Appl Biochem Biotechnol DOI 10.1007/s12010-014-0777-6
Enhanced Biodegradation of Alkane Hydrocarbons and Crude Oil by Mixed Strains and Bacterial Community Analysis Yu Chen & Chen Li & Zhengxi Zhou & Jianping Wen & Xueyi You & Youzhi Mao & Chunzhe Lu & Guangxin Huo & Xiaoqiang Jia
Received: 12 October 2013 / Accepted: 3 February 2014 # Springer Science+Business Media New York 2014
Abstract In this study, two strains, Acinetobacter sp. XM-02 and Pseudomonas sp. XM-01, were isolated from soil samples polluted by crude oil at Bohai offshore. The former one could degrade alkane hydrocarbons (crude oil and diesel, 1:4 (v/v)) and crude oil efficiently; the latter one failed to grow on alkane hydrocarbons but could produce rhamnolipid (a biosurfactant) with glycerol as sole carbon source. Compared with pure culture, mixed culture of the two strains showed higher capability in degrading alkane hydrocarbons and crude oil of which degradation rate were increased from 89.35 and 74.32±4.09 to 97.41 and 87.29±2.41 %, respectively. In the mixed culture, Acinetobacter sp. XM-02 grew fast with sufficient carbon source and produced intermediates which were subsequently utilized for the growth of Pseudomonas sp. XM-01 and then, rhamnolipid was produced by Pseudomonas sp. XM-01. Till the end of the process, Acinetobacter sp. XM-02 was inhibited by the rapid growth of Pseudomonas sp. XM-01. In addition, alkane hydrocarbon degradation rate of the mixed culture increased by 8.06 to 97.41 % compared with 87.29 % of the pure culture. The surface tension of medium dropping from 73.2×10−3 to 28.6×10−3 N/m. Based on newly found cooperation between the degrader and the coworking strain, rational Y. Chen : X. You Department of Environmental Science, School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, People’s Republic of China Y. Chen China Offshore Environmental Services Ltd., Tianjin 300452, People’s Republic of China C. Li : Z. Zhou : J. Wen : Y. Mao : C. Lu : G. Huo : X. Jia Department of Biological Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People’s Republic of China J. Wen : X. Jia Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin 300072, People’s Republic of China J. Wen : X. Jia (*) Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, People’s Republic of China e-mail: [email protected]
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investigations and optimal strategies to alkane hydrocarbons biodegradation were utilized for enhancing crude oil biodegradation. Keywords Biodegradation . Crude oil . Chromatography . Microbial growth . Biosurfactant . Waste treatment
Introduction Oil spills happened constantly, posing critical hazards for ecological system and mankind [9]. It is still a reinforcing challenge to eliminate oil pollution effectively and eco-friendly [21]. Governments, companies, and research organizations have been under an increasing pressure to develop clean and environmental technologies for treatment of the pollutants in the wake of environmental consciousness [15]. The growing environment crisis caused by oil pollution spurs focused efforts into developing effective and environment-friendly technologies for the treatment of the pollutants. Physical treatments cannot remove the pollutants completely, and the main disadvantage is too costly [19], Whereas, chemical treatments were likely to cause a second pollution to the environment due to the unexpected release of artificial toxic materials into the environment [18]. Therefore, the safe and effective biotreatment methods of oil pollution have attracted extensive attention [28]. Many strains growing on crude oil as the sole carbon source have been isolated for the biodegradation of oil [21], such as Rhodococcus [4], Acinetobacter [12], Achromobacter [2], Dietzia [5], Pseudomonas [20], etc. Among them, Acinetobacter has shown high performance for the biodegradation of crude oil [9, 14]. Acinetobacter calcoaceticus could degrade long carbon-chain n-alkane (contained more than 33 carbon atoms) and even the branched chain alkane but failed to grow on cycloalkanes [1]. Acinetobucter calcoaceticus and Alcaligenes odorans cultured in combination were more effective in degrading crude oil than their pure cultures [9]. Moreover, some strains, many of which were Pseudomonas sp., could produce biosurfactant with proper nutrients available [8, 16]. Additionally, biosurfactant, usually including rhamnolipid, saponin, and alkyl polyglucosides could overcome the difficulties in oil–cell surface contact to accelerate the biodegradation of crude oil and its production by decreasing the surface tension of medium [13, 24]. However, most studies focused on the fermentation and purification of biosurfactants with nutrient medium as well as effects of biosurfactants on crude oil or alkane hydrocarbon biodegradation. Little is known about the application and interaction mechanisms of mixed culture of biosurfactant