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Bioremediation of oil sludge contaminated soil by landfarming with added cotton stalks
International Biodeterioration & Biodegradation 106 (2016) 150e156
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Bioremediation of oil sludge contaminated soil by landfarming with added cotton stalks Shijie Wang a, b, Xiang Wang a, Chao Zhang a, Fasheng Li a, Guanlin Guo a, * a b
State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012, China Beijing Municipal Research Institute of Environmental Research, Beijing 100037, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 February 2015 Received in revised form 20 October 2015 Accepted 20 October 2015 Available online 8 November 2015
A field bioremediation of oil sludge contaminated soil was conducted by landfarming treatment with added cotton stalks in the Shengli oil field. The ability of landfarming treatment was evaluated to reduce petroleum hydrocarbons and restore soil quality. For 39-month landfarming, the initial concentration of total petroleum hydrocarbons (TPH) was 12.57 mg g 1 for oil sludge contaminated soil. The removal efficiency of TPH, saturated fraction and aromatic fraction was 68.48%, 90.04% and 85.55%, respectively. Degradation of TPH followed first order exponential decay kinetics. Soil physic-chemical properties of soil pH, saline alkali degree, nutrients, organic matters and hydrocarbon degraders were greatly improved. The results of Biolog and PCR-DGGE analysis revealed the improvement of soil microbial quantity and diversity, and the isolated predominant 23 strains showed a shift in soil community structure toward the hydrocarbon degrading species including Streptococcus sp., Shewanella sp., Bacillus sp., Pseudomonas sp., Marinobactera sp., Thermoanaerobacter sp., etc. © 2015 Published by Elsevier Ltd.
Keywords: Biodegradation Oil sludge Cotton stalk Bacterial community
1. Introduction Oil sludge was mainly from drilling, refining, transportation, and storage of crude oil, which has been classified as a priority environmental pollutant. The annual volume of oil sludge that produced by large petrochemical facilities was about 10,000 m3 (Gafarov et al., 2006). In China, the environmental risk of oil sludge contaminated sites has been verified through the investigation of vadose zone and groundwater (Jin et al., 2011). In the current technical systems of cleaning up petroleum contaminated sites with large areas, bioremediation appears more practical applicability when compared with other techniques such as solvent washing, ozonation, incineration, landfill and thermal desorption in the consideration of cost-effect and secondary pollution (Ting et al., 1999; Ouyang et al., 2005). Landfarming treatment for petroleum hydrocarbon-contaminated soils has relatively low cost ranging from US$30 to US$70 per ton (Pope and Matthews, 1993; Giasi and Morelli, 2003), and this cost decreases with the development of technology. The previous studies revealed that adding organic matters can enhance bioremediation effectiveness by improving
* Corresponding author. E-mail address: [email protected] (G. Guo). http://dx.doi.org/10.1016/j.ibiod.2015.10.014 0964-8305/© 2015 Published by Elsevier Ltd.
the soil physicochemical properties and indigenous soil microbial activity (Rhykerd et al., 1999; Kriipsalua et al., 2007). However, to date, only a few long-term landfarming projects have been implemented (Mikkonen et al., 2012). In China, there are about 28.63 million tons of cotton stalks produced by 4.57 million planting hectares every year (China Cotton Association, 2013). The available large amount of cotton stalks provides the potential as organic amendments in landfarming treatment of petroleum oil contaminated soils. Wang et al. (2012) proved the primary advantage of biopile constructed with cotton stalks in enhancing TPH removal in soil, as well as the other benefits including lowering remediation cost and reuse of cropping wastes. In this study, the landfarming treatment of oil sludge contaminated soil was conducted, and the cotton stalks were added for enhancing remediation effectiveness. The objects were to evaluate the ability of this technique to reduce petroleum hydrocarbons and improve soil physical and biological properties. For those purposes, the natural attenuation treatment was used as blank control. The parameters regarding soil quality including physicochemical properties, quantity of total heterotrophic bacteria and petroleum degrading bacteria, microbial diversity, soil residual TPH and petroleum fractions were determined before and after landfarming treatment.
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2. Materials and methods 2.1. Site characterization, contaminated soil and cotton stalk The demonstrated landfarming of oil sludge contaminated soils located in the Shengli Oil Field in Shandong Province. The site of 13,750 m2 has been used for open storage of oil sludge for over 43 years. The local climate conditions were as follows: the average annual precipitation was about 642.0 mm, and the average annual temperature was about 12.8 C ranging from 10.3 C in January to 35.6 C in July. The soil type of demonstration site was sandy clay loam that was composed of 13.7% clay (<0.002 mm), 29.6% silt (0.02e0.002 mm) and 56.7% sand (2e0.02 mm). The total petroleum hydrocarbon (TPH) content in the topsoil (0e60 cm) ranged from 0.2 to 452,000 mg kg 1. The cotton stalks used in this study were collected from the local planting region and mechanically shredded into a variety of length ranging from 10 to 30 mm. The organic matter content of cotton stalk was about 78.6% by mass ratio, and the total N, P and K content was of 1.09%, 0.15% and 1.68%, of which the available content of N, P and K was 865.6, 687.4 and 5325.1 mg g 1, respectively.
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BIOLOGTM system. In this method, the average well color development (AWCD) was introduced to reflect soil microbial activity, and Shannon index were used to reflect soil microbial diversity (Zak et al., 1994). 2.5. Soil microbial community analyses by PCR-DGGE
Oil sludge contaminated soil was mixed with cotton stalks, and the ratio of soil to cotton stalks was 1:4 (w/w). There were three 100-m2 plots prepared for landfarming treatment, and one 100-m2 plot was set for natural attenuation as blank control. Oil sludge contaminated soils and cotton stalks were mixed and laid on the plots with 30 cm layer depth. The remediation duration lasted from June 2009 to November 2012, and the 0~30 cm topsoil was tilling with the frequency of every 2 months to keep soil moisture at 20 ~ 40%.
The total soil DNA was first extracted using a Power Soil™ DNA Isolation Kit according to the manufacturer's instructions (MoBio Lab Inc., Carlsbad, CA, USA), and then the integrity and purity of DNA were measured using electrophoresis and Nanodrop 1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). PCR amplification targeting bacterial of the V3 region within 16S rDNA was performed with the 357F-GC and 517R primer set (Muyzer et al., 1993). The nested PCR was conducted for amplification of 16S rDNA using primers 27F and 1492R (Lane, 1991). Denaturing Gradient Gel Electrophoresis (DGGE) for analysis of resulting DNA fragments (15 mL) were conducted according to the method used by Muyzer et al. (1993). The denaturing gradient varied from 45% to 75% for DGGE analysis. Gels were run at 60 V for 16 h in a D-Code System (Bio-Rad) filled with 8% (w/v) polyacrylamide (acrylamide: bisacrylamide, 37:1). The gels were stained in 1:10,000 (v/v) Sybr Green I (Cambrex, Rockland, USA) nucleic acid solution for 30 min. The DGGE images were obtained from the Gel Doc XR system (Bio-RadLab, Segrate, Italy) under UV light. The selected DGGE bands were excised and purified by a TaKaRa Mini BEST DNA Fragment Purification Kit (TaKaRa Bio Inc., Shiga, Japan), and then unidirectional sequenced by the reverse primer. A sequences alignment of the purified 16S rDNA was then conducted with a BLAST program. After entering query sequences, nucleotide collection that was composed of GenBank, EMBL, DDBJ, PDB and RefSeq sequences was chose as the alignment database (http://blast.ncbi.nlm. nih.gov, the accession number (NCBI) of all 23 samples were shown in Table 2).
