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Bioremediation of petroleum-contaminated soil by biostimulation amended with biochar
International Biodeterioration & Biodegradation 85 (2013) 150e155
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International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Bioremediation of petroleum-contaminated soil by biostimulation amended with biochar Gang Qin a, *, Dan Gong b, Mei-Ying Fan c a
College of Engineering and Technology, Yangtze University, Xueyuan Road, Jingzhou 434020, Hubei, PR China School of Geophysics and Oil Resources, Yangtze University, Xueyuan Road, Jingzhou 434023, Hubei, PR China c Graduate School, Yangtze University, Xueyuan Road, Jingzhou 434023, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 June 2013 Received in revised form 3 July 2013 Accepted 11 July 2013 Available online
In this study, the effects of rice straw biochar on soil contaminant biodegradation and microbial community compositions were investigated in the laboratory during a 180-day period. The results of soil microcosm experiments showed that contaminant degradation efficiency was significantly higher in soils amended with biochar than in soils without. The adding time of biochar had apparent effects on degradation efficiency. The removal efficiencies of total petroleum hydrocarbons (TPH) were 61.2%, 77.8% and 84.8%, in the soils without biochar, amended with biochar at the beginning or the 80th day respectively. When adding biochar at the 80th day, the TPH concentration decreased to below the threshold level required for Chinese soil quality for TPH (3000 mg kg1 dry weight) in 140 days. The addition of biochar did not result in appreciable negative impacts on soil microbial community composition. It was speculated that when adding biochar at the 80th day, a large amount of metabolites could be absorbed onto the biochar, leading to significant reduction in soil toxicity and biodegradation enhancement. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Biostimulation Bioremediation MicrotoxÒ toxicity Metagenomics Sequencing
1. Introduction Spills, leaks and other releases of petroleum hydrocarbons can cause large amounts of soil spread pollution, representing a major environmental concern with serious consequences that has been drawing public concerns worldwide over the recent decades (Lu et al., 2010). Bioremediation of petroleum contaminated soils is a hot area of soil restoration research, because of its relatively low costs and environmentally friendship compared to physical and chemical processes (Grace Liu et al., 2011). This technique has been shown to be effective for petroleum contaminated soils in both laboratory and field tests (Xu and Lu, 2010; Beskoski et al., 2011; Mukherjee and Bordoloi, 2011). However, the wide practice of bioremediation in the field is limited by its low efficiency and long-term maintenance (Trindade et al., 2005). This is especially true for historically contaminated sites where the pollutants mainly consisted of complex compounds with recalcitrant chemical structures and low bioavailability (Huesemann et al., 2004). The hydrophobic
* Corresponding author. Tel./fax: þ86 716 8067521. E-mail address: [email protected] (G. Qin). 0964-8305/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibiod.2013.07.004
nature of oil retard mass transfer of air, water, and contaminants from soil particles to microorganisms, limiting the rate of uptake and metabolism of contaminants by hydrocarbon degraders (Semple et al., 2003). Some auxiliary measures such as bioslurry treatment (Lu et al., 2009), chemical oxidation (Lu et al., 2010; Gong, 2012), surfactant enhancement (Mata-Sandoval et al., 2002; Urum et al., 2006), were incorporated into biological processes to enhance pollutant bioavailability and/or to reduce substrate toxicity. Biochar produced from biomass may sequester atmospheric CO2 in soils for a long time, and thus reducing the carbon footprint of sorbent-based soil remediation in comparison with the use of coal-derived activated carbon (Bushnaf et al., 2011). Activated carbon or biochar amendments have been deployed in certain soil and sediment remediation purposes. The use of biochar could be cheaper in a remediation sense relative to AC because the waste source materials are essentially free and the production of biochar requires less energy and cost (Hale et al., 2011). The agronomic benefits of biochar in addition to sequestering C in plant based remediation may also be related to an increment of liming effects, water holding capacity, soil structure, cation exchange capacity, soil microbial activities and finally the plant growth (Glaser et al., 2002; Beesley et al., 2010). However, biochar amendment to degraded soil has
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also been shown to reduce the pollutant’s availability for microbial break-down and increase persistence (Rhodes et al., 2008). In the present work, the feasibility of the use of biochar in petroleum hydrocarbon contaminated soil remediation was assessed. To investigate the effect of adding time, biochar was amended at two different periods respectively, i.e. the beginning or the middle stage of the experiments. 2. Materials and methods 2.1. Contaminated soil The petroleum-contaminated soil was collected from an oil spill site near a tank container in Shengli Oilfield, China. For sampling, surface litters were removed and soil samples were collected to a depth of 30 cm. The soil was air-dried and sieved through a 2-mm mesh sieve, homogenized by hand with a shovel, and then stored at 4 C in the dark until used. The texture of the raw contaminated soil was classified as a clay loam, which contained (dry weight basis, d.w.): sand, 23.5%; silt, 51.2%; clay, 25.3%. The soil had the following characteristics: pH (1:2.5, soil/water ratio), 6.5; conductivity (1:10, soil/water ratio), 535 mS cm1; water holding capacity, 38.6 wt.%; humidity, 15.2 wt.%; total organic carbon (TOC), 5.42 wt.%; total nitrogen 85 mg kg1; total phosphorus 16 mg kg1; total heterotrophic bacteria, 7.50 106 colony-forming units (CFU) g1; diesel oil degrading bacteria, 3.60 104 CFU g1; total petroleum hydrocarbons (TPH), 16,300 mg kg1 (saturated hydrocarbons, 8260 mg kg1; aromatic hydrocarbons, 5130 mg kg1; polar components, 2910 mg kg1). The soil contained a significant amount and proportion of an alkane (saturated) fraction-degrading population (0.48%), suggesting that biostimulation strategy was feasible for this soil matrix. Additionally, the oil contained a relatively high percentage of aromatic fractions (31.5%). 2.2. Biochar Biochar was produced from rice straw at 500 C using a slow pyrolysis under limited oxygen according to (Lou et al., 2013). Table 1 lists the characteristics of rice straw feed. Following charring, the biochar was lightly ground and sieved to obtain a fine (<0.16 mm) size fraction, which was then rinsed with distilled water to remove the ash content and dried at 60 C for 5 days. The sample had a total surface area of 1053 m2 g1, TOC content of 83.5 wt.%, C:N:S weight ratio of 325:3.9:1, and a pH of 8.9 0.1 (1:2.5, biochar/water ratio).