producers and oil degraders [8]. Strategies to enhance biodegradation of crude oil, including amendments of nutrient and addition of biosurfactant, were prohibitive, and adding these exotic substances might produce additional stress, being a restrain of the degraders due to the booming growth of the other strains. Furthermore, utilization of single strain in crude oil biodegradation was often limited by complex realistic conditions. Biosurfactant was of great use in the biodegradation and exploitation of crude oil, meaning it has a dual role in improving the environmental and economic benefits, yet it can easily be washed away by agitation. To solve this problem, a suggestion was proposed that the biosurfactant could be mixed with nutrients to form particulate fertilizers and applied to the pollutant area or the oil well [23]. However, biosurfactants in fertilizer would be of little use in the absence of water or would be dissolved and washed away in aqueous environment. Another brilliant thought was to functionalize composite material combined with Acinetobacter venetianus 2AW (alkane-degrading bacteria), and biodegradation of alkane was facilitated by the immobilized bacterial population [12]. To enhance the effect of the degraders and the biosurfactant-producing strains, a possible alternative method is to immobilize the mixed strains
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containing the degraders and some biosurfactant-producing strains, forming indissolvable tiny particles and fertilized them into polluted areas. Thus, leaked crude oil was easy to be degraded by improving and stabilizing the local microorganism community. As most biosurfactants are secondary metabolites, and their production was activated by depletion of nutrients [3] and could be restrained by crude oil, the importance of producing biosurfactant in situ with no supplemented nutrients but crude oil was obviously revealed. Complete biodegradation of crude oil pollutants commonly depends on the synergies of bacteria in environment [6], and the mixed strains showed a greater capability of oil biodegradation compared with the pure ones [7]. Great efforts have been made in the preference of mixed degraders to the single strains, yet little is available for the mutualism between the degrader and nondegraders (functional bacteria) in oil biotreatment. Particularly, the relationships of the degraders and biosurfactant-producing strains are still a critical issue on crude oil biodegradation, and the mechanism requires a deeper insight. As for future study on in situ crude oil bioremediation, it became more valuable to investigate the interaction among strains with oil degrading and the coexisted ones. In this paper, a method of co-culturing biosurfactant-producing strain and oil degrader was developed to enhance the biodegradation rate of alkane hydrocarbons and crude oil. With investigations on consortium composition changing during biodegradation of alkane hydrocarbons, the possible reasons for these changes and the strategies to improve biodegradation were proposed.
Methods Chemicals and Reagents The oil-contaminated soil was sampled from the Boxi Offshore Oil Field, Tianjin, China. No. 0 diesel oil was purchased from a local service station of Sinopec Group and the crude oil was donated by Boxi Offshore Oil Field of China National Offshore Oil Corporation (CNOOC). Alkane hydrocarbons were a mixture of crude oil and No. 0 diesel with a volume ratio of 1:4. All other chemicals used in this study were of analytical grade. Isolation and Identification of the Strains The two bacterial strains were enriched and isolated from the soil samples. The enrichment medium comprised NaNO3, 2 g; K2HPO4·3H2O, 0.5 g; KH2PO4, 1 g; NaCl, 0.5 g; MgSO4, 0.1 g; and 10 mL trace element solution in 1 L distilled water. The trace element solution contained 1.2 % Na2EDTA·H2O, 0.2 % NaOH, 0.04 % MnSO4 4H2O, 0.04 % ZnSO4 7H2O, 0.05 % concentrated H2SO4, 0.01 % Na2M0O4·2H2O, 0.2 % FeSO4·7H2O, 0.01 % CuSO4·5H2O, 0.1 % CaCl2, and 1 % Na2SO4. Adjusting the pH value to 7.2, 100 mL of the screening media was fractionally fed into 250-mL shake flasks. Five grams soil samples and 0.5 mL alkane hydrocarbons were amended into the sterilized screening media, which were afterwards cultivated at 30 °C in a shaker with agitation of 200 rpm for 5 days. Onemilliliter culture solution was transferred into another fresh culture solution with 0.5 mL alkane hydrocarbons, and five repeats were carried out. Then, the strains were purified on agar plates with enrichment media and 1.8 % (mass ratio) agar powder. One strain designated as XM-02 was selected for its good biodegradation ability of alkane hydrocarbons. Meanwhile, biosurfactant-producing strains were isolated on blue agar plate as described previously [22], and one strain named XM-01 was selected as its greatest performance in surface tension reduction in cultivation.