2.3. Soil sampling and analysis of TPH and petroleum fractions
2.6. Data processing
The remediation plot was divided into 16 different sampling cells based on 2.5 m 2.5 m grid. The representative 20 cm surface soils were collected, and the sampling intervals were of 0 month (June, 2009), 1 month (July, 2009), 2 months (August, 2009), 4 months (October, 2009), 9 months (March, 2010), 13 months (July, 2010), 16 months (October, 2010), 26 months (August, 2011) and 39 months (September, 2012), respectively. Soil samples were freeze-dried, grounded, 2-mm mesh sieved and stored at 4 C. The accelerated solvent extractor (ASE300, Dionex, USA) and trichloromethane were used to extract petroleum hydrocarbons from 10 g soil. Referring to the previous study, the TPH content was determined by gravimetric method, and the petroleum fractions were analyzed by Thin-Layer Chromatography (MK-6S, Tokyo, Japan) (Wang et al., 2010).
All data except local mean temperature were expressed as mean ± standard deviation, and the statistical testing of the data was performed with SPSS v. 13.0. The local temperature shown in Fig. 3 was daily maximum temperature, daily mean temperature and daily minimum temperature, respectively. The correlation analysis of residual TPH in soil with remediation time and quantity of hydrocarbon degrading bacteria was performed by Metlab software (v. R2014a), and the optimal regression model with the smallest relative residual standard deviation was chosen.
2.2. Landfarming process
2.4. Enumeration of cultivable bacteria and determination of microbial activity The quantities of total heterotrophic bacteria and petroleum degrading bacteria in the soil samples were assessed by the plate count method. Tryptone Soya Agar (TSA) was used to determine the total heterotrophic bacteria (THB), and the mineral medium with the addition of sterile crude oil was used to determine the petroleum degrading bacteria (PDB) (Chaîneau et al., 1999). The colonyforming units (CFU) were recorded after incubation in the dark at 25 C on days 5 and 21, respectively. Microbial community level physiological profiles (CLPPs) were reflected by the patterns of sole-carbon-source utilization using a
3. Results and discussion 3.1. Changes of soil physicochemical properties after remediation Soil properties including texture, cation exchange capacity (CEC), nutrient status, and soil bacteria types and numbers are important factors in influencing the remediation efficiency of petroleum contaminated soils (Atlas, 1981). Significant changes of soil properties were observed after 39-month landfarming (Table 1). Previous studies showed that the improvement of soil properties facilitated biodegradation of petroleum hydrocarbons, for example, Zhang et al. (2008) found that the biodegradation rate of hydrocarbons increased as salinity decreased; and Warr et al. (2009) found that higher soil CEC stimulated the mineralization of aromatic, resin and asphaltene; and Brook et al. (2001) found that the biodegradation of hydrocarbons was enhanced if the C:P ratios were adjusted in the range of 60e800; and low salinity increases biodegradation efficiency of petroleum compounds (Garcia and
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Table 1 Changes of physicochemical properties of tested soils before and after natural attenuation and landfarming treatment. pH in Specific TOC Salinity CEC water gravity (%) (g kg 1) (cmol kg Original state 8.60 Natural 8.12 attenuation Landfarming 7.06
TNa/ANb ) (mg kg 1)
1
TPc/APd (mg kg 1)
TKe/AKf (mg kg 1)
780.00/410.60 450.00/205.00
2690.00/1420.00 12,590 2260.00/1050.00 8550
TPH (mg kg
2.74 2.42
2.33 63.04 3.02 41.7
5.44 5.89
150.00/70.50 89.20/39.70
1.44
6.74
8.06
2210.00/766.00 1860.00/880.00 5620.00/3120.00 3960
3.66
Asphaltene Saturated Aromatic Polar fraction fraction (mg g 1) ) fraction (mg kg 1) (mg g 1) (mg g 1)
1
5030 2010
3400 1760
1760 3020
2400 1760
500
490
1690
1280
* atotal nitrogen; btotal phosphorus; ctotal potassium; davailable nitrogen; eavailable phosphorus; favailable potassium.
ndez, 1996; Zheng et al., 2011). In this study, the soil salinity Herna decreased from 63.04 to 3.66 g kg 1, soil CEC increased from 5.44 to 8.06 cmol kg 1, soil total and available nutrients of N, P, K increased significantly by 1.1e13.7 times, and such the above changes provided a valuable condition for microbial growth and metabolic activity recovery. Zibilske and Materon (2005) found that 3-month decomposition of cotton stalks significantly improved soil quality. In this study, 39month landfarming also achieved other improvements of soil properties as follows, the soil specific gravity decreased from 2.74 to 1.44 g cm 3, soil TOC increased from 2.33% to 6.74%, soil pH decreased from 8.60 to 7.06, and these changes probably resulted in the improvement of soil aggregation, oxygen transport and waterholding ability. Some researchers revealed that the decomposition of organic amendments increased the bioavailability of polar and asphaltene fractions (Holman et al., 2002; Kobayashi et al., 2009) and the quantity of hydrocarbons degrading bacteria (García-Díaz et al., 2013). Liu et al. (2013) also found that the TPH degradation efficiency and 1st degradation rates reduced with the increase of soil organic matter (SOM).
3.2. Biodegradation of petroleum hydrocarbons As shown in Fig. 1, the soil residual TPH decreased by 68.48% after 39-month landfarming with addition of cotton stalks, while the soil residual TPH decreased by 32.08% after 39-month natural
attenuation treatment. The landfarming result of TPH removal was comparable with the research by Liu et al. (2010) that they found 27~46% TPH removal for 8-month field bioremediation of oil sludge. The higher TPH removal of 39-month landfarming was probably caused by added cotton stalks and timely tillage, and the previous studies revealed that the added organic amendments improved the TPH removal and soil microbial activities (Goyal et al., 1993; Rhykerd et al., 1999). Genouw et al. (1994) showed about 50% of TPH removal for 23,000 mg kg 1 petroleum oil contaminated soils, and 27% of TPH removal for 31,000 mg kg 1 petroleum oil contaminated soils, thus they concluded that the initial soil TPH content was the primary limiting factor for biodegradation. As shown in Fig. 1, the loss of TPH (Y, m%) followed a first-order exponential decay with time (X, month), and the tailing phenomena of TPH removal were also observed, but the regression parameters of degradation kinetic varied with landfarming and natural attenuation. However, the biodegradation kinetics of TPH also depended on environmental variables such as temperature, moisture, nutrient, hydrocarbons content. Sims and Sims (1999) showed the biphasic degradation kinetics for landfarming of petroleum hydrocarbons. Paudyn et al. (2008) found the TPH biodegradation at the Resolution Island site followed with the first order kinetics. Petroleum is a complex mixture composed of the four petroleum fractions (i.e. saturated fraction, aromatic fraction, polar and asphaltene), and the biodegradation abilities of petroleum fractions was closely related with their various physical and chemical properties. As shown in Fig. 2, the removals of saturated and aromatic fractions in landfarming were of 90.04% and 85.55%, while the removals of saturated and aromatic fractions in natural attenuation were of 60.04% and 48.24%. Meanwhile, the observed removals of saturated and aromatic fractions were higher than that of polar and asphaltene fractions. Light petroleum fractions of aliphatic, cycling and aromatic hydrocarbons were preferentially biodegraded (Sanscartier et al., 2009). The relative content of polar fraction showed a significant increase during the landfarming process (Fig. 2), and this change was probably due to the formation and accumulation of polar fraction caused by aerobic biodegradation of saturated and aromatic hydrocarbons (Huesemann and Moore, 1993). At another point of view, Robertson et al. (2007) revealed that polar fraction bounded to and/or sequestrated into organic compounds and soil particle surface resulted in a low efficiency of solvent extraction and biodegradation. 3.3. Microbial community variation in the bioremediation
Fig. 1. Changes of residual TPH in soil during 39-month bioremediation of natural attenuation (A) and landfarming treatment (B).