Table 1 The main characteristics of rice straw feed. Composition
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2.3. Soil microcosm experiments The soil was subjected to different treatments for an additional 180 days. For each treatment, three independent replicates (2-L glass receptacles covered with perforated parafilm) were prepared as microcosms, each containing 1000 g of soil. In all the treatments, the water content was adjusted to 60% of water holding capacity. Once a week, the microcosm contents were mixed and the soil water content was restored by controlling the weight. Every two weeks, (NH4)2SO4 and K2HPO4 were added to produce a final C:N:P ratio of 100:10:5 (Gong, 2012). Four different trials were applied in triplicate: soil was supplemented with 2% (w/w) HgCl2 to account for abiotic loss of pollutants (A); soil received no biochar (B); the soil was amended with 2% (w/w) biochar at the beginning (C), and the 80th day (D) of the experiment. 2.4. Analytical methods Oil in soil was soxhlet-extracted with dichloromethane for 16 h. The extract was condensed to 1 mL in a rotary evaporator and fractionated by silica gel column chromatography to separate saturate, aromatic and polar fractions, following the methods of Bastow et al. (2007). The different elute was evaporated to dryness under N2, and calculated gravimetrically. The measurements of n-alkanes and polycyclic aromatic hydrocarbons (PAHs) were performed by gas chromatographyemass spectrometry (GCeMS), using a Thermo-Finnigan SSQ710 GCeMS (Thermo Finnigan, San Jose, CA, USA) with a HP-5MS elastic silica capillary columns (60 m 0.25 mm 0.25 mm). The carrier gas was helium at 37 kPa. Flow velocity was 1 mL min1. The analytical conditions were: initial temperature of 50 C, with isothermal operation for 1 min; heating to 120 C at a constant rate of 20 C min1; and heating to a final temperature of 310 C at a constant rate of 4 C min1, with a 30 min isothermal. Mass spectrometer conditions were: electron impact, electron energy 70 eV; filament current 100 mA; multiplier voltage, 1200 V; full scan. Concentrations of each n-alkane were calculated based on the standard calibration curve of each corresponding standard compound (Accu Standards Inc., New Haven, CT, USA). Individual PAHs were quantified based on the retention time and m/z ratio of an authentic PAH mixed standard (SigmaeAldrich, St. Louis, MO, USA), and concentrations of each PAH were calibrated based on the standard calibration curve. 2.5. Microbiological enumeration The heterotrophic bacteria were counted on nutrient agar after incubation at 30 C for 2 days, and the results were expressed as CFU per gram of dry soil. 2.6. MicrotoxÒ toxicity assay
Proximate analysis
Component
Content (wt.%)
Component
Content (wt.%)
Cellulose Hemicellulose Lignin
57.2 29.6 13.2
Water Volatile matter Fixed carbon Ash
5.3 65.4 16.6 12.7
Elemental analysis
Alkali metal concentration
Element
Content (wt.%)
Alkali metal
Content (mg kg1)
C H N S O
38.5 4.9 1.8 0.76 54.0
Na Mg Ca K
273 1942 3105 23,875
The toxicity of soil elutriate was determined using the MicrotoxÒ bioassay according to Xu and Lu (2010). Toxicity values were the average of five replicates of each filtrate sample, expressed as EC50 (15 min, 15 C), which was defined as the effective concentration of pollutant for a 50% reduction of the luminescence of the bacterium Photobacterium phosphoreum. 2.7. DNA extraction, PCR amplification and 454 sequencing High-molecular-weight DNA from the soil was extracted with a commercially available kit (Beijing Dingguo Biotechnology Ltd., China). The 16S rDNA genes were amplified by polymerase chain reaction (PCR) in a Techgene thermocycler (FTGENE 5D, 112757-4,
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Techne Combridge Ltd., Duxford, Cambridge UK), using the forward primer 563F (50 -AYTGGGYDTAAAGNG-30 ) at the 50-end (E. coli positions 563e578) of the V4 region (239 nucleotides) and a cocktail of four equally mixed reverse primers, that is, R1 (50 TACCRGGGTHTCTAATCC-30 ), R2 (50 -TACCAGAGTATCTAATTC-30 ), R3 (50 -CTACDSRGGTMTCTAATC-30 ) and R4 (50 -TACNVGGGTATCTAATC30 ), at the 30-end of the V4 region (E. coli positions 785e802) (Murphy et al., 2010). Then DNA samples with different barcodes were mixed in equal concentration and sequenced by a Roche 454 FLX Titanium sequencer (Roche, Nutley, NJ, USA) at the Beijing Genomics Institute (Shenzhen, China). The pyrosequencing methodology used was identical to that reported by Davis et al. (2011).
sequence set of sample was aligned by Infernal (Nawrocki and Eddy, 2007) using the bacteria-alignment model in Align module of the RDP. 2.9. Statistical analysis All experiments in this study were performed in triplicate to get reliable data, and the results presented here represent the average values of three independent measurements standard deviations. The variance and significant differences among various treatments were analyzed by Student’s t-test. Data were considered to be significantly different among values if p < 0.05. All statistical analysis was performed with SPSS 13.0 for Windows.
2.8. Post-run analysis 3. Results and discussion The raw reads were treated with the Pyrosequencing Pipeline Initial Process (Cole et al., 2009) of the Ribosomal Database Project (RDP), (1) to sort those exactly matching the specific barcodes into different samples, (2) to trim off the adapters, barcodes and primers using the default parameters, and (3) to remove sequences containing ambiguous ‘N’ or shorter than 150 bps (Claesson et al., 2009). The reads selected above were defined as ‘raw reads’ for each soil sample. Taxonomic classification of the bacterial sequences of sample was carried out using the Ribosomal Database Project (RDP) Classifier. A bootstrap cutoff of 50% suggested by the RDP was applied to assign the sequences to different taxonomy levels. The normalized Trial A Trial C
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Generally, petroleum hydrocarbon biodegradation in soils is characterized by a rapid removal during the initial stage, followed by a slower and even plateau phase (Alexander, 1995). Under abiotic conditions, trial A showed a negligible decrease in soil TPH concentration (Fig. 1a), which may be due to that most of the volatile hydrocarbons had disappeared during the long-term natural attenuation. Biostimulation by nutrient amendment (trial B) caused a rapid reduction of TPH in early stage (within 60 days), followed by an apparent slowdown of biodegradation until the 180th day. At the
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Fig. 1. Time course of concentrations of TPH (a), saturates (b), aromatics (c), and polar fraction (d) in soils during 180-day bioremediation. Trial A: abiotic control; trial B: soil received no biochar; trial C: the soil was amended with 2% (w/w) biochar at the beginning; trial D: the soil was amended with 2% (w/w) biochar at the 80th day. Errors bars indicate SD of triplicate samples. The line indicates TPH legal limit for commercial-industrial use (3000 mg kg1 dry weight) in China.
