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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 biostimulationamended with biochar

Gang Qin a,*, Dan Gong b, Mei-Ying Fan c

aCollege of Engineering and Technology, Yangtze University, Xueyuan Road, Jingzhou 434020, Hubei, PR Chinab School of Geophysics and Oil Resources, Yangtze University, Xueyuan Road, Jingzhou 434023, Hubei, PR ChinacGraduate School, Yangtze University, Xueyuan Road, Jingzhou 434023, PR China

a r t i c l e i n f o

Article history:Received 17 June 2013Received in revised form3 July 2013Accepted 11 July 2013Available online

Keywords:BiostimulationBioremediationMicrotox� toxicityMetagenomicsSequencing

* Corresponding author. Tel./fax: þ86 716 8067521E-mail address: pipilang@yeah.net (G. Qin).

0964-8305/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.ibiod.2013.07.004

a b s t r a c t

In this study, the effects of rice straw biochar on soil contaminant biodegradation and microbial com-munity compositions were investigated in the laboratory during a 180-day period. The results of soilmicrocosm experiments showed that contaminant degradation efficiency was significantly higher insoils amended with biochar than in soils without. The adding time of biochar had apparent effects ondegradation 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 dayrespectively. When adding biochar at the 80th day, the TPH concentration decreased to below thethreshold level required for Chinese soil quality for TPH (3000 mg kg�1 dry weight) in 140 days. Theaddition of biochar did not result in appreciable negative impacts on soil microbial communitycomposition. It was speculated that when adding biochar at the 80th day, a large amount of metabolitescould be absorbed onto the biochar, leading to significant reduction in soil toxicity and biodegradationenhancement.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Spills, leaks and other releases of petroleum hydrocarbons cancause large amounts of soil spread pollution, representing a majorenvironmental concern with serious consequences that has beendrawing public concerns worldwide over the recent decades (Luet al., 2010).

Bioremediation of petroleum contaminated soils is a hot area ofsoil restoration research, because of its relatively low costs andenvironmentally friendship compared to physical and chemicalprocesses (Grace Liu et al., 2011). This technique has been shown tobe effective for petroleum contaminated soils in both laboratoryand field tests (Xu and Lu, 2010; Be�skoski et al., 2011; Mukherjeeand Bordoloi, 2011). However, the wide practice of bioremedia-tion in the field is limited by its low efficiency and long-termmaintenance (Trindade et al., 2005). This is especially true forhistorically contaminated sites where the pollutants mainly con-sisted of complex compounds with recalcitrant chemical structuresand low bioavailability (Huesemann et al., 2004). The hydrophobic

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nature of oil retard mass transfer of air, water, and contaminantsfrom soil particles to microorganisms, limiting the rate of uptakeand 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), surfactantenhancement (Mata-Sandoval et al., 2002; Urum et al., 2006), wereincorporated into biological processes to enhance pollutantbioavailability and/or to reduce substrate toxicity. Biochar pro-duced from biomass may sequester atmospheric CO2 in soils for along time, and thus reducing the carbon footprint of sorbent-basedsoil remediation in comparison with the use of coal-derived acti-vated carbon (Bushnaf et al., 2011). Activated carbon or biocharamendments have been deployed in certain soil and sedimentremediation purposes. The use of biochar could be cheaper in aremediation sense relative to AC because the waste source mate-rials are essentially free and the production of biochar requires lessenergy and cost (Hale et al., 2011). The agronomic benefits of bio-char in addition to sequestering C in plant based remediation mayalso be related to an increment of liming effects, water holdingcapacity, soil structure, cation exchange capacity, soil microbialactivities and finally the plant growth (Glaser et al., 2002; Beesleyet al., 2010). However, biochar amendment to degraded soil has

G. Qin et al. / International Biodeterioration & Biodegradation 85 (2013) 150e155 151

also been shown to reduce the pollutant’s availability for microbialbreak-down and increase persistence (Rhodes et al., 2008).

In the present work, the feasibility of the use of biochar in pe-troleum hydrocarbon contaminated soil remediation was assessed.To investigate the effect of adding time, biochar was amended attwo different periods respectively, i.e. the beginning or the middlestage of the experiments.