Microbial quantity is an effective index reflecting soil microbial viability and biodegradable potential. Previous studies revealed that bioremediation of hydrocarbon contaminated soils are effective only when the soil microbial quantity is higher than 103e105 CFU g 1 (Pope and Matthews, 1993; Forsyth et al., 1995), and higher microbial quantity usually promotes biodegradation process (Margesin et al., 2000; Song and Katayama, 2010). As
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TOC, etc.) probably benefited for the growth of total heterotrophic and oil degrading bacteria (Table 1). As shown in Fig. 3B, the quantity of petroleum degrading bacteria was closely related to the residual content of TPH and saturated fraction, their relationship fitted a first-order exponential equation. McGill et al. (1981) found a negative correlation between the increased quantities of soil bacteria with the loss of degradable components during bioremediation. As shown in Fig. 3 C, the significant increases of AWCD and Shannon Index of 39-month landfarming were observed when compared with that of natural attenuation and 0 month, suggesting that long-term landfarming improved soil microbial diversity and activity, and this conclusion was also proved by the researches that revealed the shift and increase of soil microbial communities after bioremediation (Baek et al., 2007; Bastian et al., 2009; Ros et al., 2010). Fig. 2. Temporal changes of residual concentration of saturated fraction, aromatic fraction, polar fraction and asphaltene fraction by natural attenuation and landfarming treatment.
shown in Fig. 3A, in the oil sludge contaminated soil, the quantity of total heterotrophic bacteria and petroleum degrading bacteria was about 7.0 105 and 9.8 104 CFU g 1 at 0 month (i.e. June 2009), and then increased to 2.25 108 CFU g 1 and 4.56 106 CFU g 1 after 39-month landfarming. The quantity of soil bacteria presented a closely correlation with the temperature (Fig. 3A), and this change trend probably affects petroleum biodegradation as ambient temperature affects both the property of spilled oil and soil microbial activity (Atlas, 1981; Venosa and Zhu, 2003). Soil condition influs et al. (2009) enced the growth of bacteria, and Castorena-Corte proved the fertilization stimulation to soil microbial quantity. After 39-month landfarming, the improved soil quality (i.e. increased nutrient content, decreased petroleum hydrocarbons, increased
3.4. PCR-DGGE analysis of soil microbial community diversity Microbial community influences the efficiency and pathway of biodegradation of contaminants (MacNaughton et al., 1999). Changes of soil microbial diversity induced by chemical pollutants can be determined by 16S rDNA amplification and DGGE analysis. As shown in Fig. 4, there were 16 bands appeared in the background soil (sample 1), 19 bands appeared in the oil sludge contaminated soil after 39-month landfarming (sample 2), 16 bands appeared in the oil sludge contaminated soil after 39-month natural attenuation (sample 3), and 14 bands appeared in the original contaminated soil before remediation (sample 4). There were 7 bands detected in all samples as the follows, the band 4 closed to Oceanobacillus sp., the band 5 closed to Marinobacter sp., the bands of 6 and 9 closed to Shewanella sp., the band 11 closed to Thermoanaerobacter sp., the band 13 closed to Thioalkalivibrio sp., the band 14 closed to Tsukamurella sp., and these microbial species what bands represented were assumed as indigenous microorganisms of
Fig. 3. Microbial quantity and activities analysis for natural attenuation and landfarming treatment: (A) changes of the total heterotrophic bacteria (THB) and petroleum degrading bacteria (PDB) with time and temperature, (B) quantity of petroleum degrading bacteria varied with the soil residual concentration of TPH and saturated fraction, (C) comparison of AWCD, Shannon index of natural attenuation and landfarming treatment at 0 and 39 month.
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Fig. 4. PCR-DGGE analysis of different soil samples (1) background soil; (2) oily sludge contaminated soil after 39-month landfarming; (3) oily sludge contaminated soil after 39-month natural attenuation treatment; (4) original oily sludge contaminated soil before remediation.
tolerated or degradative capabilities for petroleum hydrocarbons. However, microbial community changed with time and environmental conditions (Margesin and Schinner, 2001). The changes that 6 bands (i.e. band 1, 10, 12, 15, 20 and 22) were detected in sample 1 but disappeared in sample 4 revealed the effects of petroleum hydrocarbon toxicity to indigenous microorganisms (Duarte et al., 2001; Abed et al., 2002), and the re-emerging 4 bands of 10, 12, 15, 20, 22 in sample 2 and sample 3 indicated the recovery of hydrocarbon degrading microbial species after 39-month remediation of landfarming and natural attenuation. In addition, there were 6 bands of 7, 12, 18, 19, 20, 22 present in sample 3 but absent in sample 4, while there were 3 bands of 10, 16, 17 present in sample 4 but absent in sample 3, and such the differences of DGGE band patterns in the distribution of soil samples were also contributed to the difference of landfarming and natural attenuation. As shown in Table 2, the band of 8 was identified as Pseudomonas sp., the band 3 was identified as Marinobacter sp., the bands of 1, 7, 9 and 19 were identified as Shewanella sp., the bands of 15, 20 and 22 were identified as Streptococcus sp., the band 21 was identified as Bacillus sp., the band 14 was identified as Tsukamurella sp., and the above microbial species were known for oil degrading capacity (Brito et al., 2006; Al-Mailem et al., 2013). The genera Streptococcus, belonging to the class Bacilli, is facultative anaerobic Gram positive cocci (Silva et al., 2013). Due to the complex chemical composition of petroleum hydrocarbon sources, individual microorganisms metabolize only a limited range of hydrocarbon substrates, so a consortium composed of different bacterial species with overall broad enzymatic capacities is required (Mihial et al., 2006; Kim and Crowley, 2007; Schaefer and Juliane, 2007), and that consortium usually was of high biodegradation efficiency and rate of petroleum hydrocarbons as compared to strains in pure culture (Lazar et al., 1999). Vila et al. (2010) identified the bacteria responsible for the degradation of alkanes and aromatic components in a consortium that completely removed the alkane components in heavy fuel oil. Three species of Sphingomonas sp., Pseudomonas sp., Bacillus sp. were reported to be important PAH-degrading microorganisms in the contaminated soil (Uyttebroek et al., 2006), and Bacosa et al. (2010) identified a microbial consortium that preferentially
Table 2 Identification of specific bands in PCR-DGGE profiles. Band
Sample 1
Sample 2
Sample 3
Sample 4
Identified microorganism
Similarity (%)
Accession numbers of public databases
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
√ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √
√ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √
√ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √
√ √ √ √ √ √ √ √ √ √ √ √ √ √
Shewanella loihica PV-4 Thermoanaerobacter tengcongensis MB4 Marinobacter adhaerens HP15 Oceanobacillus kimchii X50 Marinobacter aquaeolei VT8 Shewanella amazonensis SB2B Haemophilusparasuis SH0165 Pseudomonas denitrificans ATCC 13867 Shewanella amazonensis WJ65 Owenweeksiahongkongensis DSM 17368 Thermoanaerobacter siderophilus SR4 Gramellaforsetii KT0803 Thioalkalivibrio sp. K90 mix chromosom Tsukamurella paurometabola DSM 20162 Streptococcus parauberis KCTC 11537 Halomonaselongata DSM 2581 Mycobacterium massiliense str. GO 06 Anaerolineathermophila UNI-1 Shewanella denitrificans OS217 Streptococcus infantarius subsp. infantarius CJ18 Bacillus megaterium DSM 319 Streptococcus gallolyticus subsp. ATCC 43143 Lactobacillus johnsonii NCC 533
97 98 99 95 98 97 99 100 99 94 97 96 97 99 98 93 98 90 98 99 99 99 100
KT865084 KT886064 KT865085 KT865086 KT865087 KT865088 KT865089 KT865090 KT865091 KT865092 KT865093 KT865095 KT865096 KT865097 KT865098 KT865099 KT865100 KT886065 KT865101 KT865102 KT865103 KT865104 KT865105
“”means the disappearance of the band; “√”means the appearance of the band.
