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Water Air Soil Pollut (2007) 185:177­184 DOI 10.1007/s11270-007-9440-y

Bioremediation of Oil Sludge in Shengli Oilfield
Shi De-qing & Zhang Jian & Gui Zhao-long & Dong Jian & Wang Tian-li & Valentina Murygina & Sergey Kalyuzhnyi

Received: 28 January 2007 / Accepted: 17 May 2007 / Published online: 13 June 2007 # Springer Science + Business Media B.V. 2007

Abstract Large quantity of dehydrated oil sludge, generated in the disposal process of oil-containing sewage in Shengli oilfield, needs to be rendered harmless to human and to the environment. Bioremediation has been accepted as an important method for the treatment of oil sludge by employing indigenous or extraneous microbial flora. The bioremediation of a dehydrated oil sludge of 960 m3 in volume was carried out in a prepared bed in Binyi oil-containing sewage disposal station, Shengli oil fields, China. Four different treatments were made to study the impact of certain process parameters on the bioremediation efficiency. Of the oil contaminants, 52.75% was degraded within 160 days when treated in a greenhouse, while the oil contaminations decreased by only 15.46% in the untreated sludge. The variations of the physical and chemical properties of
S. De-qing (*) : Z. Jian : G. Zhao-long : D. Jian : W. Tian-li Oilfield Surface Technology R&D Institute, Shengli Engineering and Consulting Co., Ltd., Dongying 257026, China e-mail: shidq@hdpu.edu.cn S. De-qing Collage of Chemistry and Chemical Engineering, China University of Petroleum, Dongying 257061, China V. Murygina : S. Kalyuzhnyi Department of Chemical Enzymology, Chemistry faculty, Moscow State University, Leninskye gory 1, build 11, 119992 Moscow, Russia

oil sludge, the amount and the functional diversity of microorganisms in sludge were characterized. The results indicated that the water-holding capacity of oil sludge, the amount and the metabolism functional diversity of microorganisms in sludge in the three treatments increased markedly compared with the control. Keywords Bioremediation . Degradation . Greenhouse . Microorganisms . Oil-containing sludge

1 Introduction The amount of oil-contaminated soil generated in the oil production process has been increasing by thousands of tons every year in Shengli oil field in China (Li et al. 2006). Parts of the contaminated soil are dehydrated oil sludge, separated from the mixture of oil, water and soil. Most of the oil sludge is piled up outdoor without any treatment, and poses a serious environmental problem. The hydrocarbons in the sludge penetrate from the top soil into the subsoil slowly, presenting a direct risk of contamination to subsoil and groundwater. On the other hand, the light hydrocarbons in the oil sludge vaporize, leaving behind a layer of oil-containing dust of soil which blows upwards to pollute the air. Therefore, the oil sludge should be treated to prevent harm to environment. Although burning of the sludge may be simple and easily adaptable, this technique has undesirable hazard in air pollution. Bioremediation of the oil sludge is believed to be an efficient, economic and


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versatile alternative to physiochemical treatments (Jackson et al. 1996; Venosa et al. 1996; Salanitro et al. 1997). Acceptance by the general public is another major advantage of this technology (Skladney and Metting 1993). Microorganisms (indigenous or extraneous) can utilize the total petroleum hydrocarbons (TPH) of crude oil as source of carbon and energy and break them down to simpler non-toxic compounds such as CO2 and H2O. But bioremediation takes a long time as the degradation efficiency of the bacteria is considerably low under natural conditions (Del'Arco and de Franca 1999; ChaÍneau et al. 2003). Therefore, some engineering processes such as addition of nutrients, watering, tilling and addition of suitable microbial flora are necessary to improve the biodegrading rate of hydrocarbons (JÜrgensen et al. 2000; Vasudevan and Rajaram 2001; Barathi and Vasudevan 2003; Kuyukina et al. 2003). In our work, four types of treatment for the oil sludge were conducted in Binyi oil-containing sewage disposal station, Shengli oil fields. Biotreatment of the oil sludge resulted in a 52.75% reduction in oil concentration after 160 days (including winter) in one treatment. It was the first attempt to remedy oilcontaining sludge by the use of microbial flora in China in an industrial scale.

