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Ex situ bioremediation of oil-contaminated soil-台湾


Journal of Hazardous Materials 176 (2010) 27–34

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Journal of Hazardous Materials
journal homepage: www.elsevier.com/locate/jhazmat

Ex situ bioremediation of oil-contaminated soil
Ta-Chen Lin a , Po-Tsen Pan b , Sheng-Shung Cheng b,c,
a

Department of Biological Science and Technology, Meiho Institute of Technology, Pingtung County, 912, Taiwan Department of Environmental Engineering, National Cheng Kung University, Tainan City 701, Taiwan c Sustainable Environment Research Center, National Cheng Kung University, Tainan City 701, Taiwan
b

a r t i c l e

i n f o

a b s t r a c t
An innovative bioprocess method, Systematic Environmental Molecular Bioremediation Technology (SEMBT) that combines bioaugmentation and biostimulation with a molecular monitoring microarray biochip, was developed as an integrated bioremediation technology to treat S- and T-series biopiles by using the landfarming operation and reseeding process to enhance the bioremediation efciency. After 28 days of the bioremediation process, diesel oil (TPHC10–C28 ) and fuel oil (TPHC10–C40 ) were degraded up to approximately 70% and 63% respectively in the S-series biopiles. When the bioaugmentation and biostimulation were applied in the beginning of bioremediation, the microbial concentration increased from approximately 105 to 106 CFU/g dry soil along with the TPH biodegradation. Analysis of microbial diversity in the contaminated soils by microarray biochips revealed that Acinetobacter sp. and Pseudomonas aeruginosa were the predominant groups in indigenous consortia, while the augmented consortia were Gordonia alkanivorans and Rhodococcus erythropolis in both series of biopiles during bioremediation. Microbial respiration as inuenced by the microbial activity reected directly the active microbial population and indirectly the biodegradation of TPH. Field experimental results showed that the residual TPH concentration in the complex biopile was reduced to less than 500 mg TPH/kg dry soil. The above results demonstrated that the SEMBT technology is a feasible alternative to bioremediate the oil-contaminated soil. Crown Copyright 2009 Published by Elsevier B.V. All rights reserved.

Article history: Received 22 May 2009 Received in revised form 24 September 2009 Accepted 19 October 2009 Available online 30 October 2009 Keywords: Hydrocarbon Landfarming Bioremediation Bioaugmentation Biostimulation

1. Introduction Soil and groundwater contamination with petroleum hydrocarbon compounds causes environmental and health concerns. This has led to increased attention to develop innovative technologies for remediation [1]. Bioremediation of petroleum hydrocarbons is an effective, economical, and environmentally friendly technology, which is considered a feasible method for treating petroleum hydrocarbon-contaminated soils [2,3]. Bioremediation is generally achieved via bioaugmentation or biostimulation or both, depending on soil conditions and the microbial community structure. The guidelines of the US EPA suggest that bioremediation is feasible when there is about 103 CFU/g soil of the microbial population. However, a low microbial population and insufcient microbial diversity affect bioremediation efciency. According to Alexander [2], bioremediation efciency is a function of the ability of the inoculated microbial degraders to remain active in the natu-

Corresponding author at: Department of Environmental Engineering, National Cheng Kung University, Tainan City 701, Taiwan. Tel.: +886 6 2757575x65827; fax: +886 6 2752790. E-mail addresses: sscheng@mail.ncku.edu.tw, robin6989@gmail.com (S.-S. Cheng).

ral environment. Therefore, increasing the ability of the inoculated microbial degraders by bioaugmentation or promoting the activity of indigenous microbial degraders by biostimulation could improve bioremediation efciency. Microbial communities should thus be monitored to promise the efciency of bioremediation. Bioaugmentation is the introduction of exogenous microorganisms into environments to accelerate bioremediation [4]. Bioaugmentation can increase pollutant removal rates by increasing the bacterial population [5,6]. In biostimulation, the soil is amended with nutrient mainly containing nitrogen and phosphorous source or biosurfactant known to enhance the TPH bioavailability at the site, thereby increasing the bioremediation efciency [1,7]. Hence, the application of bioaugmentation and biostimulation is needed to improve bioremediation efciency which is affected by the concentration and component of hydrocarbon pollution [8,9]. The oil removal efciency in a bioremediation process is mainly determined by microbial activity, which can be monitored by using molecular tools or rapid assessment packages [10]. Molecular techniques for identifying hydrocarbon-degrading bacteria have been widely used in environmental studies, especially for microarrays that rapidly grow in number. Microarray biochips, a novel technology that has been applied in the environmental eld, could offer great accuracy and sensitivity for analysis of microbial diversity [10].