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degraded the aromatic hydrocarbons in kerosene. In addition, some bacterial strains have the capacity of both hydrocarbon degrading and biosurfactants producing such as Pseudomonas aeruginosa, Halomonas sp., Rhodococcus sp. and Bacillus subtilis (Mnif et al., 2011; Peng et al., 2007), and such the microbial species were also detected in this study (Table 2). 4. Conclusions The enhanced landfarming treatment of oil sludge contaminated soil by adding cotton stalks is a useful bioremediation strategy. Compared with natural attenuation treatment, the advantages of landfarming treatment for oil sludge contaminated soil presented in the three aspects as follows: higher removals of TPH and petroleum fractions, improved soil properties, and higher soil microbial quantity and diversity. Acknowledgments The authors would also like to acknowledge the financial support provided by the Sino-Russia International Cooperation Project (Grant No. 2008DFR90550) and the National Natural Science Foundation of China (Grant No. 21207122). References € ster, J., de Beer, D., El-Nahhal, Y., Rullko € tter, J., GarciaAbed, R.M.M., Safi, N.M.D., Ko Pichel, F., 2002. Microbial diversity of a heavily polluted microbial mat and its community changes following degradation of petroleum compounds. Appl. Environ. Microbiol. 68, 1674e1683. Al-Mailem, D.M., Eliyas, M., Radwan, S.S., 2013. Oil-bioremediation potential of two hydrocarbonoclastic, diazotrophic Marinobacter strains from hypersaline areas along the Arabian Gulfcoasts. Extremophiles 17, 463e470. Atlas, R.M., 1981. Microbial degradation of petroleum hydrocarbons: an environmental perspective. Microbiol. Rev. 45, 180e209. Bacosa, H., Suto, K., Inoue, C., 2010. Preferential degradation of aromatic hydrocarbons in kerosene by a microbial consortium. Int. Biodeterior. Biodegr. 64, 702e710. Baek, K.H., Yoon, B.D., Kim, B.H., Cho, D.H., Lee, I.S., Oh, H.M., Kim, H.S., 2007. Monitoring of microbial diversity and activity during bioremediation of crude oil-contaminated soil with different treatments. J. Microbiol. Biotech. 17, 67e73. Bastian, F., Bouziri, L., Nicolardot, B., Ranjard, L., 2009. Impact of wheat stalk decomposition on successional patterns of soil microbial community structure. Soil Biol. Biochem. 41, 262e275. Brito, E.M.S., Guyoneaud, R., Goni-Urriza, M., Ranchou-Peyruse, A., Verbaere, A., 2006. Characterization of hydrocarbonoclastic bacterial communities from mangrove sediments in Guanabara Bay. Braz. Res. Microbiol. 157, 752e762. Brook, T.R., Stiver, W.H., Zyther, R.G., 2001. Biodegradation of diesel fuel in soil under various nitrogen addition regimes. Soil Sediment. Contam. 10, 539e553. s, G., Rolda n-Carrillo, T., Zapata-Pen Castorena-Corte asco, I., Reyes-Avila, J., Quej, L., Marín-Cruz, J., Olguín-Lora, P., 2009. Microcosm assays and Taguchi Ake experimental design for treatment of oil sludge containing high concentration of hydrocarbons. Bioresour. Technol. 100, 5671e5677. Chaîneau, C.H., Morel, J., Dupont, J., Bury, E., Oudot, J., 1999. Comparison of the fuel oil biodegradation potential of hydrocarbon-assimilating microorganisms isolated from a temperate agricultural soil. Sci. Total. Environ. 227, 237e247. China Cotton Association, 2013. 2013th Annual Report of China's Survey and Forecast for the National Cotton Growth and Output. http://www.china-cotton. org/article/2013/07/17/69878.php. Duarte, G.F., Alexandre, S.R., Seldin, L., de Araujo, W., van Elsas, J.D., 2001. Analysis of bacterialcommunity structure in sulfurous-oil-containing soils and detection of species carryingdibenzothiophene desulfurization (DSZ) genes. Appl. Environ. Microbiol. 67, 1052e1062. Forsyth, J.V., Tsao, Y.M., Bleam, R.D., 1995. Bioremediation: when is bioaugmentation needed? In: Hinchee, R.E., Fredrickson, J., Alleman, B.C. (Eds.), Bioaugmentation for Site Remediation. Battelle Press, Columbus, pp. 1e14. Gafarov, A.B., Panov, A.V., Filonov, A.E., Boronin, A.M., 2006. Change in the composition of a bacterial association degrading aromatic compounds during oil sludge detoxification in a continuous-flow microbial reactor. Appl. Biochem. Microbiol. 42, 160e165. Garcia, C., Hern andez, T., 1996. Influence of salinity on the biological and biochemical activity of calcciorthid soil. Plant Soil 178, 255e263. ~ a, F., BarreraGarcía-Díaz, C., Ponce-Noyola, M.T., Esparza-García, F., Rivera-Ordun s, J., 2013. PAH removal of high molecular weight by characterized bacCorte terial strains from different organic sources. Int. Biodeterior. Biodegr. 85, 311e322. Genouw, G., de Naeyer, F., van Meenen, P., van de Werf, H., de Nijs, W.,
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Simultaneous hydrocarbon biodegradation and biosurfactant production by oil field-selected bacteria. J. Appl. Microbiol. 111, 525e536. Muyzer, G., de Waal, E.C., Uitterlinden, A.G., 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rDNA. Appl. Environ. Microbiol. 59, 695e700. Ouyang, W., Liu, H., Murygina, V., Yu, Y.Y., Xiu, Z.D., Kalyuzhnyi, S., 2005. Comparison of bioaugmentation and composting for remediation of oil sludge: a fieldscale study in China. Process. Biochem. 40, 3763e3768. Paudyn, K., Rutter, A., Kerry-Rowe, R., Poland, J.S., 2008. Remediation of hydrocarbon contaminated soils in the Canadian Arctic by landfarming. Cold. Reg. Sci. Technol. 53, 702e774. Peng, F., Liu, Z., Wang, L., Shao, Z., 2007. An oil-degrading bacterium: Rodococcus erythropolis strain 3C-9 and its biosurfactants. J. Appl. Microbiol. 102, 1603e1611. Pope, D.F., Matthews, J.E., 1993. Bioremediation Using the Land Treatment Concept. USEPA/600/R-93/164. Robert S. Kerr. Environmental Research Laboratory. US Environmental Protection Agency, Ada, OK. Rhykerd, R.L., Crews, B., McInnes, K.J., Weaver, R.W., 1999. Impact of bulking agents, forced aeration, and tillage on remediation of oil-contaminated soil. Bioresour. Technol. 67, 279e285. Robertson, S.J., McGill, W.B., Massicotte, H.B., Rutherford, P.M., 2007. Petroleum hydrocarbon contamination in boreal forest soils: a mycorrhizal ecosystems perspective. Biol. Rev. 82, 213e240. ndez, T., 2010. Microbial communities Ros, M., Rodríguez, I., García, C., Herna involved in the bioremediation of an aged recalcitrant hydrocarbon polluted soil by using organic amendments. Bioresour. Technol. 101, 6916e6923. Sanscartier, D., Zeeb, B., Koch, I., Reimer, K., 2009. Bioremediation of dieselcontaminated soil by heated and humidified biopile system in cold climates. Cold. Reg. Sci. Technol. 55, 167e173. 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Wang, S.J., Guo, G.L., Yan, Z.G., Lu, G.L., Wang, Q.H., Li, F.S., 2010. The development of a method for the qualitative and quantitative determination of petroleum hydrocarbon components using thin-layer chromatography with flame ionization detection. J. Chromatogr. A 1217, 368e374. Wang, X., Wang, Q.H., Wang, S.J., Li, F.S., Guo, G.L., 2012. Effect of biostimulation on community level physiological profiles of microorganisms in field-scale biopiles composed of aged oil sludge. Bioresour. Technol. 111, 308e315. Warr, L.N., Perdrial, J.N., Lett, M.C., Heinrich-Salmeron, A., Khodja, M., 2009. Clay mineral enhanced bioremediation of marine oil pollution. App. Clay. Sci. 46, 337e345. Zhang, K., Hua, X.F., Han, H.L., Wang, J., Miao, C.C., Xu, Y.Y., Huang, Z.D., Zhang, H., Yang, J.M., Jin, W.B., Liu, Y.M., Liu, Z., 2008. Enhanced bioaugmentation of petroleum- and salt-contaminated soil using wheat straw. Chemosphere 731, 387e1392. Zak, J.C., Willing, M.R., Moorhead, D.L., Wildman, H.G., 1994. Functional diversity of microbial communities: a quantitative approach. Soil Biol. Biochem. 26, 1101e1108. Zheng, C., He, J., Wang, Y., Wang, M., Huang, Z., 2011. Hydrocarbon degradation and bioemulsifier production by thermophilic pallidus strains. Bioresour. Technol. 102, 9155e9161. Zibilske, L.M., Materon, L.A., 2005. Biochemical properties of decomposing cotton and corn stem and root residues. Soil. Sci. Soc. Am. J. 69, 378e386.