2. Materials and methods

2.1. Contaminated soil

The petroleum-contaminated soil was collected from an oil spillsite near a tank container in Shengli Oilfield, China. For sampling,surface litters were removed and soil samples were collected to adepth of 30 cm. The soil was air-dried and sieved through a 2-mmmesh sieve, homogenized by handwith a shovel, and then stored at4 �C in the dark until used. The texture of the raw contaminated soilwas 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 followingcharacteristics: pH (1:2.5, soil/water ratio), 6.5; conductivity (1:10,soil/water ratio), 535 mS cm�1; water holding capacity, 38.6 wt.%;humidity, 15.2 wt.%; total organic carbon (TOC), 5.42 wt.%; totalnitrogen 85 mg kg�1; total phosphorus 16 mg kg�1; total hetero-trophic bacteria, 7.50 � 106 colony-forming units (CFU) g�1; dieseloil degrading bacteria, 3.60 � 104 CFU g�1; total petroleum hy-drocarbons (TPH), 16,300 mg kg�1 (saturated hydrocarbons,8260 mg kg�1; aromatic hydrocarbons, 5130 mg kg�1; polar com-ponents, 2910 mg kg�1).

The soil contained a significant amount and proportion of analkane (saturated) fraction-degrading population (0.48%), sug-gesting that biostimulation strategy was feasible for this soil ma-trix. Additionally, the oil contained a relatively high percentage ofaromatic fractions (31.5%).

2.2. Biochar

Biochar was produced from rice straw at 500 �C using a slowpyrolysis under limited oxygen according to (Lou et al., 2013).Table 1 lists the characteristics of rice straw feed. Following char-ring, the biochar was lightly ground and sieved to obtain a fine(<0.16 mm) size fraction, which was then rinsed with distilledwater to remove the ash content and dried at 60 �C for 5 days. Thesample had a total surface area of 1053 m2 g�1, TOC content of83.5 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 1The main characteristics of rice straw feed.

Composition Proximate analysis

Component Content (wt.%) Component Content (wt.%)

Cellulose 57.2 Water 5.3Hemicellulose 29.6 Volatile matter 65.4Lignin 13.2 Fixed carbon 16.6

Ash 12.7

Elemental analysis Alkali metal concentration

Element Content (wt.%) Alkali metal Content (mg kg�1)

C 38.5 Na 273H 4.9 Mg 1942N 1.8 Ca 3105S 0.76 K 23,875O 54.0

2.3. Soil microcosm experiments

The soil was subjected to different treatments for an additional180 days. For each treatment, three independent replicates (2-Lglass receptacles covered with perforated parafilm) were pre-pared as microcosms, each containing 1000 g of soil. In all thetreatments, the water content was adjusted to 60% of water holdingcapacity. Once aweek, the microcosm contents weremixed and thesoil water content was restored by controlling the weight. Everytwo weeks, (NH4)2SO4 and K2HPO4 were added to produce a finalC:N:P ratio of 100:10:5 (Gong, 2012). Four different trials wereapplied in triplicate: soil was supplementedwith 2% (w/w) HgCl2 toaccount 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 andfractionated by silica gel column chromatography to separatesaturate, aromatic and polar fractions, following the methods ofBastow et al. (2007). The different elute was evaporated to drynessunder N2, and calculated gravimetrically.

The measurements of n-alkanes and polycyclic aromatic hy-drocarbons (PAHs) were performed by gas chromatographyemassspectrometry (GCeMS), using a Thermo-Finnigan SSQ710 GCeMS(Thermo Finnigan, San Jose, CA, USA) with a HP-5MS elastic silicacapillary columns (60m� 0.25 mm� 0.25 mm). The carrier gas washelium at 37 kPa. Flow velocity was 1 mL min�1. The analyticalconditions were: initial temperature of 50 �C, with isothermaloperation for 1 min; heating to 120 �C at a constant rate of 20 �Cmin�1; and heating to a final temperature of 310 �C at a constantrate of 4 �C min�1, with a 30 min isothermal. Mass spectrometerconditions were: electron impact, electron energy 70 eV; filamentcurrent 100 mA; multiplier voltage, 1200 V; full scan.