0304-3894/$ – see front matter. Crown Copyright 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2009.10.080

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The landfarming method used in the bioremediation of oil-contaminated soil is an effective, economic and promising technology for cleaning up hydrocarbon-contaminated soil [3]. Turning the soil regularly, provides oxygen transportation needed for biostimulation and increases the opportunity of contact by mixing microbes with oil-pollutants and water. Since microorganisms play a vital role in bioremediation process, they should be monitored with an accurate molecular biotechnology. Therefore, in this study an innovative bioprocess technology, Systematic Environmental Molecular Biotechnology (SEMBT), was developed for eld applications in treating petroleum oil-contaminated soil. This bioprocess included bioaugmentation and biostimulation using the landfarming procedure with the operational strategy of reseeding previous biopile soils in series. Molecular microarray biotechnology was used for monitoring during the bioremediation process. The integrated operational strategy of SEMBT improves the biodegradation of hydrocarbon as well as bioremediation efciency. 2. Materials and methods 2.1. KH-100 site description The KH-100 site is located near the harbor of Kaohsiung City, southern Taiwan. The site has a storage tank station that has had an oil leak from the past ten years, mainly diesel oil and fuel oil. The mean daily temperature of the operational time was 30 ± 10 C during bioremediation. Total average rainfall was 1800 mm in per year, and mostly concentrated from May to August. The annual mean air humidity was approximately 77%. 2.2. Soil biopile Experimental soil was collected from two sites and divided into two series of biopiles (S- and T-series) with different levels of TPH concentration containing diesel oil and fuel oil. Each series consisted of four small-scale biopiles (S0, S1, S2, S3 and T0, T1, T2, T3), three treated biopiles, and one control or untreated biopile (S0 and T0). The biopile size was approximately 4 m (L) × 3 m (W) × 2 m (H) with a soil volume of approximately 20 m3 . Experimental soil biopiles were rst analyzed and were then subjected to treatment using bioaugmentation with strains of Gordonia alkanivorans CC-JG39, Rhodococcus erythropolis CC-BC11, Acinetobacter junii CC-FH2 and Exiguobacterium aurantiacum CC-LSH4-1, as well as biostimulation with biosurfactant Rhamnolipid (RL) produced by Pseudomonas aeruginosa. The biopiles were sampled by using a composite sampling method. Measurement of soil moisture (%) and pH followed the procedures of soil analysis [11]. 2.3. Microbial assay A bioassay was carried out by using the total plate count as the quantitative estimation of enumeration, while a qualitative assay was accomplished by monitoring molecular DNA using microarray biochips with intergenic spacers (ITS) [12]. Enumeration of the bacterial plate count for soil samples followed the methods described in Gallego et al. [13]. The microarray biochips method consisted of the amplication by nested PCR of the ribosomal DNA intergenic spacers (ITS) regions of DNA extracted from contaminated soil. An oligonucleotide array was applied to directly detect bacteria in diesel and fuel oil-contaminated soil. 2.4. Total petroleum hydrocarbon analysis Total petroleum hydrocarbons (TPH) were extracted from the soil samples by using dichloromethane following the procedure recommended in US EPA test methods 3550B [11]. The organic

phase was passed through a cartridge lled with anhydrous sodium sulfate (Na2 SO4 , Sigma) to remove residual water and concentrated to near-dryness under a vacuum. The concentrate was re-dissolved with 2 ml dichloromethane and then concentrated to 1 ml by a N2 purge. The samples were quantied by using a gas chromatograph with an Agilent DB-1 fused silica capillary column (type RTX-5; 30 m long, I.D. 0.53 mm, D.F. 1.5 m; Restek, Bellefonte, USA) and ame ionization detector (GC-FID, PerkinElmer GC model no. 8310) as described by Mohn and Stewart [14].