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The morphological, physiological, and phylogenetic properties of the strains were assayed based on 16S rDNA analysis, demonstrated as foregoing documents [17]. Analysis of 16S rDNA sequences was performed under standard reaction conditions with Taq DNA polymerase. Both primers synthesis (27F: 5′-AGA GTT TGA TCC TGG CTC AG-3′ and 1492R: 5′GGT TAC CTT GTT ACG ACT T-3′) and gene sequencing were performed by Beijing Genomics Institute (China). Identification and homology search were carried out with BLAST program at the National Center for Biotechnology Information (NCBI) Website (http://blast. ncbi.nlm.nih.gov). GenBank accession numbers for the 16S rDNA sequences were obtained, and phylogenetic trees of the strains were established by MEGA 5.05 with NJ method. Physiological and biochemical appraisals of the strains were conducted with biochemical appraisal kits (nonfermentative bacteria biochemical coding assessor GYZ-15n, Microbial Reagent Co., Ltd., Hangzhou, China). Analysis of Composition of Mixed Culture During Hydrocarbon Degradation The crude oil was heterogeneous, hardly dissoluble, and unevenly dispersed [14]. The crude oil was diluted by a fourfold volume of diesel and then used as the sole carbon source for screening the degrader and the strains’ synergy analysis. The culture media was the same as the screening media, and cell growth was determined spectrophotometrically and valued in OD600. Four groups of experiments were performed: group 1, a control group without strains inoculated; group 2, Pseudomonas sp. XM-01 with initial OD600 value of 0.01 was inoculated; group 3, the culture media was inoculated by Acinetobacter sp. XM-02 with an identical initial OD600; and group 4, Pseudomonas sp. XM-01 and Acinetobacter sp. XM-02 were both inoculated with OD600 value of 0.1 separately; all the screening media were added with 0.5 % (v/v) alkane hydrocarbons as the sole carbon source and energy supply. Two milliliters of the media was fetched in the process of cultivation, and cell growth was conducted by colony forming unit (CFU) detected every 8 h. The culture was diluted to its 10−6, 10−7, 10−8, and 10−9 and 100 μL of the diluted solution was plated on LB agar plates which were incubated at 30 °C for 3 days. The two strains in mixed strains were distinguished by the different colonial morphology. All the plates were in quintuplicate. GC-MS Analysis of the Substrates Five samples of groups 3 and 4 were taken in the 5-day experiment. In particular, the culture media were internally marked by 10 % octane and then sampled, extracted, and analyzed by gas chromatography and mass spectrometer (GC-MS) system Agilent 6890 (Agilent Technologies Co. Ltd., USA) with HP-5MS capillary column (maximum temperature, 325 °C; rated length, 30.0 m; nominal inside diameter, 250.00 μm; nominal thickness of the film, 0.25 μm, the initial flow, 1.0 mL min−1; rated initial pressure, 7.63 psi; and an average flow speed, 36 cm s−1), the carrier gas was helium with a total flow of 24.0 mL min−1. The initial oven temperature was set as 50 °C for 0.5 min as the balance time and then heated up till 310 °C with a linear rate of 10 °C min−1. Mixed with 1,000 times the volume of n-hexane, a uniform and flowing phase was formed and 1 μL of the miscible liquids were injected. Characterization of the Biosurfactants Produced in the Mixed Cultures Surface tension of the four groups described above was measured every 8 h by the surface tension measurement with a full-automatic surface tensiometer JK99B (Zhongchen Digital Technology Equipment Co., Ltd., Shanghai, China). The biosurfactant produced after
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biodegradation by the mixed strains (group 4) was extracted as [16] described, identified, and analyzed by thin chromatography (TLC) and LC-MS spectrum [11]. Biodegradation of Crude Oil by the Single and Mixed Strains The crude oil biodegradation media were the same as the screening media, but the carbon source was replaced by 5 % (v/v) crude oil. The single strains were purified in tube, collected, and diluted by sterilized water to be dispersed as concentrated bacterial suspension. Group 1, Acinetobacter sp. XM-02 with an initial OD600 of 0.1 was inoculated into 100 mL media in 250 mL flasks; group 2, Acinetobacter sp. XM-02 and Pseudomonas sp. XM-01 with initial OD600 of 0.1 separately were both inoculated into the media. After being cultivated under a rotatation speed of 200 rpm at 30 °C for 10 days, the residual crude oil was extracted by methods described before [26]. All the experiments were carried out in quintuplicate. To validate the effect of the mixed strains, repeat crude oil biodegradation was carried out for a new round. The mixed strains after the first biodegradation were harvested by centrifugation and washed twice with sterile mineral culture. Cells were resuspended, and three groups of experiments for identifying the components transformation were redone. Group 1, cells harvested from crude oil biodegradation by the mixed strains in round one were inoculated into 100 mL fresh sterile mineral culture; group 2, cells from round one and washed Pseudomonas sp. XM-01 were inoculated into 100 mL fresh sterile mineral culture; group 3, cells from round one and washed Acinetobacter sp. XM-02 were inoculated into 100 mL fresh sterile mineral culture. Five milliliters of crude oil were added into these three cultures, final cell concentrations were all quantified to OD600 of 0.1, and re-cultivation was undertaken under a rotation speed of 200 rpm at 30 °C for 10 days. GC-MS for Crude Oil Crude oil in control and the residual oil after biodegradation of the pure and mixed strains were extracted and analyzed by GC-MS, as described above.