Concentrations of each n-alkane were calculated based on thestandard calibration curve of each corresponding standard com-pound (Accu Standards Inc., New Haven, CT, USA). Individual PAHswere quantified based on the retention time and m/z ratio of anauthentic PAHmixed standard (SigmaeAldrich, St. Louis, MO, USA),and concentrations of each PAH were calibrated based on thestandard calibration curve.

2.5. Microbiological enumeration

The heterotrophic bacteria were counted on nutrient agar afterincubation at 30 �C for 2 days, and the results were expressed asCFU per gram of dry soil.

2.6. Microtox� toxicity assay

The toxicity of soil elutriate was determined using the Micro-tox� bioassay according to Xu and Lu (2010). Toxicity values werethe average of five replicates of each filtrate sample, expressed asEC50 (15 min, 15 �C), which was defined as the effective concen-tration of pollutant for a 50% reduction of the luminescence of thebacterium Photobacterium phosphoreum.

2.7. DNA extraction, PCR amplification and 454 sequencing

High-molecular-weight DNA from the soil was extracted with acommercially available kit (Beijing Dingguo Biotechnology Ltd.,China). The 16S rDNA genes were amplified by polymerase chainreaction (PCR) in a Techgene thermocycler (FTGENE 5D, 112757-4,

G. Qin et al. / International Biodeterioration & Biodegradation 85 (2013) 150e155152

Techne Combridge Ltd., Duxford, Cambridge UK), using the forwardprimer 563F (50-AYTGGGYDTAAAGNG-30) at the 50-end (E. colipositions 563e578) of the V4 region (239 nucleotides) and acocktail of four equally mixed reverse primers, that is, R1 (50-TACCRGGGTHTCTAATCC-30), R2 (50-TACCAGAGTATCTAATTC-30), R3(50-CTACDSRGGTMTCTAATC-30) and R4 (50-TACNVGGGTATCTAATC-30), at the 30-end of the V4 region (E. coli positions 785e802)(Murphy et al., 2010). Then DNA samples with different barcodeswere mixed in equal concentration and sequenced by a Roche 454FLX Titanium sequencer (Roche, Nutley, NJ, USA) at the BeijingGenomics Institute (Shenzhen, China). The pyrosequencing meth-odology used was identical to that reported by Davis et al. (2011).

2.8. Post-run analysis

The raw reads were treated with the Pyrosequencing PipelineInitial Process (Cole et al., 2009) of the Ribosomal Database Project(RDP), (1) to sort those exactly matching the specific barcodes intodifferent samples, (2) to trim off the adapters, barcodes and primersusing the default parameters, and (3) to remove sequences con-taining ambiguous ‘N’ or shorter than 150 bps (Claesson et al.,2009). The reads selected above were defined as ‘raw reads’ foreach soil sample.

Taxonomic classification of the bacterial sequences of samplewas carried out using the Ribosomal Database Project (RDP) Clas-sifier. A bootstrap cutoff of 50% suggested by the RDPwas applied toassign the sequences to different taxonomy levels. The normalized

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Fig. 1. Time course of concentrations of TPH (a), saturates (b), aromatics (c), and polar frareceived no biochar; trial C: the soil was amended with 2% (w/w) biochar at the beginninindicate � SD of triplicate samples. The line indicates TPH legal limit for commercial-indus

sequence set of samplewas aligned by Infernal (Nawrocki and Eddy,2007) using the bacteria-alignment model in Align module of theRDP.

2.9. Statistical analysis

All experiments in this study were performed in triplicate to getreliable data, and the results presented here represent the averagevalues of three independent measurements � standard deviations.The variance and significant differences among various treatmentswere analyzed by Student’s t-test. Data were considered to besignificantly different among values if p < 0.05. All statisticalanalysis was performed with SPSS 13.0 for Windows.

