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Biodegradation of Crude Oil in Contaminated Soils by Free and Immobilized Microorganisms
Pedosphere 22(5): 717–725, 2012 ISSN 1002-0160/CN 32-1315/P c 2012 Soil Science Society of China Published by Elsevier B.V. and Science Press
Biodegradation of Crude Oil in Contaminated Soils by Free and Immobilized Microorganisms∗1 WANG Zhen-Yu1 , XU Ying1,2 , WANG Hao-Yun3 , ZHAO Jian1,4 , GAO Dong-Mei1 , LI Feng-Min1 and B. XING1,4,∗2 1 College
of Environmental Science and Engineering, Ocean University of China, Qingdao 266100 (China) of Animal Science and Veterinary Medicine, Qingdao Agricultural University, Qingdao 266109 (China) 3 Chemical and Mineral Metal Material Testing Center, Tianjin Entry-Exit Inspection and Quarantine Bureau, Tianjin 300456 (China) 4 Department of Plant, Soil and Insect Sciences, University of Massachusetts, Amherst MA 01003 (USA) 2 College
(Received October 2, 2011; revised January 16, 2012)
ABSTRACT The efficiencies of free and immobilized bacterial cultures of petroleum hydrocarbon degraders were evaluated and compared in this study. Hydrocarbon-degrading microbial communities with high tolerance to and high degrading ability of crude oil were obtained from the soil contaminated with crude oil in the Yellow River Delta. Then, the microbial cells were immobilized in sodium alginate (SA) beads and sodium alginate-diatomite (SAD) beads. The biodegradation of crude oil in soil by immobilized cells was compared with that by free cells at three inoculation concentrations, 1 × 104 colony forming units (cfu) kg−1 (low concentration, L), 5 × 104 cfu kg−1 (medium concentration, M), and 1 × 105 cfu kg−1 (high concentration, H). At 20 d after inoculation, the maximum degradation rate in the immobilized systems reached 29.8% (SAD-M), significantly higher (P < 0.05) than that of the free cells (21.1%), and the SAD beads showed greater degradation than the SA beads. Moreover, both microbial populations and total microbial activity reached significantly higher level (P < 0.05) in the immobilized systems than free cell systems at a same initial inoculation amount. The scanning electronic microscope (SEM) images also confirmed the advantages of the immobilized microstructure of SAD beads. The enhanced degradation and bacterial growth in the SAD beads indicated the high potential of SAD beads as an effective option for bioremediation of crude oil-contaminated soils in the Yellow River Delta. Key Words:
bacterial growth, degradation rate, hydrocarbon degraders, immobilized cells, sodium alginate-diatomite beads
Citation: Wang, Z. Y., Xu, Y., Wang, H. Y., Zhao, J., Gao, D. M., Li, F. M. and Xing, B. 2012. Biodegradation of crude oil in contaminated soils by free and immobilized microorganisms. Pedosphere. 22(5): 717–725.
INTRODUCTION Persistent crude oil exploration in the Yellow River Delta poses a serious threat to the environment, ecosystems, and human health due to enhanced soil contamination (Peng et al., 2009; Wang et al., 2011). Crude oil is composed of complex hydrocarbons with low bioavailability and is persistent in soil. Bioremediation to clean up petroleum hydrocarboncontaminated soil was considered to be beneficial over physical and chemical methods because of its costeffectiveness along with low environmental impact (Margesin and Schinner, 2001; Wang et al., 2008). However, the success of most bioaugmentation experiments depends on the survival of the inoculated cells (J´ez´equel and Lebeau, 2008). The inability of intro∗1 Supported
duced non-indigenous microorganisms to compete with the indigenous microflora and predators severely limits biodegradation (Mallory et al., 1983; J´ez´equel and Lebeau, 2008). Thus, the use of indigenous microorganisms to degrade petroleum hydrocarbons is important and has been well documented in both terrestrial and aquatic ecosystems (Margesin and Schinner, 2001; Norman et al., 2004; Vaccar et al., 2005; Wang et al., 2008). Immobilization of cells protects the cells from predation and natural competition with the soil microflora and is known to be more effective in shielding perturbations of environmental conditions such as pH and toxic compounds in comparison to suspension culture (J´ez´equel and Lebeau, 2008; Zhang et al., 2008). Immobilization has been achieved in a number of ways by
by the National Natural Science Foundation of China (No. 41073067), the Key Program of the Ministry of Education, China (No. 308016), and the National Major Special Technological Programme Concerning Water Pollution Control and Management of China (No. 2009ZX07010-008). ∗2 Corresponding author. E-mail: [email protected].
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physical adsorption, ion-coagulation, cross-linking, and/or entrapment methods in a variety of matrices (Su et al., 2006), such as sodium alginate (Bazot and Lebeau, 2009; Ya˜ nez-Ocampo et al., 2009), polyvinyl alcohol (PVA) (Cunninghama et al., 2004), activated carbon, zeolite (Liang et al., 2009), and vermiculite (Su et al., 2006). Among all the matrices, alginate extracted from macro-algae, a natural polymer, is most popularly used for the preparation of gel beads with low cost and mild conditions of immobilization (Mofidi et al., 2000; Massalha et al., 2007), along with its easy degradability and low-toxicity. Therefore, alginate is suitable to be used as an immobilization matrix for bioremediation of contaminated soils. The microorganisms immobilized in alginate beads have been used in hostile environments and the beads provide nutrients and appropriate conditions, such as facilitating the transfer of oxygen which is crucial for rapid hydrocarbon mineralization, to allow rapid bioremediation in contaminated systems (Cunninghama et al., 2004; Sathesh Prabua and Thatheyus, 2007; Vancov et al., 2007; Bazot and Lebeau, 2009; Ya˜ nezOcampo et al., 2009). The slow release of inoculants from the alginate beads also favored their proficiency in contaminant removal (Vancov et al., 2007; Bazot and Lebeau, 2009). However, the dense gel layers of the alginate beads hinder mass transfer of substrates, pollutants, and degraded products, which restricts their potential application in bioremediation (Zhang et al., 2008). The incorporation of some adsorbents into alginate beads facilitated the transport of pollutants towards both the surface and interior regions (Zhang et al., 2008). Diatomite, mainly consisting of SiO2 (80%–90%), formed from dead diatoms in seas and lakes has been approved as a food-grade material by the U.S. Food and Drug Administration (FDA) (Xiong and Peng, 2008). Diatomite with high porosity (80%–85% voids) (Xiong and Peng, 2008) can enhance the porosity and improve mass transfer property of alginate beads if incorporated. Moreover, the diatomite released into the soil after alginate degradation may improve the soil quality. Hence, the immobilization of functional microorganisms using alginate and diatomite together is a good prospective method to bioremediate contaminated soil systems. There are few data on the advantages of such immobilization for crude oilcontaminated soil remediation by microorganisms. Especially, in the oilfields of the Yellow River Delta, the area of contaminated soil is large. The immobilization matrix used in remediation should be degraded naturally and non-toxic.
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In the present study, sodium alginate and diatomite were selected because of their natural origin and many advantages. The aim of the present study was to compare the efficiency of free and immobilized inoculants of the indigenous petroleum hydrocarbon-degrading bacteria in removing crude oil from a soil of the Yellow River Delta. We studied the influence of the inoculum amount and the amendment of diatomite to sodium alginate beads on the contamination removal, microbial activity, and degrading microorganism colonization. The preservation condition of immobilized beads was also analyzed. MATERIALS AND METHODS Contaminated soil samples The contaminated soil was collected from the vicinity of an oil well in the Yellow River Delta, China. The soil was silty clay, and the concentration of petroleum hydrocarbon of the soil was about 10 g kg−1 . The basic physical and chemical properties of the soil (0–20 cm) were as follows: pH of 8.17, organic matter of 4.9 g kg−1 , salinity of 14.5 g kg−1 NaCl, total N of 25 g kg−1 , and total P of 0.4 g kg−1 . Petroleum hydrocarbon-degrading microorganisms The enrichment and isolation of petroleum hydrocarbon-degrading microorganisms were conducted immediately after the soil was collected. Five grams of the soil samples were inoculated in a 150-mL sterilized mineral salt medium (MSM) containing 1 g L−1 K2 HPO4 , 1 g L−1 KH2 PO4 , 1 g L−1 (NH4 )2 SO4 , 15 g L−1 NaCl, 0.2 g L−1 MgSO4 , 0.02 g L−1 CaCl2 and a trace of FeCl3 . After incubation and serial subculturing (eight transfers), the mixed hydrocarbon-degrading microbial communities were obtained after 80 d of enrichment. For isolation, a combination of serial dilution and steak plate method was used to obtain single colonies of pure cultures. The isolated dominant bacterial cultures were identified by polymerase chain reaction (PCR) amplification and sequencing of the chromosomal DNA, which was completed by the Shanghai GeneCore BioTechnologies Co., Ltd. Preparation of immobilized bacteria Cell suspension. The mixed petroleum hydrocarbon-degrading microorganisms grew in the MSM containing 20 mg mL−1 crude oil as a sole carbon source, and inoculated for 60 h (exponent growth period). Then, the cells were centrifuged and the supernatant was discarded. The pellet was washed and suspended in solution of 8.5 g L−1 NaCl.
