.8%, silt 42.7% and clay 56.4%; organic matter content of 0.8% (mass loss on ignition); C:N ratio of 8:1; pH of 6.2) was used in this experimental study. Plastic vessels (10 L capacity) measuring 35 cm in height and 40 cm in diameter were used to contain the treatments. The vessels had plastic piping (7.5 mm diameter) inserted and sealed into three holes in the base, which were attached to an air compressor, and aerated for 15 min every 24 h. No further manual mixing was undertaken throughout the study. Silvaperl washed gravel (obtained from a building supplies store) was washed in water and laid to a thickness of 3 cm in the base of each vessel (to promote more uniform air dissipation). The gravel was then covered with a perforated plastic sheet that allowed air to percolate up from the base and disallowed the migration of earthworms into the gravel. Soil and/or compost was rehydrated and manually mixed into the experimental vessel. The mass of the contaminated soil (at 70% of maximum WHC) was kept constant at 2 kg (wet weight), equivalent to 1.82 kg dry weight. The compost substrate (at 70% of maximum WHC) was added as wet weight at the ratios of: 0.5 (1 kg) and 2 (4 kg). This equated to 0.78 kg and 3.16 kg dry weight, respectively. The contaminated soil and the compost only treatments contained only 2 kg (wet weight) of either contaminated soil or compost, respectively. Adult earthworms (Dendrobaena veneta) were depurated (laid on damp filter paper in an enclosed plastic petri dish) for 24 h prior to use. Ten earthworms per kg of material (each depurated earthworm weighing 1.0 0.2 g) were added to each of the required treatments and replicates. The earthworms were combined with the material (resulting in 1:100 biomass:material w/w ratio) and perforated lids were sealed around the vessels. Water was added periodically to maintain levels at 70% of maximum WHC. It was noted from prior research (data not shown) that D. veneta, when compared to other widely used earthworm species (in this instance Lumbricus rubellus and Eisenia fetida), outperformed them in terms of survival and tolerance of hydrocarbon contaminated soils (up to
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3 g kg1 total PAH). It was thus felt justifiable to utilise D. veneta within this study to ensure survival for the incubation period. 2.2. Hydrocarbon and +PAH analysis Extractable Petroleum Hydrocarbons (EPH – C10–C40) were P quantified by GC-FID and PAH by GC–MS. Initially, samples (5 g; n ¼ 3 of all treatments) were extracted using an end over end shake extraction method with 50:50 (Hexane:Acetone) at a soil:solvent ratio of 1:10 for 1 h. GC–FID analysis was undertaken using a Hewlett Packard 5890/6890 FID with a Zebron ZB-1 (Phenomenex, UK) fused silica capillary column (15 m 0.32 mm 1 mm) using helium at a constant pressure of 15 psi at an initial flow of 1.5 ml min1 with nitrogen at 30 ml min1, hydrogen at 30 ml min1 and air at 300 ml min1 and a split injection (2:1 ratio) of 2.0 ml at a flow of 7.1 ml min1. The column oven was set at 60 C for 1.0 min, 60–325 C programmed at 15 C min1 and 325 C for 2.0 min. GC–MS was undertaken using a Hewlett Packard 6890 Gas Chromatograph with HP7683 series injector and HP7683 series autosampler and a Hewlett Packard 5973 MSD Mass Selective Detector. A fused silica capillary column was used with measurements of 15 m 0.25 mm 0.1 mm using helium at a constant flow of 1 ml min1 and a pulsed splitless injection of 1.0 ml at 20 ml min1. The column oven was set at 49 C for 1.25 min, 49– 250 C at 25 C min1, 250–300 C at 35 C min1 and 300–340 C at 60 C min1. 2.3. Assessment of catabolic activity by
14
C-respirometry
Schott bottles (250 ml) were adapted such that a glass vial (7 ml) could be suspended from the lid. This vial contained GF/A filter paper (20 mm 20 mm) and 1 M sodium hydroxide (1 ml; supplied by Merck, UK) to ‘trap’ mineralised 14CO2 (Allan et al., 2007). Samples (10 g) to be screened for catabolic activity (contaminated soil, compost, 1:0.5 and 1:2 (soil:compost (wt/wt)) were slurried in respirometers with sterile distilled water (30 ml)). 14C-9-Phenanthrene, 14C-1-hexadecane and 14C-UL-toluene (added as individual compounds) (all supplied by Sigma, UK; radioactive and chemical purity >95%) were spiked into the slurried respirometers, using toluene as the carrier solvent, such that 100 ml of spike delivered 200 Bq per respirometer. Respirometers were continuously shaken at 100 rpm using an IKA Labortechnik KS 501 digital flatbed shaker and sampled periodically until 648 h (27 d) assay time had elapsed. Spiking efficiency was determined to be >94 1.0%. Ultima Gold (6 ml) was added to changed vials, which were stored in darkness for a minimum of 48 h prior to analysis. Trapped 14CO2 was determined by liquid scintillation counting, using a Canberra Packard Tri-Carb 2900TR liquid scintillation counter for 10 min per sample. 3. Results 3.1. Earthworm survival Following the initial incubation period, prior to the catabolism study, it was noted that earthworm survival was highest in the compost treatments (70 12%) and lowest in the contaminated soil control (15 13.2%). Following addition of compost to the soil, the treatments showed significantly higher survival with increasing compost amounts (p < 0.05). Survival in the 1:0.5 and 1:2 treatments were 23 8.5% and 64 15%, respectively. Thus, as the contaminated soil content increased, earthworm mortality increased, which was attributed to soil toxicity and the possibility of inappropriate food quality and availability, as previously noted as detrimental factors (Edwards and Bohlen, 1996; Schaefer and Filser, 2007).
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Whilst reproduction was not considered quantitatively, juvenile earthworms were recovered at 84 d from all treatments, indicating reproduction had occurred. In addition to this, cocoons were also noted in all treatments, further indicating that toxicity was not so great as to inhibit reproduction.
materials were combined, levels of 14C-phenanthrene catabolic activity were significantly (p < 0.05) higher (68.5 14.0% and 68.4 14.1% in the 1:0.5 and 1:2 treatments, respectively) with respect to the soil only control. 3.4. Temporal changes in catabolic activity in the soil only controls
3.2. Hydrocarbon loss In general terms, both the addition of compost and the addition of earthworms to contaminated soil, 1:0.5 and 1:2 treatments P resulted in enhanced loss of EPH (C10–C40) and PAH over the 84 d P incubation period (Table 1). EPH values have had PAH values P subtracted, in order to reflect losses exclusive of PAH. Whilst both the NW and W contaminated soil only treatments saw significant (p < 0.05) losses following the incubation period (with respect to their start concentrations), they retained the greatest residual EPH P and PAH concentrations of all the treatments studied. Addition of compost without earthworms at both ratios (1:0.5 and 1:2) resulted in significantly (p < 0.05) enhanced losses of both EPH and P PAH with respect to the soil only control. Finally, where compost and earthworms were used in combination, further significant P (p < 0.05) reductions in both EPH and PAH concentrations were observed. The relative extents of loss of hydrocarbons from the treatments are discussed in subsequent sections with respect to how they relate to levels of catabolic activity within uncombined (soil and compost) and combined (1:0.5 and 1:2) treatments at study start (0 d) and end (84 d). 3.3. Catabolic activity in unblended substrates and mixtures at 0 d The level of 14C-hexadecane catabolic activity (Fig. 1A) was not significantly different (p > 0.05) in the soil (41.3 þ 0.6%) and compost (43.3 þ 6.5%) only treatments. Upon combination of contaminated soil and compost, no significant increase (p > 0.05) in 14 C-hexadecane catabolic activity was observed in the 1:2 mixture (43.8 þ 2.8%). However, 14C-hexadecane catabolic activity was significantly (p < 0.05) increased in the 1:0.5 treatment (54.0 þ 4.4%) with respect to the soil only, compost only and 1:2 treatments. The level of 14C-toluene catabolic activity (Fig. 1B) in the contaminated soil only control was the lowest of all compounds/ treatments (8.8 2.2%). However, the level of 14C-toluene catabolic activity was significantly (p < 0.05) higher, almost three times higher (32.8 12.0%), in the compost only control. When soil and compost were combined, both mixed treatments indicated significantly (p < 0.05) higher levels of 14C-toluene catabolic activity (27.0 2.2% and 35.7 4.0%, in the 1:0.5 and 1:2 treatments, respectively) that were comparable to that of the compost only control. The level of 14C-phenanthrene catabolic activity (Fig. 1C) was observed to be high in the soil only treatment (48.7 2.5%) and higher still in the compost only treatment (58.7 6.4%). When Table 1 P EPH and PAH residual concentrations (% of start concentration) for contaminated soil (CS), 1:0.5 and 1:2 treatments at 84 d in the absence (NW) and presence (W) of earthworms P Treatment EPH residual concentration PAH residual concentration (% of start concentration) (% of start concentration)
CS 1:0.5 1:2
84 d NW
84 d W
84 d NW
84 d W
55.4 5.3Aa 19.0 5.0Ba 11.5 3.0Ca
33.4 4.6Ab 15.8 4.0Ba 5.0 2.4Cb
69.2 6.4Aa 40.9 9.3Ba 20.1 1.3Ca
65.3 9.3Aa 17.9 3.4Bb 7.7 4.9Cb
Same upper case letters within columns for each compound represent no significant difference (p>0.05). Same lower case letters between columns for either EPH or P PAH represent no significant difference between with and without earthworm treatments (p > 0.05).