2.5. Biogas analysis Biogases were measured by using a gas chromatograph (model GC-8A, Shimadzu, Japan) equipped with a stainless steel column (3 m × 1/8 in. I.D.; stationary phase: Carbosieve SII) and a thermal conductivity detector (TCD). Gases were sampled by using 1 l collection bag (CAT#232-01, SKC) per week before turning over the biopiles periodically. After the biopiles were turned over, a 2 m porous pile was inserted into the bottom of each biopile to collect soil biogas randomly. The analytical method is referred to the literature [15]. Soil temperature in the soil was measured simultaneously with microbial respiration.

2.6. Bioremediation process design The concept of the proposed bioremediation process was based on the combination of bioaugmentation and biostimulation with operational strategy using a landfarming procedure by reseeding previous 4 m3 biopile soil input biopiles in the beginning to enhance the increase in bacterial population. Our experiment was conducted in the biopile, which is 1.8–2.0 m high at the center, by using landfarming strategy with a plough machine per week. The biopile soil was periodically turned over with approximately volume of 0.5 m3 by landfarming. The bacterial community was monitored by a microarray biochip during the operational period.

2.7. Statistical analysis An analysis of variance (ANOVA) was performed to test the difference of initial and nal TPH concentrations between the treated experiment biopiles and untreated control biopiles.

3. Results and discussion 3.1. Characterization of contaminated soil The characterization of soil is presented in Table 1. Two series of biopiles (S0, S1, S2, S3 and T0, T1, T2, T3) had different TPH levels, in which the THP concentrations in the S-series biopiles were half of that in the T-series biopiles. TPHC10–C28 and TPHC10–C40 are regulated under the Taiwan EPA guideline. According to the carbon number of hydrocarbons, the components of TPHC10–C40 basically can be divided into low molecular weight as diesel oil (TPHC10–C28 ) and high molecular weight as heavy oil (TPHC28–C40 ), the data of TPHC28–C40 can be approximately estimated by subtracting the concentration. The concentrations of diesel oil were similar in both the S- and T-series biopiles. Therefore, there were more fractions of high-molecular-weight heavy oil in the T-series biopiles. The microbial populations in both series of biopiles were about 105 CFU/g dry soil and the microbial diversities were similar. Among them, Pseudomonas putida only appeared in the T-series biopiles. Many of these are well known to be efcient fuel oil or diesel-degraders [4,6,16,17].

T.-C. Lin et al. / Journal of Hazardous Materials 176 (2010) 27–34 Table 1 Characterization of contaminated soils. Parameters TPHC10–C28 (mg/kg) TPHC10–C40 (mg/kg) Soil texture Total N (%) Total P (%) Total organic matter (%) Total organic carbon (%) EC (dS/m) pH Total plate count (CFU/g dry soil) Bacterial diversity S-series soil T-series soil

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1020–2200 1800–2790 2200–4260 5850–7580 Sandy Loamy sandy 0.030 ± 0.002 0.052 ± 0.005 0.046 ± 0.004 0.049 ± 0.007 1.9 ± 0.5 2.1 ± 0.4 1.1 ± 0.3 1.3 ± 0.2 0.54 ± 0.04 1.4 ± 0.3 7.0 ± 0.3 7.2 ± 0.2 5 (3.3–6.7) × 105 (2.2–6.3) × 10 Acinetobactor junii, Gordonia alkanivorans, Rhodococcu Acinetobactor junii, Gordonia alkanivorans, Rhodococcu erythropolis, Acinetobacter sp., Gordonia desulfuricans, erythropolis, Acinetobacter sp., Gordonia desulfuricans, Pseudomonas sp., Pseudomonas aeruginosa, Ralstonia pickettiPseudomonas sp., Pseudomonas aeruginosa, Pseudomonas pudita, Ralstonia picketti