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Cell immobilization. The immobilization of petroleum hydrocarbon-degrading microorganisms was conducted using sodium alginate (SA) beads and sodium alginate-diatomite (SAD) beads. The immobilization procedure was adapted from Sathesh Prabua and Thatheyus (2007) and Ya˜ nez-Ocampo et al. (2009), with 60 g L−1 sodium alginate solution or the mixed solution of 60 g L−1 sodium alginate and 20 g L−1 diatomite. Degradation experiment To assess the efficiency of mixed petroleum hydrocarbon-degrading microbial communities to degrade crude oil, 10 mL inoculum (optical density = 0.7 at 600 nm) was added into 150 mL of MSM with the concentrations of crude oil at 10, 20, and 30 mg mL−1 . The extent of degradation of crude oil was determined by gas chromatography (GC) (Chung and King, 2001). Cells immobilized in SA and SAD beads and free cells (F) were respectively inoculated in pots (30 cm diameter and 30 cm depth) containing 10 kg of contaminated soil. Pots without any added bacteria were used as the control. For each pot with inoculated bacteria, 3 cell concentration gradients were set: 1 × 104 colony forming units (cfu) kg−1 (low concentration, L), 5 × 104 cfu kg−1 (medium concentration, M) and 1 × 105 cfu kg−1 (high concentration, H). To simplify the expression, SA, SAD, and free beads with these 3 concentration levels were defined as SA-L, SA-M and SA-H; SAD-L, SAD-M and SAD-H; and Free-L, FreeM and Free-H, respectively. Nutrient solution was supplemented to each pot, up to a C:N:P ratio of 100:10:1. The experiment was carried out outside for 70 d, and the moisture content was kept at 60% water-holding capacity (WHC) according to a pre-experiment (data not shown). During the 70 d of experiment, samples were taken 7 times at 0, 10, 20, 30, 40, 50, and 70 d from each treatment for analyses of petroleum hydrocarbon concentration, microbial activity, and the population of degrading microorganisms. Sample analyses Physicochemical characteristics of the soil. pH (in 0.01 mol L−1 CaCl2 ) was measured at a 1:2.5 soil to solution ratio. Organic matter with the Walkley and Black dichromate oxidation method, total nitrogen with the Kjeldahl method, and total phosphorus by a spectrophotometer after NaOH digestion were analyzed according to Bao (2000). Electrical conductivity (EC) was determined using a 1:5 soil to water suspen-
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sion, which was converted to EC values in saturation extract (ECe ) and expressed as salinity (Muhammad et al., 2008). Soil moisture content was determined gravimetrically by drying at 105 ◦ C for 24 h. The waterholding capacity (WHC) was determined by soaking the soil samples in water for 2 h followed by draining for 2 h (Vanhala, 2002). Oil concentration of the soil. The extraction and determination of crude oil were carried out by the method from EPA3550B (US EPA, 1996) and GB17378.5-1998 (Ocean Monitoring Regulation Committee, 1998). The concentration of oil was determined using GC (GC-2010, Shimadzu Co., Japan). Bacterial population and activity. The hydrocarbon-degrading microorganisms was enumerated by the most probable number (MPN) procedure of Wrenn and Venosa (1996) and Cunninghama et al. (2004), with the addition of diesel oil as the soil carbon source. Total microbial activity was determined with fluorescein diacetate (FDA) method (Green et al., 2006; Wang et al., 2010). The FDA hydrolytic activity was measured from the absorbance of supernatant at 490 nm (A490 ) using a spectrophotometer. Cell growth analysis and immobilized bead preservation The cell concentration was determined by measuring optical density (OD) at 600 nm using a spectrophotometer and the corresponding cell number was measured with an epifluorescence microscope (Wang et al., 2008). Cell count immobilized in each bead was estimated with MSM agar plate (Ascon-Cabrera and Lebeault, 1995). The cells immobilized in SAD beads were observed by scanning electronic microscope (SEM, S-4800, Hitachi, Japan). The beads were fixed in 2.5% glutaraldehyde according to the method of Su et al. (2006). The method of immobilized bead preservation was adopted from Trevors et al. (1993) and Vancov et al. (2007). Glycerol (200 mL L−1 ), trehalose (200 g L−1 ) and skim milk (200 g L−1 ) were supplemented to the mixtures of sodium alginate (60 g L−1 ) and diatomite (20 g L−1 ) beads. Fresh beads were preserved by freezing-drying, air-drying, and freezing. The cell viability was also determined through dissolving the beads in 10 mL of sterile EDTA solution (0.1 mg L−1 ) (Br´eant et al., 2002; Bazot and Lebeau, 2009). Statistical analysis Every test was repeated three times. Statistical analyses were performed with SPSS for Windows 12.0. The significance of the various parameters was tested
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by one-way analysis of variance (ANOVA) using the least significance difference (LSD) test. RESULTS AND DISCUSSION Crude oil degradation potentials of hydrocarbon-degrading microorganisms The efficiency of the indigenous microbial community to transform crude oil was determined at 7 and 14 d of incubation (Figs. 1 and 2). The cultures with 10 and 20 mg mL−1 of crude oil had higher removal ratios (90% and 88%) and cell numbers (4.2 × 108 and 6.3 × 108 ) at 14 d than the culture with 30 mg mL−1 of crude oil. The increase in substrate concentration up to 30 mg mL−1 of crude oil showed moderately toxic to at least some microorganisms of the culture which had the microbial cell counts of 1.0 × 108 and 2.4 × 108 and the degradation percentages of crude oil of 28% and 50% after 7 and 14 d of incubation, respectively.
Fig. 2 Microbial cell counts in the biodegradation systems with the initial concentrations of crude oil of 10, 20, and 30 mg mL−1 at 7 and 14 d of incubation.
These indicated that the indigenous microbial community showed high tolerance to and transformation ability of crude oil (Vancov et al., 2007). Then, 3 dominant single colonies which could play a major role in biodegradation of crude oil were isolated and identified as Microbacterium foliorum, Gordonia alkanivorans, and Mesorhizobiu. Biodegradation of crude oil in soil by immobilized and free cells
Fig. 1 Degradation of crude oil by hydrocarbon-degrading microbial communities at 7 and 14 d of incubation with the initial concentrations of crude oil of 10, 20, and 30 mg mL−1 .
Crude oil was degraded more quickly by the immobilized cells (SAD and SA) than the free cells at the same inoculant concentrations (Fig. 3 a, b, c). When the inoculation density was 1 × 104 cfu kg−1 (low concentration, L) and 5 × 104 cfu kg−1 (medium concentration, M), the degradation rates of immobilized cells were significantly higher than those of the free cells. To simplify data presentation, the removal rates of crude
Fig. 3 Crude oil degradation by immobilized cells in sodium alginate-diatomite (SAD) and sodium alginate (SA) beads and free cells (F) at different initial inoculation amounts: 1 × 104 cfu kg−1 (low concentration, L) (a), 5 × 104 cfu kg−1 (medium concentration, M) (b), and 1 × 105 cfu kg−1 (high concentration, H) (c). The removal rate of crude oil = the removal rate of crude oil of soil with free or immobilized cells – the removal rate of crude oil of the control.