3. Results and discussion

3.1. TPH biodegradation in soils

Generally, petroleum hydrocarbon biodegradation in soils ischaracterizedbya rapid removal during the initial stage, followedbyaslower and even plateau phase (Alexander, 1995). Under abiotic con-ditions, trial A showed a negligible decrease in soil TPH concentration(Fig. 1a), which may be due to that most of the volatile hydrocarbonshad disappeared during the long-term natural attenuation.

Biostimulation by nutrient amendment (trial B) caused a rapidreduction of TPH in early stage (within 60 days), followed by anapparent slowdown of biodegradation until the 180th day. At the

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ction (d) in soils during 180-day bioremediation. Trial A: abiotic control; trial B: soilg; trial D: the soil was amended with 2% (w/w) biochar at the 80th day. Errors barstrial use (3000 mg kg�1 dry weight) in China.

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Fig. 2. Total removal percentages of n-alkanes and 16 priority PAHs after 180 days oftreatment. 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 atthe 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]fluo-ranthene, indeno[1,2,3-cd]pyrene, and dibenz[ah]anthracene.

G. Qin et al. / International Biodeterioration & Biodegradation 85 (2013) 150e155 153

end of process, the total removal efficiency of TPH for trial B was61.2% (from 16,300 mg kg�1 at t ¼ 0 day to 6420 mg kg�1 at t ¼ 180days). Actually, TPH removal reached a plateau at the late stage ofthe experiment, and no apparent changes in TPH concentrationswere observed since the 120th day (Fig. 1a). It is known that pe-troleum hydrocarbons cannot be completely metabolized by mi-croorganisms to CO2 and H2O, and always leaves more or lesscomplex residues (mainly recalcitrant compounds andmetabolites)(Atlas, 1995). These compounds often become increasingly lessbioavailable with the passing of time due to their low solubility inwater and their sequestration by soils (Alexander, 1995). Addi-tionally, high concentrations of fatty acids generated by petroleumbiooxidation could exert ecotoxicity to soil microflora and thusimpact further biodegradation processes (Lu et al., 2010).

The addition of 2% biochar at the beginning of the experimentdid not result in significantly greater reduction in the TPH con-centration until the 60th day relative to the soil without biochar(Fig. 1a). Afterwards, biodegradation process was accelerated andTPH level in trial C decreased to 3625 mg kg�1 after 180 days oftreatment, 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 declinedto 3870 mg kg�1, and the biodegradation rate had also sloweddown during the late period.

Interestingly, when adding biochar at day 80, bioremediationwas promoted significantly (Fig. 1a). Since the 140th day, TPHconcentration was significantly lower in trial D than in trial C,reaching a concentration below the threshold level required forChinese soil quality for TPH (3000 mg kg�1 d.w.). At the end of theexperiment, TPH concentration in trial D decreased to2480 mg kg�1, corresponding to a total removal efficiency of 84.8%.These results indicated that adding time of biochar had apparentinfluence on biodegradation.

A few investigations have demonstrated that black carbon(including activated carbon and biochar) could reduce contaminantbioavailability and biodegradation in soil (Zhang et al., 2005;Rhodes et al., 2008). In these studies, pollutant concentrations insoil were generally lower (<500 mg kg�1), and apparent seques-tering effects may occur, leading to reduction in pollutant mobilityand bioavailability. Contrastingly, Vasilyeva et al. (2010) found thatadding activated carbon helped overcome toxicity of poly-chlorinated biphenyls tomicroorganisms, and Hale et al. (2011) alsoreported that biochar amendment had a stimulatory effect on PAHbiodegradation in soil. In the present study, the reduction in themobility and bioaccessibility of soil contaminants caused by bio-char addition may be lower, since the TPH concentration wasrelatively high. Xia et al. (2010) held that biochar generated underlower temperatures may absorb contaminants via a partitionmechanism that is relatively more accessible to microbes thanadsorption dominant processes, which appear in biochar producedat higher temperatures. Interestingly, it was found that phenan-threne adsorbed to black carbon was more bioavailable than pre-dicted by chemical extraction (Rhodes et al., 2008). It washypothesized that microorganisms adhered to black carbon parti-cles, and degraded phenanthrene absorbed onto the surface.