3.2. TPH biodegradation There are some differences between S- and T-series biopiles in the biodegradation curves of TPH including TPHC10–C28 and TPHC10–C40 as shown in Figs. 1 and 2. There seems to be two different biodegradation mechanisms that might involve the concentration and components of TPH [6,18]. In the S-series biopiles, two distinct phases are present in the bioremediation process, whereas a directly decreasing trend is present in the T-series biopiles. For both diesel and fuel oil, the biodegradation curves of TPH in the Sseries biopiles rapidly decrease before 60 days in the rst phase of bioremediation followed by a slow decrease phase, which remained stable from then on up to 240 days in the second phase of bioremediation. As shown in Fig. 1, the rst phase occurred between days 0 and 60, and after day 60 the second phase was seen. There

were two different degradation efciencies in both the S1 and S2 biopiles. The degradation efciency in the rst phase was higher and the degradation curve in second phase became at after day 60. This is due to low-molecular-weight diesel oil being easily biodegraded in the rst phase, whereas high-molecular-weight heavy oil was difcult to biodegrade in the second phase. The percentages of diesel in the S-series biopiles were higher than those in the T-series biopiles, leading to fast biodegradation in the rst phase of bioremediation. Due to an initially low TPH concentration in the S3 biopile, the biodegradation curve of TPH showed a directly decreasing trend similar to those of the T-series biopiles, in which the percentages of diesel were relatively low. Therefore, it is reasonable to assume that the biodegradation time and degree were effected by the fraction of TPH components and concentration [6]. Two phases of biodegradation efciencies occurred in S-series biopiles which contain high fraction of diesel, whereas only single phase occurred

Fig. 1. The biodegradation curves of (A) TPHC10–C40 and (B) TPHC10–C28 in the S-series biopiles.

Fig. 2. The biodegradation curves of (A) TPHC10–C40 and (B) TPHC10–C28 in the T-series biopiles.

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Table 2 Proles of TPH removal in the S- and T-series biopiles on day 28 in the rst phase of bioremediation. Biopile Day 0 Day 28 Removal Removal % 1120 2080 2700 780 960 2020 2350 930 920 1060 1510 420 510 990 1130 380 30 58 63 35 15 27 32 16 42 60 70 41 20 35 44 21 Removal rate mg/kg dry soil-day 40 74 96 28 34 72 84 33 33 38 54 15 18 35 40 14

mg/kg dry soil TPHC10–C40 S0 S1 S2 S3 T0 T1 T2 T3 TPHC10–C28 S0 S1 S2 S3 T0 T1 T2 T3 3690 3560 4260 2200 6310 7580 7380 5850 2200 1780 2150 1020 2520 2790 2550 1800 2570 1480 1560 1420 5350 5560 5030 4920 1280 720 640 600 2010 1800 1420 1420