With respect to the 0 d NW contaminated soil only treatments, levels of catabolic activity were noted to be high in the case of 14 C-hexadecane (41.3 0.6%) (Fig. 2A) and 14C-phenanthrene (48.7 2.5%) (Fig. 2C), but much lower in the case of toluene (8.8 2.2%) (Fig. 2B). Following 84 d, the levels of catabolic activity decreased by 64.6% (to less than half the original level) for 14 C-hexadecane (p < 0.05), while only a small decrease (p > 0.05) was observed in the level of 14C-phenanthrene catabolic activity (a decrease of 7.7%). In contrast, the levels of 14C-toluene catabolic activity increased markedly by 51.8% (to approximately twice the original level) (p < 0.05). Following the 84 d treatment period, the NW EPH concentrations (Table 1) were observed to drop to 55.4 5.3% of their start concentrations (loss of 26.8%) in the soil only controls. In contrast, P over the same period, the PAH concentrations decreased to a lesser extent to 69.2 þ 6.4% (loss of 19.5%) in the soil only controls, mirroring, to some extent the losses of these representative 14 C-hydrocarbons. Where earthworms had been present within the contaminated soil only treatments (Fig. 2), levels of catabolic activity were not found to be significantly different (p > 0.05), when compared to the NW treatments at 84 d, for both 14C-hexadecane (19.1 þ 1.8%) (Fig. 2A) and 14C-toluene (19.7 þ 1.3%) (Fig. 2B). However, in the case of 14C-phenanthrene (Fig. 2C), a positive and significant (p < 0.05) influence on catabolic activity (58.0 þ 1.6%) was observed when earthworms were present. 3.5. Influence of compost ratio and earthworm presence on catabolic activity at 84 d Following the incubation period of 84 d (Table 1), it was observed that for all compounds maximum levels of catabolic activity were present in treatments that contained contaminated soil, compost and earthworms together. With respect to 14C-hexadecane (Fig. 3A), the NW 1:0.5 and 1:2 contaminated soil:compost treatments resulted in a significantly (p < 0.05) greater catabolic ability in comparison to the NW and W soil only control, suggestive of the beneficial use of compost alone even at its lowest ratio. However, no significant difference (p > 0.05) was observed between extents of mineralisation in the NW 1:0.5 and 1:2 soil and compost treatments (28.2 1.4% and 29.4 1.3%, respectively). The 1:0.5 and 1:2 soil:compost W treatments (for 84 d) indicted significantly higher (p < 0.05) levels of 14C-hexadecane catabolic activity, with respect to the NW and W soil only controls and the NW 1:0.5 and 1:2 treatments. The increase was marked, with mineralisation extents of 68.6 1.7% and 53.2 2.6%, for 1:0.5 and 1:2, respectively. These results were noted to be much higher than that in the W control soil treatment (19.1 1.8%). Considering 14C-toluene (Fig. 3B), the same general trends were observed wherein 1:0.5 and 1:2 soil:compost substrates without earthworms, resulted in a significant (p < 0.05) increase in toluene catabolic activity, with respect to the NW and W soil only control treatments. Again, as was the case for 14C-hexadecane, no significant differences (p > 0.05) were observed between levels of catabolic activity in the 1:0.5 and 1:2 soil:compost substrates (33.7 5.3% and 37.9 1.0%, respectively). The 1:0.5 and 1:2 W soil:compost treatments indicted significantly higher (p < 0.05) levels of 14C-toluene catabolic activity with
Mineralisation (% Relative to start activity)
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100 80
A
CS Compost 1:0.5 1:2
60 a
b b
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B
b
c b
a
C
b
b a
a
40 a 20 0
Fig. 1. Catabolic activity (% mineralisation) in contaminated soil (CS), compost, 1:0.5 and 1:2 treatments at 0 d for 14C-hexadecane (A), 14C-toluene (B) and Error bars represent SE of the mean. Same lower case letters signify no significant difference (p > 0.05) between extents of mineralisation.
respect to the NW and W soil only treatments/controls. This increase in catabolic activity was marked, with mineralisation extents of 68.6 1.7% and 56.2 2.1% for 1:05 and 1:2, respectively. These results were again noted to be much higher than in the W soil only treatment (16.7 1.3%). It was again observed that the 1:0.5 and 1:2 substrates which had had earthworms incubated within them for 84 d had significantly (p < 0.05) greater catabolic activity than in comparison to 0 d, whilst the same treatments without earthworms determined lower catabolic activity than at 0 d. Finally, considering 14C-phenanthrene (Fig. 3C), it was noted that compost applied to the contaminated soil at the ratios of 1:0.5 and 1:2 significantly increased (p < 0.05) levels of 14C-phenanthrene catabolic activity with respect to the NW and W soil only treatments. Although significantly different (p < 0.05) to each other, the 1:0.5 and 1:2 soil:compost treatments indicated similar levels of catabolic activity (67.7 1.7% and 73.0 0.9%, respectively). Again, the W 1:0.5 and 1:2 contaminated soil:compost treatments resulted in the highest levels of 14C-phenanthrene catabolic activity observed. The increase was marked, with levels of catabolic activity of 85.9 1.3% and 73.0 2.6% for 1:05 and 1:2, respectively. These results were again noted to be much higher than that in the W soil treatment (58.0 1.7%). 4. Discussion 4.1. Catabolic activity in unblended substrates and mixtures at 0 d
Mineralisation (% relative to start activity)
It was noted that levels of 14C-hexadecane catabolic activity were high in all substrates and mixtures (>40%). This is to be expected on account of hexadecane (C16) being a lighter straight chained alkane, susceptible to degradation by beta-oxidation (Alexander, 1999); a ubiquitous catabolic pathway. High levels of hexadecane catabolic activity are consistent with other works stating the preferential, or ease of, degradation of hexadecane, or
80
60
C-phenanthrene (C).
similar light alkanes (Walker and Colwell, 1976; Atlas, 1995; Kastner et al., 1995; Olsen et al., 1999; Prince et al., 2007). We submit that the enhanced catabolism was likely to be due to the compost’s incumbent positive characteristics such as microbial numbers and diversity, moisture retaining ability, nutrient input and increased aerobicity (Semple et al., 2001). Although other research has shown hydrocarbon losses when utilising compost (Kastner et al., 1995; Kastner and Mahro, 1996; Namkoong et al., 2002; Sasek et al., 2003; Antizar-Ladislao et al., 2004) it was noted that in our results there was a lack of a lag period; with 14C-hexadecane catabolic activity being well established at 0 d. The observed toluene catabolic activity was initially low, but such observed extents of toluene degradation are not uncommon in mixed gasoline treatments (Yerushalmi and Guiot, 1998); with toluene degradation by fungi, an important microbial component of compost, being previously noted (Yadav and Reddy, 1993; Prenafeta-Boldu et al., 2002). The 14C-phenanthrene losses were in keeping with previous results that noted catabolic competence for phenanthrene in both compost and soil–compost mixtures (Kastner and Mahro, 1996; Cajthaml et al., 2002; Reid et al., 2002; Sasek et al., 2003; Puglisi et al., 2006). Overall, these results indicate that levels of catabolic activity, with respect to 14C-hexadecane and 14C-phenanthrene, were already high in the contaminated soil while levels of toluene catabolic activity were initially low. Given the history of the contamination (petrol filling station forecourt), pre-exposure to aliphatic hydrocarbons, PAHs and to a lesser extent toluene, support the observed levels of catabolic activity in the soil. With respect to all compounds, levels of catabolic activity were higher in the soil plus compost treatments when compared to the soil only treatments. Finally, mixing soil with compost resulted in increased catabolic activity for all substrates; the degrees of enhancement being greatest where soil only catabolic activity levels were lowest, i.e. enhancement in catabolic activity in the order 14C-toluene > 14 C-phenanthrene > 14C-hexadecane.
A
0d 84 d NW 84 d W
14
B
b a
C
a
a 40 b 20
b a
b
b
0 Fig. 2. Catabolic activity (% mineralisation) for CS at 0 d and 84 d (NW and W) for 14C-hexadecane (A), 14C-toluene (B) and mean. Same lower case letters signify no significant difference (p > 0.05) between extents of mineralisation.
14
C-phenanthrene (C). Error bars represent SE of the
Z.A. Hickman, B.J. Reid / Soil Biology & Biochemistry 40 (2008) 2970–2976
Mineralisation (% relative to start activity)
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100 80
b
a
b
a
a
C
b
b a
40
0
b
b
60
20
B
A
NW W
a* a
a*
b
CS
a* a
1:0.5
1:2
a*
a
CS
1:0.5
1:2
CS
1:0.5
1:2
Fig. 3. Catabolic activity in CS, 1:0.5 and 1:2 treatments at 84 d for 14C-hexadecane NW and W (A), 14C-toluene NW and W (B) and 14C-phenanthrene NW and W (C). Error bars represent SE of the mean. Same lower case letters signify no significant differences (p > 0.05) between NW and W treatments. An asterisk signifies no significant difference between corresponding treatments.