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Bioremediation of oil sludge contaminated soil by landfarming with added cotton stalks Shijie Wang a, b, Xiang Wang a, Chao Zhang a, Fasheng Li a, Guanlin Guo a, * a b
State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012, China Beijing Municipal Research Institute of Environmental Research, Beijing 100037, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 February 2015 Received in revised form 20 October 2015 Accepted 20 October 2015 Available online 8 November 2015
A field bioremediation of oil sludge contaminated soil was conducted by landfarming treatment with added cotton stalks in the Shengli oil field. The ability of landfarming treatment was evaluated to reduce petroleum hydrocarbons and restore soil quality. For 39-month landfarming, the initial concentration of total petroleum hydrocarbons (TPH) was 12.57 mg g 1 for oil sludge contaminated soil. The removal efficiency of TPH, saturated fraction and aromatic fraction was 68.48%, 90.04% and 85.55%, respectively. Degradation of TPH followed first order exponential decay kinetics. Soil physic-chemical properties of soil pH, saline alkali degree, nutrients, organic matters and hydrocarbon degraders were greatly improved. The results of Biolog and PCR-DGGE analysis revealed the improvement of soil microbial quantity and diversity, and the isolated predominant 23 strains showed a shift in soil community structure toward the hydrocarbon degrading species including Streptococcus sp., Shewanella sp., Bacillus sp., Pseudomonas sp., Marinobactera sp., Thermoanaerobacter sp., etc. © 2015 Published by Elsevier Ltd.
Keywords: Biodegradation Oil sludge Cotton stalk Bacterial community
1. Introduction Oil sludge was mainly from drilling, refining, transportation, and storage of crude oil, which has been classified as a priority environmental pollutant. The annual volume of oil sludge that produced by large petrochemical facilities was about 10,000 m3 (Gafarov et al., 2006). In China, the environmental risk of oil sludge contaminated sites has been verified through the investigation of vadose zone and groundwater (Jin et al., 2011). In the current technical systems of cleaning up petroleum contaminated sites with large areas, bioremediation appears more practical applicability when compared with other techniques such as solvent washing, ozonation, incineration, landfill and thermal desorption in the consideration of cost-effect and secondary pollution (Ting et al., 1999; Ouyang et al., 2005). Landfarming treatment for petroleum hydrocarbon-contaminated soils has relatively low cost ranging from US$30 to US$70 per ton (Pope and Matthews, 1993; Giasi and Morelli, 2003), and this cost decreases with the development of technology. The previous studies revealed that adding organic matters can enhance bioremediation effectiveness by improving
* Corresponding author. E-mail address: [email protected] (G. Guo). http://dx.doi.org/10.1016/j.ibiod.2015.10.014 0964-8305/© 2015 Published by Elsevier Ltd.
the soil physicochemical properties and indigenous soil microbial activity (Rhykerd et al., 1999; Kriipsalua et al., 2007). However, to date, only a few long-term landfarming projects have been implemented (Mikkonen et al., 2012). In China, there are about 28.63 million tons of cotton stalks produced by 4.57 million planting hectares every year (China Cotton Association, 2013). The available large amount of cotton stalks provides the potential as organic amendments in landfarming treatment of petroleum oil contaminated soils. Wang et al. (2012) proved the primary advantage of biopile constructed with cotton stalks in enhancing TPH removal in soil, as well as the other benefits including lowering remediation cost and reuse of cropping wastes. In this study, the landfarming treatment of oil sludge contaminated soil was conducted, and the cotton stalks were added for enhancing remediation effectiveness. The objects were to evaluate the ability of this technique to reduce petroleum hydrocarbons and improve soil physical and biological properties. For those purposes, the natural attenuation treatment was used as blank control. The parameters regarding soil quality including physicochemical properties, quantity of total heterotrophic bacteria and petroleum degrading bacteria, microbial diversity, soil residual TPH and petroleum fractions were determined before and after landfarming treatment.
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2. Materials and methods 2.1. Site characterization, contaminated soil and cotton stalk The demonstrated landfarming of oil sludge contaminated soils located in the Shengli Oil Field in Shandong Province. The site of 13,750 m2 has been used for open storage of oil sludge for over 43 years. The local climate conditions were as follows: the average annual precipitation was about 642.0 mm, and the average annual temperature was about 12.8 C ranging from 10.3 C in January to 35.6 C in July. The soil type of demonstration site was sandy clay loam that was composed of 13.7% clay (<0.002 mm), 29.6% silt (0.02e0.002 mm) and 56.7% sand (2e0.02 mm). The total petroleum hydrocarbon (TPH) content in the topsoil (0e60 cm) ranged from 0.2 to 452,000 mg kg 1. The cotton stalks used in this study were collected from the local planting region and mechanically shredded into a variety of length ranging from 10 to 30 mm. The organic matter content of cotton stalk was about 78.6% by mass ratio, and the total N, P and K content was of 1.09%, 0.15% and 1.68%, of which the available content of N, P and K was 865.6, 687.4 and 5325.1 mg g 1, respectively.
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BIOLOGTM system. In this method, the average well color development (AWCD) was introduced to reflect soil microbial activity, and Shannon index were used to reflect soil microbial diversity (Zak et al., 1994). 2.5. Soil microbial community analyses by PCR-DGGE
Oil sludge contaminated soil was mixed with cotton stalks, and the ratio of soil to cotton stalks was 1:4 (w/w). There were three 100-m2 plots prepared for landfarming treatment, and one 100-m2 plot was set for natural attenuation as blank control. Oil sludge contaminated soils and cotton stalks were mixed and laid on the plots with 30 cm layer depth. The remediation duration lasted from June 2009 to November 2012, and the 0~30 cm topsoil was tilling with the frequency of every 2 months to keep soil moisture at 20 ~ 40%.