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oil were calculated as the degradation rate of the treatment samples minus that of the control. The concentration of crude oil in all treatments decreased quickly at the beginning of the incubation. At 20 d, the degradation rate was the maximum in the immobilized systems up to 29.8% (SAD-M) of crude oil in contrast to 21.2% by the free cells. At that time, the degradation rate of the SAD cultures was about 2 times higher than that of the free cell culture at the low inoculation density of 1 × 104 cfu kg−1 (L). The degradation rate decreased after 20 d, which was mainly due to efficient utilization of labile hydrocarbon sources by degrading microorganisms at the beginning, leading to the high percentage of removal at 20 d. Then, the remained recalcitrant hydrocarbons served as the carbon source, which slowed down the removal rate (Jørgensen et al., 2000). In addition, the expense of nutrients and products of toxic intermediates were also likely to inhibit the growth of degrading microorganisms, resulting in drop in the degradation percentage of crude oil in soil (Bento et al., 2005). Effect of SAD on biodegradation Alginate could increase bacterial survival in soil compared with free cells (Trevors et al., 1993; Cassidy et al., 1997). Previous researches have shown that immobilized cells in SA showed a faster removal rate of contaminants than free cells (Sathesh Prabua and Thatheyus, 2007; Vancov et al., 2007; Bazot and Lebeau, 2009; Ya˜ nez-Ocampo et al., 2009). This could be induced by complex factors, such as protection of carriers from deleterious effects (Su et al., 2006), high affinity between immobilization materials and substrates (Wilson and Bradley, 1996), and acceleration of oxygen transfer (Liang et al., 2009), which led to increment in cell count and availability of the substrates (Su et al., 2006). Our results showed that the SAD treatments presented greater degradation than the SA
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treatments. At 20 d, the highest removal reached 29.8% (SAD-M) and 25.6% (SA-M). At the end of experiment, the SAD treatments had the greatest degradation of 46.3% (SAD-M), 3.4% higher than the SA treatments (Fig. 3b). Diatomite incorporated into alginate beads could improve mass transfer property of the alginate beads (Xiong and Peng, 2008), which subsequently enhanced the removal rate of contaminants. In addition, when alginate was degraded, diatomite would effectively improve the pore structure of soil, promoting the aerobic biodegradation of petroleum hydrocarbons. The immobilization of cells in SAD beads was confirmed by scanning electron microscope (SEM) and the images are shown in Fig. 4. Cells were well distributed in the interior of the SAD beads. It can also be seen that the SAD beads had large porosity, which permitted excellent mass transport of oxygen, nutrients, and degradation substrates. It was reported that the dense gel layer structure in the SA beads hindered the efficient transfer of oxygen, which limited aerobic strains of degrading microorganism community (Zhang et al., 2008; Ya˜ nez-Ocampo et al., 2009). Our results showed that diatomite was an excellent amendment for alginate beads for improving porosity and mass transfer property and thus efficiently improving the growth of immobilized cells and the bioremediation Effect of initial inoculation concentration on biodegradation The initial inoculation amount on biodegradation was also investigated (Fig. 3a, c). The immobilized cells at the low inoculation concentration (1 × 104 cfu kg−1 ) had a higher degradation rate than the free cells at the high inoculation concentration (1 × 105 cfu kg−1 ). At 20 d, the degradation rates of SAD-L and F-H were 27.3% and 21.2%, respectively. However, the advantage of the immobilization treatments with the high
Fig. 4 Scanning electronic microscope (SEM) micrographs (× 10 000) of interior structure of sodium alginate-diatomite (SAD) beads without cells (a) and SAD beads with immobilized cells (b).
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inoculation concentration (1 × 105 cfu kg−1 ) was not significant (P < 0.05) (Fig. 3c). The rapid metabolization with a high concentration of hydrocarbondegrading microorganisms caused the limitation of oxygen and nutrients, which restricted the propagation size of microorganisms (Cunninghama et al., 2004; Chaˆıneau et al., 2005). Therefore, the inoculation concentration could affect the degradation rate, and the efficient level of inoculation concentration was selected at less than 5 × 104 cfu kg−1 of soil. Hydrocarbon-degrading microorganism populations and activity Large numbers of hydrocarbon degraders and active microbial communities were keys to successful bioremediation. The changes of microorganism numbers and activity in soil during degradation are shown
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in Figs. 5 and 6. The results showed that the free indigenous microbial cultures quickly adapted to the environment and presented a rapid multiplication and increase of total microbial activity. In the immobilized systems, inoculant formulation with SAD or SA beads showed similar growth kinetics of degrading microorganisms and total microbial activity. At the early stage, microbial growth and replication within the matrix may release some microorganisms to the surrounding environment (Su et al., 2006), resulting in low degrading-microorganism population and activity in soil. After 10 d, both microbial population and total microbial activity increased rapidly in the SAD and SA systems, resulting from both massive cell releases associating with bead degradation and their subsequent growth in the soil systems (Lebeau et al., 1997; Br´eant et al., 2002; Bazot and Lebeau, 2009). At approxima-
Fig. 5 Petroleum hydrocarbon-degrading microorganism populations in soil inoculated with immobilized cells in sodium alginatediatomite (SAD) beads and sodium alginate (SA) beads and with free cells (F) at different initial inoculation amounts of 1 × 104 cfu kg−1 (low concentration, L) (a) and 5 × 104 cfu kg−1 (medium concentration, M) (b), and 1 × 105 cfu kg−1 (high concentration, H) (c). The population of petroleum hydrocarbon-degrading microorganisms was the population of the soil sample with free or immobilized cells minus that of the control. MPN = most probable number.
Fig. 6 Microbial activity (expressed as fluorescein diacetate (FDA) hydrolyzed) in soil inoculated with immobilized cells in sodium alginate-diatomite (SAD) beads and sodium alginate (SA) beads and with free cells (F) at different initial inoculation amounts of 1 × 104 cfu kg−1 (low concentration, L) (a), 5 × 104 cfu kg−1 (medium concentration, M) (b), and 1 × 105 cfu kg−1 (high concentration, H) (c). The microbial activity in soil was the microbial activity of the soil samples with free or immobilized cells minus that of the control.
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tely 20 d, both microbial population and total microbial activity were significantly higher (P < 0.05) than those for the free cells at a same initial inoculation amount. The maximum degrading-microorganism numbers were 6.1 × 108 and 3.6 × 108 cfu g−1 for the SAD-M and SA-M treatments, respectively, in contrast to 1.8 × 107 cfu g−1 of the free cell treatments. These indicated that the high rate of reproduction of the introduced microbes in beads, especially in the SAD beads, could be due to the carriers that accelerate the oxygen transfer and provide protection and a favorable niche for microorganisms to utilize hydrocarbons (Liang et al., 2009). After 20 d, degrading microorganism population and total microbial activity dropped and reached equilibrium in all treatments at 30 d, and the degrading microorganisms in the immobilization systems were one order of magnitude more than those of the free cell systems. This was possibly due to the fact that the bulk of labile hydrocarbons and the other nutrients had been consumed before 20 d, along with the production of toxic intermediates which were likely to have an inhibitory influence upon their proliferation and activity at later stages (Bento et al., 2005). Preservation of immobilized cells for potential application The immobilized cells had a great potential in bioremediation of crude oil-contaminated soils. For field application, the preservation of immobilized cells should also be considered. The cell survival in various bead formulations was assessed and compared with wet beads for a 60-d period. Wet beads could support stable cell survival for only about 15 d at 4 ◦ C (Fig. 7), while the other beads could support it for a 60-d period. Moreover, air drying was the best preservation method because it had little effect on cell viability or
Fig. 7 Cell survival in various beads protected by trehalose (T), glycerol (G) or skim milk (SM): wet beads in sterile water at 4 ◦ C (CK), air-dried beds (AD), freeze-dried beds (FD), and frozen beds (F).