4.2. Temporal changes in catabolic activity in the soil only controls Both the 84 d treatment results and the catabolic assessment results reflect the fact that when the contaminated soil was initially added to the treatment vessels, the indigenous microbial populations had a preferential environment (inputted oxygen and moisture) in which to rapidly multiply. The reduced 14C-hexadecane catabolic activity at 84 d for the NW treatment would suggest a shift in microbial populations towards other hydrocarbon degradation as the concentration of this substrate reduced; thus highlighting the complex relationship that exists between degrader communities, their hydrocarbon preferences and soil contaminant loadings. Relevantly to this study, Walker and Colwell (1976) identified mineralisation rates of 14C-hydrocarbons to be in the order of 14C-hexadecane > 14C-naphthalene > 14C-toluene. They stated that 14C-naphthalene and 14C-hexadecane mineralisation extents were similar, although 14C-hexadecane rates were much higher. It should be borne in mind that degradation extents and rates of individual classes of hydrocarbons, or of individual hydrocarbons are not straightforward, especially when present as a complex mixture such as with diesel and petroleum contaminations. Such preferential degradation has previously been noted by Walker and Colwell (1976), Atlas (1995) and Geerdink et al. (1996). With respect to preferential hydrocarbon utilisation, Atlas (1995) established the following relationship between hydrocarbon structure and resistance to biodegradation: n-alkanes < branched and cyclic alkanes < aromatics < polar compounds, stating that within each structural class, alkyl branching or substitution will further complicate degradation ability. More recently, Prince et al. (2007) determined that during aerobic degradation of gasoline, n-alkanes, iso-alkanes, simple and alkylated aromatic compounds were the most readily degraded, followed by the smaller n-alkanes and iso-alkanes and napththenes. This research is complemented by others, who also observed similar orders of degradation (Foght et al., 1990; Leahy and Colwell, 1990; Robinson et al., 1990; Olsen et al., 1999). In light of the reduction (and of the putative limitation) in easily degradable hydrocarbons, catabolic activity for the more recalcitrant compounds e.g. 14C-phenanthrene and 14C-toluene had been maintained or enhanced, respectively. The limited 14C-toluene degradation at 0 d was attributed to the assumed low start toluene degrader numbers. Whilst the BTEX group can represent a large proportion of commercial petroleum (Marchall et al., 2003), these compounds are volatile and following soil extraction and subsequent manual mixing and preparation, it is conceivable (although not measured) that only minimal percentage of toluene start concentration remained (Liste et al., 2002). The increased toluene
catabolic ability at 84 d reflects the fact that hydrocarbon losses were extensive during the 84 d incubation period and as a consequence (discussed above) more labile substrates will have been depleted. This, in combination with an improved incubation environment, will have promoted the catabolic activity for more recalcitrant compounds, and specifically, toluene. Relevantly, Yerushalmi and Guiot (1998) reported toluene degradation to be much slower in mixed gasoline when compared to its degradation as a sole substrate. Relevantly, earthworms are efficient at grinding soil into smaller fractions via their digestion mechanisms (Bolan and Baskaran, 1996) and it is hypothesised that such actions can result in the release of previously bound contaminants (Barois et al., 1993; Gevao et al., 2001), thus making them available for microbial degradation. 4.3. Influence of compost ratio and earthworm presence on catabolic activity at 84 d Interestingly, the 1:0.5 and 1:2 soil:compost W treatments had significantly (p < 0.05) greater 14C-hexadecane and 14C-toluene catabolic activity, at 84 d, in comparison to 0 d, whilst the same treatments without earthworms determined lower catabolic activity than at 0 d. Thus, earthworm inclusion dictated continued and increased soil/compost catabolic activity whilst a decline was observed in the without earthworm treatments. The increased catabolic activity may partly be attributed to earthworm digestion processes fragmenting soil particles, enhancement of hydrocarbon degraders, provision of increased nutrients to degraders and inoculation of the egested casts with microorganisms (Edwards and Bohlen, 1996). The resultant casts have been reported to be both nutrient and microbially rich and therefore would have assisted in the proliferation of microbial numbers and activity (Edwards and Bohlen, 1996; Brown and Doube, 2004). The decline in catabolic activity can be related to the changes in hydrocarbon concentrations (Table 1) and thus reflected changes in the soil microbial numbers, diversity and activity. With respect to all compounds, these results indicated that the addition of compost to contaminated soil in the ratio of 1:0.5 and 1:2 subsequently determined significantly greater hydrocarbon losses in comparison to the contaminated soil only (Table 1). Whilst compost’s inherent abiotic and biotic properties were likely to have determined observed mineralisation extents, as is supported by previous work (Kastner et al., 1995; Namkoong et al., 2002; Reid et al., 2002; Antizar-Ladislao et al., 2004; Sasek et al., 2003), compost addition to contaminated soil was not as effective at promoting extensive mineralisation as was the co-application of compost and earthworms.
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The combined addition of earthworms and compost to contaminated soil resulted in the greatest enhancement in catabolic activity, with enhancement factors, relative to the soil only control, of 3.6 times, 3.5 times and 1.5 times for 14C-hexadecane, 14 C-toluene and 14C-phenanthrene, respectively. It is suggested that earthworm presence sufficiently altered (biologically, chemically and physically) the nature of the substrate to determine significantly increased catabolic activity. It follows that where hydrocarbon catabolic activity has been enhanced, biodegradation (assuming favourable conditions) would also be enhanced. Importantly, the earthworm effects would include the promotion of both the compost’s and the contaminated soil’s catabolically active microorganisms, via mechanisms previously discussed. In all cases, the most extensive mineralisation was observed in the 1:0.5 soil:compost substrates which had been exposed to earthworms. It is hypothesised that at this soil:compost ratio, and at the density of earthworms used, they would not be as effective at soil avoidance as would be otherwise expected in the 1:2 ratio, and thus interaction with the contaminated soil would be more extensive. Thus, it is conceivable that greater ingestion of contaminated material would be achieved in the 1:0.5 W treatments with respect to the 1:2 W treatments (in which contaminated material was dispersed in a larger volume of matrix) and the soil only treatments (where greater toxicity was manifested in increased mortality and reduced earthworm–soil interaction). On account of greater ingestion of contaminated soil in the 1:0.5 W treatments earthworm-soil-microbes interaction would have been maximised; thus, promoting the greatest enhancement in hydrocarbon catabolic activity. 4.4. Conclusion The addition of compost in combination with earthworms to hydrocarbon contaminated soil significantly enhanced catabolic activity. Indeed, levels of catabolic activity in these treatments were significantly greater than in the compost only treatments and the enhancements even more pronounced than in the earthworm only treatments. Thus, these results collectively support the hypothesis that synergy does exist between compost addition and earthworm presence in the promotion of catabolic activity for a range of organic contaminants. This synergy might be attributable to the compost providing diverse microbial populations with the capacity to degrade hydrocarbons, an input of limiting nutrients, a matrix conducive to providing aerobic conditions and moisture retaining capacities. In addition, the earthworms provide a vector for mixing, aerating and redistributing promoted catabolically active microorganisms, and to the release of sequestered contaminants through their digestive actions. As a consequence of greatly enhanced levels of hydrocarbon catabolic activity, significantly lower biodegradation end points were achieved. Thus, the inclusion of earthworms within composting bioremediation strategies is supported. References Alexander, M., 1999. Biodegradation and Bioremediation. Academic Press. Alexander, M., 2000. Aging, bioavailability, and overestimation of risk from environmental pollutants. Environmental Science and Technology 34, 4259–4265. Allan, I.J., Semple, K.T., Hare, R., Reid, B.J., 2007. Cyclodextrin enhanced biodegradation of polycyclic aromatic hydrocarbons and phenols in contaminated soil slurries. Environmental Science and Technology 41, 5498–5504. Antizar-Ladislao, B., Lopez-Real, J., Beck, A.J., 2004. Bioremediation of polycyclic aromatic hydrocarbons (PAHs) contaminated soil using composting approaches. Critical Reviews in Environmental Science and Technology 34, 249–289. Atlas, R.M., 1981. Microbial degradation of petroleum hydrocarbons: an environmental perspective. Microbiology and Molecular Biology Reviews 45, 180–209.