in the T-series biopile with low fraction of diesel. The TPH biodegradation of two phases in our experiments coincides with the level of the TPH concentration, as reported by Thomassin-Lacroix [6]. As shown in Table 2, during the rst 28 days when about 60% of the total amount of TPHC10–C40 was removed, the TPHC10–C40 removal rates in the S1 and S2 biopiles were approximately 74 and 96 mg TPH/kg of dry soil per day, respectively. In contrast, the TPHC10–C40 removal rate of the S0 control biopile was approximately 40 mg TPH/kg of dry soil per day during the rst 28 days when approximately 30% of the total amount of TPH was removed. The results show that bioaugmentation and biostimulation with reseeding strategy performed well in the rst month. The total amounts of TPHC10–C40 removed (%) in the S-series biopiles were about twice compared with those in the T-series biopiles, while the TPH removal rates were similar. This shows that landfarming technology performed more efciently in the S-series biopiles with a high fraction of diesel, than in the T-series biopile with a low fraction of diesel. The TPHC10–C40 removal rate of S3 biopiles was approximately 28 mg TPH/kg of dry soil per day during the rst 28 days, giving a 35% removal of TPH. Although the TPHC10–C40 removal rate in biopile S3 was less than that in the biopile S0, the TPHC10–C40 removals (%) of biopile S3 was higher than that in the biopile S0 during the 28 days. This might be due to the low biodegradation and high fraction of high-molecular-weight heavy oil in biopile S3 resisting to microbial attack. Consequently, the biodegradation curve of TPH shows a slowly decreasing trend as shown in Fig. 1 [19]. Due to both limited factors of the initial concentration and high fraction of high-molecular-weight heavy oil, the degradation efciency of biopile S3 was found to be a little better than that of biopile S0. When the TPHC10–C40 peak of S3 biopile at initial (no. S30727) and the 62th (no. S30927) day of bioremediation were compared, we observed that the diesel was biodegraded while the heavy oil was not. As shown in Fig. 3(B), the TPHC28–C40 was hardly degradable and the biodegrading curve in the second phase of S-series became at after 62 days (Fig. 1). A possible explanation for the phenomenon is the inability of inoculation to degrade the particular hydrocarbons present in the contaminated soil such as an unresolved complex mixture (UCM) [20]. Another reason is the inability of inoculation to attack the pollutant adsorption on the soil, because hydrocarbons bind strongly to humic substances and to clay minerals [21,22]. Therefore, the degradation efciencies of biopiles S1 and S2 were better than that in the S3 biopile or in con-

Fig. 3. Comparison of the TPH chromatogram showing TPHC10–C40 portions in S3 biopile at (A) 0th day (no. S30727) and (B) 62th day (no. S30927). The TPHC10–C28 peaks refer to low-molecular-weight hydrocarbons and the TPHC28–C40 peaks refer to high-molecular-weight hydrocarbons.

trol biopile (S0). Hence, landfarming technology using the strategy with reseeding process can shorten treatment time and improve the bioremediation efciency. ANOVA refers to an analysis of variance, which is frequently used in statistics. The removal rates were higher in the treated biopiles (S1 and S2) than the control S0 in the rst phase of bioremediation at 5% level of signicance. There were signicant differences in the nal TPHC10–C40 concentrations between treated experiment biopiles and untreated control biopiles in both series of biopiles (ANOVA with = 0.05). There was a signicant effect of bioaugmentation and biostimulation in the rst phase of bioremediation. Although nal TPHC10–C40 concentrations of all treated S-series biopiles were in the range of 200–600 mg/kg of dry soil at the end of the treatment period (240 days), the level of TPHC10–C40 in the treated S-series biopiles reduced below the legal TPH concentration (1000 mg/kg dry soil) regulated by the Taiwan government after 100 days. More than 150 days were needed for the untreated control of the S-series biopiles to reduce to less than 1000 mg/kg dry soil. This shows that SEMBT can shorten treatment time by half (Fig. 1). ANOVA was also applied to test variability among the all biopiles for TPHC10–C40 biodegradation. Results of this statistical analysis indicated that there were signicant differences in the nal TPHC10–C40 concentrations between treated experiment biopiles and untreated control biopiles in both series of biopiles ( = 0.05) [6]. Hence, the achieved end point TPH in S-series biopiles of this experiment was within limitations of Taiwan EPA regulation. 3.3. Microbial investigations on bioremediation biopile 3.3.1. Enumeration of microbial population As shown in Table 1, the populations were in the range of 2.2–6.7 × 105 CFU/g soil at the beginning of bioremediation in both the series of bipoles. The supplement of bioaugmentation and biostimulation at the rst phase of bioremediation resulted in a higher count (106 –107 CFU/g soil) in the experiment group, compared to that within the control group (105 CFU/g soil), as shown in Fig. 4.