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survival in the beads (about 0.1 log cfu per bead lower for protection agent of glycerol and trehalose) compared to the wet beads (at 4 ◦ C), while a minute reduction (about 0.2–0.3 log cfu per bead) was found in the freeze-dried and frozen beads. The freeze-dried and air-dried beads displayed considerable weight loss, size diminution, hardness increase, and significant shrinkage compared with the wet beads and immediately frozen beads (Vancov et al., 2007). The wet beads limit their potential use in field-scale bioremediation because of inconvenient storage, transportation, and application. Glycerol and trehalose, which offered effective protection for cells during drying and freezing, were proved to be suitable protecting agents compared to skim milk in the present study. The addition of skim milk as an amendment to beads remarkably raised the immobilized cell number up to 4.5 log cfu per bead. The increased cell number could be explained by the fact that after the formation of immobilized beads, they were completely gelled in 0.1 mg L−1 calcium chloride sterile solution for a long time period (12 h) and the immobilized cells would quickly reproduce by utilizing skim milk during the gelation, which induced rupturing of the beads. As a result, skim milk was not suitable as an amendment for our beads to provide protection. Given the cell activity (Cassidy et al., 1996), size, and weight (Vancov et al., 2007), air-dried beads protected by glycerol or trehalose were recommended for field application to degrade crude oil-contaminated soil. CONCLUSIONS Mixed hydrocarbon-degrading microbial communities were obtained and immobilized in sodium alginate (SA) beads and sodium alginate-diatomite (SAD) beads. The cells immobilized in SA and SAD beads resulted in higher removal of crude oil than the free cells at the same inoculation concentrations. The SAD treatments had advantages over the SA treatments. The immobilized cells at a low inoculation concentration (1 × 104 cfu kg−1 ) displayed a higher degradation rate of crude oil than the free cells at a high inoculation concentration (1 × 105 cfu kg−1 ). At about 20 d, microbial populations and total microbial activity reached the highest level and both parameters of the immobilization treatments were significantly higher (P < 0.05) than those of the free cells at the same initial inoculation amounts. Air-dried beads showed a great potential for long-term storage, feasible handling, and field application. Therefore, use of SAD beads to immobilize indigenous microbes for the biore-
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mediation of crude oil-contaminated soil in the Yellow River Delta could be a feasible and promising option. ACKNOWLEDGEMENTS The authors are grateful to Ms. HU Wen-Wen, Dr. Farhana Maqbool, Ms. DAI Yan-Hui, and Mr. XU Zhenhua, Ocean University of China, for their technical assistance. REFERENCES Ascon-Cabrera, M. A. and Lebeault, J. M. 1995. Cell hydrophobicity influencing the activity/stability of xenobioticdegrading microorganisms in a continuous biphasic aqueousorganic system. J. Ferment. Bioeng. 80: 270–275. Bao, S. D. 2000. Soil and Agricultural Chemistry Analysis (in Chinese). Agriculture Press, Beijing. Bazot, S. and Lebeau, T. 2009. Effect of immobilization of a bacterial consortium on diuron dissipation and community dynamics. Bioresour. Technol. 100: 4257–4261. Bento, F. M., Camargo, F. A. O., Okeke, B. C. and Frankenberger, W. T. 2005. Comparative bioremediation of soils contaminated with diesel oil by natural attenuation, biostimulation and bioaugmentation. Bioresour. Technol. 96: 1049–1055. Br´ eant, D., J´ ez´ equel, K. and Lebeau, T. 2002. Optimisation of the cell release from immobilised cells of Bacillus simplex cultivated in culture media enriched with Cd2+ : influence of Cd2+ , inoculum size, culture medium and alginate beads characteristics. Biotechnol. Lett. 24: 1237–1241. Cassidy, M. B., Lee, H. and Trevors, J. T. 1996. Environmental applications of immobilized microbial cells: a review. J. Ind. Microbiol. 16: 79–101. Cassidy, M. B., Lee, H. and Trevors, J. T. 1997. Survival and activity of lac-lux marked Pseudomonas aeruginosa UG2Lr cells encapsulated in κ-carrageenan over four years at 4 ◦ C. J. Microbiol. Methods. 30: 167–170. Chaˆıneau, C. H., Rougeux, G., Y´epr´ emian, C. and Oudot, J. 2005. Effects of nutrient concentration on the biodegradation of crude oil and associated microbial populations in the soil. Soil Biol. Biochem. 37: 1490–1497. Chung, W. K. and King, G. M. 2001. Isolation, characterization, and polyaromatic hydrocarbon degradation potential of aerobic bacteria from marine macrofaunal burrow sediments and description of Lutibacterium anuloederans gen. nov., sp. nov., and Cycloclasticus spirillensus sp. nov. Appl. Environ. Microbiol. 67: 5585–5592. Cunningham, C. J., Ivshina, I. B., Lozinsky, V. I., Kuyukina, M. S. and Philp, J. C. 2004. Bioremediation of diesel-contaminated soil by microorganisms immobilised in polyvinyl alcohol. Int. Biodeterior. Biodegrad. 54: 167– 174. US Environmental Protection Agency (US EPA). 1996. EPA 3550B Ultrasonic Extraction. Revision 2. US EPA, Washington, D. C. Green, V. S., Stott, D. E. and Diack, M. 2006. Assay for fluorescein diacetate hydrolytic activity: Optimization for soil samples. Soil Biol. Biochem. 38: 693–701. J´ ez´ equel, K. and Lebeau, T. 2008. Soil bioaugmentation by free and immobilized bacteria to reduce potentially phytoavailable cadmium. Bioresour. Technol. 99: 690–698.
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Jørgensen, K. S., Puustinen, J. and Suortti, A. M. 2000. Bioremediation of petroleum hydrocarbon-contaminated soil by composting in biopiles. Environ. Pollut. 107: 245–254. Lebeau, T., Jouenne, T. and Junter, G. A. 1997. Simultaneous fermentation of glucose and xylose by pure and mixed cultures of Saccharomyces cerevisiae and Candida shehatae immobilized in a two-chambered bioreactor. Enzyme Microb. Technol. 21: 265–272. Liang, Y. T., Zhang, X., Dai, D. J. and Li, G. H. 2009. Porous biocarrier-enhanced biodegradation of crude oil contaminated soil. Int. Biodeterior. Biodegrad. 63: 80–87. Mallory, L. M., Yuk, C. S., Liang, L. N. and Alexander, M. 1983. Alternative prey: a mechanism for elimination of bacterial species by protozoa. Appl. Environ. Microbiol. 46: 1073– 1079. Margesin, R. and Schinner, F. 2001. Bioremediation (natural attenuation and biostimulation) of diesel-oil-contaminated soil in an alpine glacier skiing area. Appl. Environ. Microbiol. 67: 3127–3133. Massalha, N., Basheer, S. and Sabbah, I. 2007. Effect of adsorption and bead size of immobilized biomass on the rate of biodegradation of phenol at high concentration levels. Ind. Eng. Chem. Res. 46: 6820–6824. Mofidi, N., Aghai-Moghadam, M. and Sarbolouki, M. N. 2000. Mass preparation and characterization of alginate microspheres. Process Biochem. 35: 885–888. Muhammad, S., M¨ uller, T. and Joergensen, R. G. 2008. Relationships between soil biological and other soil properties in saline and alkaline arable soils from the Pakistani Punjab. J. Arid Environ. 72: 448–457. Norman, R. S., Moeller, P., McDonald, T. J. and Morris, P. J. 2004. Effect of pyocyanin on a crude-oil-degrading microbial community. Appl. Environ. Microbiol. 70: 4004–4011. Ocean Monitoring Regulation Committee. 1998. GB17378.51998. The Specification for Marine Monitoring: Part 5. Sediment Analysis (in Chinese). Ocean Press, Beijing. Peng, S. W., Zhou, Q. X., Cai, Z. and Zhang, Z. N. 2009. Phytoremediation of petroleum contaminated soils by Mirabilis Jalapa L. in a greenhouse plot experiment. J. Hazard. Mater. 168: 1490–1496. Sathesh Prabua, C. and Thatheyus, A. J. 2007. Biodegradation of acrylamide employing free and immobilized cells of Pseudomonas aeruginosa. Int. Biodeterior. Biodegrad. 60: 69–73. Su, D., Li, P. J., Frank, S. and Xiong, X. Z. 2006. Biodegradation of benzo[a]pyrene in soil by Mucor sp. SF06 and Bacillus sp. SB02 co-immobilized on vermiculite. J. Environ. Sci. 18: 1204–1209. Trevors, J. T., Van Elsas, J. D., Lee, H. and Wolters, A. C. 1993. Survival of alginate encapsulated Pseudomonas fluorescens cells in soil. Appl. Microbiol. Biotech. 39: 637–643. Vacca, D. J., Bleam, W. F. and Hickey, W. J. 2005. Isolation of soil bacteria adapted to degrade humic acid-sorbed phenanthrene. Appl. Environ. Microbiol. 71: 3797–3805. Vancov, T., Jury, K., Rice, N., Van Zwieten, L. and Morris, S. 2007. Enhancing cell survival of atrazine degrading Rhodococcus erythropolis NI86/21 cells encapsulated in alginate beads. J. Appl. Microbiol. 102: 212–220. Vanhala, P. 2002. Seasonal variation in the soil respiration rate in coniferous forest soils. Soil Biol. Biochem. 34: 1375– 1379. Wang, Z. Y., Gao, D. M., Li, F. M., Zhao, J., Xin, Y. Z., Simkins, S. and Xing, B. S. 2008. Petroleum hydrocarbon degradation potential of soil bacteria native to the Yellow River Delta. Pedosphere. 18: 707–716.