Results and Discussion Isolation and Characterization of the Strains Strains XM-01 and XM-02 were isolated from soil samples polluted by crude oil for years. XM-01 was gram negative, different in size, club shaped, with one apical seta on one end, and nonsporeformer by micro-examination; colonies cultivated for 3 days were brown, round, smooth, semi-humid, and serrated edges; this strain was arginine, citrate, urea, and oxidase positive, indicating the possibility of belonging to Pseudomonas sp. It was verified that XM-01 could produce rhamnolipid (a biosurfactant) with glycerol as sole carbon source. Strain XM-02 was proved to be a good degrader for alkane hydrocarbons as well as crude oil. It was gram negative, but the crystal violet was not easy to fade from its cell wall, with ability to make capsule, nonflagellum, nonsporeformer, arranged in pairs, small, and slender; colonies on agar media cultivated for 3 days were generally pale yellow, round, semitransparent, moist, and glossy with neat edge. The strain is urea positive and nitrate-reducing negative (Table 1), suggesting that it might be one of Acinetobacter (by checking on the nonfermentative bacteria biochemical appraisal code book).
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Table 1 Physiology and biochemistry appraisals of the strain XM-01 and XM-02
“+” positive, “−” negative
Item
XM-01
XM-02
Arginine
+
−
Ornithine Lysine
− −
− −
Citrate
+
−
Nitrate reducing
−
−
Urea
+
+
Aesculin
−
−
Imdole
−
−
ONPG
−
−
Sucrose Maltose
− −
− −
Xylose
−
−
Hydrogen sulfide
−
−
Glucose
+
−
Oxidase
+
−
16S rDNA sequences analysis showed XM-01 (with the GenBank accession number being JX093569) belong to Pseudomonas sp and XM-02 (GenBank accession number, JX093571) belongs to Acinetobacter sp (as shown in Fig. 1) Alkane Hydrocarbons Biodegradation Changes in Bacterial Species Composition in Mixed Culture The four groups of experiments were carried out during 5 days cultivation in alkane hydrocarbons; cell concentrations and the strains’ composition of the mixed strains were measured every 8 h (as shown in Fig. 2). For group 1, there was no bacteria inoculated and the OD600 remained zero till the end; for group 2, cells grew a little bit and then the growth was inhibited at the 16th hour. The culture remained clear but turned a bit light green, and the viable cells stayed invariable till the end, showing that the Pseudomonas sp. XM-01 entered a rapid growth period by utilizing the biodegradable components in alkane hydrocarbons; for group 3, cells entered the logarithmic period and grew rapidly after being delayed for 16 h. The maximum OD600 value was above 5.0 at the 80th hour, proving its effective biodegradation of the alkane hydrocarbons. The culture solution turned light brown since the 32th hour and then became increasingly darker. For group 4, cell grew slowly in the first 16 h, which was almost the same as group 3. Afterward, cell concentration of group 4 increased more sharply than group 3, and the color turned light brown at the 24th hour, 12 h earlier than Acinetobacter sp alone. At the 40th hour, cells began to grow gently with a mild increase till the 64th hour, and the color turned darker in the process. Another rapid cell growth period was observed after the 64th hour, and the color of the media turned greener, and bubbles started to appear. Based on these results, a model was proposed: Acinetobacter sp. XM-02 in group 3 grew well and degraded alkane hydrocarbons effectively, whereas the growth pattern of Pseudomonas sp. XM-01 and Acinetobacter sp. XM-02 in group 4 was shown to be more interesting: Acinetobacter sp. XM-02 (the degrader of alkane hydrocarbons) grew rapidly with sufficient alkane hydrocarbons at the beginning, and Pseudomonas sp. XM-01 (the biosurfactant
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Fig. 1 Phylogenetic trees of Pseudomonas sp. XM-01 and Acinetobacter sp. XM-02
producer) began to increase with the intermediates metabolized by the biodegradation of alkane hydrocarbons (after the 16th hour). With the prosperity of Pseudomonas sp. XM-01, the degrader was restrained gradually and the color of the media was changed by the rapid growth of Pseudomonas sp. XM-01 (from lighter brown to darker green). After the 64th hour,
Fig. 2 Cell growth of the four groups in alkane hydrocarbon biodegradation
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faster growing Pseudomonas sp. XM-01 began to dominate the mixed strains and a secondary growth came into being. With the changing culture condition, it became more suitable for Pseudomonas sp. XM-01 and more restraining for Acinetobacter sp. XM-02, leading to the decline of the degrader and the booming of the biosurfactant producer. As the growth of Pseudomonas sp. XM-01 stabilized, biosurfactant (identified as glycolipids, results are shown in “Identification of Biosurfactant Produced”) began to synthetize, causing the reduction in surface tension and decrease in content of heavy components in alkane hydrocarbons. The conclusion could be verified with the appearance of adding alkane hydrocarbons into the media of group 4 after 120 h, and there was no biodegradation of alkane hydrocarbons observed (meaning the available cells were biosurfactant producer and no degrader left). While with a supply of Acinetobacter sp. XM-02, alkane hydrocarbons was once again degradable and the color of the mixed culture (green present) turned brown and then green again, the same as the appearance of group 4 previously. This verification consolidated the conclusion of the interaction between the two strains in alkane hydrocarbon biodegradation. As a speculative conclusion on the changing pattern of the mixed strains in biodegradation was drawn, it is necessary to study the transformation of cell composition in the mixed strains. The two strains behaved dissimilarly in the process of alkane hydrocarbon co-treating based on CFU counts shown in Fig. 3. The development of Acinetobacter sp. XM-02 presents a common microorganism growth curve with four roughly legible growth periods. Whereas, Pseudomonas sp. XM-01 keeps booming since the 24th hour and peaks at the 104th hour. As shown in Fig. 3, the study of the two strains’ cell growth pattern was discovered: Acinetobacter sp. XM-02 began to boom while Pseudomonas sp. XM-01 grew next to nothing. The culture was still clear with few cells and intermediates in the first 16 h. With more alkane hydrocarbons biodegraded, Pseudomonas sp. XM-01 started to play a more important role in the biodegradation process, as metabolic activity of Pseudomonas sp. XM-01 gets stronger, the secondary metabolism of Pseudomonas sp. XM-01 would commence and the secondary metabolite, rhamnolipid, was produced. Rhamnolipid, a kind of glycolipida biosurfactant, could make the alkane