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of 4 m3 soil from biopile S1 in the beginning. This phenomenon also occurred in the T-series biopiles T1 and T2. Therefore, the strategy with reseeding process performed well due to increasing microbial population. Hence, bioaugmentation and biostimulation increased the microbial population in the beginning, which resulted in rapid TPH biodegradation in the rst phase of bioremediation [23,24]. With bioaugmentation and biostimulation, the population count was above 5.0 × 106 CFU/g dry soil in the rst phase of bioremediation. In the second phase, however, it decreased to below 5.0 × 106 CFU/g dry soil due to the easily biodegradable diesel consumed and left the difcult biodegradable heavy oil. Microbial inoculation was deemed necessary since suitable HC-degrading bacteria were not found in sufcient numbers in the on-site samples prior to landfarming [6]. The TPH biodegradation was slower in the T-series because the microbial population could not utilize the lower quantity of diesel (32–37% of TPH) as a potential nutrient source; the S-series microbial population successfully utilized the more abundant diesel (46–60% of TPH) as a potential nutrient source. In the second phase, the TPH biodegradations were slower in all S-series biopiles. There might be a certain threshold for microbial populations to utilize TPHC28–C40 [25]. For example, isoprenoids pristane, phytane, and cyclo-alkanes like resin composed of UCM ware partially or completely resistant to microbial attack [19,26].

Fig. 4. Number proles of microbial population in (A) S-series and (B) T-series soil during bioremediation.

In both series of biopiles, the initial population counts were about 2.2 × 105 CFU/g dry soil then increased to 6.3 × 107 CFU/g dry soil when supplemented with bioaugmentation and biostimulation at the rst phase of bioremediation. The bacterial number in the S2 biopile was higher than that in the S1 biopile due to the reseeding approach at the beginning; the similar results were also obtained in T1 and T2 biopiles. The growth proles of the microbial population reected the TPH biodegradation compared to the control biopiles, as shown especially in S1, S2, T1, and T2 biopiles. Microbial population seems to be lower and constant in the S-series biopiles after 150 days of bioremediation, which is owing to the limited available carbon source in the soil. The uctuation of microbial population was small after 150 days of bioremediation. Similar changes in microbial population were found in T-series biopile. The T0 biopile (control) showed the lowest bacterial number when compared with other biopiles, which corresponded to the TPH biodegradation efciency. Thus, an immediate increase in the population density of indigenous microbes could ensure rapid degradation of the pollutants [23]. Hence, the best bioaugmentation performance can be achieved by using pre-selected bacteria that increase in abundance. With the increase of a specic microbial community and biosurfactant addition, this approach could improve TPH biodegradation and reduce the cleanup time substantially. In the statistical analysis, the bacterial numbers in biopile S1 and S2 were one order of magnitude (P < 0.05) higher than that in biopile S0 after supplementation with bioaugmentation and biostimulation at the beginning. The microbial population of biopile S2 increased half an order of magnitude (P < 0.05) higher than that of biopile S1 mainly due to the reseeding

3.3.2. Microbial community analysis with microarray identication The microbial community was monitored by a microarray biochip and revealed the abundance of microbial diversity in the primitive soil in both series of biopiles. As shown in Table 3, ve indigenous bacteria (i.e. Acinetobactor sp., G. desulfuricans, Pseudomonas sp., P. aeruginosa, and R. Picketti) and four augmented ones (i.e. A. junii, G. alkanivorans, and R. erythropolis) were initially detected in both series of biopiles. Microbial diversity was high during the rst phase of bioremediation and microbial growth was prosperous due to bioaugmentation and biostimulation with the reseeding strategy. Therefore, TPH was rapidly removed by bacteria in the rst phase of bioremediation. During the rst 4 months of bioremediation, the ve indigenous bacteria and four augmented bacteria monitored by the microarray biochip were still detected to a larger extent in the S-series biopiles, lasting to the ending of bioremediation, with the exception of S0 and S3 biopiles, in which E. aurantiacum and G. desulfuricans disappeared at the end. The strain E. aurantiacum being rst screened from oil-contaminated soil, is here reported as a hydrocarbon assimilator capable of degrading heavy oil hydrocarbons, and disappeared in the S-series biopiles at last might be due to less fraction of heavy oil. Both bacteria with oil degrading activities disappeared at the nal stage and this might affect the efciency of bioremediation. As carbon is the key factor governing microbial growth in soil and produces functional diversity of soil microbes [27]. We found both bacteria with oil degrading activities disappeared at the nal stage when the available carbon has depleted and this might affect the efciency of bioremediation (Fig. 1). Acinetobacter sp. and P. aeruginosa were the predominant groups in indigenous consortia, while the augmented consortia were G. alkanivorans and R. erythropolis in the S-series of biopiles during bioremediation. Six indigenous bacteria (i.e. Acinetobacter sp., G. desulfaricans, Pseudomonas sp., P. aeruginosa, P. pudita and R. picketti) and four augmented bacteria (i.e. A. junii, G. alkanivorans, R. erythropolis and E. aurantiacum) monitored by the mircroarray biochip were found in the T-series biopile on sites (Table 3). Most of them have been reported as hydrocarbon degraders [28]. There were some differences between the S- and T-series biopiles. For instance, P. putida as a PAH-degrading bacterium [29], was a distinct species found