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Wang, Z. Y., Xin, Y. Z., Gao, D. M., Li, F. M., Morgan, J. and Xing, B. S. 2010. Microbial community characteristics in a degraded wetland of the Yellow River Delta. Pedosphere. 20: 466–478. Wang, Z. Y., Xu, Y., Zhao, J., Li, F. M., Gao, D. M. and Xing, B. S. 2011. Remediation of petroleum contaminated soils through composting and rhizosphere degradation. J. Hazard. Mater. 190: 677–685. Wilson, N. G. and Bradley, G. 1996. Enhanced degradation of petrol (Slovene diesel) in an aqueous system by immobilized Pseudomonas fluorescens. J. Appl. Microbiol. 80: 99–104. Wrenn, B. A. and Venosa, A. D. 1996. Selective enumeration of aromatic and aliphatic hydrocarbon degrading bacteria by a most-probable-number procedure. Can. J. Microbiol. 42:
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252–258. Xiong, W. H. and Peng, J. 2008. Development and characterization of ferrihydrite-modified diatomite as a phosphorus adsorbent. Water Res. 42: 4869–4877. Ya˜ nez-Ocampo, G., Sanchez-Salinas, E., Jimenez-Tobon, G. A., Penninckx, M. and Ortiz-Hern´ adez, M. L. 2009. Removal of two organophosphate pesticides by a bacterial consortium immobilized in alginate or tezontle. J. Hazard. Mater. 168: 1554–1561. Zhang, K., Xu, Y. Y., Hua, X. F., Han, H. L., Wang, J. N., Wang, J., Liu, Y. M. and Liu, Z. 2008. An intensified degradation of phenanthrene with macroporous alginate-lignin beads immobilized Phanerochaete chrysosporium. Biochem. Eng. J. 41: 251–257.
Biodegradation of Crude Oil in Contaminated Soils by Free and Immobilized Microorganisms∗1 WANG Zhen-Yu1 , XU Ying1,2 , WANG Hao-Yun3 , ZHAO Jian1,4 , GAO Dong-Mei1 , LI Feng-Min1 and B. XING1,4,∗2 1 College
of Environmental Science and Engineering, Ocean University of China, Qingdao 266100 (China) of Animal Science and Veterinary Medicine, Qingdao Agricultural University, Qingdao 266109 (China) 3 Chemical and Mineral Metal Material Testing Center, Tianjin Entry-Exit Inspection and Quarantine Bureau, Tianjin 300456 (China) 4 Department of Plant, Soil and Insect Sciences, University of Massachusetts, Amherst MA 01003 (USA) 2 College
(Received October 2, 2011; revised January 16, 2012)
ABSTRACT The efficiencies of free and immobilized bacterial cultures of petroleum hydrocarbon degraders were evaluated and compared in this study. Hydrocarbon-degrading microbial communities with high tolerance to and high degrading ability of crude oil were obtained from the soil contaminated with crude oil in the Yellow River Delta. Then, the microbial cells were immobilized in sodium alginate (SA) beads and sodium alginate-diatomite (SAD) beads. The biodegradation of crude oil in soil by immobilized cells was compared with that by free cells at three inoculation concentrations, 1 × 104 colony forming units (cfu) kg−1 (low concentration, L), 5 × 104 cfu kg−1 (medium concentration, M), and 1 × 105 cfu kg−1 (high concentration, H). At 20 d after inoculation, the maximum degradation rate in the immobilized systems reached 29.8% (SAD-M), significantly higher (P < 0.05) than that of the free cells (21.1%), and the SAD beads showed greater degradation than the SA beads. Moreover, both microbial populations and total microbial activity reached significantly higher level (P < 0.05) in the immobilized systems than free cell systems at a same initial inoculation amount. The scanning electronic microscope (SEM) images also confirmed the advantages of the immobilized microstructure of SAD beads. The enhanced degradation and bacterial growth in the SAD beads indicated the high potential of SAD beads as an effective option for bioremediation of crude oil-contaminated soils in the Yellow River Delta. Key Words:
bacterial growth, degradation rate, hydrocarbon degraders, immobilized cells, sodium alginate-diatomite beads
Citation: Wang, Z. Y., Xu, Y., Wang, H. Y., Zhao, J., Gao, D. M., Li, F. M. and Xing, B. 2012. Biodegradation of crude oil in contaminated soils by free and immobilized microorganisms. Pedosphere. 22(5): 717–725.
INTRODUCTION Persistent crude oil exploration in the Yellow River Delta poses a serious threat to the environment, ecosystems, and human health due to enhanced soil contamination (Peng et al., 2009; Wang et al., 2011). Crude oil is composed of complex hydrocarbons with low bioavailability and is persistent in soil. Bioremediation to clean up petroleum hydrocarboncontaminated soil was considered to be beneficial over physical and chemical methods because of its costeffectiveness along with low environmental impact (Margesin and Schinner, 2001; Wang et al., 2008). However, the success of most bioaugmentation experiments depends on the survival of the inoculated cells (J´ez´equel and Lebeau, 2008). The inability of intro∗1 Supported
duced non-indigenous microorganisms to compete with the indigenous microflora and predators severely limits biodegradation (Mallory et al., 1983; J´ez´equel and Lebeau, 2008). Thus, the use of indigenous microorganisms to degrade petroleum hydrocarbons is important and has been well documented in both terrestrial and aquatic ecosystems (Margesin and Schinner, 2001; Norman et al., 2004; Vaccar et al., 2005; Wang et al., 2008). Immobilization of cells protects the cells from predation and natural competition with the soil microflora and is known to be more effective in shielding perturbations of environmental conditions such as pH and toxic compounds in comparison to suspension culture (J´ez´equel and Lebeau, 2008; Zhang et al., 2008). Immobilization has been achieved in a number of ways by
by the National Natural Science Foundation of China (No. 41073067), the Key Program of the Ministry of Education, China (No. 308016), and the National Major Special Technological Programme Concerning Water Pollution Control and Management of China (No. 2009ZX07010-008). ∗2 Corresponding author. E-mail: [email protected].