Fig. 3 Growth curve of available cells in the mixed cultures
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hydrocarbons more biodegradable. Pseudomonas sp. XM-01 and its productions favor the biodegradation of alkane hydrocarbons in this stage. With the decline of alkane hydrocarbons and fast-growing Pseudomonas sp. XM-01, the predominance of Acinetobacter sp. XM-02 was challenged by Pseudomonas sp. XM-01 which kept flourishing with the intermediates as its substrates, resulting in more biosurfactant produced; however, the available cells of Acinetobacter sp. XM-02 were of low metabolic activity and declined gradually, and the color of the media was changing from brown to green (the characteristic color of Pseudomonas sp. XM-01 in culture). Finally, the available cells of Acinetobacter sp. XM-02 decreased to zero and Pseudomonas sp. XM-01 began to slow down, at this phase, the media color was green and the surface tension of the media was reduced to 28.6×10−3 N/m, generating a lot of bubbles. To verify the consortium composition changing above, 0.5 mL of fresh alkane hydrocarbons was added into the culture media of group 4 after 120 h, and it showed a negative degradation of the substrates. However, when the media was further added with a batch of fresh Acinetobacter sp. XM-02, growing observation of cells and biodegradation of the alkane hydrocarbons showed up again. This indicates: (1) the only one that was left behind of group 4 was Pseudomonas sp. XM01 at the end of the experiment; (2) the tolerance nature but not the degradation ability to alkane hydrocarbons by Pseudomonas sp. XM-01; and (3) the importance of alkane hydrocarbons to the degrader. Thus, a hypothetical model could be made: the main factor for the decrease even extinction of available Acinetobacter sp. XM-02 was the lack of substrates: alkane hydrocarbons, but not the toxicity of mass-growing Pseudomonas sp. XM-01. Identification of Biosurfactant Produced To state the biosurfactant production in the biodegradation of alkane hydrocarbons, surface tensions of the four groups were measured (as shown in Fig. 4). No changing of surface tension was observed in group 1. As for group 2, the surface tension dropped a little to 64.7× 10−3 N/m till the 56th hour and remained steady afterwards, meaning a slight growth of Pseudomonas sp. XM-01 (see Fig. 2). Acinetobacter sp. XM-02 could degrade the vast majority of alkane hydrocarbons rapidly in 5 days; the surface tension of the media dropped slowly and smoothly to an indistinctive terminal value of 51.4×10−3 N/m. All the three groups produced no bubbles in the surface of the media. In group 4, surface tension of the media dropped dramatically since the 8th hour and became stable at the 96th hour. It dropped to 28.6×10−3 N/m, which was rarely found in biodegradation of alkane hydrocarbons. Crude biosurfactant of group 4 was collected and identified as an affiliation to glycolipids. Further investigation of the glycolipids by TLC and LC-MS spectrum confirmed it was rhamnolipid. Previously reported substrates used for biosurfactant fermentations by Pseudomonas sp. were mannitol [16], soybean oil, peanut oil [23], or glucose [3]. There were few reports on the production of biosurfactant by cultivation in the media with diesel and the crude oil as the sole carbon source [16, 23]. Generation of rhamnolipids with alkane hydrocarbons as substrates was uncommon to Pseudomonas sp. and a great part was taken by the degrader at this process. One possible reason could be that some metabolic intermediate substances was utilized by Pseudomonas sp. XM-01, and the mixed oil–water phases promoted the composition changes in cell wall and the biosynthesis of rhamnolipid as its stress reaction to oil. Alkane Hydrocarbons Biodegradation Analysis Alkane hydrocarbons degraded by the pure and the mixed strains, namely groups 3 and 4 were sampled and extracted by n-hexane, analyzed by GC-MS. Examination of alkane hydrocarbons was carried out by the integral area comparison out of gas chromatogram. The
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Fig. 4 Surface tension of the four groups in alkane hydrocarbon biodegradation
degradation rate of alkane hydrocarbons were 89.35 and 97.41 % in groups 3 and 4, respectively. To investigate the biodegradation kinetically, cultures were sampled when cell concentration changed greatly was chosen as its potential transformation and indication of the tremendous changing in substrate. As shown in Fig. 5, GC-MS analysis of the extractions at each sampling point indicated the reducing tendency of alkane hydrocarbons, as well as its variations in different components. These results show that biodegradation of alkane hydrocarbons by Acinetobacter sp. XM-02 was carried out steadily; however, the biodegradation by the mixed strains was different, more than 80 % of the substrates were biodegraded by the 80th hour, and the process went slow thereafter. This phenomenon corresponded with the consortium composition changing of the mixed strains when the available cells of Acinetobacter sp. XM-02 began to decrease dramatically and Pseudomonas sp. XM-01 kept soaring and producing biosurfactants. Another conclusion which had been reported repeatedly could be confirmed by the