G. Qin et al. / International Biodeterioration & Biodegradation 85 (2013) 150e155 Trial B
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end of process, the total removal efficiency of TPH for trial B was 61.2% (from 16,300 mg kg1 at t ¼ 0 day to 6420 mg kg1 at t ¼ 180 days). Actually, TPH removal reached a plateau at the late stage of the experiment, and no apparent changes in TPH concentrations were observed since the 120th day (Fig. 1a). It is known that petroleum hydrocarbons cannot be completely metabolized by microorganisms to CO2 and H2O, and always leaves more or less complex residues (mainly recalcitrant compounds and metabolites) (Atlas, 1995). These compounds often become increasingly less bioavailable with the passing of time due to their low solubility in water and their sequestration by soils (Alexander, 1995). Additionally, high concentrations of fatty acids generated by petroleum biooxidation could exert ecotoxicity to soil microflora and thus impact further biodegradation processes (Lu et al., 2010). The addition of 2% biochar at the beginning of the experiment did not result in significantly greater reduction in the TPH concentration until the 60th day relative to the soil without biochar (Fig. 1a). Afterwards, biodegradation process was accelerated and TPH level in trial C decreased to 3625 mg kg1 after 180 days of treatment, corresponding to a total removal efficiency of 77.8%, which was significantly higher than that of trial B (p < 0.05). Actually, at the 160th day, TPH concentration in trial C had declined to 3870 mg kg1, and the biodegradation rate had also slowed down during the late period. Interestingly, when adding biochar at day 80, bioremediation was promoted significantly (Fig. 1a). Since the 140th day, TPH concentration was significantly lower in trial D than in trial C, reaching a concentration below the threshold level required for Chinese soil quality for TPH (3000 mg kg1 d.w.). At the end of the experiment, TPH concentration in trial D decreased to 2480 mg kg1, corresponding to a total removal efficiency of 84.8%. These results indicated that adding time of biochar had apparent influence on biodegradation. A few investigations have demonstrated that black carbon (including activated carbon and biochar) could reduce contaminant bioavailability and biodegradation in soil (Zhang et al., 2005; Rhodes et al., 2008). In these studies, pollutant concentrations in soil were generally lower (<500 mg kg1), and apparent sequestering effects may occur, leading to reduction in pollutant mobility and bioavailability. Contrastingly, Vasilyeva et al. (2010) found that adding activated carbon helped overcome toxicity of polychlorinated biphenyls to microorganisms, and Hale et al. (2011) also reported that biochar amendment had a stimulatory effect on PAH biodegradation in soil. In the present study, the reduction in the mobility and bioaccessibility of soil contaminants caused by biochar addition may be lower, since the TPH concentration was relatively high. Xia et al. (2010) held that biochar generated under lower temperatures may absorb contaminants via a partition mechanism that is relatively more accessible to microbes than adsorption dominant processes, which appear in biochar produced at higher temperatures. Interestingly, it was found that phenanthrene adsorbed to black carbon was more bioavailable than predicted by chemical extraction (Rhodes et al., 2008). It was hypothesized that microorganisms adhered to black carbon particles, and degraded phenanthrene absorbed onto the surface. Existing studies demonstrate complex effects of black carbon addition on soil bioremediation of organic pollutants, which could be related to certain factors including the origin and production method of black carbon, soil characteristics, contaminant types and microbial properties, etc.
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Fig. 2. Total removal percentages of n-alkanes and 16 priority PAHs after 180 days of treatment. Trial B: soil received no biochar; trial C: the soil was amended with 2% (w/ w) biochar at the beginning; trial D: the soil was amended with 2% (w/w) biochar at the 80th day. The 16 priority PAHs are: naphthalene, acenaphthene, acenaphthylene, fluorene, phenanthrene, anthracene, pyrene, fluoranthrene, benzo[ghi]perylene, benz [a]anthracene, chrysene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, indeno[1,2,3-cd]pyrene, and dibenz[ah]anthracene.
biodegradation of saturates was observed in the biochar amended soil. The concentrations of saturates decreased from 8260 mg kg1 at t ¼ 0 day to 1200, 553 and 612 mg kg1 at t ¼ 180 days, in trial B, C and D respectively, corresponding to removal efficiencies of 85.5%, 93.2% and 92.6%, respectively. It can be found that the addition of biochar slightly enhanced biodegradation of saturates, and adding time of biochar had no apparent impact on degradation efficiency of saturated fractions. Bushnaf et al. (2011) found that biodegradation of linear, cyclic and branched alkanes were more rapid in the soil amended with biochar than that without. As shown in Fig. 2c, adding biochar significantly increased degradation efficiencies of aromatics. The concentrations of aromatics decreased from 5130 mg kg1 at t ¼ 0 day to 1810, 881 and 1025 mg kg1 at t ¼ 180 days, in trial B, C and D respectively, corresponding to removal efficiencies of 64.7%, 82.8% and 80.0%, respectively. It can be observed that adding time of biochar had no apparent effect on degradation efficiency of aromatic fractions. Fig. 2d shows a real increase in the concentrations of polar fractions in the soils after the start-up of incubation, which was ascribed to the accumulation of metabolic intermediates. Adding biochar significantly increased degradation efficiencies of polar fractions, especially when amending biochar at day 80. The concentrations of polar fractions decreased from 2910 mg kg1 at t ¼ 0 day to 2192 and 843 mg kg1 at t ¼ 180 days in trial C and D respectively, corresponding to removal efficiencies of 24.7% and 71.0%, respectively. It has been shown that polar fractions in crude oil are partially or completely resistant to microbial degradation (Pollard et al., 1999). Nevertheless, it has also been reported that a 30% maximum biodegradation of polar compounds can appear in optimal cultures Chaillan et al., 2006). In this study, the addition of biochar at the 80th day can greatly enhance biodegradation of polar fractions. Based on the above results, it can be concluded that, biodegradation of polar fractions is crucial for the success of bioremediation practice of oil contaminated soils. In this study, the lower TPH removal in trial B was mainly due to the accumulation of metabolites.
3.2. Degradation of saturated, aromatic and polar fractions
3.3. Degradation of n-alkanes and PAHs
The concentrations of saturate, aromatic and polar fractions with time are presented in Fig. 2bed, respectively. A more rapid
GCeMS analysis was performed on the extracted oil samples before and after 180 days of incubation, respectively. Total removal
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percentages of n-alkanes (C8eC40) and 16 priority PAHs were calculated and shown in Fig. 2. After 180 days of treatment, n-alkanes and PAHs in trial B showed concentration decreases of 88.6% and 45.3%, respectively. The removal efficiencies of n-alkanes in trial C and D were 95.3% and 94.1%, respectively. Approximately 61.4% and 59.5% of PAHs were removed in trial C and D, respectively. The addition of biochar enhanced removal of n-alkanes and PAHs. In general, the removal efficiencies of PAHs were lower than that of aromatic fractions, indicating that the 16 priority PAHs were more recalcitrant within the aromatic compounds in the oils. 3.4. Total bacterial count A statistically significant difference was observed for the total counts of soil heterotrophic bacteria 180 days after the incubation, with trial D having the highest cell count of 1.31 109 CFU per gram of dry soil weight, 5.1 times higher than trial B, which had the lowest cell count (2.56 108 CFU g1) (Fig. 3a). This result showed that biochar amendment overall was not detrimental to aerobic microbial activity. It is known that biochar reduces bioaccessibility, chemical activity and ecotoxicity of organic compounds to soil microorganisms (Ogbonnaya and Semple, 2013). Moreover, biochar nutrient properties can improve plant growth and microbial activity and have been shown to enhance biodegradation of bioaccessible contaminants (Ogbonnaya and Semple, 2013).
(a) 1.40E+009
3.5. MicrotoxÒ toxicity MicrotoxÒ analysis was performed over the course of the study to monitor toxicity changes in the incubated soils and the results are shown in Fig. 3b. In trial B and D, the MicrotoxÒ toxicity first increased during the initial phase of biodegradation, but started to decrease after 40 days of incubation. In trial C, the toxicity declined during the initial 20 days. Higher EC50 values at day 180 in trial C and D suggested an overall reduction in soil toxicity. Significantly lower toxicity in soil (p < 0.05) was observed in trial D relative to trial B and C. It can be observed that the change trends of soil toxicity generally coincided with that of the concentrations of polar compounds. Thus, it can be speculated that some toxic metabolites were produced during biodegradation of petroleum compounds. In the present work, polar compounds could be more efficiently biodegraded in the soil amended with biochar at day 80 than that at the beginning (Fig. 1). We speculated that, when adding biochar at the beginning, the absorption sites on the biochar surfaces would be occupied mainly by petroleum hydrocarbons. As biodegradation proceeded, the absorption capacity of biochar for metabolites was limited. However, when adding biochar at the 80th day, a large amount of metabolites could be absorbed onto the biochar, because at this point the concentrations of petroleum hydrocarbons had decreased remarkably and a great quantity of metabolites had been accumulated. Thereupon, the addition of biochar at day 80 could result in significant reduction in soil toxicity and thus promote biodegradation. However, we could not verify this speculation by MicrotoxÒ toxicity assay, because the tiny size of biochar particles made it impossible to separate biochar from the soil after mixing and incubation.