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Atlas, R.M., 1995. Bioremediation of petroleum pollutants. International Biodeterioration & Biodegradation 35, 317–327. Barois, I., Villemin, G., Lavelle, P., Toutain, F., 1993. Transformation of the soil structure through Pontoscolex corethrurus (Oligochaeta) intestinal tract. Geoderma 56, 57–66. Bolan, N.S., Baskaran, S., 1996. Characteristics of earthworm casts affecting herbicide sorption and movement. Biology and Fertility of Soils 22, 367–372. Boopathy, R., 2000. Factors limiting bioremediation technologies. Bioresource Technology 74, 63–67. Brown, G.G., Doube, B.M., 2004. In: Edwards, C.A. (Ed.), Earthworm Ecology, second ed. CRC Press, Boca Raton, FL, pp. 213–240. Cajthaml, T., Bhatt, M., Sasek, V., Mateju, V., 2002. Bioremediation of PAHcontaminated soil by composting: a case study. Folia Microbiologica 47, 696– 700. Contreras-Ramos, S.M., Alvarez-Bernal, D., Dendooven, L., 2006. Eisenia fetida increased removal of polycyclic aromatic hydrocarbons from soil. Environmental Pollution 141, 396–401. Edwards, C.A., Bohlen, C.J., 1996. Biology and Ecology of Earthworms, third ed. Chapman & Hall, London. Foght, J.M., Fedorak, P.M., Westlake, D.W.S., 1990. Mineralization of [14C] hexadecane and [14C] phenanthrene in crude oil: specificity among bacterial isolates. Canadian Journal of Microbiology 36, 169–175. Geerdink, M.J., Van Loosdrecht, M.C.M., Luyben, K.C.A.M., 1996. Biodegradability of diesel oil. Biodegradation 7, 73–81. Gevao, B., Mordaunt, C., Semple, K.T., Piearce, T.G., Jones, K.C., 2001. Bioavailability of nonextractable (bound) pesticide residues to earthworms. Environmental Science and Technology 35, 501–507. Hickman, Z.A., Reid, B.J., 2008. Earthworm assisted bioremediation of organic contaminants. Environment International, doi:10.1016/j.envint.2008.02.013. Kastner, M., Lotter, S., Heerenklage, J., BreuerJammali, M., Stegmann, R., Mahro, B., 1995. Fate of 14C-labeled anthracene and hexadecane in compost-manured soil. Applied Microbiology and Biotechnology 43, 1128–1135. Kastner, M., Mahro, B., 1996. Microbial degradation of polycyclic aromatic hydrocarbons in soils affected by the organic matrix of compost. Applied Microbiology and Biotechnology 44, 668–675. Kersante, A., Martin-Laurent, F., Soulas, G., Binet, F., 2006. Interactions of earthworms with atrazine-degrading bacteria in an agricultural soil. FEMS Microbiology Ecology 57, 192–205. Lavelle, P., Pashanasi, B., Charpentier, F., Gilot, C., Rossii, J.-P., Derouard, L., Andre, J., Ponge, J.-F., Bernier, N., 2004. In: Edwards, C.A. (Ed.), Earthworm Ecology, second ed. CRC Press, Boca Raton, FL, pp. 145–160. Leahy, J.G., Colwell, R.R., 1990. Microbial degradation of hydrocarbons in the environment. Microbiological Reviews 54, 305–315. Liste, H.-H., Quinones-Rivera, A., Tang, J., 2002. Availability of aged toluene and decane for biodegradation in soil. Water, Air, and Soil Pollution 133, 227–234. Marchall, R., Penet, R., Solana-Serena, S., Vandecasteele, J.P., 2003. Gasoline and oil biodegradation. Oil & Gas Science and Technology 58, 441–448. Namkoong, W., Hwang, E.-Y., Park, J.S., Choi, J.-Y., 2002. Bioremediation of dieselcontaminated soil with composting. Environmental Pollution 119, 23–31. Olsen, J.J., Mills, G.L., Herbert, B.E., Morris, P.J., 1999. Biodegradation rates of separated diesel components. Environmental Toxicology and Chemistry 18, 2448– 2453. Ostberg, T.L., Jonsson, A.P., Lundstrom, U.S., 2007. Enhanced degradation of n-hexadecane in diesel fuel contaminated soil by the addition of fermented whey. Soil and Sediment Contamination 16, 221–232. Prenafeta-Boldu, F.X., Vervoot, J., Grotenhuis, J.T.C., Van-Groenestijn, J.W., 2002. Substrate interactions during the biodegradation of benzene, toluene, ethylbenzene, and xylene (BTEX) hydrocarbons by the fungus Cladophialophora sp. strain T1. Applied and Environmental Microbiology 68, 2660–2665. Prince, R.C., Parkerton, T.F., Lee, C., 2007. The primary aerobic biodegradation of gasoline hydrocarbons. Environmental Science and Technology 41, 3316– 3321. Puglisi, E., Cappa, F., Fragoulis, G., Trevisan, M., Del Re, A.A.M., 2006. Bioavailability and degradation of phenanthrene in compost amended soils. Chemosphere 67, 548–556. Reid, B.J., Jones, K.C., Semple, K.T., 2000. Bioavailability of persistent organic pollutants in soils and sediments – a perspective on mechanisms, consequences and assessment. Environmental Pollution 108, 103–112. Reid, B.J., Fermor, T.R., Semple, K.T., 2002. Induction of PAH-catabolism in mushroom compost and its use in the biodegradation of soil-associated phenanthrene. Environmental Pollution 118, 65–73. Robinson, K.G., Farmer, W.S., Novak, J.Y., 1990. Availability of sorbed toluene in soils for biodegradation by acclimated bacteria. Water Research 24, 345–350. Romantschuck, M., Sarand, I., Petanen, T., Peltola, R., Jonsson-Vihanne, M., Koivula, T., Yrjala, K., Haahtela, K., 2000. Means to improve the effect of in-situ bioremediation of contaminated soil: an overview of novel approaches. Environmental Pollution 107, 179–185. Sasek, V., Bhatt, M., Cajthaml, T., Malachova, K., Lednicka, D., 2003. Compostmediated removal of polycyclic aromatic hydrocarbons from contaminated soil. Archives of Environmental Contamination and Toxicology 44, 336–342. Schaefer, M., Filser, J., 2007. The influence of earthworms and organic additives on the biodegradation of oil contaminated soil. Applied Soil Ecology 36, 53– 62. Semple, K.T., Reid, B.J., Fermor, T.R., 2001. Impact of composting strategies on the treatment of soils contaminated with organic pollutants. Environmental Pollution 112, 269–283.
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Semple, K.T., Morriss, W.W.J., Paton, G.I., 2003. Bioavailability of hydrophobic organic contaminants in soils: fundamental concepts and techniques for analysis. European Journal of Soil Science 54, 809–818. Tharaken, J., Tomlinson, D., Addagada, A., Shafagati, A., 2006. Biotransforamtion of PCBs in contaminated sludge: potential for novel biological technologies. Engineering in Life Sciences 6, 43–50. Verma, A., Pillai, M.K.K., 1991. Bioavailability of soil-bound residues of DDT and HCH to earthworms. Current Science 61, 840–843.
Walker, J.D., Colwell, R.R., 1976. Measuring the potential of hydrocarbon-degrading bacteria. Applied and Environmental Microbiology 31, 189–197. Yadav, J.S., Reddy, C.A., 1993. Degradation of benzene, toluene, ethylbenzene, and xylenes (BTEX) by the lignen-degrading basidiomycete Phanerochaete chrysosporium. Applied and Environmental Microbiology 59, 756–762. Yerushalmi, L., Guiot, S.R., 1998. Kinetics of biodegradation of gasoline and its hydrocarbon constituents. Applied Microbiology and Biotechnology 49, 475– 481.
Contents lists available at ScienceDirect
Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
Increased microbial catabolic activity in diesel contaminated soil following addition of earthworms (Dendrobaena veneta) and compost Zachary A. Hickman, Brian J. Reid* School of Environmental Sciences, University of East Anglia, Norwich, Norfolk NR4 7TJ, UK
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 March 2008 Received in revised form 12 June 2008 Accepted 13 August 2008 Available online 24 September 2008
This study sought to assess the influence of compost and earthworms (Dendrobaena veneta) upon the level of hydrocarbon catabolism in petroleum contaminated forecourt soil (extractable petroleum hydrocarbons (EPH) 10 þ 1.8 g kg1 and total 16 United States Environment Protection Agency (USEPA) polycyclic aromatic hydrocarbons (PAH) 1.62 0.5 g kg1). The catabolic activity of the indigenous microorganisms within uncombined materials (soil and compost) and within the combined treatments (soil plus compost; either with or without earthworms) was assessed by 14C-radiorespirometry (14Chexadecane, 14C-toluene and 14C-phenanthrene). Maximum levels of catabolic activity were observed (at the end of the incubation period; 84 d) for all three compounds in the combined contaminated soil, compost and earthworm mixtures. Significant (p < 0.05) enhancement factors (relative to the soil only control) in catabolic activity in the combined treatments (soil:compost (1:0.5)) of 3.6 times, 1.5 times and 3.5 times were observed for 14C-hexadecane, 14C-phenanthrene and 14C-toluene, respectively; with maximum levels of catabolic activity for these substrates being 68.6 1.7%, 37.9 5.3% and 85.9 1.3%. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Earthworm Hydrocarbon Catabolism Diesel Compost
1. Introduction Catabolically competent microorganisms are often present within soils contaminated with organic compounds. However, biodegradation may be limited on account of: low compound availability/accessibility, oxygen limitations, pH, nutrient limitations, suboptimal C:N:P ratio, temperature, inappropriate moisture conditions, toxic levels of contaminants (or co-contaminants), and/ or inadequate concentrations of terminal electron acceptors (Atlas, 1981; Alexander, 2000; Boopathy, 2000; Reid et al., 2000; Romantschuck et al., 2000; Semple et al., 2003). Thus, bioremediation methodologies often require the optimisation or enhancement of such environmental and biological conditions. It is widely recognised that earthworms can significantly and positively affect the soil environment in terms of soil organic matter dynamics and turnover, improved soil structure, improved soil fertility (Edwards and Bohlen, 1996; Lavelle et al., 2004; Kersante et al., 2006), breakdown of soil particles, substrate aeration and moisture retention and drainage (Edwards and Bohlen, 1996). Many of these actions and factors, such as the excretion of protein rich mucous, reworking and fragmentation of carbon and deposition of cast excreta are stimulators for soil microorganisms (Edwards and Bohlen, 1996; Brown and Doube, 2004). Thus, within
* Corresponding author. Tel.: þ44 1603 592357; fax: þ44 1603 591327. E-mail address: [email protected] (B.J. Reid). 0038-0717/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2008.08.016
the arena of bioremediation, the use of earthworms to improve soil conditions and to subsequently promote microbial numbers, diversity and activity should realise benefits for levels of catabolic activity and subsequent enhancement of organic contaminant biodegradation. Indeed, recent work has shown that earthworms, through their biological, chemical and physical actions upon soils, can assist in increased losses of PAHs (Contreras-Ramos et al., 2006), crude oils (Schaefer and Filser, 2007) and PCBs (Tharaken et al., 2006). A recent article (Hickman and Reid, 2008) provides a review of these manuscripts and the wider literature with respect to how earthworms might assist bioremediation. Beyond the isolated application of earthworms for the bioremediation of hydrocarbon contaminated soil, the co-application of both compost and earthworms is putatively advantageous. Whilst the compost provides additional microbial numbers and diversity, nutrients, pH buffering, and improved moisture retention (Semple et al., 2001), earthworm digestive actions result in greater soil particle surface areas, which could theoretically improve accessibility of bound or sequestered contaminants to degrader microorganisms (Verma and Pillai, 1991; Gevao et al., 2001). Significantly, the release of previously bound contaminants (Gevao et al., 2001), providing they are subsequently degraded, would facilitate attainment of lower bioremediation end points. This work relates the influence of treatment type (namely, contaminated soil only, compost only, and soil:compost mixtures (in ratios 1:0.5 and 1:2 wt/wt), in the presence and absence of earthworms) to observed levels of catabolic activity assessed by
Z.A. Hickman, B.J. Reid / Soil Biology & Biochemistry 40 (2008) 2970–2976 14 C-hydrocarbon respirometry. Ultimately, this work sought to establish if earthworms and compost, as individual additions and in combination with each other, enhanced hydrocarbon catabolic activity. Catabolic potential was assessed by 14C-radiorespirometry using three representative hydrocarbons, namely, 14C-hexadecane, 14 C-toluene and 14C-phenanthrene. These hydrocarbons are commonly found in petroleum impacted soils and have previously been used in other studies (Foght et al., 1990; Robinson et al., 1990; Geerdink et al., 1996; Prenafeta-Boldu et al., 2002; Reid et al., 2002; Ostberg et al., 2007).