The total soil DNA was first extracted using a Power Soil™ DNA Isolation Kit according to the manufacturer's instructions (MoBio Lab Inc., Carlsbad, CA, USA), and then the integrity and purity of DNA were measured using electrophoresis and Nanodrop 1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). PCR amplification targeting bacterial of the V3 region within 16S rDNA was performed with the 357F-GC and 517R primer set (Muyzer et al., 1993). The nested PCR was conducted for amplification of 16S rDNA using primers 27F and 1492R (Lane, 1991). Denaturing Gradient Gel Electrophoresis (DGGE) for analysis of resulting DNA fragments (15 mL) were conducted according to the method used by Muyzer et al. (1993). The denaturing gradient varied from 45% to 75% for DGGE analysis. Gels were run at 60 V for 16 h in a D-Code System (Bio-Rad) filled with 8% (w/v) polyacrylamide (acrylamide: bisacrylamide, 37:1). The gels were stained in 1:10,000 (v/v) Sybr Green I (Cambrex, Rockland, USA) nucleic acid solution for 30 min. The DGGE images were obtained from the Gel Doc XR system (Bio-RadLab, Segrate, Italy) under UV light. The selected DGGE bands were excised and purified by a TaKaRa Mini BEST DNA Fragment Purification Kit (TaKaRa Bio Inc., Shiga, Japan), and then unidirectional sequenced by the reverse primer. A sequences alignment of the purified 16S rDNA was then conducted with a BLAST program. After entering query sequences, nucleotide collection that was composed of GenBank, EMBL, DDBJ, PDB and RefSeq sequences was chose as the alignment database (http://blast.ncbi.nlm. nih.gov, the accession number (NCBI) of all 23 samples were shown in Table 2).
2.3. Soil sampling and analysis of TPH and petroleum fractions
2.6. Data processing
The remediation plot was divided into 16 different sampling cells based on 2.5 m 2.5 m grid. The representative 20 cm surface soils were collected, and the sampling intervals were of 0 month (June, 2009), 1 month (July, 2009), 2 months (August, 2009), 4 months (October, 2009), 9 months (March, 2010), 13 months (July, 2010), 16 months (October, 2010), 26 months (August, 2011) and 39 months (September, 2012), respectively. Soil samples were freeze-dried, grounded, 2-mm mesh sieved and stored at 4 C. The accelerated solvent extractor (ASE300, Dionex, USA) and trichloromethane were used to extract petroleum hydrocarbons from 10 g soil. Referring to the previous study, the TPH content was determined by gravimetric method, and the petroleum fractions were analyzed by Thin-Layer Chromatography (MK-6S, Tokyo, Japan) (Wang et al., 2010).
All data except local mean temperature were expressed as mean ± standard deviation, and the statistical testing of the data was performed with SPSS v. 13.0. The local temperature shown in Fig. 3 was daily maximum temperature, daily mean temperature and daily minimum temperature, respectively. The correlation analysis of residual TPH in soil with remediation time and quantity of hydrocarbon degrading bacteria was performed by Metlab software (v. R2014a), and the optimal regression model with the smallest relative residual standard deviation was chosen.
2.2. Landfarming process
2.4. Enumeration of cultivable bacteria and determination of microbial activity The quantities of total heterotrophic bacteria and petroleum degrading bacteria in the soil samples were assessed by the plate count method. Tryptone Soya Agar (TSA) was used to determine the total heterotrophic bacteria (THB), and the mineral medium with the addition of sterile crude oil was used to determine the petroleum degrading bacteria (PDB) (Chaîneau et al., 1999). The colonyforming units (CFU) were recorded after incubation in the dark at 25 C on days 5 and 21, respectively. Microbial community level physiological profiles (CLPPs) were reflected by the patterns of sole-carbon-source utilization using a
3. Results and discussion 3.1. Changes of soil physicochemical properties after remediation Soil properties including texture, cation exchange capacity (CEC), nutrient status, and soil bacteria types and numbers are important factors in influencing the remediation efficiency of petroleum contaminated soils (Atlas, 1981). Significant changes of soil properties were observed after 39-month landfarming (Table 1). Previous studies showed that the improvement of soil properties facilitated biodegradation of petroleum hydrocarbons, for example, Zhang et al. (2008) found that the biodegradation rate of hydrocarbons increased as salinity decreased; and Warr et al. (2009) found that higher soil CEC stimulated the mineralization of aromatic, resin and asphaltene; and Brook et al. (2001) found that the biodegradation of hydrocarbons was enhanced if the C:P ratios were adjusted in the range of 60e800; and low salinity increases biodegradation efficiency of petroleum compounds (Garcia and
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Table 1 Changes of physicochemical properties of tested soils before and after natural attenuation and landfarming treatment. pH in Specific TOC Salinity CEC water gravity (%) (g kg 1) (cmol kg Original state 8.60 Natural 8.12 attenuation Landfarming 7.06
TNa/ANb ) (mg kg 1)
1
TPc/APd (mg kg 1)
TKe/AKf (mg kg 1)
780.00/410.60 450.00/205.00
2690.00/1420.00 12,590 2260.00/1050.00 8550
TPH (mg kg
2.74 2.42
2.33 63.04 3.02 41.7
5.44 5.89
150.00/70.50 89.20/39.70
1.44
6.74
8.06
2210.00/766.00 1860.00/880.00 5620.00/3120.00 3960
3.66
Asphaltene Saturated Aromatic Polar fraction fraction (mg g 1) ) fraction (mg kg 1) (mg g 1) (mg g 1)
1
5030 2010
3400 1760
1760 3020
2400 1760
500
490
1690
1280
* atotal nitrogen; btotal phosphorus; ctotal potassium; davailable nitrogen; eavailable phosphorus; favailable potassium.
ndez, 1996; Zheng et al., 2011). In this study, the soil salinity Herna decreased from 63.04 to 3.66 g kg 1, soil CEC increased from 5.44 to 8.06 cmol kg 1, soil total and available nutrients of N, P, K increased significantly by 1.1e13.7 times, and such the above changes provided a valuable condition for microbial growth and metabolic activity recovery. Zibilske and Materon (2005) found that 3-month decomposition of cotton stalks significantly improved soil quality. In this study, 39month landfarming also achieved other improvements of soil properties as follows, the soil specific gravity decreased from 2.74 to 1.44 g cm 3, soil TOC increased from 2.33% to 6.74%, soil pH decreased from 8.60 to 7.06, and these changes probably resulted in the improvement of soil aggregation, oxygen transport and waterholding ability. Some researchers revealed that the decomposition of organic amendments increased the bioavailability of polar and asphaltene fractions (Holman et al., 2002; Kobayashi et al., 2009) and the quantity of hydrocarbons degrading bacteria (García-Díaz et al., 2013). Liu et al. (2013) also found that the TPH degradation efficiency and 1st degradation rates reduced with the increase of soil organic matter (SOM).