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Time (days) Fig. 3. (a) Total microbial cell count for the soil samples taken after 180 days. (b) Time course of EC50 value of the soil samples during the 180-day period. Trial B: soil received no biochar; trial C: the soil was amended with 2% (w/w) biochar at the beginning; trial D: the soil was amended with 2% (w/w) biochar at the 80th day.
Fig. 4. Bacterial community taxonomic composition before and after 180 days of incubation based on metagenomic sequencing. (a) Community composition at the phylum/class level based on all annotated fragments. (b) Pseudomonas relative abundance based on all annotated fragments. Trial B: soil received no biochar; trial C: the soil was amended with 2% (w/w) biochar at the beginning; trial D: the soil was amended with 2% (w/w) biochar at the 80th day.
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3.6. Metagenomic sequencing Biostimulation treatment resulted in the increment of the abundance of several bacterial phylum/classes, like Gammaproteobacteria, Actinobacteria, Sphingomonadales and Alphaproteobacteria (Fig. 4a). In contrast, bioremediation process resulted in a reduction in the abundance of Acidobacteria, Bacteroidetes, Chlorobi, Chloroflexi, Cyanobacteria, Burkholderiales, Planctomycetes and Deltaproteobacteria. The addition of biochar produced relatively similar taxonomic profiles at the phylum/class level, but with some interesting variations. Gammaproteobacteria, Acidobacteria, Chlorobi and Chloroflexi increased through the time course while Actinobacteria and Alphaproteobacteria decreased (Fig. 4a). In the t ¼ 180 days samples, Gammaproteobacteria comprised up to 38% of the total community. At the phylum/class level, the samples after biotreatment showed a diversified taxonomic profile, with a clear dominance of any of Gammaproteobacteria. The high abundance of Gammaproteobacteria in the t ¼ 180 days samples was mainly due to Pseudomonas species (Fig. 4b). The relative abundance of Pseudomonas increased after incubation. In general, Fig. 4 shows that biochar amendment did not significantly alter bacterial community compositions. This indicates that biochar in the present study had no apparent negative effect on soil microorganism populations. Pseudomonas, Rhodococcus, Caulobacter and sphingomonads are well-known hydrocarbon degraders, which tend to be enriched in well aerated soils with larger amounts of nitrogen nutrients and oil. Pseudomonas species were hypothesized to be one of the major alkane and aromatic hydrocarbon degraders in petroleum contaminated soils (Haines et al., 2002). 4. Conclusions This work demonstrated the feasibility of a bioremediation process using biostimulation with biochar amendment for petroleum-contaminated soil. The results of soil microcosm experiments showed that the soil amended with rice straw biochar resulted in a better improvement in pollutant removal, compared with biostimulation alone. When adding biochar at the 80th day, the TPH concentration decreased to below the threshold level required for Chinese soil quality for TPH (3000 mg kg1 dry weight) in 140 days. The results showed the applicability in use of biochar for remediation of oil-contaminated soil. The addition of biochar did not cause appreciable negative influences on soil microflora. Therefore, rice straw biochar may act as an efficient soil remediation additive. References Alexander, M., 1995. How toxic are toxic chemicals in soil? Environmental Science and Technology 29, 2713e2717. Atlas, R.M., 1995. Petroleum biodegradation and oil spill bioremediation. Marine Pollution Bulletin 31, 178e182. Bastow, T.P., van Aarssen, B.G.K., Lang, D., 2007. Rapid small-scale separation of saturate, aromatic and polar components in petroleum. Organic Geochemistry 38, 1235e1250. Beesley, L., Moreno-Jiménez, E., Gomez-Eyles, J.L., 2010. Effects of biochar and greenwaste compost amendments on mobility, bioavailability and toxicity of inorganic and organic contaminants in a multi-element polluted soil. Environmental Pollution 158, 2282e2287. Beskoski, V.P., Gojgi c-Cvijovi c, G., Mili c, J., Ili c, M., Mileti c, S., Solevi c, T., Vrvi c, M.M., 2011. Ex situ bioremediation of a soil contaminated by mazut (heavy residual fuel oil) e a field experiment. Chemosphere 83, 34e40. Bushnaf, K.M., Puricelli, S., Saponaro, S., Werner, D., 2011. Effect of biochar on the fate of volatile petroleum hydrocarbons in an aerobic sandy soil. Journal of Contaminant Hydrology 126, 208e215. Chaillan, F., Chaîneau, C.H., Point, V., Saliot, A., Oudot, J., 2006. Factors inhibiting bioremediation of soil contaminated with weathered oils and drill cuttings. Environmental Pollution 144, 255e265.