GC-MS results: the biodegradation of short-chain alkane hydrocarbons was prioritized and the heavy ones remained gradually decomposable [25]. Compositional variation of the two degradation patterns at some time points was studied. At the 24th hour, the degradation rate of C17 was 31.42 % by Acinetobacter sp. XM-02. However, it turned to 24.01 % when degraded by the mixed strains (as shown in Fig. 6), but this trend was broken as the degradation rate by the mixed strains increased sharper and exceeded the pure strain at the 80th hour. Finally, the degradation rates of the pure and mixed strains were 88.85 and 93.12 %, respectively. Other components exhibited the same regular pattern. It was during the 48th, 104th, and 120th hours when the biodegradation rates of C11, C23, and C30 that group 4 exceeded group 3, respectively. In addition, nearly all components were more extensively degraded by the mixed strains than the pure ones at the end of the experiment. Disintegration effects of the longer-chain hydrocarbons biodegraded by the mixed strains were faster, and the increasing tendency went sharper after the 80th hour. This anastomosis indicated that the availability for alkane hydrocarbons biodegradation was improved when exposed to the rising concentrations of biosurfactant. As shown in Fig. 6, peak
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Fig. 5 GC-MS analysis of hydrocarbon biodegradation by the single and mixed strains
areas of C11 were degraded by the mixed strains at the 24th hour, and the hours after which were all less than the peak area degraded by the pure strain. Components of alkane hydrocarbons showed the same degradation pattern as the C11. As the components of alkane hydrocarbons got longer, more time was needed for the over-biodegradation by the mixed strains. It indicated that heavier hydrocarbons showed a much greater resistance to the surface-active agent than the light ones. Crude Oil Biodegradation After degradation for 10 days, 5.13±2.05 % crude oil was lost by abiotic factors, 74.32± 4.09 % of crude oil was biodegraded by Acinetobacter sp. XM-02 alone, and 87.29±2.41 % of crude oil was degraded by the mixed strains, 12.83 % higher than the pure strain (as shown in Fig. 7). The degradable ability of crude oil was remarkably enhanced by the synergy between Acinetobacter sp. XM-02 and Pseudomonas sp. XM-01. The promotion of crude oil biodegradation by mixed culture was of great utility value together with nutriment addition. A batch experiment was conducted as described in “Biodegradation of Crude Oil by the Single and Mixed Strains” to study how the two strains in the mixed culture acted with each other. Just as expected, biodegradation rates of crude oil in 10 days showed great divergences for these three groups: 47.31, 49.26, and 88.54 % of crude oil was decomposed in groups 1, 2, and 3, respectively. Biodegradation rates in groups 1 and 2 were much lower than the first round, indicating the constituent of the mixed strains had already changed after 10 days biodegradation in round one; results of groups 1 and 2 were in a little difference which demonstrated little affection on the addition of biosurfactant-producing strain after round one. However, biodegradation rate in group 3 was much higher than groups 1 and 2, which showed
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Fig. 6 GC-MS analysis of C11/C14/C17/C23 biodegradation by the single and mixed strains
that the degrading strain added in round 2 played a significant role in the mixed culture. These experimental results verified that the composition of the mixed strains after round one had been changed. The amount of the degrader was reduced but not vanished, which is why more
Fig. 7 Crude oil biodegradation by the single and mixed strains
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than 45 % of the crude oil was still degraded in group 1. However, the biomass and activity of the degrader were inhibited by Pseudomonas sp. XM-01 at the end of the 10-day experiment, so the degradation rate was far lower in the second round than the first round. This reduction was due to the recession of the degrader but not the biosurfactant-producing strain, which was also confirmed by the low degradation rate in group 2, whose composition was similar to group 1 and the significant enhancement of group 3 (group 1 amended with Acinetobacter sp. XM-02), whose colony structure was reconstructed to its similarity with the first-round experiment. The experiments above confirmed that the degrader strain declined but the biosurfactant-producing strain flourished in the process of crude oil biodegradation, which was the same as the findings drawn in “Changes in Bacterial Species Composition in Mixed Culture.” The actual system is more complex; this article only has carried on the preliminary work in laboratory. However, the research provided us with a meaningful idea for enhancing biodegradation of crude oil and its derivatives: by designing the program rationally and controlling the local consortium composition dynamically with nutriment addition to increase the degradability of crude oil, where more research needs to be done in the future. There are plenty of different hydrocarbon compounds in crude oil, leading different biodegradation patterns for different compounds. Basically, the biodegradable order of components in crude oil: n-alkanes>i-alkanes>low molecular weight aromatics>high molecular weight aromatics and cyclic alkanes [10, 27]. Crude oil before and after biodegradation were sampled and analyzed by GC-MS (the result is shown in Fig. 8). Short-chain hydrocarbons were nearly all degraded, and the long-chain ones remained hard to decompose. Interestingly, less heavy components of crude oil biodegraded by the mixed strains were retained than those biodegraded by Acinetobacter sp. XM-02 alone. This condition could be explained by the function of Pseudomonas sp. XM-01 and the biosurfactant produced by it, which was also a confirmation to the conclusion drawn in Fig. 5.