Fig. 1 The layout drawing of the prepared bed

2 Materials and Methods 2.1 Site and Experiment Scale Bioremediation experiments of oil sludge were undertaken in the territory of the Shengli oil field (Shandong Province, China) owned by the SINOPEC Company. A 2400 m2 prepared bed outlined by concrete was built for the disposal of the oil sludge generated from the Binyi oil-containing sewage disposal station. The thickness of the dehydrated sludge in the prepared bed was 400 mm, and the total volume of the sludge was 960 m3. 2.2 Construction of the Prepared Bed The prepared bed was 80 â 30 m in size, and 0.7 m in height. The layout drawing is shown in Fig. 1. The prepared bed was divided into two equal parts by a ditch. A percolate water collection pool was built at the end of the bed. A green house 15 â 6 m in size was

constructed at the corner of the bed to explore the possibility of bioremediation in cold winter. A small cell of 4 m2 was built as control. Four different treatments were designed in the prepared bed. In treatment B, one type of commercial bacterium and nutrient were added into the soil. Treatment A was in the small-scale greenhouse, with the same operation as in treatment B. In treatment C, nutrient was added to stimulate the biodegradation activity of indigenous microorganisms. And treatment D is the control without any operation. To prevent seepage of oil into the ground water, the bottom of the prepared bed was covered by a 5-mm thick layer of geomembrane. A set of drainpipes was laid on the geomembrane to collect the percolate and was covered with an 100-mm layer of gravels. On top of the gravel layer was laid an 100-mm layer of sand, on which the oil sludge sits. 2.3 Pretreatment of the Oil Sludge The dehydrated oil sludge collected from storage pit was loaded into the prepared bed. The oil sludge had heavy clay texture and low oxygen diffusivity. In


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order to enhance aeration and water-holding capacity of the sludge, organic and inorganic bulking materials (woodchips and sand) were added. The content of woodchips in the sludge was 5.0% (w/w) and that of sand was 10% (w/w). Urea was provided as a nitrogen source, and potassium dihydrogen phosphate as a phosphorus source. The ratio of C, N and P in the oil sludge was 100:10:1 after the fertilizers had been added. Same pretreatments were conducted in treatments A, B and C. In the control treatment D no pretreatment was done. 2.4 Bioremediation Process Microorganism obtained from Russia named "Rhoder" was sown to treatment A and B. The "Rhoder" preparation (Patent RF No. 2174496) has been used for bioremediation of oil-contaminated soils in Russia (Murygina et al. 2000, 2005; Ouyang et al. 2006). The application amount was approximately 1 kg of "Rhoder" per ton of oil sludge. Over the course of the experiment, the landfarming cells were tilled twice a week to maintain high level of oxygen in the sludge. Water was added after tilling to maintain a moisture level of 40% in the sludge. 2.5 Analytical Methods Oil sludge in different treatment was sampled at different stage of bioremediation. Five samples were taken in treatment A and D, and 15 samples in treatment B and C. Physical and chemical properties of the oil sludge, such as water-holding capacity, pH value, total nitrogen, hydrolyzed nitrogen, available phosphorus and potassium, were characterized by well-known methods used in the agricultural chemistry. The oil content in sludge samples was determined gravimetrically in amount of TPH extracted by chloroform (Christofi et al. 1998; Capelli et al. 2001). Oil fraction analysis was performed using a thin-layer chromatography (TLC) (Yan-gen 2000; Kuyukina et al. 2003). In this analysis, soil samples were extracted in a 3:1 mixture of dichloromethane-pentane. The pentanesoluble fractions were applied onto the TLC, and consecutively eluted with n-hexane to separate the saturated hydrocarbons, dichloromethane-pentane (55:45) to separate the aromatics, and dichloromethane-methanol (98:2) to separate the non-hydrocarbons. The elutes were detected by an FID, and the composition deter-

mined by the calculated peak area. Resin and asphaltene content of samples (pentane-insoluble fractions) were determined gravimetrically. The method of dilution plating on agar plates was used to monitor the number of bacteria and actinomycetes in the sludge samples (Mesarch and Nies 1997). The method of most-probable-number was used to count the number of hydrocarbon degrading microorganisms and that of aromatic hydrocarbon degrading microorganisms (Wrenn and Venosa 1996). The microbial metabolism function diversity in soil was assessed using BIOLOG GN microplates, which contained 95 different carbon sources (Yang et al. 2000). A 10-3 diluted soil sample was prepared by serial dilutions of suspended wet soil in phosphate buffer solution. One hundred fifty microliters of the supernatant was added to each well of the BIOLOG plates. The plates were incubated at 30°C for 5 days. The color development in the wells was measured as absorbance at 590 nm at 0, 20, 24, 30, 36, 48, 72, 96 and 120 h using a microplate reader. The average well color development (AWCD) of the plate can be calculated by the following equation: hX i. AWCD ¼ ÏCi þ R÷ 95 Where Ci is the absorbance of each well except the control well; R is the absorbance of the control.