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Table 3 Microbial diversity detected by microarray in both series of biopiles during bioremediation. Bacteria Day 0 S0/T00 Augmented bacteria A. junii E. aurantiacum G. alkanivorans R. erythropolis Indigenous bacteria Acinetobacter sp. G. desulfuricans Pseudomonas sp. P. aeruginosa R. picketti P. pudita +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ /+ S1/T1 +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ /+ S2/T2 +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ /+ S3/T3 +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ /+ Day 120 S0/T00 +/+ /+ +/+ +/+ +/+ / +/+ +/+ /+ /+ S1/T1 +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ /+ S2/T2 +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ /+ S3/T3 +/+ /+ +/+ +/+ +/+ /+ +/+ +/+ +/+ /+ Day 240 S0/T0 +/ / +/+ +/+ +/+ / /+ +/+ / / S1/T1 +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ /+ S2/T2 +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ /+ S3/T3 +/+ /+ +/+ +/+ +/+ /+ / +/+ +/ /+

Note: +, Detectable; , non-detectable.

in the T-series biopiles. Hence, we expected the biodegradation curves of TPH to be different for the biopiles since the fractions of TPH component and the microbial communities were different. The predominant groups of indigenous and augmented consortia in the T-series biopiles were the same as those in the S-series biopiles during bioremediation [30]. Among them, bacteria from the genus Acinetobacter are one of the most active strains in the assimilation of saturates and aromatics [19]. Bacteria from the genus Gordonia with the dioxygenase gene have been reported to degrade polyaromatic hydrocarbon compounds [29]. Bacteria of the genus Rhodococcus have been reported to assimilate n-alkanes and more than 90% of the branched alkanes [19]. These bacterial strains represent hydrocarbon (HC)-degrading genera [19]. 3.4. Microbial respiration The microbial respiration as inuenced by the microbial activity reects directly the microbial population and indirectly the biodegradation of TPH [30]. The observed oxygen concentration rst decreased with time as oxygen was consumed by microbial respiration, while the carbon dioxide concentration increased with time as carbon dioxide was produced by the microbial respiration in soil (Fig. 5). The biogas analysis shows only a small different trend in both S- and T-series of biopiles. On day 60, the S-series biopiles were relatively higher in CO2 concentration and lower in O2 concentration, and this phenomenon was similar to that on day 90 in the T-series biopiles. These results reect directly the microbial population and indirectly the biodegradation of TPH (Figs. 1, 2 and 4). The biodegradation model consisting of two phases in S1 and S2 biopiles leads to the highest CO2 production at day 60, and it was proposed that higher fractions of diesel might be present in the pollutants in these two biopiles. Another biodegradation model consisting of only one phase was seen in S0 and S3 biopiles, which leads to the delay of the highest CO2 production at day 90. This phenomenon can also be found in the CO2 production patterns in T-series biopiles, owing to the lower fractions of diesel in the pollutants in these biopiles. The entrapped air was utilized for oxygen uptake and CO2 release due to microbial respiration during the period of landfarming. Landfarming method provided aerobic conditions to microbial consortium for TPH degradation. The degradation of TPH involves the oxidation of hydrocarbon by oxygenase, for which oxygen is required [31]. Therefore, the degradation of TPH was directly related to the respiration of microbial populations in the soil [32]. Only one phase was observed in the T-series during bioremediation, which might be due to a mass transfer limitation of the oxygen diffusion [33]. The effect of oxygen limitation on the microbial