2. Materials and methods Treatments included soil and compost mixtures at ratios of 1:0.5 and 1:2 (wt/wt), in addition to contaminated soil only and compost only control treatments. For 84 d, half of the treatments were incubated with earthworms (W) (ten earthworms per kg material), and half without earthworms (NW) (n ¼ 5 of each treatment). Samples were taken at 0 d and 84 d for 14C-respirometer assays. 2.1. Experimental set-up Mature compost (C:N ratio of 19:1; moisture content of 70% of maximum water holding capacity (WHC); pH of 7.2) was produced from shredded leaves, grass and other green material (50%) and stalks and woodier materials (50%) that underwent rapid composting utilising an enclosed force aerated system. The composted material continued to ‘mature’ outdoors for approximately 4 months prior to use. Aged petroleum contaminated soil (EPH 10 1.8 g kg1; total 16 USEPA PAH 1.62 þ 0.5 g kg1 (hereafter referred to as SPAH); sand 0.8%, silt 42.7% and clay 56.4%; organic matter content of 0.8% (mass loss on ignition); C:N ratio of 8:1; pH of 6.2) was used in this experimental study. Plastic vessels (10 L capacity) measuring 35 cm in height and 40 cm in diameter were used to contain the treatments. The vessels had plastic piping (7.5 mm diameter) inserted and sealed into three holes in the base, which were attached to an air compressor, and aerated for 15 min every 24 h. No further manual mixing was undertaken throughout the study. Silvaperl washed gravel (obtained from a building supplies store) was washed in water and laid to a thickness of 3 cm in the base of each vessel (to promote more uniform air dissipation). The gravel was then covered with a perforated plastic sheet that allowed air to percolate up from the base and disallowed the migration of earthworms into the gravel. Soil and/or compost was rehydrated and manually mixed into the experimental vessel. The mass of the contaminated soil (at 70% of maximum WHC) was kept constant at 2 kg (wet weight), equivalent to 1.82 kg dry weight. The compost substrate (at 70% of maximum WHC) was added as wet weight at the ratios of: 0.5 (1 kg) and 2 (4 kg). This equated to 0.78 kg and 3.16 kg dry weight, respectively. The contaminated soil and the compost only treatments contained only 2 kg (wet weight) of either contaminated soil or compost, respectively. Adult earthworms (Dendrobaena veneta) were depurated (laid on damp filter paper in an enclosed plastic petri dish) for 24 h prior to use. Ten earthworms per kg of material (each depurated earthworm weighing 1.0 0.2 g) were added to each of the required treatments and replicates. The earthworms were combined with the material (resulting in 1:100 biomass:material w/w ratio) and perforated lids were sealed around the vessels. Water was added periodically to maintain levels at 70% of maximum WHC. It was noted from prior research (data not shown) that D. veneta, when compared to other widely used earthworm species (in this instance Lumbricus rubellus and Eisenia fetida), outperformed them in terms of survival and tolerance of hydrocarbon contaminated soils (up to
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3 g kg1 total PAH). It was thus felt justifiable to utilise D. veneta within this study to ensure survival for the incubation period. 2.2. Hydrocarbon and +PAH analysis Extractable Petroleum Hydrocarbons (EPH – C10–C40) were P quantified by GC-FID and PAH by GC–MS. Initially, samples (5 g; n ¼ 3 of all treatments) were extracted using an end over end shake extraction method with 50:50 (Hexane:Acetone) at a soil:solvent ratio of 1:10 for 1 h. GC–FID analysis was undertaken using a Hewlett Packard 5890/6890 FID with a Zebron ZB-1 (Phenomenex, UK) fused silica capillary column (15 m 0.32 mm 1 mm) using helium at a constant pressure of 15 psi at an initial flow of 1.5 ml min1 with nitrogen at 30 ml min1, hydrogen at 30 ml min1 and air at 300 ml min1 and a split injection (2:1 ratio) of 2.0 ml at a flow of 7.1 ml min1. The column oven was set at 60 C for 1.0 min, 60–325 C programmed at 15 C min1 and 325 C for 2.0 min. GC–MS was undertaken using a Hewlett Packard 6890 Gas Chromatograph with HP7683 series injector and HP7683 series autosampler and a Hewlett Packard 5973 MSD Mass Selective Detector. A fused silica capillary column was used with measurements of 15 m 0.25 mm 0.1 mm using helium at a constant flow of 1 ml min1 and a pulsed splitless injection of 1.0 ml at 20 ml min1. The column oven was set at 49 C for 1.25 min, 49– 250 C at 25 C min1, 250–300 C at 35 C min1 and 300–340 C at 60 C min1. 2.3. Assessment of catabolic activity by
14
C-respirometry
Schott bottles (250 ml) were adapted such that a glass vial (7 ml) could be suspended from the lid. This vial contained GF/A filter paper (20 mm 20 mm) and 1 M sodium hydroxide (1 ml; supplied by Merck, UK) to ‘trap’ mineralised 14CO2 (Allan et al., 2007). Samples (10 g) to be screened for catabolic activity (contaminated soil, compost, 1:0.5 and 1:2 (soil:compost (wt/wt)) were slurried in respirometers with sterile distilled water (30 ml)). 14C-9-Phenanthrene, 14C-1-hexadecane and 14C-UL-toluene (added as individual compounds) (all supplied by Sigma, UK; radioactive and chemical purity >95%) were spiked into the slurried respirometers, using toluene as the carrier solvent, such that 100 ml of spike delivered 200 Bq per respirometer. Respirometers were continuously shaken at 100 rpm using an IKA Labortechnik KS 501 digital flatbed shaker and sampled periodically until 648 h (27 d) assay time had elapsed. Spiking efficiency was determined to be >94 1.0%. Ultima Gold (6 ml) was added to changed vials, which were stored in darkness for a minimum of 48 h prior to analysis. Trapped 14CO2 was determined by liquid scintillation counting, using a Canberra Packard Tri-Carb 2900TR liquid scintillation counter for 10 min per sample. 3. Results 3.1. Earthworm survival Following the initial incubation period, prior to the catabolism study, it was noted that earthworm survival was highest in the compost treatments (70 12%) and lowest in the contaminated soil control (15 13.2%). Following addition of compost to the soil, the treatments showed significantly higher survival with increasing compost amounts (p < 0.05). Survival in the 1:0.5 and 1:2 treatments were 23 8.5% and 64 15%, respectively. Thus, as the contaminated soil content increased, earthworm mortality increased, which was attributed to soil toxicity and the possibility of inappropriate food quality and availability, as previously noted as detrimental factors (Edwards and Bohlen, 1996; Schaefer and Filser, 2007).