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physical adsorption, ion-coagulation, cross-linking, and/or entrapment methods in a variety of matrices (Su et al., 2006), such as sodium alginate (Bazot and Lebeau, 2009; Ya˜ nez-Ocampo et al., 2009), polyvinyl alcohol (PVA) (Cunninghama et al., 2004), activated carbon, zeolite (Liang et al., 2009), and vermiculite (Su et al., 2006). Among all the matrices, alginate extracted from macro-algae, a natural polymer, is most popularly used for the preparation of gel beads with low cost and mild conditions of immobilization (Mofidi et al., 2000; Massalha et al., 2007), along with its easy degradability and low-toxicity. Therefore, alginate is suitable to be used as an immobilization matrix for bioremediation of contaminated soils. The microorganisms immobilized in alginate beads have been used in hostile environments and the beads provide nutrients and appropriate conditions, such as facilitating the transfer of oxygen which is crucial for rapid hydrocarbon mineralization, to allow rapid bioremediation in contaminated systems (Cunninghama et al., 2004; Sathesh Prabua and Thatheyus, 2007; Vancov et al., 2007; Bazot and Lebeau, 2009; Ya˜ nezOcampo et al., 2009). The slow release of inoculants from the alginate beads also favored their proficiency in contaminant removal (Vancov et al., 2007; Bazot and Lebeau, 2009). However, the dense gel layers of the alginate beads hinder mass transfer of substrates, pollutants, and degraded products, which restricts their potential application in bioremediation (Zhang et al., 2008). The incorporation of some adsorbents into alginate beads facilitated the transport of pollutants towards both the surface and interior regions (Zhang et al., 2008). Diatomite, mainly consisting of SiO2 (80%–90%), formed from dead diatoms in seas and lakes has been approved as a food-grade material by the U.S. Food and Drug Administration (FDA) (Xiong and Peng, 2008). Diatomite with high porosity (80%–85% voids) (Xiong and Peng, 2008) can enhance the porosity and improve mass transfer property of alginate beads if incorporated. Moreover, the diatomite released into the soil after alginate degradation may improve the soil quality. Hence, the immobilization of functional microorganisms using alginate and diatomite together is a good prospective method to bioremediate contaminated soil systems. There are few data on the advantages of such immobilization for crude oilcontaminated soil remediation by microorganisms. Especially, in the oilfields of the Yellow River Delta, the area of contaminated soil is large. The immobilization matrix used in remediation should be degraded naturally and non-toxic.
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In the present study, sodium alginate and diatomite were selected because of their natural origin and many advantages. The aim of the present study was to compare the efficiency of free and immobilized inoculants of the indigenous petroleum hydrocarbon-degrading bacteria in removing crude oil from a soil of the Yellow River Delta. We studied the influence of the inoculum amount and the amendment of diatomite to sodium alginate beads on the contamination removal, microbial activity, and degrading microorganism colonization. The preservation condition of immobilized beads was also analyzed. MATERIALS AND METHODS Contaminated soil samples The contaminated soil was collected from the vicinity of an oil well in the Yellow River Delta, China. The soil was silty clay, and the concentration of petroleum hydrocarbon of the soil was about 10 g kg−1 . The basic physical and chemical properties of the soil (0–20 cm) were as follows: pH of 8.17, organic matter of 4.9 g kg−1 , salinity of 14.5 g kg−1 NaCl, total N of 25 g kg−1 , and total P of 0.4 g kg−1 . Petroleum hydrocarbon-degrading microorganisms The enrichment and isolation of petroleum hydrocarbon-degrading microorganisms were conducted immediately after the soil was collected. Five grams of the soil samples were inoculated in a 150-mL sterilized mineral salt medium (MSM) containing 1 g L−1 K2 HPO4 , 1 g L−1 KH2 PO4 , 1 g L−1 (NH4 )2 SO4 , 15 g L−1 NaCl, 0.2 g L−1 MgSO4 , 0.02 g L−1 CaCl2 and a trace of FeCl3 . After incubation and serial subculturing (eight transfers), the mixed hydrocarbon-degrading microbial communities were obtained after 80 d of enrichment. For isolation, a combination of serial dilution and steak plate method was used to obtain single colonies of pure cultures. The isolated dominant bacterial cultures were identified by polymerase chain reaction (PCR) amplification and sequencing of the chromosomal DNA, which was completed by the Shanghai GeneCore BioTechnologies Co., Ltd. Preparation of immobilized bacteria Cell suspension. The mixed petroleum hydrocarbon-degrading microorganisms grew in the MSM containing 20 mg mL−1 crude oil as a sole carbon source, and inoculated for 60 h (exponent growth period). Then, the cells were centrifuged and the supernatant was discarded. The pellet was washed and suspended in solution of 8.5 g L−1 NaCl.
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Cell immobilization. The immobilization of petroleum hydrocarbon-degrading microorganisms was conducted using sodium alginate (SA) beads and sodium alginate-diatomite (SAD) beads. The immobilization procedure was adapted from Sathesh Prabua and Thatheyus (2007) and Ya˜ nez-Ocampo et al. (2009), with 60 g L−1 sodium alginate solution or the mixed solution of 60 g L−1 sodium alginate and 20 g L−1 diatomite. Degradation experiment To assess the efficiency of mixed petroleum hydrocarbon-degrading microbial communities to degrade crude oil, 10 mL inoculum (optical density = 0.7 at 600 nm) was added into 150 mL of MSM with the concentrations of crude oil at 10, 20, and 30 mg mL−1 . The extent of degradation of crude oil was determined by gas chromatography (GC) (Chung and King, 2001). Cells immobilized in SA and SAD beads and free cells (F) were respectively inoculated in pots (30 cm diameter and 30 cm depth) containing 10 kg of contaminated soil. Pots without any added bacteria were used as the control. For each pot with inoculated bacteria, 3 cell concentration gradients were set: 1 × 104 colony forming units (cfu) kg−1 (low concentration, L), 5 × 104 cfu kg−1 (medium concentration, M) and 1 × 105 cfu kg−1 (high concentration, H). To simplify the expression, SA, SAD, and free beads with these 3 concentration levels were defined as SA-L, SA-M and SA-H; SAD-L, SAD-M and SAD-H; and Free-L, FreeM and Free-H, respectively. Nutrient solution was supplemented to each pot, up to a C:N:P ratio of 100:10:1. The experiment was carried out outside for 70 d, and the moisture content was kept at 60% water-holding capacity (WHC) according to a pre-experiment (data not shown). During the 70 d of experiment, samples were taken 7 times at 0, 10, 20, 30, 40, 50, and 70 d from each treatment for analyses of petroleum hydrocarbon concentration, microbial activity, and the population of degrading microorganisms. Sample analyses Physicochemical characteristics of the soil. pH (in 0.01 mol L−1 CaCl2 ) was measured at a 1:2.5 soil to solution ratio. Organic matter with the Walkley and Black dichromate oxidation method, total nitrogen with the Kjeldahl method, and total phosphorus by a spectrophotometer after NaOH digestion were analyzed according to Bao (2000). Electrical conductivity (EC) was determined using a 1:5 soil to water suspen-
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sion, which was converted to EC values in saturation extract (ECe ) and expressed as salinity (Muhammad et al., 2008). Soil moisture content was determined gravimetrically by drying at 105 ◦ C for 24 h. The waterholding capacity (WHC) was determined by soaking the soil samples in water for 2 h followed by draining for 2 h (Vanhala, 2002). Oil concentration of the soil. The extraction and determination of crude oil were carried out by the method from EPA3550B (US EPA, 1996) and GB17378.5-1998 (Ocean Monitoring Regulation Committee, 1998). The concentration of oil was determined using GC (GC-2010, Shimadzu Co., Japan). Bacterial population and activity. The hydrocarbon-degrading microorganisms was enumerated by the most probable number (MPN) procedure of Wrenn and Venosa (1996) and Cunninghama et al. (2004), with the addition of diesel oil as the soil carbon source. Total microbial activity was determined with fluorescein diacetate (FDA) method (Green et al., 2006; Wang et al., 2010). The FDA hydrolytic activity was measured from the absorbance of supernatant at 490 nm (A490 ) using a spectrophotometer. Cell growth analysis and immobilized bead preservation The cell concentration was determined by measuring optical density (OD) at 600 nm using a spectrophotometer and the corresponding cell number was measured with an epifluorescence microscope (Wang et al., 2008). Cell count immobilized in each bead was estimated with MSM agar plate (Ascon-Cabrera and Lebeault, 1995). The cells immobilized in SAD beads were observed by scanning electronic microscope (SEM, S-4800, Hitachi, Japan). The beads were fixed in 2.5% glutaraldehyde according to the method of Su et al. (2006). The method of immobilized bead preservation was adopted from Trevors et al. (1993) and Vancov et al. (2007). Glycerol (200 mL L−1 ), trehalose (200 g L−1 ) and skim milk (200 g L−1 ) were supplemented to the mixtures of sodium alginate (60 g L−1 ) and diatomite (20 g L−1 ) beads. Fresh beads were preserved by freezing-drying, air-drying, and freezing. The cell viability was also determined through dissolving the beads in 10 mL of sterile EDTA solution (0.1 mg L−1 ) (Br´eant et al., 2002; Bazot and Lebeau, 2009). Statistical analysis Every test was repeated three times. Statistical analyses were performed with SPSS for Windows 12.0. The significance of the various parameters was tested
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by one-way analysis of variance (ANOVA) using the least significance difference (LSD) test. RESULTS AND DISCUSSION Crude oil degradation potentials of hydrocarbon-degrading microorganisms The efficiency of the indigenous microbial community to transform crude oil was determined at 7 and 14 d of incubation (Figs. 1 and 2). The cultures with 10 and 20 mg mL−1 of crude oil had higher removal ratios (90% and 88%) and cell numbers (4.2 × 108 and 6.3 × 108 ) at 14 d than the culture with 30 mg mL−1 of crude oil. The increase in substrate concentration up to 30 mg mL−1 of crude oil showed moderately toxic to at least some microorganisms of the culture which had the microbial cell counts of 1.0 × 108 and 2.4 × 108 and the degradation percentages of crude oil of 28% and 50% after 7 and 14 d of incubation, respectively.