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Gong, X.B., 2012. Remediation of weathered petroleum oil-contaminated soil using a combination of biostimulation and modified Fenton oxidation. International Biodeterioration & Biodegradation 70, 89e95. Grace Liu, P.W., Chang, T.C., Whang, L.M., Kao, C.H., Pan, P.T., Cheng, S.S., 2011. Bioremediation of petroleum hydrocarbon contaminated soil: effects of strategies and microbial community shift. International Biodeterioration & Biodegradation 65, 1119e1127. Haines, J.R., Herrmann, R., Lee, K., Cobanli, S., Blaise, C., 2002. Microbial population analysis as a measure of ecosystem restoration. Bioremediation Journal 6, 283e296. Hale, S., Hanley, K., Lehmann, J., Zimmerman, A., Cornelissen, G., 2011. Effects of chemical, biological, and physical aging as well as soil addition on the sorption of pyrene to activated carbon and biochar. Environmental Science & Technology 45, 10445e10453. Huesemann, M.H., Hausmann, T.S., Fortman, T.J., 2004. Does bioavailability limit biodegradation? A comparison of hydrocarbon biodegradation and desorption rates in aged soils. Biodegradation 15, 261e274. Lou, L., Liu, F., Yue, Q., Chen, F., Yang, Q., Hu, B., Chen, Y., 2013. Influence of humic acid on the sorption of pentachlorophenol by aged sediment amended with rice-straw biochar. Applied Geochemistry 33, 76e83. Lu, M., Zhang, Z., Sun, S., Wang, Q., Zhong, W., 2009. Enhanced degradation of bioremediation residues in petroleum contaminated-soil using a two-liquid phase bioslurry reactor. Chemosphere 77, 161e168. Lu, M., Zhang, Z., Qiao, W., Wei, X., Guan, Y., Ma, Q., Guan, Y., 2010. Remediation of petroleum-contaminated soil after composting by sequential treatment with Fenton-like oxidation and biodegradation. Bioresource Technology 101, 2106e 2113. Mata-Sandoval, J.C., Karns, J., Torrents, A., 2002. Influence of rhamnolipids and Triton X-100 on the desorption of pesticides from soils. Environmental Science & Technology 36, 4669e4675. Mukherjee, A.K., Bordoloi, N.K., 2011. Bioremediation and reclamation of soil contaminated with petroleum oil hydrocarbons by exogenously seeded bacterial consortium: a pilot-scale study. Environmental Science and Pollution Research 18, 471e478. Murphy, E., Cotter, P., Healy, S., Marques, T., O’Sullivan, O., Fouhy, F., Clarke, S., O’Toole, P., Quigley, E., Stanton, C., Ross, P., O’Doherty, R., Shanahan, F., 2010. Composition and energy harvesting capacity of the gut microbiota: relationship to diet, obesity and time in mouse models. Gut 59, 1635e1642. Nawrocki, E.P., Eddy, S.R., 2007. Query-dependent banding (QDB) for faster RNA similarity searches. PLoS Computational Biology 3, e56. Ogbonnaya, U., Semple, K.T., 2013. Impact of biochar on organic contaminants in soil: a tool for mitigating risk? Agronomy 3, 349e375. Pollard, S.J.T., Whittaker, M., Risden, G.C., 1999. The fate of heavy oil wastes in soil microcosms. I, a performance assessment of biotransformation indices. Science of the Total Environment 226, 1e22. Rhodes, A.H., Carlin, A., Semple, K.T., 2008. Impact of black carbon in the extraction and mineralization of phenanthrene in soil. Environmental Science and Technology 42, 740e745. Semple, K.T., Morriss, A.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, 809e818. Trindade, P.V.O., Sobral, L.G., Rizzo, A.C.L., Leite, S.G.F., Soriano, A.U., 2005. Bioremediation of a weathered and a recently oil-contaminated soils from Brazil: a comparison study. Chemosphere 58, 515e522. Urum, K., Grigson, S., Pekdemir, T., McMenamy, S., 2006. A comparison of the efficiency of different surfactants for removal of crude oil from contaminated soils. Chemosphere 62, 1403e1410. Vasilyeva, G.K., Strijakova, E.R., Nikolaeva, S.N., Lebedev, A.T., Shea, P.J., 2010. Dynamics of PCB removal and detoxification in historically contaminated soils amended with activated carbon. 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Contents lists available at ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Bioremediation of petroleum-contaminated soil by biostimulation amended with biochar Gang Qin a, *, Dan Gong b, Mei-Ying Fan c a
College of Engineering and Technology, Yangtze University, Xueyuan Road, Jingzhou 434020, Hubei, PR China School of Geophysics and Oil Resources, Yangtze University, Xueyuan Road, Jingzhou 434023, Hubei, PR China c Graduate School, Yangtze University, Xueyuan Road, Jingzhou 434023, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 June 2013 Received in revised form 3 July 2013 Accepted 11 July 2013 Available online
In this study, the effects of rice straw biochar on soil contaminant biodegradation and microbial community compositions were investigated in the laboratory during a 180-day period. The results of soil microcosm experiments showed that contaminant degradation efficiency was significantly higher in soils amended with biochar than in soils without. The adding time of biochar had apparent effects on degradation efficiency. The removal efficiencies of total petroleum hydrocarbons (TPH) were 61.2%, 77.8% and 84.8%, in the soils without biochar, amended with biochar at the beginning or the 80th day respectively. When adding biochar at the 80th day, the TPH concentration decreased to below the threshold level required for Chinese soil quality for TPH (3000 mg kg1 dry weight) in 140 days. The addition of biochar did not result in appreciable negative impacts on soil microbial community composition. It was speculated that when adding biochar at the 80th day, a large amount of metabolites could be absorbed onto the biochar, leading to significant reduction in soil toxicity and biodegradation enhancement. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Biostimulation Bioremediation MicrotoxÒ toxicity Metagenomics Sequencing
1. Introduction Spills, leaks and other releases of petroleum hydrocarbons can cause large amounts of soil spread pollution, representing a major environmental concern with serious consequences that has been drawing public concerns worldwide over the recent decades (Lu et al., 2010). Bioremediation of petroleum contaminated soils is a hot area of soil restoration research, because of its relatively low costs and environmentally friendship compared to physical and chemical processes (Grace Liu et al., 2011). This technique has been shown to be effective for petroleum contaminated soils in both laboratory and field tests (Xu and Lu, 2010; Beskoski et al., 2011; Mukherjee and Bordoloi, 2011). However, the wide practice of bioremediation in the field is limited by its low efficiency and long-term maintenance (Trindade et al., 2005). This is especially true for historically contaminated sites where the pollutants mainly consisted of complex compounds with recalcitrant chemical structures and low bioavailability (Huesemann et al., 2004). The hydrophobic
* Corresponding author. Tel./fax: þ86 716 8067521. E-mail address: [email protected] (G. Qin). 0964-8305/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibiod.2013.07.004
nature of oil retard mass transfer of air, water, and contaminants from soil particles to microorganisms, limiting the rate of uptake and metabolism of contaminants by hydrocarbon degraders (Semple et al., 2003). Some auxiliary measures such as bioslurry treatment (Lu et al., 2009), chemical oxidation (Lu et al., 2010; Gong, 2012), surfactant enhancement (Mata-Sandoval et al., 2002; Urum et al., 2006), were incorporated into biological processes to enhance pollutant bioavailability and/or to reduce substrate toxicity. Biochar produced from biomass may sequester atmospheric CO2 in soils for a long time, and thus reducing the carbon footprint of sorbent-based soil remediation in comparison with the use of coal-derived activated carbon (Bushnaf et al., 2011). Activated carbon or biochar amendments have been deployed in certain soil and sediment remediation purposes. The use of biochar could be cheaper in a remediation sense relative to AC because the waste source materials are essentially free and the production of biochar requires less energy and cost (Hale et al., 2011). The agronomic benefits of biochar in addition to sequestering C in plant based remediation may also be related to an increment of liming effects, water holding capacity, soil structure, cation exchange capacity, soil microbial activities and finally the plant growth (Glaser et al., 2002; Beesley et al., 2010). However, biochar amendment to degraded soil has
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also been shown to reduce the pollutant’s availability for microbial break-down and increase persistence (Rhodes et al., 2008). In the present work, the feasibility of the use of biochar in petroleum hydrocarbon contaminated soil remediation was assessed. To investigate the effect of adding time, biochar was amended at two different periods respectively, i.e. the beginning or the middle stage of the experiments. 2. Materials and methods 2.1. Contaminated soil The petroleum-contaminated soil was collected from an oil spill site near a tank container in Shengli Oilfield, China. For sampling, surface litters were removed and soil samples were collected to a depth of 30 cm. The soil was air-dried and sieved through a 2-mm mesh sieve, homogenized by hand with a shovel, and then stored at 4 C in the dark until used. The texture of the raw contaminated soil was classified as a clay loam, which contained (dry weight basis, d.w.): sand, 23.5%; silt, 51.2%; clay, 25.3%. The soil had the following characteristics: pH (1:2.5, soil/water ratio), 6.5; conductivity (1:10, soil/water ratio), 535 mS cm1; water holding capacity, 38.6 wt.%; humidity, 15.2 wt.%; total organic carbon (TOC), 5.42 wt.%; total nitrogen 85 mg kg1; total phosphorus 16 mg kg1; total heterotrophic bacteria, 7.50 106 colony-forming units (CFU) g1; diesel oil degrading bacteria, 3.60 104 CFU g1; total petroleum hydrocarbons (TPH), 16,300 mg kg1 (saturated hydrocarbons, 8260 mg kg1; aromatic hydrocarbons, 5130 mg kg1; polar components, 2910 mg kg1). The soil contained a significant amount and proportion of an alkane (saturated) fraction-degrading population (0.48%), suggesting that biostimulation strategy was feasible for this soil matrix. Additionally, the oil contained a relatively high percentage of aromatic fractions (31.5%). 2.2. Biochar Biochar was produced from rice straw at 500 C using a slow pyrolysis under limited oxygen according to (Lou et al., 2013). Table 1 lists the characteristics of rice straw feed. Following charring, the biochar was lightly ground and sieved to obtain a fine (<0.16 mm) size fraction, which was then rinsed with distilled water to remove the ash content and dried at 60 C for 5 days. The sample had a total surface area of 1053 m2 g1, TOC content of 83.5 wt.%, C:N:S weight ratio of 325:3.9:1, and a pH of 8.9 0.1 (1:2.5, biochar/water ratio).