3 Results and Discussions 3.1 Characterization of the Oil Sludge Before Remediation The water content of the oil sludge was as high as 43.1% even after a dewatering progress with plate-andframe filter press. Analytical results indicated that the

Table 1 The physical and chemical properties of oil sludge Untreated pH (H2O) Total nitrogen (g·kg-1) Hydrolyzed nitrogen (mg·kg-1) Available phosphorus (g·kg-1) Available potassium (mg·kg-1) Water-holding capacity (%) 7.30 ± 0.04 1.10 ± 0.09 44±4 0.79 ± 0.23 175 ± 20 5.1 ± 0.6 Pretreated 7.23 ± 0.04 2.05 ± 0.60 86±8 46 ± 11 764 ± 47 56 ± 8


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3.2 Analysis of the Bioremediation Results 3.2.1 The Variation of the Oil Content in the Sludge The "Rhoder" microorganism preparation was added to the oil sludge on October 21, 2005. Tilling and watering were conducted in the next 40 days. The average air temperature in the last 10 days of November in Shengli oil field was 5.9°C, below the required temperature of the "Rhoder". Therefore, the bioremediation for treatments B and C had to stop at the end of November. However, the bioremediation proceeded as usual in treatment A in the greenhouse. The oil contents in the different treatments have changed significantly in the 40-day remediation process, as shown in Fig. 2. The air temperature went up and reached 6.4°C at the first 10 days of March, 2006. Operation of bioremediation was performed as before. Sampling and analyzing were carried out on March 30. By then, 160 days had passed from the beginning of the bioremediation. The variation of the oil content of the sludge during this time is also shown in Fig. 2. As can be seen from the figure, the degradation amount of oil in different treatments follows this sequence: A > B > C > D. The same amount of "Rhoder" was added into treatment A and B. But the average air and ground temperatures of treatment A were 10°C higher than those of treatment B because A was done in the greenhouse. In addition, the air humidity in the greenhouse was far greater than that outside. The warm and humid environment was in favor of the growth of the microorganism in the sludge (Whyte et al. 1998, 2001; Gibb et al. 2001; Delillea et al. 2004), and helped to improve the degradation activity of related enzymes in the microbes. Consequently, the oil content in the sludge in treatment A was the lowest. Although the environment conditions and the operations such as tilling and watering for treatment B and C were the same, the "Rhoder" was inoculated into treatment B, while indigenous micro-

Fig. 2 The variation of oil content in the sludge of the four treatments

concentration of petroleum contaminants in the sludge was 129,600 mg·kg-1. Group composition analysis showed the following composition: 36.35% saturated alkanes, 26.47% aromatics, 26.89% non-hydrocarbons, and 2.56% resins and asphaltenes. Generally speaking, the natural conditions of oilcontaining sludge are not conducive to the growth of microorganisms. The carbon content is so high that the ratio of C:N:P is out of balance. According to previous studies (Del'Arco and de Franca 1999; ChaÍneau et al. 2003; Ayotamuno et al. 2006), the absence of fertilizer would result in low degradation rate for the oil sludge. Because of this, urea and potassium dihydrogen phosphate were cast into the sludge as supplements. The physical and chemical properties of untreated and pretreated oil sludge are shown in Table 1. There was no marked change in the pH value. But the content of nutrient increased evidently because of the addition of fertilizers. The water-holding capacity of oil sludge increased from 5.15 to 56.98% after the addition of bulking materials because they are low density materials that increase the porosity of the soil, and help form water-stable aggregates (Vasudevan and Rajaram 2001).

Table 2 The degradation amount of different group components in sludge after 160-day bioremediation

Treatment

Degradation amount (g·kg-1) Saturated alkane Arene 14.4 ± 1.8 14.4 ± 2.0 7.7 ± 0.9 7.0 ± 1.8 Non-hydrocarbon 7.2 6.8 3.5 1.8 ± ± ± ± 1.1 1.3 1.0 0.5 Resin and asphaltene 0.25 0.22 0.15 0.09 ± ± ± ± 0.05 0.04 0.04 0.03

A B C D

36.3 27.1 17.9 11.1

± ± ± ±

2.7 2.7 3.6 2.9


Water Air Soil Pollut (2007) 185:177­184 Table 3 The amount of different kinds of microorganisms in sludge at different bioremediation stages Treatment Remediation time (days) 40 160 40 160 40 160 40 160 Bacteria (â106 cfu/g) 475 ± 77 64 ± 16 252 ± 33 19 ± 7 218 ± 45 14 ± 1.1 8.0 ± 2.2 1.1 ± 0.3 Actinomycetes (â103 cfu/g) 289 ± 10 152 ± 38 149 ± 20 46 ± 13 62 ± 12 43 ± 11 1.25 ± 0.27 1.2 ± 0.35 Hydrocarbon degradation microorganisms (â105 cells/g) 2375 ± 497 64 ± 19 1920 ± 238 31 ± 8 298 ± 86 29 ± 13 55 ± 17 5.0 ± 1.6