Fig. 2 Microbial cell counts in the biodegradation systems with the initial concentrations of crude oil of 10, 20, and 30 mg mL−1 at 7 and 14 d of incubation.
These indicated that the indigenous microbial community showed high tolerance to and transformation ability of crude oil (Vancov et al., 2007). Then, 3 dominant single colonies which could play a major role in biodegradation of crude oil were isolated and identified as Microbacterium foliorum, Gordonia alkanivorans, and Mesorhizobiu. Biodegradation of crude oil in soil by immobilized and free cells
Fig. 1 Degradation of crude oil by hydrocarbon-degrading microbial communities at 7 and 14 d of incubation with the initial concentrations of crude oil of 10, 20, and 30 mg mL−1 .
Crude oil was degraded more quickly by the immobilized cells (SAD and SA) than the free cells at the same inoculant concentrations (Fig. 3 a, b, c). When the inoculation density was 1 × 104 cfu kg−1 (low concentration, L) and 5 × 104 cfu kg−1 (medium concentration, M), the degradation rates of immobilized cells were significantly higher than those of the free cells. To simplify data presentation, the removal rates of crude
Fig. 3 Crude oil degradation by immobilized cells in sodium alginate-diatomite (SAD) and sodium alginate (SA) beads and free cells (F) at different initial inoculation amounts: 1 × 104 cfu kg−1 (low concentration, L) (a), 5 × 104 cfu kg−1 (medium concentration, M) (b), and 1 × 105 cfu kg−1 (high concentration, H) (c). The removal rate of crude oil = the removal rate of crude oil of soil with free or immobilized cells – the removal rate of crude oil of the control.
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oil were calculated as the degradation rate of the treatment samples minus that of the control. The concentration of crude oil in all treatments decreased quickly at the beginning of the incubation. At 20 d, the degradation rate was the maximum in the immobilized systems up to 29.8% (SAD-M) of crude oil in contrast to 21.2% by the free cells. At that time, the degradation rate of the SAD cultures was about 2 times higher than that of the free cell culture at the low inoculation density of 1 × 104 cfu kg−1 (L). The degradation rate decreased after 20 d, which was mainly due to efficient utilization of labile hydrocarbon sources by degrading microorganisms at the beginning, leading to the high percentage of removal at 20 d. Then, the remained recalcitrant hydrocarbons served as the carbon source, which slowed down the removal rate (Jørgensen et al., 2000). In addition, the expense of nutrients and products of toxic intermediates were also likely to inhibit the growth of degrading microorganisms, resulting in drop in the degradation percentage of crude oil in soil (Bento et al., 2005). Effect of SAD on biodegradation Alginate could increase bacterial survival in soil compared with free cells (Trevors et al., 1993; Cassidy et al., 1997). Previous researches have shown that immobilized cells in SA showed a faster removal rate of contaminants than free cells (Sathesh Prabua and Thatheyus, 2007; Vancov et al., 2007; Bazot and Lebeau, 2009; Ya˜ nez-Ocampo et al., 2009). This could be induced by complex factors, such as protection of carriers from deleterious effects (Su et al., 2006), high affinity between immobilization materials and substrates (Wilson and Bradley, 1996), and acceleration of oxygen transfer (Liang et al., 2009), which led to increment in cell count and availability of the substrates (Su et al., 2006). Our results showed that the SAD treatments presented greater degradation than the SA
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treatments. At 20 d, the highest removal reached 29.8% (SAD-M) and 25.6% (SA-M). At the end of experiment, the SAD treatments had the greatest degradation of 46.3% (SAD-M), 3.4% higher than the SA treatments (Fig. 3b). Diatomite incorporated into alginate beads could improve mass transfer property of the alginate beads (Xiong and Peng, 2008), which subsequently enhanced the removal rate of contaminants. In addition, when alginate was degraded, diatomite would effectively improve the pore structure of soil, promoting the aerobic biodegradation of petroleum hydrocarbons. The immobilization of cells in SAD beads was confirmed by scanning electron microscope (SEM) and the images are shown in Fig. 4. Cells were well distributed in the interior of the SAD beads. It can also be seen that the SAD beads had large porosity, which permitted excellent mass transport of oxygen, nutrients, and degradation substrates. It was reported that the dense gel layer structure in the SA beads hindered the efficient transfer of oxygen, which limited aerobic strains of degrading microorganism community (Zhang et al., 2008; Ya˜ nez-Ocampo et al., 2009). Our results showed that diatomite was an excellent amendment for alginate beads for improving porosity and mass transfer property and thus efficiently improving the growth of immobilized cells and the bioremediation Effect of initial inoculation concentration on biodegradation The initial inoculation amount on biodegradation was also investigated (Fig. 3a, c). The immobilized cells at the low inoculation concentration (1 × 104 cfu kg−1 ) had a higher degradation rate than the free cells at the high inoculation concentration (1 × 105 cfu kg−1 ). At 20 d, the degradation rates of SAD-L and F-H were 27.3% and 21.2%, respectively. However, the advantage of the immobilization treatments with the high
Fig. 4 Scanning electronic microscope (SEM) micrographs (× 10 000) of interior structure of sodium alginate-diatomite (SAD) beads without cells (a) and SAD beads with immobilized cells (b).
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inoculation concentration (1 × 105 cfu kg−1 ) was not significant (P < 0.05) (Fig. 3c). The rapid metabolization with a high concentration of hydrocarbondegrading microorganisms caused the limitation of oxygen and nutrients, which restricted the propagation size of microorganisms (Cunninghama et al., 2004; Chaˆıneau et al., 2005). Therefore, the inoculation concentration could affect the degradation rate, and the efficient level of inoculation concentration was selected at less than 5 × 104 cfu kg−1 of soil. Hydrocarbon-degrading microorganism populations and activity Large numbers of hydrocarbon degraders and active microbial communities were keys to successful bioremediation. The changes of microorganism numbers and activity in soil during degradation are shown
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in Figs. 5 and 6. The results showed that the free indigenous microbial cultures quickly adapted to the environment and presented a rapid multiplication and increase of total microbial activity. In the immobilized systems, inoculant formulation with SAD or SA beads showed similar growth kinetics of degrading microorganisms and total microbial activity. At the early stage, microbial growth and replication within the matrix may release some microorganisms to the surrounding environment (Su et al., 2006), resulting in low degrading-microorganism population and activity in soil. After 10 d, both microbial population and total microbial activity increased rapidly in the SAD and SA systems, resulting from both massive cell releases associating with bead degradation and their subsequent growth in the soil systems (Lebeau et al., 1997; Br´eant et al., 2002; Bazot and Lebeau, 2009). At approxima-
Fig. 5 Petroleum hydrocarbon-degrading microorganism populations in soil inoculated with immobilized cells in sodium alginatediatomite (SAD) beads and sodium alginate (SA) beads and with free cells (F) at different initial inoculation amounts of 1 × 104 cfu kg−1 (low concentration, L) (a) and 5 × 104 cfu kg−1 (medium concentration, M) (b), and 1 × 105 cfu kg−1 (high concentration, H) (c). The population of petroleum hydrocarbon-degrading microorganisms was the population of the soil sample with free or immobilized cells minus that of the control. MPN = most probable number.