Table 1 The main characteristics of rice straw feed. Composition
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2.3. Soil microcosm experiments The soil was subjected to different treatments for an additional 180 days. For each treatment, three independent replicates (2-L glass receptacles covered with perforated parafilm) were prepared as microcosms, each containing 1000 g of soil. In all the treatments, the water content was adjusted to 60% of water holding capacity. Once a week, the microcosm contents were mixed and the soil water content was restored by controlling the weight. Every two weeks, (NH4)2SO4 and K2HPO4 were added to produce a final C:N:P ratio of 100:10:5 (Gong, 2012). Four different trials were applied in triplicate: soil was supplemented with 2% (w/w) HgCl2 to account for abiotic loss of pollutants (A); soil received no biochar (B); the soil was amended with 2% (w/w) biochar at the beginning (C), and the 80th day (D) of the experiment. 2.4. Analytical methods Oil in soil was soxhlet-extracted with dichloromethane for 16 h. The extract was condensed to 1 mL in a rotary evaporator and fractionated by silica gel column chromatography to separate saturate, aromatic and polar fractions, following the methods of Bastow et al. (2007). The different elute was evaporated to dryness under N2, and calculated gravimetrically. The measurements of n-alkanes and polycyclic aromatic hydrocarbons (PAHs) were performed by gas chromatographyemass spectrometry (GCeMS), using a Thermo-Finnigan SSQ710 GCeMS (Thermo Finnigan, San Jose, CA, USA) with a HP-5MS elastic silica capillary columns (60 m 0.25 mm 0.25 mm). The carrier gas was helium at 37 kPa. Flow velocity was 1 mL min1. The analytical conditions were: initial temperature of 50 C, with isothermal operation for 1 min; heating to 120 C at a constant rate of 20 C min1; and heating to a final temperature of 310 C at a constant rate of 4 C min1, with a 30 min isothermal. Mass spectrometer conditions were: electron impact, electron energy 70 eV; filament current 100 mA; multiplier voltage, 1200 V; full scan. Concentrations of each n-alkane were calculated based on the standard calibration curve of each corresponding standard compound (Accu Standards Inc., New Haven, CT, USA). Individual PAHs were quantified based on the retention time and m/z ratio of an authentic PAH mixed standard (SigmaeAldrich, St. Louis, MO, USA), and concentrations of each PAH were calibrated based on the standard calibration curve. 2.5. Microbiological enumeration The heterotrophic bacteria were counted on nutrient agar after incubation at 30 C for 2 days, and the results were expressed as CFU per gram of dry soil. 2.6. MicrotoxÒ toxicity assay
Proximate analysis
Component
Content (wt.%)
Component
Content (wt.%)
Cellulose Hemicellulose Lignin
57.2 29.6 13.2
Water Volatile matter Fixed carbon Ash
5.3 65.4 16.6 12.7
Elemental analysis
Alkali metal concentration
Element
Content (wt.%)
Alkali metal
Content (mg kg1)
C H N S O
38.5 4.9 1.8 0.76 54.0
Na Mg Ca K
273 1942 3105 23,875
The toxicity of soil elutriate was determined using the MicrotoxÒ bioassay according to Xu and Lu (2010). Toxicity values were the average of five replicates of each filtrate sample, expressed as EC50 (15 min, 15 C), which was defined as the effective concentration of pollutant for a 50% reduction of the luminescence of the bacterium Photobacterium phosphoreum. 2.7. DNA extraction, PCR amplification and 454 sequencing High-molecular-weight DNA from the soil was extracted with a commercially available kit (Beijing Dingguo Biotechnology Ltd., China). The 16S rDNA genes were amplified by polymerase chain reaction (PCR) in a Techgene thermocycler (FTGENE 5D, 112757-4,
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Techne Combridge Ltd., Duxford, Cambridge UK), using the forward primer 563F (50 -AYTGGGYDTAAAGNG-30 ) at the 50-end (E. coli positions 563e578) of the V4 region (239 nucleotides) and a cocktail of four equally mixed reverse primers, that is, R1 (50 TACCRGGGTHTCTAATCC-30 ), R2 (50 -TACCAGAGTATCTAATTC-30 ), R3 (50 -CTACDSRGGTMTCTAATC-30 ) and R4 (50 -TACNVGGGTATCTAATC30 ), at the 30-end of the V4 region (E. coli positions 785e802) (Murphy et al., 2010). Then DNA samples with different barcodes were mixed in equal concentration and sequenced by a Roche 454 FLX Titanium sequencer (Roche, Nutley, NJ, USA) at the Beijing Genomics Institute (Shenzhen, China). The pyrosequencing methodology used was identical to that reported by Davis et al. (2011).
sequence set of sample was aligned by Infernal (Nawrocki and Eddy, 2007) using the bacteria-alignment model in Align module of the RDP. 2.9. Statistical analysis All experiments in this study were performed in triplicate to get reliable data, and the results presented here represent the average values of three independent measurements standard deviations. The variance and significant differences among various treatments were analyzed by Student’s t-test. Data were considered to be significantly different among values if p < 0.05. All statistical analysis was performed with SPSS 13.0 for Windows.
2.8. Post-run analysis 3. Results and discussion The raw reads were treated with the Pyrosequencing Pipeline Initial Process (Cole et al., 2009) of the Ribosomal Database Project (RDP), (1) to sort those exactly matching the specific barcodes into different samples, (2) to trim off the adapters, barcodes and primers using the default parameters, and (3) to remove sequences containing ambiguous ‘N’ or shorter than 150 bps (Claesson et al., 2009). The reads selected above were defined as ‘raw reads’ for each soil sample. Taxonomic classification of the bacterial sequences of sample was carried out using the Ribosomal Database Project (RDP) Classifier. A bootstrap cutoff of 50% suggested by the RDP was applied to assign the sequences to different taxonomy levels. The normalized Trial A Trial C
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Generally, petroleum hydrocarbon biodegradation in soils is characterized by a rapid removal during the initial stage, followed by a slower and even plateau phase (Alexander, 1995). Under abiotic conditions, trial A showed a negligible decrease in soil TPH concentration (Fig. 1a), which may be due to that most of the volatile hydrocarbons had disappeared during the long-term natural attenuation. Biostimulation by nutrient amendment (trial B) caused a rapid reduction of TPH in early stage (within 60 days), followed by an apparent slowdown of biodegradation until the 180th day. At the
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Fig. 1. Time course of concentrations of TPH (a), saturates (b), aromatics (c), and polar fraction (d) in soils during 180-day bioremediation. Trial A: abiotic control; trial B: soil received no biochar; trial C: the soil was amended with 2% (w/w) biochar at the beginning; trial D: the soil was amended with 2% (w/w) biochar at the 80th day. Errors bars indicate SD of triplicate samples. The line indicates TPH legal limit for commercial-industrial use (3000 mg kg1 dry weight) in China.