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Whilst reproduction was not considered quantitatively, juvenile earthworms were recovered at 84 d from all treatments, indicating reproduction had occurred. In addition to this, cocoons were also noted in all treatments, further indicating that toxicity was not so great as to inhibit reproduction.
materials were combined, levels of 14C-phenanthrene catabolic activity were significantly (p < 0.05) higher (68.5 14.0% and 68.4 14.1% in the 1:0.5 and 1:2 treatments, respectively) with respect to the soil only control. 3.4. Temporal changes in catabolic activity in the soil only controls
3.2. Hydrocarbon loss In general terms, both the addition of compost and the addition of earthworms to contaminated soil, 1:0.5 and 1:2 treatments P resulted in enhanced loss of EPH (C10–C40) and PAH over the 84 d P incubation period (Table 1). EPH values have had PAH values P subtracted, in order to reflect losses exclusive of PAH. Whilst both the NW and W contaminated soil only treatments saw significant (p < 0.05) losses following the incubation period (with respect to their start concentrations), they retained the greatest residual EPH P and PAH concentrations of all the treatments studied. Addition of compost without earthworms at both ratios (1:0.5 and 1:2) resulted in significantly (p < 0.05) enhanced losses of both EPH and P PAH with respect to the soil only control. Finally, where compost and earthworms were used in combination, further significant P (p < 0.05) reductions in both EPH and PAH concentrations were observed. The relative extents of loss of hydrocarbons from the treatments are discussed in subsequent sections with respect to how they relate to levels of catabolic activity within uncombined (soil and compost) and combined (1:0.5 and 1:2) treatments at study start (0 d) and end (84 d). 3.3. Catabolic activity in unblended substrates and mixtures at 0 d The level of 14C-hexadecane catabolic activity (Fig. 1A) was not significantly different (p > 0.05) in the soil (41.3 þ 0.6%) and compost (43.3 þ 6.5%) only treatments. Upon combination of contaminated soil and compost, no significant increase (p > 0.05) in 14 C-hexadecane catabolic activity was observed in the 1:2 mixture (43.8 þ 2.8%). However, 14C-hexadecane catabolic activity was significantly (p < 0.05) increased in the 1:0.5 treatment (54.0 þ 4.4%) with respect to the soil only, compost only and 1:2 treatments. The level of 14C-toluene catabolic activity (Fig. 1B) in the contaminated soil only control was the lowest of all compounds/ treatments (8.8 2.2%). However, the level of 14C-toluene catabolic activity was significantly (p < 0.05) higher, almost three times higher (32.8 12.0%), in the compost only control. When soil and compost were combined, both mixed treatments indicated significantly (p < 0.05) higher levels of 14C-toluene catabolic activity (27.0 2.2% and 35.7 4.0%, in the 1:0.5 and 1:2 treatments, respectively) that were comparable to that of the compost only control. The level of 14C-phenanthrene catabolic activity (Fig. 1C) was observed to be high in the soil only treatment (48.7 2.5%) and higher still in the compost only treatment (58.7 6.4%). When Table 1 P EPH and PAH residual concentrations (% of start concentration) for contaminated soil (CS), 1:0.5 and 1:2 treatments at 84 d in the absence (NW) and presence (W) of earthworms P Treatment EPH residual concentration PAH residual concentration (% of start concentration) (% of start concentration)
CS 1:0.5 1:2
84 d NW
84 d W
84 d NW
84 d W
55.4 5.3Aa 19.0 5.0Ba 11.5 3.0Ca
33.4 4.6Ab 15.8 4.0Ba 5.0 2.4Cb
69.2 6.4Aa 40.9 9.3Ba 20.1 1.3Ca
65.3 9.3Aa 17.9 3.4Bb 7.7 4.9Cb
Same upper case letters within columns for each compound represent no significant difference (p>0.05). Same lower case letters between columns for either EPH or P PAH represent no significant difference between with and without earthworm treatments (p > 0.05).
With respect to the 0 d NW contaminated soil only treatments, levels of catabolic activity were noted to be high in the case of 14 C-hexadecane (41.3 0.6%) (Fig. 2A) and 14C-phenanthrene (48.7 2.5%) (Fig. 2C), but much lower in the case of toluene (8.8 2.2%) (Fig. 2B). Following 84 d, the levels of catabolic activity decreased by 64.6% (to less than half the original level) for 14 C-hexadecane (p < 0.05), while only a small decrease (p > 0.05) was observed in the level of 14C-phenanthrene catabolic activity (a decrease of 7.7%). In contrast, the levels of 14C-toluene catabolic activity increased markedly by 51.8% (to approximately twice the original level) (p < 0.05). Following the 84 d treatment period, the NW EPH concentrations (Table 1) were observed to drop to 55.4 5.3% of their start concentrations (loss of 26.8%) in the soil only controls. In contrast, P over the same period, the PAH concentrations decreased to a lesser extent to 69.2 þ 6.4% (loss of 19.5%) in the soil only controls, mirroring, to some extent the losses of these representative 14 C-hydrocarbons. Where earthworms had been present within the contaminated soil only treatments (Fig. 2), levels of catabolic activity were not found to be significantly different (p > 0.05), when compared to the NW treatments at 84 d, for both 14C-hexadecane (19.1 þ 1.8%) (Fig. 2A) and 14C-toluene (19.7 þ 1.3%) (Fig. 2B). However, in the case of 14C-phenanthrene (Fig. 2C), a positive and significant (p < 0.05) influence on catabolic activity (58.0 þ 1.6%) was observed when earthworms were present. 3.5. Influence of compost ratio and earthworm presence on catabolic activity at 84 d Following the incubation period of 84 d (Table 1), it was observed that for all compounds maximum levels of catabolic activity were present in treatments that contained contaminated soil, compost and earthworms together. With respect to 14C-hexadecane (Fig. 3A), the NW 1:0.5 and 1:2 contaminated soil:compost treatments resulted in a significantly (p < 0.05) greater catabolic ability in comparison to the NW and W soil only control, suggestive of the beneficial use of compost alone even at its lowest ratio. However, no significant difference (p > 0.05) was observed between extents of mineralisation in the NW 1:0.5 and 1:2 soil and compost treatments (28.2 1.4% and 29.4 1.3%, respectively). The 1:0.5 and 1:2 soil:compost W treatments (for 84 d) indicted significantly higher (p < 0.05) levels of 14C-hexadecane catabolic activity, with respect to the NW and W soil only controls and the NW 1:0.5 and 1:2 treatments. The increase was marked, with mineralisation extents of 68.6 1.7% and 53.2 2.6%, for 1:0.5 and 1:2, respectively. These results were noted to be much higher than that in the W control soil treatment (19.1 1.8%). Considering 14C-toluene (Fig. 3B), the same general trends were observed wherein 1:0.5 and 1:2 soil:compost substrates without earthworms, resulted in a significant (p < 0.05) increase in toluene catabolic activity, with respect to the NW and W soil only control treatments. Again, as was the case for 14C-hexadecane, no significant differences (p > 0.05) were observed between levels of catabolic activity in the 1:0.5 and 1:2 soil:compost substrates (33.7 5.3% and 37.9 1.0%, respectively). The 1:0.5 and 1:2 W soil:compost treatments indicted significantly higher (p < 0.05) levels of 14C-toluene catabolic activity with
Mineralisation (% Relative to start activity)
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100 80
A
CS Compost 1:0.5 1:2
60 a
b b
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B
b
c b
a
C
b
b a
a
40 a 20 0
Fig. 1. Catabolic activity (% mineralisation) in contaminated soil (CS), compost, 1:0.5 and 1:2 treatments at 0 d for 14C-hexadecane (A), 14C-toluene (B) and Error bars represent SE of the mean. Same lower case letters signify no significant difference (p > 0.05) between extents of mineralisation.
respect to the NW and W soil only treatments/controls. This increase in catabolic activity was marked, with mineralisation extents of 68.6 1.7% and 56.2 2.1% for 1:05 and 1:2, respectively. These results were again noted to be much higher than in the W soil only treatment (16.7 1.3%). It was again observed that the 1:0.5 and 1:2 substrates which had had earthworms incubated within them for 84 d had significantly (p < 0.05) greater catabolic activity than in comparison to 0 d, whilst the same treatments without earthworms determined lower catabolic activity than at 0 d. Finally, considering 14C-phenanthrene (Fig. 3C), it was noted that compost applied to the contaminated soil at the ratios of 1:0.5 and 1:2 significantly increased (p < 0.05) levels of 14C-phenanthrene catabolic activity with respect to the NW and W soil only treatments. Although significantly different (p < 0.05) to each other, the 1:0.5 and 1:2 soil:compost treatments indicated similar levels of catabolic activity (67.7 1.7% and 73.0 0.9%, respectively). Again, the W 1:0.5 and 1:2 contaminated soil:compost treatments resulted in the highest levels of 14C-phenanthrene catabolic activity observed. The increase was marked, with levels of catabolic activity of 85.9 1.3% and 73.0 2.6% for 1:05 and 1:2, respectively. These results were again noted to be much higher than that in the W soil treatment (58.0 1.7%). 4. Discussion 4.1. Catabolic activity in unblended substrates and mixtures at 0 d
Mineralisation (% relative to start activity)
It was noted that levels of 14C-hexadecane catabolic activity were high in all substrates and mixtures (>40%). This is to be expected on account of hexadecane (C16) being a lighter straight chained alkane, susceptible to degradation by beta-oxidation (Alexander, 1999); a ubiquitous catabolic pathway. High levels of hexadecane catabolic activity are consistent with other works stating the preferential, or ease of, degradation of hexadecane, or
80
60
C-phenanthrene (C).