Fig. 6 Microbial activity (expressed as fluorescein diacetate (FDA) hydrolyzed) in soil inoculated with immobilized cells in sodium alginate-diatomite (SAD) beads and sodium alginate (SA) beads and with free cells (F) at different initial inoculation amounts of 1 × 104 cfu kg−1 (low concentration, L) (a), 5 × 104 cfu kg−1 (medium concentration, M) (b), and 1 × 105 cfu kg−1 (high concentration, H) (c). The microbial activity in soil was the microbial activity of the soil samples with free or immobilized cells minus that of the control.
CRUDE OIL BIODEGRADATION IN CONTAMINATED SOILS
tely 20 d, both microbial population and total microbial activity were significantly higher (P < 0.05) than those for the free cells at a same initial inoculation amount. The maximum degrading-microorganism numbers were 6.1 × 108 and 3.6 × 108 cfu g−1 for the SAD-M and SA-M treatments, respectively, in contrast to 1.8 × 107 cfu g−1 of the free cell treatments. These indicated that the high rate of reproduction of the introduced microbes in beads, especially in the SAD beads, could be due to the carriers that accelerate the oxygen transfer and provide protection and a favorable niche for microorganisms to utilize hydrocarbons (Liang et al., 2009). After 20 d, degrading microorganism population and total microbial activity dropped and reached equilibrium in all treatments at 30 d, and the degrading microorganisms in the immobilization systems were one order of magnitude more than those of the free cell systems. This was possibly due to the fact that the bulk of labile hydrocarbons and the other nutrients had been consumed before 20 d, along with the production of toxic intermediates which were likely to have an inhibitory influence upon their proliferation and activity at later stages (Bento et al., 2005). Preservation of immobilized cells for potential application The immobilized cells had a great potential in bioremediation of crude oil-contaminated soils. For field application, the preservation of immobilized cells should also be considered. The cell survival in various bead formulations was assessed and compared with wet beads for a 60-d period. Wet beads could support stable cell survival for only about 15 d at 4 ◦ C (Fig. 7), while the other beads could support it for a 60-d period. Moreover, air drying was the best preservation method because it had little effect on cell viability or
Fig. 7 Cell survival in various beads protected by trehalose (T), glycerol (G) or skim milk (SM): wet beads in sterile water at 4 ◦ C (CK), air-dried beds (AD), freeze-dried beds (FD), and frozen beds (F).
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survival in the beads (about 0.1 log cfu per bead lower for protection agent of glycerol and trehalose) compared to the wet beads (at 4 ◦ C), while a minute reduction (about 0.2–0.3 log cfu per bead) was found in the freeze-dried and frozen beads. The freeze-dried and air-dried beads displayed considerable weight loss, size diminution, hardness increase, and significant shrinkage compared with the wet beads and immediately frozen beads (Vancov et al., 2007). The wet beads limit their potential use in field-scale bioremediation because of inconvenient storage, transportation, and application. Glycerol and trehalose, which offered effective protection for cells during drying and freezing, were proved to be suitable protecting agents compared to skim milk in the present study. The addition of skim milk as an amendment to beads remarkably raised the immobilized cell number up to 4.5 log cfu per bead. The increased cell number could be explained by the fact that after the formation of immobilized beads, they were completely gelled in 0.1 mg L−1 calcium chloride sterile solution for a long time period (12 h) and the immobilized cells would quickly reproduce by utilizing skim milk during the gelation, which induced rupturing of the beads. As a result, skim milk was not suitable as an amendment for our beads to provide protection. Given the cell activity (Cassidy et al., 1996), size, and weight (Vancov et al., 2007), air-dried beads protected by glycerol or trehalose were recommended for field application to degrade crude oil-contaminated soil. CONCLUSIONS Mixed hydrocarbon-degrading microbial communities were obtained and immobilized in sodium alginate (SA) beads and sodium alginate-diatomite (SAD) beads. The cells immobilized in SA and SAD beads resulted in higher removal of crude oil than the free cells at the same inoculation concentrations. The SAD treatments had advantages over the SA treatments. The immobilized cells at a low inoculation concentration (1 × 104 cfu kg−1 ) displayed a higher degradation rate of crude oil than the free cells at a high inoculation concentration (1 × 105 cfu kg−1 ). At about 20 d, microbial populations and total microbial activity reached the highest level and both parameters of the immobilization treatments were significantly higher (P < 0.05) than those of the free cells at the same initial inoculation amounts. Air-dried beads showed a great potential for long-term storage, feasible handling, and field application. Therefore, use of SAD beads to immobilize indigenous microbes for the biore-
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mediation of crude oil-contaminated soil in the Yellow River Delta could be a feasible and promising option. ACKNOWLEDGEMENTS The authors are grateful to Ms. HU Wen-Wen, Dr. Farhana Maqbool, Ms. DAI Yan-Hui, and Mr. XU Zhenhua, Ocean University of China, for their technical assistance. REFERENCES Ascon-Cabrera, M. A. and Lebeault, J. M. 1995. Cell hydrophobicity influencing the activity/stability of xenobioticdegrading microorganisms in a continuous biphasic aqueousorganic system. J. Ferment. Bioeng. 80: 270–275. Bao, S. D. 2000. Soil and Agricultural Chemistry Analysis (in Chinese). Agriculture Press, Beijing. Bazot, S. and Lebeau, T. 2009. Effect of immobilization of a bacterial consortium on diuron dissipation and community dynamics. Bioresour. Technol. 100: 4257–4261. Bento, F. M., Camargo, F. A. O., Okeke, B. C. and Frankenberger, W. T. 2005. Comparative bioremediation of soils contaminated with diesel oil by natural attenuation, biostimulation and bioaugmentation. Bioresour. Technol. 96: 1049–1055. Br´ eant, D., J´ ez´ equel, K. and Lebeau, T. 2002. Optimisation of the cell release from immobilised cells of Bacillus simplex cultivated in culture media enriched with Cd2+ : influence of Cd2+ , inoculum size, culture medium and alginate beads characteristics. Biotechnol. Lett. 24: 1237–1241. Cassidy, M. B., Lee, H. and Trevors, J. T. 1996. Environmental applications of immobilized microbial cells: a review. J. Ind. Microbiol. 16: 79–101. Cassidy, M. B., Lee, H. and Trevors, J. T. 1997. Survival and activity of lac-lux marked Pseudomonas aeruginosa UG2Lr cells encapsulated in κ-carrageenan over four years at 4 ◦ C. J. Microbiol. Methods. 30: 167–170. Chaˆıneau, C. H., Rougeux, G., Y´epr´ emian, C. and Oudot, J. 2005. Effects of nutrient concentration on the biodegradation of crude oil and associated microbial populations in the soil. Soil Biol. Biochem. 37: 1490–1497. Chung, W. K. and King, G. M. 2001. Isolation, characterization, and polyaromatic hydrocarbon degradation potential of aerobic bacteria from marine macrofaunal burrow sediments and description of Lutibacterium anuloederans gen. nov., sp. nov., and Cycloclasticus spirillensus sp. nov. Appl. Environ. Microbiol. 67: 5585–5592. Cunningham, C. J., Ivshina, I. B., Lozinsky, V. I., Kuyukina, M. S. and Philp, J. C. 2004. Bioremediation of diesel-contaminated soil by microorganisms immobilised in polyvinyl alcohol. Int. Biodeterior. Biodegrad. 54: 167– 174. US Environmental Protection Agency (US EPA). 1996. EPA 3550B Ultrasonic Extraction. Revision 2. US EPA, Washington, D. C. Green, V. S., Stott, D. E. and Diack, M. 2006. Assay for fluorescein diacetate hydrolytic activity: Optimization for soil samples. Soil Biol. Biochem. 38: 693–701. J´ ez´ equel, K. and Lebeau, T. 2008. Soil bioaugmentation by free and immobilized bacteria to reduce potentially phytoavailable cadmium. Bioresour. Technol. 99: 690–698.
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