3.2. Biodegradation of petroleum hydrocarbons As shown in Fig. 1, the soil residual TPH decreased by 68.48% after 39-month landfarming with addition of cotton stalks, while the soil residual TPH decreased by 32.08% after 39-month natural
attenuation treatment. The landfarming result of TPH removal was comparable with the research by Liu et al. (2010) that they found 27~46% TPH removal for 8-month field bioremediation of oil sludge. The higher TPH removal of 39-month landfarming was probably caused by added cotton stalks and timely tillage, and the previous studies revealed that the added organic amendments improved the TPH removal and soil microbial activities (Goyal et al., 1993; Rhykerd et al., 1999). Genouw et al. (1994) showed about 50% of TPH removal for 23,000 mg kg 1 petroleum oil contaminated soils, and 27% of TPH removal for 31,000 mg kg 1 petroleum oil contaminated soils, thus they concluded that the initial soil TPH content was the primary limiting factor for biodegradation. As shown in Fig. 1, the loss of TPH (Y, m%) followed a first-order exponential decay with time (X, month), and the tailing phenomena of TPH removal were also observed, but the regression parameters of degradation kinetic varied with landfarming and natural attenuation. However, the biodegradation kinetics of TPH also depended on environmental variables such as temperature, moisture, nutrient, hydrocarbons content. Sims and Sims (1999) showed the biphasic degradation kinetics for landfarming of petroleum hydrocarbons. Paudyn et al. (2008) found the TPH biodegradation at the Resolution Island site followed with the first order kinetics. Petroleum is a complex mixture composed of the four petroleum fractions (i.e. saturated fraction, aromatic fraction, polar and asphaltene), and the biodegradation abilities of petroleum fractions was closely related with their various physical and chemical properties. As shown in Fig. 2, the removals of saturated and aromatic fractions in landfarming were of 90.04% and 85.55%, while the removals of saturated and aromatic fractions in natural attenuation were of 60.04% and 48.24%. Meanwhile, the observed removals of saturated and aromatic fractions were higher than that of polar and asphaltene fractions. Light petroleum fractions of aliphatic, cycling and aromatic hydrocarbons were preferentially biodegraded (Sanscartier et al., 2009). The relative content of polar fraction showed a significant increase during the landfarming process (Fig. 2), and this change was probably due to the formation and accumulation of polar fraction caused by aerobic biodegradation of saturated and aromatic hydrocarbons (Huesemann and Moore, 1993). At another point of view, Robertson et al. (2007) revealed that polar fraction bounded to and/or sequestrated into organic compounds and soil particle surface resulted in a low efficiency of solvent extraction and biodegradation. 3.3. Microbial community variation in the bioremediation
Fig. 1. Changes of residual TPH in soil during 39-month bioremediation of natural attenuation (A) and landfarming treatment (B).
Microbial quantity is an effective index reflecting soil microbial viability and biodegradable potential. Previous studies revealed that bioremediation of hydrocarbon contaminated soils are effective only when the soil microbial quantity is higher than 103e105 CFU g 1 (Pope and Matthews, 1993; Forsyth et al., 1995), and higher microbial quantity usually promotes biodegradation process (Margesin et al., 2000; Song and Katayama, 2010). As
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TOC, etc.) probably benefited for the growth of total heterotrophic and oil degrading bacteria (Table 1). As shown in Fig. 3B, the quantity of petroleum degrading bacteria was closely related to the residual content of TPH and saturated fraction, their relationship fitted a first-order exponential equation. McGill et al. (1981) found a negative correlation between the increased quantities of soil bacteria with the loss of degradable components during bioremediation. As shown in Fig. 3 C, the significant increases of AWCD and Shannon Index of 39-month landfarming were observed when compared with that of natural attenuation and 0 month, suggesting that long-term landfarming improved soil microbial diversity and activity, and this conclusion was also proved by the researches that revealed the shift and increase of soil microbial communities after bioremediation (Baek et al., 2007; Bastian et al., 2009; Ros et al., 2010). Fig. 2. Temporal changes of residual concentration of saturated fraction, aromatic fraction, polar fraction and asphaltene fraction by natural attenuation and landfarming treatment.
shown in Fig. 3A, in the oil sludge contaminated soil, the quantity of total heterotrophic bacteria and petroleum degrading bacteria was about 7.0 105 and 9.8 104 CFU g 1 at 0 month (i.e. June 2009), and then increased to 2.25 108 CFU g 1 and 4.56 106 CFU g 1 after 39-month landfarming. The quantity of soil bacteria presented a closely correlation with the temperature (Fig. 3A), and this change trend probably affects petroleum biodegradation as ambient temperature affects both the property of spilled oil and soil microbial activity (Atlas, 1981; Venosa and Zhu, 2003). Soil condition influs et al. (2009) enced the growth of bacteria, and Castorena-Corte proved the fertilization stimulation to soil microbial quantity. After 39-month landfarming, the improved soil quality (i.e. increased nutrient content, decreased petroleum hydrocarbons, increased
3.4. PCR-DGGE analysis of soil microbial community diversity Microbial community influences the efficiency and pathway of biodegradation of contaminants (MacNaughton et al., 1999). Changes of soil microbial diversity induced by chemical pollutants can be determined by 16S rDNA amplification and DGGE analysis. As shown in Fig. 4, there were 16 bands appeared in the background soil (sample 1), 19 bands appeared in the oil sludge contaminated soil after 39-month landfarming (sample 2), 16 bands appeared in the oil sludge contaminated soil after 39-month natural attenuation (sample 3), and 14 bands appeared in the original contaminated soil before remediation (sample 4). There were 7 bands detected in all samples as the follows, the band 4 closed to Oceanobacillus sp., the band 5 closed to Marinobacter sp., the bands of 6 and 9 closed to Shewanella sp., the band 11 closed to Thermoanaerobacter sp., the band 13 closed to Thioalkalivibrio sp., the band 14 closed to Tsukamurella sp., and these microbial species what bands represented were assumed as indigenous microorganisms of
Fig. 3. Microbial quantity and activities analysis for natural attenuation and landfarming treatment: (A) changes of the total heterotrophic bacteria (THB) and petroleum degrading bacteria (PDB) with time and temperature, (B) quantity of petroleum degrading bacteria varied with the soil residual concentration of TPH and saturated fraction, (C) comparison of AWCD, Shannon index of natural attenuation and landfarming treatment at 0 and 39 month.
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Fig. 4. PCR-DGGE analysis of different soil samples (1) background soil; (2) oily sludge contaminated soil after 39-month landfarming; (3) oily sludge contaminated soil after 39-month natural attenuation treatment; (4) original oily sludge contaminated soil before remediation.