Conclusions Two strains isolated from areas polluted by crude oil were identified and nominated as Pseudomonas sp. XM-01 and Acinetobacter sp. XM-02. Pseudomonas sp. XM-01 with a biosurafactant-producing ability (identified as rhamnolipid) could tolerate alkane hydrocarbons but could not degrade them. Acinetobacter sp. XM-02 was the degrader and it could reduce 74.32±4.09 % of crude oil in 10 days. Enhancement of crude oil biodegraded was achieved by the mixed strains at a biodegradation rate of
Fig. 8 GC-MS analysis of crude oil biodegradation by the single and mixed strains
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87.29±2.41 %. Moreover, it was found that almost all of the components in alkane hydrocarbons obtained higher degradation rates by the mixed strain than by the single strain. The effect of the biosurfactant produced on the enhancement of short-chain alkane hydrocarbons biodegradation was more remarkable. Studies on the composition changing of the mixed strains in alkane hydrocarbons biodegradation were carried out, indicating that growth of Pseudomonas sp. XM-01 was firstly inhibited and then boosted by the metabolites degraded by Acinetobacter sp. XM-02; meanwhile, biosurfactant identified as rhamnolipid produced by Pseudomonas sp. XM-01 increased the degradation of alkane hydrocarbons. Finally, with the fast growth of Pseudomonas sp. XM-01 and the decrease of available substrates, the degrader was restrained. These observations were in accordance with the crude oil biodegradation in the two round experiments. These results indicated the enhancing ability of the biosurfactant-producing strain in crude oil biodegradation. In conclusion, with the mixed bacterial cultures isolated and new strategies proposed in this study, promising enhancement of crude oil biodegradation was achieved, which would be a potential way for bioremediation of crude oilcontaminated soil and water. Acknowledgments The authors wish to acknowledge the financial support provided by the National Key Basic Research Program of China (No. 2014CB745100), the Natural Science Foundation of Tianjin, the Seed Foundation of Tianjin University, the Program of Introducing Talents of Discipline to Universities (No. B06006), the Science and Technology Project from China National Offshore Oil Corporation (CNOOC-KJ 125 ZDXM 25JAB NFCY 2013-01) and the Science and Technology Project of Tianjin Binhai New Area (2012-XJR23017).
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Appl Biochem Biotechnol 10. Leahy, J. G., & Colwell, R. R. (1990). Microbial degradation of hydrocarbons in the environment. Microbiological Reviews, 54(3), 305–315. 11. Lotfabad, T. B., Abassi, H., Ahmadkhaniha, R., Roostaazad, R., Masoomi, F., Zahiri, H. S., Ahmadian, G., Vali, H., & Noghabi, K. A. (2010). Structural characterization of a rhamnolipid-type biosurfactant produced by Pseudomonas aeruginosa MR01: enhancement of di-rhamnolipid proportion using gamma irradiation. Colloids and Surfaces B-Biointerfaces, 81(2), 397–405. 12. Luckarift, H. R., Sizemore, S. R., Farrington, K. E., Fulmer, P. A., Biffinger, J. C., Nadeau, L. J., & Johnson, G. R. (2011). Biodegradation of medium chain hydrocarbons by Acinetobacter venetianus 2AW immobilized to hair-based adsorbent mats. Biotechnology Progress, 27(6), 1580–1587. 13. Mehdi, H., & Giti, E. (2008). Investigation of alkane biodegradation using the microtiter plate method and correlation between biofilm formation, biosurfactant production and crude oil biodegradation. International Biodeterioration & Biodegradation, 62(2), 170–178. 14. Mishra, S., Sarma, P. M., & Lal, B. (2004). Crude oil degradation efficiency of a recombinant Acinetobacter baumannii strain and its survival in crude oil-contaminated soil microcosm. Fems Microbiology Letters, 235(2), 323–331. 15. Mohanty, S., & Mukherji, S. (2012). Alteration in cell surface properties of Burkholderia spp. during surfactant-aided biodegradation of petroleum hydrocarbons. Applied Microbiology and Biotechnology, 94(1), 193–204. 16. Nayak, A. S., Vijaykumar, M. H., & Karegoudar, T. B. (2009). Characterization of biosurfactant produced by Pseudoxanthomonas sp PNK-04 and its application in bioremediation. International Biodeterioration & Biodegradation, 63(1), 73–79. 