similar light alkanes (Walker and Colwell, 1976; Atlas, 1995; Kastner et al., 1995; Olsen et al., 1999; Prince et al., 2007). We submit that the enhanced catabolism was likely to be due to the compost’s incumbent positive characteristics such as microbial numbers and diversity, moisture retaining ability, nutrient input and increased aerobicity (Semple et al., 2001). Although other research has shown hydrocarbon losses when utilising compost (Kastner et al., 1995; Kastner and Mahro, 1996; Namkoong et al., 2002; Sasek et al., 2003; Antizar-Ladislao et al., 2004) it was noted that in our results there was a lack of a lag period; with 14C-hexadecane catabolic activity being well established at 0 d. The observed toluene catabolic activity was initially low, but such observed extents of toluene degradation are not uncommon in mixed gasoline treatments (Yerushalmi and Guiot, 1998); with toluene degradation by fungi, an important microbial component of compost, being previously noted (Yadav and Reddy, 1993; Prenafeta-Boldu et al., 2002). The 14C-phenanthrene losses were in keeping with previous results that noted catabolic competence for phenanthrene in both compost and soil–compost mixtures (Kastner and Mahro, 1996; Cajthaml et al., 2002; Reid et al., 2002; Sasek et al., 2003; Puglisi et al., 2006). Overall, these results indicate that levels of catabolic activity, with respect to 14C-hexadecane and 14C-phenanthrene, were already high in the contaminated soil while levels of toluene catabolic activity were initially low. Given the history of the contamination (petrol filling station forecourt), pre-exposure to aliphatic hydrocarbons, PAHs and to a lesser extent toluene, support the observed levels of catabolic activity in the soil. With respect to all compounds, levels of catabolic activity were higher in the soil plus compost treatments when compared to the soil only treatments. Finally, mixing soil with compost resulted in increased catabolic activity for all substrates; the degrees of enhancement being greatest where soil only catabolic activity levels were lowest, i.e. enhancement in catabolic activity in the order 14C-toluene > 14 C-phenanthrene > 14C-hexadecane.
A
0d 84 d NW 84 d W
14
B
b a
C
a
a 40 b 20
b a
b
b
0 Fig. 2. Catabolic activity (% mineralisation) for CS at 0 d and 84 d (NW and W) for 14C-hexadecane (A), 14C-toluene (B) and mean. Same lower case letters signify no significant difference (p > 0.05) between extents of mineralisation.
14
C-phenanthrene (C). Error bars represent SE of the
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Mineralisation (% relative to start activity)
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100 80
b
a
b
a
a
C
b
b a
40
0
b
b
60
20
B
A
NW W
a* a
a*
b
CS
a* a
1:0.5
1:2
a*
a
CS
1:0.5
1:2
CS
1:0.5
1:2
Fig. 3. Catabolic activity in CS, 1:0.5 and 1:2 treatments at 84 d for 14C-hexadecane NW and W (A), 14C-toluene NW and W (B) and 14C-phenanthrene NW and W (C). Error bars represent SE of the mean. Same lower case letters signify no significant differences (p > 0.05) between NW and W treatments. An asterisk signifies no significant difference between corresponding treatments.
4.2. Temporal changes in catabolic activity in the soil only controls Both the 84 d treatment results and the catabolic assessment results reflect the fact that when the contaminated soil was initially added to the treatment vessels, the indigenous microbial populations had a preferential environment (inputted oxygen and moisture) in which to rapidly multiply. The reduced 14C-hexadecane catabolic activity at 84 d for the NW treatment would suggest a shift in microbial populations towards other hydrocarbon degradation as the concentration of this substrate reduced; thus highlighting the complex relationship that exists between degrader communities, their hydrocarbon preferences and soil contaminant loadings. Relevantly to this study, Walker and Colwell (1976) identified mineralisation rates of 14C-hydrocarbons to be in the order of 14C-hexadecane > 14C-naphthalene > 14C-toluene. They stated that 14C-naphthalene and 14C-hexadecane mineralisation extents were similar, although 14C-hexadecane rates were much higher. It should be borne in mind that degradation extents and rates of individual classes of hydrocarbons, or of individual hydrocarbons are not straightforward, especially when present as a complex mixture such as with diesel and petroleum contaminations. Such preferential degradation has previously been noted by Walker and Colwell (1976), Atlas (1995) and Geerdink et al. (1996). With respect to preferential hydrocarbon utilisation, Atlas (1995) established the following relationship between hydrocarbon structure and resistance to biodegradation: n-alkanes < branched and cyclic alkanes < aromatics < polar compounds, stating that within each structural class, alkyl branching or substitution will further complicate degradation ability. More recently, Prince et al. (2007) determined that during aerobic degradation of gasoline, n-alkanes, iso-alkanes, simple and alkylated aromatic compounds were the most readily degraded, followed by the smaller n-alkanes and iso-alkanes and napththenes. This research is complemented by others, who also observed similar orders of degradation (Foght et al., 1990; Leahy and Colwell, 1990; Robinson et al., 1990; Olsen et al., 1999). In light of the reduction (and of the putative limitation) in easily degradable hydrocarbons, catabolic activity for the more recalcitrant compounds e.g. 14C-phenanthrene and 14C-toluene had been maintained or enhanced, respectively. The limited 14C-toluene degradation at 0 d was attributed to the assumed low start toluene degrader numbers. Whilst the BTEX group can represent a large proportion of commercial petroleum (Marchall et al., 2003), these compounds are volatile and following soil extraction and subsequent manual mixing and preparation, it is conceivable (although not measured) that only minimal percentage of toluene start concentration remained (Liste et al., 2002). The increased toluene
catabolic ability at 84 d reflects the fact that hydrocarbon losses were extensive during the 84 d incubation period and as a consequence (discussed above) more labile substrates will have been depleted. This, in combination with an improved incubation environment, will have promoted the catabolic activity for more recalcitrant compounds, and specifically, toluene. Relevantly, Yerushalmi and Guiot (1998) reported toluene degradation to be much slower in mixed gasoline when compared to its degradation as a sole substrate. Relevantly, earthworms are efficient at grinding soil into smaller fractions via their digestion mechanisms (Bolan and Baskaran, 1996) and it is hypothesised that such actions can result in the release of previously bound contaminants (Barois et al., 1993; Gevao et al., 2001), thus making them available for microbial degradation. 4.3. Influence of compost ratio and earthworm presence on catabolic activity at 84 d Interestingly, the 1:0.5 and 1:2 soil:compost W treatments had significantly (p < 0.05) greater 14C-hexadecane and 14C-toluene catabolic activity, at 84 d, in comparison to 0 d, whilst the same treatments without earthworms determined lower catabolic activity than at 0 d. Thus, earthworm inclusion dictated continued and increased soil/compost catabolic activity whilst a decline was observed in the without earthworm treatments. The increased catabolic activity may partly be attributed to earthworm digestion processes fragmenting soil particles, enhancement of hydrocarbon degraders, provision of increased nutrients to degraders and inoculation of the egested casts with microorganisms (Edwards and Bohlen, 1996). The resultant casts have been reported to be both nutrient and microbially rich and therefore would have assisted in the proliferation of microbial numbers and activity (Edwards and Bohlen, 1996; Brown and Doube, 2004). The decline in catabolic activity can be related to the changes in hydrocarbon concentrations (Table 1) and thus reflected changes in the soil microbial numbers, diversity and activity. With respect to all compounds, these results indicated that the addition of compost to contaminated soil in the ratio of 1:0.5 and 1:2 subsequently determined significantly greater hydrocarbon losses in comparison to the contaminated soil only (Table 1). Whilst compost’s inherent abiotic and biotic properties were likely to have determined observed mineralisation extents, as is supported by previous work (Kastner et al., 1995; Namkoong et al., 2002; Reid et al., 2002; Antizar-Ladislao et al., 2004; Sasek et al., 2003), compost addition to contaminated soil was not as effective at promoting extensive mineralisation as was the co-application of compost and earthworms.
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The combined addition of earthworms and compost to contaminated soil resulted in the greatest enhancement in catabolic activity, with enhancement factors, relative to the soil only control, of 3.6 times, 3.5 times and 1.5 times for 14C-hexadecane, 14 C-toluene and 14C-phenanthrene, respectively. It is suggested that earthworm presence sufficiently altered (biologically, chemically and physically) the nature of the substrate to determine significantly increased catabolic activity. It follows that where hydrocarbon catabolic activity has been enhanced, biodegradation (assuming favourable conditions) would also be enhanced. Importantly, the earthworm effects would include the promotion of both the compost’s and the contaminated soil’s catabolically active microorganisms, via mechanisms previously discussed. In all cases, the most extensive mineralisation was observed in the 1:0.5 soil:compost substrates which had been exposed to earthworms. It is hypothesised that at this soil:compost ratio, and at the density of earthworms used, they would not be as effective at soil avoidance as would be otherwise expected in the 1:2 ratio, and thus interaction with the contaminated soil would be more extensive. Thus, it is conceivable that greater ingestion of contaminated material would be achieved in the 1:0.5 W treatments with respect to the 1:2 W treatments (in which contaminated material was dispersed in a larger volume of matrix) and the soil only treatments (where greater toxicity was manifested in increased mortality and reduced earthworm–soil interaction). On account of greater ingestion of contaminated soil in the 1:0.5 W treatments earthworm-soil-microbes interaction would have been maximised; thus, promoting the greatest enhancement in hydrocarbon catabolic activity. 4.4. Conclusion The addition of compost in combination with earthworms to hydrocarbon contaminated soil significantly enhanced catabolic activity. Indeed, levels of catabolic activity in these treatments were significantly greater than in the compost only treatments and the enhancements even more pronounced than in the earthworm only treatments. Thus, these results collectively support the hypothesis that synergy does exist between compost addition and earthworm presence in the promotion of catabolic activity for a range of organic contaminants. This synergy might be attributable to the compost providing diverse microbial populations with the capacity to degrade hydrocarbons, an input of limiting nutrients, a matrix conducive to providing aerobic conditions and moisture retaining capacities. In addition, the earthworms provide a vector for mixing, aerating and redistributing promoted catabolically active microorganisms, and to the release of sequestered contaminants through their digestive actions. As a consequence of greatly enhanced levels of hydrocarbon catabolic activity, significantly lower biodegradation end points were achieved. Thus, the inclusion of earthworms within composting bioremediation strategies is supported. References Alexander, M., 1999. Biodegradation and Bioremediation. Academic Press. Alexander, M., 2000. Aging, bioavailability, and overestimation of risk from environmental pollutants. Environmental Science and Technology 34, 4259–4265. Allan, I.J., Semple, K.T., Hare, R., Reid, B.J., 2007. Cyclodextrin enhanced biodegradation of polycyclic aromatic hydrocarbons and phenols in contaminated soil slurries. Environmental Science and Technology 41, 5498–5504. Antizar-Ladislao, B., Lopez-Real, J., Beck, A.J., 2004. Bioremediation of polycyclic aromatic hydrocarbons (PAHs) contaminated soil using composting approaches. Critical Reviews in Environmental Science and Technology 34, 249–289. Atlas, R.M., 1981. Microbial degradation of petroleum hydrocarbons: an environmental perspective. Microbiology and Molecular Biology Reviews 45, 180–209.