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Arene degradation microorganisms (â105 cells/g) 350 ± 60 44 ± 16 148 ± 36 15 ± 4 57 ± 17 10 ± 2 2.9 ± 1.4 4.6 ± 1.3

A B C D

organisms filled the role of degrading the contaminants in treatment C. The oil content in treatment B decreased from 110,160 mg·kg-1 to 60,800 mg·kg-1 in 160 days, and to 81,040 mg·kg-1 in treatment C. This comparison indicated that the "Rhoder" showed a better bioremediation effect than the indigenous microorganisms. In the same period, the oil content in the control (treatment D) decreased from 129,600 to 109,560 mg·kg-1. The degradation efficiency of oil contaminants in treatment A was 52.75%, but only 15.46% for the control. It should be noted that the bioremediation was carried out in the winter. The degradation rate of the oil should be higher if the process were conducted in the summer. The degradation amounts of different kinds of petroleum components have been measured and shown in Table 2. The degradation amount of saturated alkanes was the highest among all the petroleum components. However, the degradation of arenes was much lower as aromatic hydrocarbons are more resistant to microbial degradation (Gogoi et al. 2003; ChaÍneau et al. 2003). The degradation of resins and asphaltenes

was the lowest as they are often considered to be nonbiodegradable (Milne et al. 1998). 3.2.2 The Variation of the Amount of Cultivable Microorganisms Bioremediation is the degradation process of petroleum hydrocarbon by microorganisms. So it would help to understand the course of bioremediation by studying the variation of the amount of cultivable microorganisms, especially the amount of hydrocarbon degrading microbes. The amounts of bacteria, actinomycetes, hydrocarbon degrading microorganisms and arene degrading microorganisms were measured after 40 days and 160 days of bioremediation, respectively, as shown in Table 3. The results showed that the increase of these microorganisms in treatment A, B and C was 1­2 orders of magnitude more than in treatment D. It also can be seen from the table that the increase of these microorganisms in the greenhouse was the highest among all the treatments. The results also demonstrated

Table 4 The physical and chemical properties of oil sludge at different bioremediation stages Treatment Remediation time (days) pH(H2O) Total nitrogen (g·kg-1) Hydrolyzed nitrogen (mg·kg-1) 101 ± 14 134 ± 16 82 ± 21 81 ± 11 80 ± 15 79 ± 14 45 ± 4 44 ± 2 Available phosphorus (mg·kg-1) 112 ± 31 116 ± 37 51 ± 14 305 ± 71 42 ± 11 16 ± 5 1.1 ± 0.3 1.2 ± 0.3 Available potassium (mg·kg-1) 1260 ± 110 1270 ± 260 769 ± 39 782 ± 37 725 ± 52 603 ± 112 195 ± 12 264 ± 5 Water-holding capacity (%)

A B C D

40 160 40 160 40 160 40 160

7.41 7.60 7.43 7.56 7.46 7.59 7.48 7.59

± ± ± ± ± ± ± ±

0.05 0.13 0.08 0.08 0.04 0.04 0.04 0.33

2.89 2.99 2.01 1.98 1.89 1.92 1.12 1.28

± ± ± ± ± ± ± ±

0.45 0.40 0.45 0.41 0.26 0.28 0.12 0.16

63 ± 6.6 71 ± 1.9 59 ± 6.7 62 ± 5.0 57 ± 13.4 59 ± 20.6 7.8 ± 1.7 9.2 ± 2.3


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1.5 1.2 Treatment A Treatment B Treatment C Treatment D

Water Air Soil Pollut (2007) 185:177­184

AWCD

0.9 0.6 0.3 0 0 24 48 72 96 120

Time (h)

Fig. 3 The comparison of AWCD at different incubation times for each treatment

that temperature was one of the most important factors in bioremediation in winter. The increase of actinomycete was significant in treatment A and B compared with treatment C and D. Many kinds of organics can be degraded by actinomycete (Essien and Udosen 2000). It also can be seen that the quantity of microbes decreased remarkably at the end of bioremediation process by comparing the data of 40 and 160 days. This should be the result of low temperature in the winter. 3.2.3 The Variation of Physical and Chemical Properties of the Sludge Table 4 shows the physical and chemical properties of the oil sludge in different bioremediation stages. Comparing the data in Table 1 and Table 4, we can see that the pH value varied little and stayed in the range from 7.20 to 7.60 in the whole process. The content of N, P and K changed little and remained in the appropriate level for microorganisms growth. The water-holding capacity for all treatments increased with time, which indicates that the content of hydrophobic substance (i.e. the petroleum hydrocarbon) decreased and thus it was easier for the soil to take up water than before (Mishra et al. 2001).