Existing studies demonstrate complex effects of black carbonaddition on soil bioremediation of organic pollutants, which couldbe related to certain factors including the origin and productionmethod of black carbon, soil characteristics, contaminant types andmicrobial properties, etc.

3.2. Degradation of saturated, aromatic and polar fractions

The concentrations of saturate, aromatic and polar fractionswith time are presented in Fig. 2bed, respectively. A more rapid

biodegradation of saturates was observed in the biochar amendedsoil. The concentrations of saturates decreased from 8260 mg kg�1

at t ¼ 0 day to 1200, 553 and 612 mg kg�1 at t ¼ 180 days, in trial B,C and D respectively, corresponding to removal efficiencies of85.5%, 93.2% and 92.6%, respectively. It can be found that theaddition of biochar slightly enhanced biodegradation of saturates,and adding time of biochar had no apparent impact on degradationefficiency of saturated fractions. Bushnaf et al. (2011) found thatbiodegradation of linear, cyclic and branched alkanes were morerapid in the soil amended with biochar than that without.

As shown in Fig. 2c, adding biochar significantly increaseddegradation efficiencies of aromatics. The concentrations of aro-matics decreased from 5130 mg kg�1 at t ¼ 0 day to 1810, 881 and1025 mg kg�1 at t ¼ 180 days, in trial B, C and D respectively, cor-responding to removal efficiencies of 64.7%, 82.8% and 80.0%,respectively. It can be observed that adding time of biochar had noapparent effect on degradation efficiency of aromatic fractions.

Fig. 2d shows a real increase in the concentrations of polar frac-tions in the soils after the start-up of incubation,whichwas ascribedto the accumulation of metabolic intermediates. Adding biocharsignificantly increased degradation efficiencies of polar fractions,especially when amending biochar at day 80. The concentrations ofpolar fractions decreased from2910mgkg�1 at t¼ 0day to2192 and843 mg kg�1 at t ¼ 180 days in trial C and D respectively, corre-sponding to removal efficiencies of 24.7% and 71.0%, respectively. Ithas been shown that polar fractions in crude oil are partially orcompletely resistant to microbial degradation (Pollard et al., 1999).Nevertheless, it has also been reported that a 30% maximumbiodegradation of polar compounds can appear in optimal culturesChaillan et al., 2006). In this study, the addition of biochar at the80thday can greatly enhance biodegradation of polar fractions.

Based on the above results, it can be concluded that, biodegra-dation of polar fractions is crucial for the success of bioremediationpractice of oil contaminated soils. In this study, the lower TPHremoval in trial Bwasmainly due to theaccumulationofmetabolites.

3.3. Degradation of n-alkanes and PAHs

GCeMS analysis was performed on the extracted oil samplesbefore and after 180 days of incubation, respectively. Total removal

G. Qin et al. / International Biodeterioration & Biodegradation 85 (2013) 150e155154

percentages of n-alkanes (C8eC40) and 16 priority PAHs werecalculated and shown in Fig. 2. After 180 days of treatment, n-al-kanes and PAHs in trial B showed concentration decreases of 88.6%and 45.3%, respectively. The removal efficiencies of n-alkanes intrial C and D were 95.3% and 94.1%, respectively. Approximately61.4% and 59.5% of PAHswere 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 ofaromatic fractions, indicating that the 16 priority PAHs were morerecalcitrant within the aromatic compounds in the oils.

3.4. Total bacterial count

A statistically significant difference was observed for the totalcounts of soil heterotrophic bacteria 180 days after the incubation,with trial D having the highest cell count of 1.31�109 CFU per gramof dry soil weight, 5.1 times higher than trial B, which had thelowest cell count (2.56 � 108 CFU g�1) (Fig. 3a). This result showedthat biochar amendment overall was not detrimental to aerobicmicrobial activity. It is known that biochar reduces bioaccessibility,chemical activity and ecotoxicity of organic compounds to soilmicroorganisms (Ogbonnaya and Semple, 2013). Moreover, biocharnutrient properties can improve plant growth and microbial ac-tivity and have been shown to enhance biodegradation of bio-accessible contaminants (Ogbonnaya and Semple, 2013).