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end of process, the total removal efficiency of TPH for trial B was 61.2% (from 16,300 mg kg1 at t ¼ 0 day to 6420 mg kg1 at t ¼ 180 days). Actually, TPH removal reached a plateau at the late stage of the experiment, and no apparent changes in TPH concentrations were observed since the 120th day (Fig. 1a). It is known that petroleum hydrocarbons cannot be completely metabolized by microorganisms to CO2 and H2O, and always leaves more or less complex residues (mainly recalcitrant compounds and metabolites) (Atlas, 1995). These compounds often become increasingly less bioavailable with the passing of time due to their low solubility in water and their sequestration by soils (Alexander, 1995). Additionally, high concentrations of fatty acids generated by petroleum biooxidation could exert ecotoxicity to soil microflora and thus impact further biodegradation processes (Lu et al., 2010). The addition of 2% biochar at the beginning of the experiment did not result in significantly greater reduction in the TPH concentration until the 60th day relative to the soil without biochar (Fig. 1a). Afterwards, biodegradation process was accelerated and TPH level in trial C decreased to 3625 mg kg1 after 180 days of treatment, corresponding to a total removal efficiency of 77.8%, which was significantly higher than that of trial B (p < 0.05). Actually, at the 160th day, TPH concentration in trial C had declined to 3870 mg kg1, and the biodegradation rate had also slowed down during the late period. Interestingly, when adding biochar at day 80, bioremediation was promoted significantly (Fig. 1a). Since the 140th day, TPH concentration was significantly lower in trial D than in trial C, reaching a concentration below the threshold level required for Chinese soil quality for TPH (3000 mg kg1 d.w.). At the end of the experiment, TPH concentration in trial D decreased to 2480 mg kg1, corresponding to a total removal efficiency of 84.8%. These results indicated that adding time of biochar had apparent influence on biodegradation. A few investigations have demonstrated that black carbon (including activated carbon and biochar) could reduce contaminant bioavailability and biodegradation in soil (Zhang et al., 2005; Rhodes et al., 2008). In these studies, pollutant concentrations in soil were generally lower (<500 mg kg1), and apparent sequestering effects may occur, leading to reduction in pollutant mobility and bioavailability. Contrastingly, Vasilyeva et al. (2010) found that adding activated carbon helped overcome toxicity of polychlorinated biphenyls to microorganisms, and Hale et al. (2011) also reported that biochar amendment had a stimulatory effect on PAH biodegradation in soil. In the present study, the reduction in the mobility and bioaccessibility of soil contaminants caused by biochar addition may be lower, since the TPH concentration was relatively high. Xia et al. (2010) held that biochar generated under lower temperatures may absorb contaminants via a partition mechanism that is relatively more accessible to microbes than adsorption dominant processes, which appear in biochar produced at higher temperatures. Interestingly, it was found that phenanthrene adsorbed to black carbon was more bioavailable than predicted by chemical extraction (Rhodes et al., 2008). It was hypothesized that microorganisms adhered to black carbon particles, and degraded phenanthrene absorbed onto the surface. Existing studies demonstrate complex effects of black carbon addition on soil bioremediation of organic pollutants, which could be related to certain factors including the origin and production method of black carbon, soil characteristics, contaminant types and microbial properties, etc.
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Fig. 2. Total removal percentages of n-alkanes and 16 priority PAHs after 180 days of treatment. Trial B: soil received no biochar; trial C: the soil was amended with 2% (w/ w) biochar at the beginning; trial D: the soil was amended with 2% (w/w) biochar at the 80th day. The 16 priority PAHs are: naphthalene, acenaphthene, acenaphthylene, fluorene, phenanthrene, anthracene, pyrene, fluoranthrene, benzo[ghi]perylene, benz [a]anthracene, chrysene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, indeno[1,2,3-cd]pyrene, and dibenz[ah]anthracene.
biodegradation of saturates was observed in the biochar amended soil. The concentrations of saturates decreased from 8260 mg kg1 at t ¼ 0 day to 1200, 553 and 612 mg kg1 at t ¼ 180 days, in trial B, C and D respectively, corresponding to removal efficiencies of 85.5%, 93.2% and 92.6%, respectively. It can be found that the addition of biochar slightly enhanced biodegradation of saturates, and adding time of biochar had no apparent impact on degradation efficiency of saturated fractions. Bushnaf et al. (2011) found that biodegradation of linear, cyclic and branched alkanes were more rapid in the soil amended with biochar than that without. As shown in Fig. 2c, adding biochar significantly increased degradation efficiencies of aromatics. The concentrations of aromatics decreased from 5130 mg kg1 at t ¼ 0 day to 1810, 881 and 1025 mg kg1 at t ¼ 180 days, in trial B, C and D respectively, corresponding to removal efficiencies of 64.7%, 82.8% and 80.0%, respectively. It can be observed that adding time of biochar had no apparent effect on degradation efficiency of aromatic fractions. Fig. 2d shows a real increase in the concentrations of polar fractions in the soils after the start-up of incubation, which was ascribed to the accumulation of metabolic intermediates. Adding biochar significantly increased degradation efficiencies of polar fractions, especially when amending biochar at day 80. The concentrations of polar fractions decreased from 2910 mg kg1 at t ¼ 0 day to 2192 and 843 mg kg1 at t ¼ 180 days in trial C and D respectively, corresponding to removal efficiencies of 24.7% and 71.0%, respectively. It has been shown that polar fractions in crude oil are partially or completely resistant to microbial degradation (Pollard et al., 1999). Nevertheless, it has also been reported that a 30% maximum biodegradation of polar compounds can appear in optimal cultures Chaillan et al., 2006). In this study, the addition of biochar at the 80th day can greatly enhance biodegradation of polar fractions. Based on the above results, it can be concluded that, biodegradation of polar fractions is crucial for the success of bioremediation practice of oil contaminated soils. In this study, the lower TPH removal in trial B was mainly due to the accumulation of metabolites.