T h e r e w e r e s o m e d i ff e r e n c e s i n p h y s i c a l a n d chemical properties of the sludge among the treatments. The content of nutrient in treatment A was the highest. Possible reason for the higher nitrogen content in treatment A could be bacterial N2-fixation, while higher available nutrients can be attributed to higher water solubility of corresponding salts at higher temperature. Since there was not any operation for treatment D, the concentrations of N, P and K in the sludge were the lowest. The water-holding capacity for the four treatments followed the same order: the highest for treatment A, and the lowest for treatment D. It can be concluded that the degradation of the hydrophobic hydrocarbon was higher for treatment B than for treatment C by comparing the difference in water-holding capacity between the two treatments. 3.2.4 Comparison of the Microbial Functional Diversity in Different Treatments The BIOLOG system can detect the utilization of specific carbon sources by the bacterial communities using a set of 95 different carbon compounds present on either Gram(+) or Gram(-) microplates. The BIOLOG method has many attributes: relatively simple, fast and economical, and its data acquisition can be automated using a microplate reader and applicable software. In our work, the BIOLOG system was used to study the microbial metabolism function in different treatments. The AWCD at different incubation times for each treatment can be observed in Fig. 3. The result shows that the AWCD value follows this order: treatment A > treatment B > treatment C > treatment D, which was consistent with the order of the microorganisms' quantities and the degradation effectiveness of oil in the four treatments. In addition, the AWCD values for treatment A, B and C were much higher than the value for treatment D. This indicates that adding extraneous microorganisms and taking measures such as

Table 5 The diversity indices of microorganisms in different treatments after 160-day bioremediation Treatment A B C D Shannon index 4.380 4.303 4.204 2.969 ± ± ± ± 0.033 0.041 0.051 0.265 Shannon evenness 0.968 0.956 0.951 0.718 ± ± ± ± 0.008 0.006 0.016 0.071 Gini index 0.986 0.985 0.979 0.913 ± ± ± ± 0.000 .001 0.006 0.032 McIntosh index 10.282 ± 0.542 9.171 ± 0.457 6.093 ± 1.369 2.242 ± 0.578 McIntosh evenness 0.986 0.981 0.963 0.812 ± ± ± ± 0.003 0.002 0.015 0.066


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watering and tilling could increase the metabolism activity of microorganisms effectively. The influence of different treatment processes on the microbial metabolism functional diversity was analyzed by determining the ability of the communities to oxidize various carbon sources in BIOLOG system. Shannon index, Shannon evenness, Gini index, McIntosh index and McIntosh evenness were used to characterize functional diversity in a community in this work. The results are shown in Table 5. As can be seen from the table, the three indices and two evennesses in treatment A, B and C were all much higher than in treatment D. This demonstrates that the microbial metabolism functions in the three treatments were more diverse than in the untreated cell (Zak et al. 1994). The higher function diversity indices in treatment A testifies that the environment conditions in the greenhouse were more suitable for the growth of microbes than in other treatments in the winter. However, it should be noted that the function diversity index of microbes is based on the cultivable microorganisms, and does not apply to the uncultivable microorganisms in soil.

4. The physical and chemical properties of the oil sludge changed after bioremediation. The waterholding capacity of soil increased evidently. 5. The amount, the degradation activity and the metabolism functional diversity of microorganisms in oil sludge increased to various degrees in different treatments.

Acknowledgments Financial support for this work from SINOPEC Company and technology support from Moscow State University are hereby gratefully acknowledged.

References
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4 Conclusions The following conclusions can be drawn from this work: 1. Addition of extraneous microorganisms can improve the bioremediation process. The biodegradation effectiveness of TPH was 44.73% in treatment B. In contrast, treatment C, which had the same operation but without the extraneous microorganisms, was only 26.43%. 2. Appropriate operations (addition of woodchips and nutrient, tilling and watering) can enhance the biodegradation activity of the indigenous microbes. The biodegradation effectiveness of TPH for the control without any operation was only 15.46%, much lower than those for the other treatments. 3. Greenhouse makes bioremediation of contaminated soil in the winter possible. The temperature and humidity in the greenhouse were much higher than outside. As a result, the degradation effectiveness of TPH in the sludge was the highest among all the treatments.


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