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3.5. Microtox� toxicity

Microtox� analysis was performed over the course of the studyto monitor toxicity changes in the incubated soils and the resultsare shown in Fig. 3b. In trial B and D, the Microtox� toxicity firstincreased during the initial phase of biodegradation, but started todecrease after 40 days of incubation. In trial C, the toxicitydeclined during the initial 20 days. Higher EC50 values at day 180in trial C and D suggested an overall reduction in soil toxicity.Significantly lower toxicity in soil (p < 0.05) was observed in trialD relative to trial B and C. It can be observed that the changetrends of soil toxicity generally coincided with that of the con-centrations of polar compounds. Thus, it can be speculated thatsome toxic metabolites were produced during biodegradation ofpetroleum compounds.

In the present work, polar compounds could be more efficientlybiodegraded in the soil amendedwith biochar at day 80 than that atthe beginning (Fig. 1). We speculated that, when adding biochar atthe beginning, the absorption sites on the biochar surfaces wouldbe occupied mainly by petroleum hydrocarbons. As biodegradationproceeded, the absorption capacity of biochar for metabolites waslimited. However, when adding biochar at the 80th day, a largeamount of metabolites could be absorbed onto the biochar, becauseat this point the concentrations of petroleum hydrocarbons haddecreased remarkably and a great quantity of metabolites had beenaccumulated. Thereupon, the addition of biochar at day 80 couldresult in significant reduction in soil toxicity and thus promotebiodegradation. However, we could not verify this speculation byMicrotox� toxicity assay, because the tiny size of biochar particlesmade it impossible to separate biochar from the soil after mixingand incubation.

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G. Qin et al. / International Biodeterioration & Biodegradation 85 (2013) 150e155 155

3.6. Metagenomic sequencing

Biostimulation treatment resulted in the increment of theabundance of several bacterial phylum/classes, like Gammaproteo-bacteria, Actinobacteria, Sphingomonadales and Alphaproteobacteria(Fig. 4a). In contrast, bioremediation process resulted in a reductionin the abundance of Acidobacteria, Bacteroidetes, Chlorobi, Chloro-flexi, Cyanobacteria, Burkholderiales, Planctomycetes and Deltapro-teobacteria. The addition of biochar produced relatively similartaxonomic profiles at the phylum/class level, but with some inter-esting variations. Gammaproteobacteria, Acidobacteria, Chlorobi andChloroflexi increased through the time course while Actinobacteriaand Alphaproteobacteria decreased (Fig. 4a). In the t ¼ 180 dayssamples, Gammaproteobacteria comprised up to 38% of the totalcommunity. At the phylum/class level, the samples after bio-treatment showed a diversified taxonomic profile, with a cleardominance of any of Gammaproteobacteria. The high abundance ofGammaproteobacteria in the t ¼ 180 days samples was mainly dueto Pseudomonas species (Fig. 4b). The relative abundance of Pseu-domonas increased after incubation.

In general, Fig. 4 shows that biochar amendment did not signif-icantly alter bacterial community compositions. This indicates thatbiochar in the present study had no apparent negative effect on soilmicroorganism populations. Pseudomonas, Rhodococcus, Caulo-bacter and sphingomonads are well-known hydrocarbon degraders,which tend to be enriched inwell aerated soils with larger amountsof nitrogen nutrients and oil. Pseudomonas species were hypothe-sized to be one of the major alkane and aromatic hydrocarbon de-graders in petroleum contaminated soils (Haines et al., 2002).

4. Conclusions

This work demonstrated the feasibility of a bioremediationprocess using biostimulation with biochar amendment forpetroleum-contaminated soil. The results of soil microcosm ex-periments showed that the soil amended with rice straw biocharresulted in a better improvement in pollutant removal, comparedwith biostimulation alone. When adding biochar at the 80th day,the TPH concentration decreased to below the threshold levelrequired for Chinese soil quality for TPH (3000mg kg�1 dry weight)in 140 days. The results showed the applicability in use of biocharfor remediation of oil-contaminated soil. The addition of biochardid not cause appreciable negative influences on soil microflora.Therefore, rice straw biochar may act as an efficient soil remedia-tion additive.

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