tolerated or degradative capabilities for petroleum hydrocarbons. However, microbial community changed with time and environmental conditions (Margesin and Schinner, 2001). The changes that 6 bands (i.e. band 1, 10, 12, 15, 20 and 22) were detected in sample 1 but disappeared in sample 4 revealed the effects of petroleum hydrocarbon toxicity to indigenous microorganisms (Duarte et al., 2001; Abed et al., 2002), and the re-emerging 4 bands of 10, 12, 15, 20, 22 in sample 2 and sample 3 indicated the recovery of hydrocarbon degrading microbial species after 39-month remediation of landfarming and natural attenuation. In addition, there were 6 bands of 7, 12, 18, 19, 20, 22 present in sample 3 but absent in sample 4, while there were 3 bands of 10, 16, 17 present in sample 4 but absent in sample 3, and such the differences of DGGE band patterns in the distribution of soil samples were also contributed to the difference of landfarming and natural attenuation. As shown in Table 2, the band of 8 was identified as Pseudomonas sp., the band 3 was identified as Marinobacter sp., the bands of 1, 7, 9 and 19 were identified as Shewanella sp., the bands of 15, 20 and 22 were identified as Streptococcus sp., the band 21 was identified as Bacillus sp., the band 14 was identified as Tsukamurella sp., and the above microbial species were known for oil degrading capacity (Brito et al., 2006; Al-Mailem et al., 2013). The genera Streptococcus, belonging to the class Bacilli, is facultative anaerobic Gram positive cocci (Silva et al., 2013). Due to the complex chemical composition of petroleum hydrocarbon sources, individual microorganisms metabolize only a limited range of hydrocarbon substrates, so a consortium composed of different bacterial species with overall broad enzymatic capacities is required (Mihial et al., 2006; Kim and Crowley, 2007; Schaefer and Juliane, 2007), and that consortium usually was of high biodegradation efficiency and rate of petroleum hydrocarbons as compared to strains in pure culture (Lazar et al., 1999). Vila et al. (2010) identified the bacteria responsible for the degradation of alkanes and aromatic components in a consortium that completely removed the alkane components in heavy fuel oil. Three species of Sphingomonas sp., Pseudomonas sp., Bacillus sp. were reported to be important PAH-degrading microorganisms in the contaminated soil (Uyttebroek et al., 2006), and Bacosa et al. (2010) identified a microbial consortium that preferentially
Table 2 Identification of specific bands in PCR-DGGE profiles. Band
Sample 1
Sample 2
Sample 3
Sample 4
Identified microorganism
Similarity (%)
Accession numbers of public databases
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
√ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √
√ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √
√ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √
√ √ √ √ √ √ √ √ √ √ √ √ √ √
Shewanella loihica PV-4 Thermoanaerobacter tengcongensis MB4 Marinobacter adhaerens HP15 Oceanobacillus kimchii X50 Marinobacter aquaeolei VT8 Shewanella amazonensis SB2B Haemophilusparasuis SH0165 Pseudomonas denitrificans ATCC 13867 Shewanella amazonensis WJ65 Owenweeksiahongkongensis DSM 17368 Thermoanaerobacter siderophilus SR4 Gramellaforsetii KT0803 Thioalkalivibrio sp. K90 mix chromosom Tsukamurella paurometabola DSM 20162 Streptococcus parauberis KCTC 11537 Halomonaselongata DSM 2581 Mycobacterium massiliense str. GO 06 Anaerolineathermophila UNI-1 Shewanella denitrificans OS217 Streptococcus infantarius subsp. infantarius CJ18 Bacillus megaterium DSM 319 Streptococcus gallolyticus subsp. ATCC 43143 Lactobacillus johnsonii NCC 533
97 98 99 95 98 97 99 100 99 94 97 96 97 99 98 93 98 90 98 99 99 99 100
KT865084 KT886064 KT865085 KT865086 KT865087 KT865088 KT865089 KT865090 KT865091 KT865092 KT865093 KT865095 KT865096 KT865097 KT865098 KT865099 KT865100 KT886065 KT865101 KT865102 KT865103 KT865104 KT865105
“”means the disappearance of the band; “√”means the appearance of the band.
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degraded the aromatic hydrocarbons in kerosene. In addition, some bacterial strains have the capacity of both hydrocarbon degrading and biosurfactants producing such as Pseudomonas aeruginosa, Halomonas sp., Rhodococcus sp. and Bacillus subtilis (Mnif et al., 2011; Peng et al., 2007), and such the microbial species were also detected in this study (Table 2). 4. Conclusions The enhanced landfarming treatment of oil sludge contaminated soil by adding cotton stalks is a useful bioremediation strategy. Compared with natural attenuation treatment, the advantages of landfarming treatment for oil sludge contaminated soil presented in the three aspects as follows: higher removals of TPH and petroleum fractions, improved soil properties, and higher soil microbial quantity and diversity. Acknowledgments The authors would also like to acknowledge the financial support provided by the Sino-Russia International Cooperation Project (Grant No. 2008DFR90550) and the National Natural Science Foundation of China (Grant No. 21207122). References € ster, J., de Beer, D., El-Nahhal, Y., Rullko € tter, J., GarciaAbed, R.M.M., Safi, N.M.D., Ko Pichel, F., 2002. Microbial diversity of a heavily polluted microbial mat and its community changes following degradation of petroleum compounds. Appl. Environ. Microbiol. 68, 1674e1683. Al-Mailem, D.M., Eliyas, M., Radwan, S.S., 2013. Oil-bioremediation potential of two hydrocarbonoclastic, diazotrophic Marinobacter strains from hypersaline areas along the Arabian Gulfcoasts. Extremophiles 17, 463e470. Atlas, R.M., 1981. Microbial degradation of petroleum hydrocarbons: an environmental perspective. Microbiol. Rev. 45, 180e209. Bacosa, H., Suto, K., Inoue, C., 2010. Preferential degradation of aromatic hydrocarbons in kerosene by a microbial consortium. Int. Biodeterior. Biodegr. 64, 702e710. Baek, K.H., Yoon, B.D., Kim, B.H., Cho, D.H., Lee, I.S., Oh, H.M., Kim, H.S., 2007. Monitoring of microbial diversity and activity during bioremediation of crude oil-contaminated soil with different treatments. J. Microbiol. Biotech. 17, 67e73. Bastian, F., Bouziri, L., Nicolardot, B., Ranjard, L., 2009. Impact of wheat stalk decomposition on successional patterns of soil microbial community structure. Soil Biol. Biochem. 41, 262e275. Brito, E.M.S., Guyoneaud, R., Goni-Urriza, M., Ranchou-Peyruse, A., Verbaere, A., 2006. Characterization of hydrocarbonoclastic bacterial communities from mangrove sediments in Guanabara Bay. Braz. Res. Microbiol. 157, 752e762. Brook, T.R., Stiver, W.H., Zyther, R.G., 2001. Biodegradation of diesel fuel in soil under various nitrogen addition regimes. Soil Sediment. Contam. 10, 539e553. s, G., Rolda n-Carrillo, T., Zapata-Pen Castorena-Corte asco, I., Reyes-Avila, J., Quej, L., Marín-Cruz, J., Olguín-Lora, P., 2009. Microcosm assays and Taguchi Ake experimental design for treatment of oil sludge containing high concentration of hydrocarbons. Bioresour. Technol. 100, 5671e5677. Chaîneau, C.H., Morel, J., Dupont, J., Bury, E., Oudot, J., 1999. Comparison of the fuel oil biodegradation potential of hydrocarbon-assimilating microorganisms isolated from a temperate agricultural soil. Sci. Total. Environ. 227, 237e247. China Cotton Association, 2013. 2013th Annual Report of China's Survey and Forecast for the National Cotton Growth and Output. http://www.china-cotton. org/article/2013/07/17/69878.php. Duarte, G.F., Alexandre, S.R., Seldin, L., de Araujo, W., van Elsas, J.D., 2001. Analysis of bacterialcommunity structure in sulfurous-oil-containing soils and detection of species carryingdibenzothiophene desulfurization (DSZ) genes. Appl. Environ. Microbiol. 67, 1052e1062. Forsyth, J.V., Tsao, Y.M., Bleam, R.D., 1995. Bioremediation: when is bioaugmentation needed? In: Hinchee, R.E., Fredrickson, J., Alleman, B.C. (Eds.), Bioaugmentation for Site Remediation. Battelle Press, Columbus, pp. 1e14. Gafarov, A.B., Panov, A.V., Filonov, A.E., Boronin, A.M., 2006. Change in the composition of a bacterial association degrading aromatic compounds during oil sludge detoxification in a continuous-flow microbial reactor. Appl. Biochem. Microbiol. 42, 160e165. Garcia, C., Hern andez, T., 1996. Influence of salinity on the biological and biochemical activity of calcciorthid soil. Plant Soil 178, 255e263. ~ a, F., BarreraGarcía-Díaz, C., Ponce-Noyola, M.T., Esparza-García, F., Rivera-Ordun s, J., 2013. PAH removal of high molecular weight by characterized bacCorte terial strains from different organic sources. Int. Biodeterior. Biodegr. 85, 311e322. Genouw, G., de Naeyer, F., van Meenen, P., van de Werf, H., de Nijs, W.,
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