Fig. 5. Biogas proles of microbial respiration in (A) S-series and (B) T-series soil during bioremediation. Symbol: (—), Carbon dioxide production; (- - -), oxygen consumption.

activity led to a slow biodegradation of TPH during the bioremediation. This indicates that the activities of bacteria were hindered in the T-series biopiles. At the middle stage of the landfarming process, when most of the easily biodegradable hydrocarbons present in the soil have been degraded by the microorganisms existing in the soil, the ratio of CO2 concentration to O2 concentration gradually decreased and then leveled off. This implies that the efciency of the TPH minimization involves the component of TPH, and the microbial respiration reects the bioremediation efciency. Microorganisms prefer the more easily available component of TPH over the less easily degradable heavy oil [34]. Some differences

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were observed between the S- and T-series biopiles during the initial stages of the TPH biodegradation. In the S-series biopiles, about 60 mg O2 /kg dry soil consumed and about 60 mg CO2 /kg dry soil produced were responsible for about 80% TPHC10–C28 removal during the rst 60 days of bioremediation. In the T-series biopiles, about 50 mg O2 /kg dry soil consumed and about 50 mg CO2 /kg dry soil produced accounted for about 60% TPHC10–C28 removal during the rst 90 days of bioremediation. Both the O2 consumption and CO2 production in the T-series biopiles were less than those in the Sseries biopiles. This indicates that the biogas assay directly reects the microbial activities in soil in accordance to the TPH degradation. Lee et al. [35] also monitored the O2 utilization and CO2 production pattern during biodegradation to measure the biodegradation rate of a diesel fuel in in situ bioremediation. It is essential to maintain an aerobic condition by optimizing the environmental condition for achieving improved results in biostimulation of TPHs in open eld experiments [5]. Addition of the microbial consortia can increase the degrading microorganisms present in the biopiles, which will provide a short-term benet. How to shorten the time for bioremediation is the main goal in such kinds of these experiments. By denition, bioaugmentation corresponds to an increase in the gene pool and genetic diversity of the site [36]. By using bioaugmentation strategy it was possible to reach better degradation when compared with natural attenuation or biostimulation during bioremediation process [23]. Although on some occasions it has been shown that only biostimulation works in the degradation of pollutants, the lack of microorganisms in the late phase (second phase) of bioremediation owing to unavailable or hard degradable hydrocarbons may lead long time to remediate the soil. Therefore bioaugmentation was needed when degrading microorganisms were in low number or diversity, or inadequate microbial populations were present in the oil-contaminated soils. 4. Conclusions This study presented an innovative bioremediation method, the Systematic Environmental Molecular Bioremediation Technology (SEMBT), for biopiles by combining bioaugmentation and biostimulation with the reseeding strategy. The diesel-contamination was efciently removed to about 70% by bioremediation of biopiles over a period of 28 days. The degradation and removal rates of TPH in the S-series biopiles were 10% higher than those in the T-series biopiles. During the initial stages of bioremediation, applied bioaugmentation and biostimulation increased the TPHC10–C40 degradation removal by 16% on the average over the control. Monitoring the microbial population quantitatively and integrating microarray identication qualitatively during the bioremediation process proved to be benecial. Biogases assay indicates that biodegradation of TPH is directly related to the microbial respiration. The microbial population size of 106 CFU/g soil with abundance of 8 different genera improved the TPH degradation in the experimental and control groups. Such microbial consortia with high and constant biodegradation ability can be used for industrial applications of bioremediation. Hence, the SEMBT shows potential applications in ex situ bioremediation. Acknowledgements This work was supported by grants from the Department of Industry Technology, Ministry of Economic Affairs, R.O.C., under the contract no. 97-EC-17-A10-S1-0013, and by grants from the Kaohsiung Renery plant, Oil-renery Division, China Petroleum Corporation, R.O.C., and the Sustainable Environment Research Center, National Cheng Kung University, Taiwan.

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