3.2. Degradation of saturated, aromatic and polar fractions
3.3. Degradation of n-alkanes and PAHs
The concentrations of saturate, aromatic and polar fractions with time are presented in Fig. 2bed, respectively. A more rapid
GCeMS analysis was performed on the extracted oil samples before and after 180 days of incubation, respectively. Total removal
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percentages of n-alkanes (C8eC40) and 16 priority PAHs were calculated and shown in Fig. 2. After 180 days of treatment, n-alkanes and PAHs in trial B showed concentration decreases of 88.6% and 45.3%, respectively. The removal efficiencies of n-alkanes in trial C and D were 95.3% and 94.1%, respectively. Approximately 61.4% and 59.5% of PAHs were removed in trial C and D, respectively. The addition of biochar enhanced removal of n-alkanes and PAHs. In general, the removal efficiencies of PAHs were lower than that of aromatic fractions, indicating that the 16 priority PAHs were more recalcitrant within the aromatic compounds in the oils. 3.4. Total bacterial count A statistically significant difference was observed for the total counts of soil heterotrophic bacteria 180 days after the incubation, with trial D having the highest cell count of 1.31 109 CFU per gram of dry soil weight, 5.1 times higher than trial B, which had the lowest cell count (2.56 108 CFU g1) (Fig. 3a). This result showed that biochar amendment overall was not detrimental to aerobic microbial activity. It is known that biochar reduces bioaccessibility, chemical activity and ecotoxicity of organic compounds to soil microorganisms (Ogbonnaya and Semple, 2013). Moreover, biochar nutrient properties can improve plant growth and microbial activity and have been shown to enhance biodegradation of bioaccessible contaminants (Ogbonnaya and Semple, 2013).
(a) 1.40E+009
3.5. MicrotoxÒ toxicity MicrotoxÒ analysis was performed over the course of the study to monitor toxicity changes in the incubated soils and the results are shown in Fig. 3b. In trial B and D, the MicrotoxÒ toxicity first increased during the initial phase of biodegradation, but started to decrease after 40 days of incubation. In trial C, the toxicity declined during the initial 20 days. Higher EC50 values at day 180 in trial C and D suggested an overall reduction in soil toxicity. Significantly lower toxicity in soil (p < 0.05) was observed in trial D relative to trial B and C. It can be observed that the change trends of soil toxicity generally coincided with that of the concentrations of polar compounds. Thus, it can be speculated that some toxic metabolites were produced during biodegradation of petroleum compounds. In the present work, polar compounds could be more efficiently biodegraded in the soil amended with biochar at day 80 than that at the beginning (Fig. 1). We speculated that, when adding biochar at the beginning, the absorption sites on the biochar surfaces would be occupied mainly by petroleum hydrocarbons. As biodegradation proceeded, the absorption capacity of biochar for metabolites was limited. However, when adding biochar at the 80th day, a large amount of metabolites could be absorbed onto the biochar, because at this point the concentrations of petroleum hydrocarbons had decreased remarkably and a great quantity of metabolites had been accumulated. Thereupon, the addition of biochar at day 80 could result in significant reduction in soil toxicity and thus promote biodegradation. However, we could not verify this speculation by MicrotoxÒ toxicity assay, because the tiny size of biochar particles made it impossible to separate biochar from the soil after mixing and incubation.
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Time (days) Fig. 3. (a) Total microbial cell count for the soil samples taken after 180 days. (b) Time course of EC50 value of the soil samples during the 180-day period. Trial B: soil received no biochar; trial C: the soil was amended with 2% (w/w) biochar at the beginning; trial D: the soil was amended with 2% (w/w) biochar at the 80th day.
Fig. 4. Bacterial community taxonomic composition before and after 180 days of incubation based on metagenomic sequencing. (a) Community composition at the phylum/class level based on all annotated fragments. (b) Pseudomonas relative abundance based on all annotated fragments. Trial B: soil received no biochar; trial C: the soil was amended with 2% (w/w) biochar at the beginning; trial D: the soil was amended with 2% (w/w) biochar at the 80th day.
G. Qin et al. / International Biodeterioration & Biodegradation 85 (2013) 150e155
3.6. Metagenomic sequencing Biostimulation treatment resulted in the increment of the abundance of several bacterial phylum/classes, like Gammaproteobacteria, Actinobacteria, Sphingomonadales and Alphaproteobacteria (Fig. 4a). In contrast, bioremediation process resulted in a reduction in the abundance of Acidobacteria, Bacteroidetes, Chlorobi, Chloroflexi, Cyanobacteria, Burkholderiales, Planctomycetes and Deltaproteobacteria. The addition of biochar produced relatively similar taxonomic profiles at the phylum/class level, but with some interesting variations. Gammaproteobacteria, Acidobacteria, Chlorobi and Chloroflexi increased through the time course while Actinobacteria and Alphaproteobacteria decreased (Fig. 4a). In the t ¼ 180 days samples, Gammaproteobacteria comprised up to 38% of the total community. At the phylum/class level, the samples after biotreatment showed a diversified taxonomic profile, with a clear dominance of any of Gammaproteobacteria. The high abundance of Gammaproteobacteria in the t ¼ 180 days samples was mainly due to Pseudomonas species (Fig. 4b). The relative abundance of Pseudomonas increased after incubation. In general, Fig. 4 shows that biochar amendment did not significantly alter bacterial community compositions. This indicates that biochar in the present study had no apparent negative effect on soil microorganism populations. Pseudomonas, Rhodococcus, Caulobacter and sphingomonads are well-known hydrocarbon degraders, which tend to be enriched in well aerated soils with larger amounts of nitrogen nutrients and oil. Pseudomonas species were hypothesized to be one of the major alkane and aromatic hydrocarbon degraders in petroleum contaminated soils (Haines et al., 2002). 4. Conclusions This work demonstrated the feasibility of a bioremediation process using biostimulation with biochar amendment for petroleum-contaminated soil. The results of soil microcosm experiments showed that the soil amended with rice straw biochar resulted in a better improvement in pollutant removal, compared with biostimulation alone. When adding biochar at the 80th day, the TPH concentration decreased to below the threshold level required for Chinese soil quality for TPH (3000 mg kg1 dry weight) in 140 days. The results showed the applicability in use of biochar for remediation of oil-contaminated soil. The addition of biochar did not cause appreciable negative influences on soil microflora. Therefore, rice straw biochar may act as an efficient soil remediation additive. References Alexander, M., 1995. How toxic are toxic chemicals in soil? Environmental Science and Technology 29, 2713e2717. Atlas, R.M., 1995. Petroleum biodegradation and oil spill bioremediation. Marine Pollution Bulletin 31, 178e182. Bastow, T.P., van Aarssen, B.G.K., Lang, D., 2007. Rapid small-scale separation of saturate, aromatic and polar components in petroleum. Organic Geochemistry 38, 1235e1250. Beesley, L., Moreno-Jiménez, E., Gomez-Eyles, J.L., 2010. Effects of biochar and greenwaste compost amendments on mobility, bioavailability and toxicity of inorganic and organic contaminants in a multi-element polluted soil. Environmental Pollution 158, 2282e2287. Beskoski, V.P., Gojgi c-Cvijovi c, G., Mili c, J., Ili c, M., Mileti c, S., Solevi c, T., Vrvi c, M.M., 2011. Ex situ bioremediation of a soil contaminated by mazut (heavy residual fuel oil) e a field experiment. Chemosphere 83, 34e40. Bushnaf, K.M., Puricelli, S., Saponaro, S., Werner, D., 2011. Effect of biochar on the fate of volatile petroleum hydrocarbons in an aerobic sandy soil. Journal of Contaminant Hydrology 126, 208e215. Chaillan, F., Chaîneau, C.H., Point, V., Saliot, A., Oudot, J., 2006. Factors inhibiting bioremediation of soil contaminated with weathered oils and drill cuttings. Environmental Pollution 144, 255e265.
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