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Atlas, R.M., 1995. Bioremediation of petroleum pollutants. International Biodeterioration & Biodegradation 35, 317–327. Barois, I., Villemin, G., Lavelle, P., Toutain, F., 1993. Transformation of the soil structure through Pontoscolex corethrurus (Oligochaeta) intestinal tract. Geoderma 56, 57–66. Bolan, N.S., Baskaran, S., 1996. Characteristics of earthworm casts affecting herbicide sorption and movement. Biology and Fertility of Soils 22, 367–372. Boopathy, R., 2000. Factors limiting bioremediation technologies. Bioresource Technology 74, 63–67. Brown, G.G., Doube, B.M., 2004. In: Edwards, C.A. (Ed.), Earthworm Ecology, second ed. CRC Press, Boca Raton, FL, pp. 213–240. Cajthaml, T., Bhatt, M., Sasek, V., Mateju, V., 2002. Bioremediation of PAHcontaminated soil by composting: a case study. Folia Microbiologica 47, 696– 700. Contreras-Ramos, S.M., Alvarez-Bernal, D., Dendooven, L., 2006. Eisenia fetida increased removal of polycyclic aromatic hydrocarbons from soil. Environmental Pollution 141, 396–401. Edwards, C.A., Bohlen, C.J., 1996. Biology and Ecology of Earthworms, third ed. Chapman & Hall, London. Foght, J.M., Fedorak, P.M., Westlake, D.W.S., 1990. Mineralization of [14C] hexadecane and [14C] phenanthrene in crude oil: specificity among bacterial isolates. Canadian Journal of Microbiology 36, 169–175. Geerdink, M.J., Van Loosdrecht, M.C.M., Luyben, K.C.A.M., 1996. Biodegradability of diesel oil. Biodegradation 7, 73–81. Gevao, B., Mordaunt, C., Semple, K.T., Piearce, T.G., Jones, K.C., 2001. Bioavailability of nonextractable (bound) pesticide residues to earthworms. Environmental Science and Technology 35, 501–507. Hickman, Z.A., Reid, B.J., 2008. Earthworm assisted bioremediation of organic contaminants. Environment International, doi:10.1016/j.envint.2008.02.013. Kastner, M., Lotter, S., Heerenklage, J., BreuerJammali, M., Stegmann, R., Mahro, B., 1995. Fate of 14C-labeled anthracene and hexadecane in compost-manured soil. Applied Microbiology and Biotechnology 43, 1128–1135. Kastner, M., Mahro, B., 1996. Microbial degradation of polycyclic aromatic hydrocarbons in soils affected by the organic matrix of compost. Applied Microbiology and Biotechnology 44, 668–675. Kersante, A., Martin-Laurent, F., Soulas, G., Binet, F., 2006. Interactions of earthworms with atrazine-degrading bacteria in an agricultural soil. FEMS Microbiology Ecology 57, 192–205. Lavelle, P., Pashanasi, B., Charpentier, F., Gilot, C., Rossii, J.-P., Derouard, L., Andre, J., Ponge, J.-F., Bernier, N., 2004. In: Edwards, C.A. (Ed.), Earthworm Ecology, second ed. CRC Press, Boca Raton, FL, pp. 145–160. Leahy, J.G., Colwell, R.R., 1990. Microbial degradation of hydrocarbons in the environment. Microbiological Reviews 54, 305–315. Liste, H.-H., Quinones-Rivera, A., Tang, J., 2002. Availability of aged toluene and decane for biodegradation in soil. Water, Air, and Soil Pollution 133, 227–234. Marchall, R., Penet, R., Solana-Serena, S., Vandecasteele, J.P., 2003. Gasoline and oil biodegradation. Oil & Gas Science and Technology 58, 441–448. Namkoong, W., Hwang, E.-Y., Park, J.S., Choi, J.-Y., 2002. Bioremediation of dieselcontaminated soil with composting. Environmental Pollution 119, 23–31. Olsen, J.J., Mills, G.L., Herbert, B.E., Morris, P.J., 1999. Biodegradation rates of separated diesel components. Environmental Toxicology and Chemistry 18, 2448– 2453. Ostberg, T.L., Jonsson, A.P., Lundstrom, U.S., 2007. Enhanced degradation of n-hexadecane in diesel fuel contaminated soil by the addition of fermented whey. Soil and Sediment Contamination 16, 221–232. Prenafeta-Boldu, F.X., Vervoot, J., Grotenhuis, J.T.C., Van-Groenestijn, J.W., 2002. Substrate interactions during the biodegradation of benzene, toluene, ethylbenzene, and xylene (BTEX) hydrocarbons by the fungus Cladophialophora sp. strain T1. Applied and Environmental Microbiology 68, 2660–2665. Prince, R.C., Parkerton, T.F., Lee, C., 2007. The primary aerobic biodegradation of gasoline hydrocarbons. Environmental Science and Technology 41, 3316– 3321. Puglisi, E., Cappa, F., Fragoulis, G., Trevisan, M., Del Re, A.A.M., 2006. Bioavailability and degradation of phenanthrene in compost amended soils. Chemosphere 67, 548–556. Reid, B.J., Jones, K.C., Semple, K.T., 2000. Bioavailability of persistent organic pollutants in soils and sediments – a perspective on mechanisms, consequences and assessment. Environmental Pollution 108, 103–112. Reid, B.J., Fermor, T.R., Semple, K.T., 2002. Induction of PAH-catabolism in mushroom compost and its use in the biodegradation of soil-associated phenanthrene. Environmental Pollution 118, 65–73. Robinson, K.G., Farmer, W.S., Novak, J.Y., 1990. Availability of sorbed toluene in soils for biodegradation by acclimated bacteria. Water Research 24, 345–350. Romantschuck, M., Sarand, I., Petanen, T., Peltola, R., Jonsson-Vihanne, M., Koivula, T., Yrjala, K., Haahtela, K., 2000. Means to improve the effect of in-situ bioremediation of contaminated soil: an overview of novel approaches. Environmental Pollution 107, 179–185. Sasek, V., Bhatt, M., Cajthaml, T., Malachova, K., Lednicka, D., 2003. Compostmediated removal of polycyclic aromatic hydrocarbons from contaminated soil. Archives of Environmental Contamination and Toxicology 44, 336–342. Schaefer, M., Filser, J., 2007. The influence of earthworms and organic additives on the biodegradation of oil contaminated soil. Applied Soil Ecology 36, 53– 62. Semple, K.T., Reid, B.J., Fermor, T.R., 2001. Impact of composting strategies on the treatment of soils contaminated with organic pollutants. Environmental Pollution 112, 269–283.
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Semple, K.T., Morriss, W.W.J., Paton, G.I., 2003. Bioavailability of hydrophobic organic contaminants in soils: fundamental concepts and techniques for analysis. European Journal of Soil Science 54, 809–818. Tharaken, J., Tomlinson, D., Addagada, A., Shafagati, A., 2006. Biotransforamtion of PCBs in contaminated sludge: potential for novel biological technologies. Engineering in Life Sciences 6, 43–50. Verma, A., Pillai, M.K.K., 1991. Bioavailability of soil-bound residues of DDT and HCH to earthworms. Current Science 61, 840–843.
Walker, J.D., Colwell, R.R., 1976. Measuring the potential of hydrocarbon-degrading bacteria. Applied and Environmental Microbiology 31, 189–197. Yadav, J.S., Reddy, C.A., 1993. Degradation of benzene, toluene, ethylbenzene, and xylenes (BTEX) by the lignen-degrading basidiomycete Phanerochaete chrysosporium. Applied and Environmental Microbiology 59, 756–762. Yerushalmi, L., Guiot, S.R., 1998. Kinetics of biodegradation of gasoline and its hydrocarbon constituents. Applied Microbiology and Biotechnology 49, 475– 481.