17. Obayori, U. S., Adebusoye, S. A., Adewale, A. O., Oyetibo, G. O., Oluyemi, O. O., Amokun, R. A., & Ilori, M. O. (2009). Differential degradation of crude oil (Bonny Light) by four Pseudomonas strains. Journal of Environmental Sciences China, 21(2), 243–248. 18. Okieimen, C. O., & Okieimen, F. E. (2002). Effect of natural rubber processing sludge on the degradation of crude oil hydrocarbons in soil. Bioresource Technology, 82(1), 95–97. 19. Ollis, D. (1992). Slick solution for oil spills. Nature, 358, 453–454. 20. Salam, L. B., Obayori, O. S., Akashoro, O. S., & Okogie, G. O. (2011). Biodegradation of bonny light crude oil by bacteria isolated from contaminated soil. International Journal of Agriculture and Biology, 13(2), 245–250. 21. Sei, K., Sugimoto, Y., Mori, K., Maki, H., & Kohno, T. (2003). Monitoring of alkane-degrading bacteria in a sea-water microcosm during crude oil degradation by polymerase chain reaction based on alkane-catabolic genes. Environmental Microbiology, 5(6), 517–522. 22. Siegmund, I., & Wagner, F. (1991). New method for detecting rhamnolipids excreted by Pseudomonas species during growth on mineral agar. Biotechnology Techniques, 5(4), 265–268. 23. Thavasi, R., Jayalakshmi, S., & Banat, I. M. (2011). Application of biosurfactant produced from peanut oil cake by Lactobacillus delbrueckii in biodegradation of crude oil. Bioresource Technology, 102(3), 3366– 3372. 24. Thavasi, R., Jayalakshmi, S., & Banat, I. M. (2011). Effect of biosurfactant and fertilizer on biodegradation of crude oil by marine isolates of Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa. Bioresource Technology, 102(2), 772–778. 25. Vyas, T. K., & Dave, B. P. (2007). Effect of crude oil concentrations, temperature and pH on growth and degradation of crude oil by marine bacteria. Indian Journal of Marine Sciences, 36(1), 76–85. 26. Wang, X. B., Chi, C. Q., Nie, Y., Tang, Y. Q., Tan, Y., Wu, G., & Wu, X. L. (2011). Degradation of petroleum hydrocarbons (C6-C40) and crude oil by a novel Dietzia strain. Bioresource Technology, 102(17), 7755–7761. 27. Wang, Z., & Fingas, M. F. (2003). Development of oil hydrocarbon fingerprinting and identification techniques. Marine Pollution Bulletin, 47(9), 423–452. 28. Yerima, M. B., Balogun, A. A., Farouq, A. A., & Muhammad, S. (2009). Laboratory based degradation of light crude oil by aquatic phycomycetes. African Journal of Biotechnology, 8(16), 3851–3853.
Enhanced Biodegradation of Alkane Hydrocarbons and Crude Oil by Mixed Strains and Bacterial Community Analysis Yu Chen & Chen Li & Zhengxi Zhou & Jianping Wen & Xueyi You & Youzhi Mao & Chunzhe Lu & Guangxin Huo & Xiaoqiang Jia
Received: 12 October 2013 / Accepted: 3 February 2014 # Springer Science+Business Media New York 2014
Abstract In this study, two strains, Acinetobacter sp. XM-02 and Pseudomonas sp. XM-01, were isolated from soil samples polluted by crude oil at Bohai offshore. The former one could degrade alkane hydrocarbons (crude oil and diesel, 1:4 (v/v)) and crude oil efficiently; the latter one failed to grow on alkane hydrocarbons but could produce rhamnolipid (a biosurfactant) with glycerol as sole carbon source. Compared with pure culture, mixed culture of the two strains showed higher capability in degrading alkane hydrocarbons and crude oil of which degradation rate were increased from 89.35 and 74.32±4.09 to 97.41 and 87.29±2.41 %, respectively. In the mixed culture, Acinetobacter sp. XM-02 grew fast with sufficient carbon source and produced intermediates which were subsequently utilized for the growth of Pseudomonas sp. XM-01 and then, rhamnolipid was produced by Pseudomonas sp. XM-01. Till the end of the process, Acinetobacter sp. XM-02 was inhibited by the rapid growth of Pseudomonas sp. XM-01. In addition, alkane hydrocarbon degradation rate of the mixed culture increased by 8.06 to 97.41 % compared with 87.29 % of the pure culture. The surface tension of medium dropping from 73.2×10−3 to 28.6×10−3 N/m. Based on newly found cooperation between the degrader and the coworking strain, rational Y. Chen : X. You Department of Environmental Science, School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, People’s Republic of China Y. Chen China Offshore Environmental Services Ltd., Tianjin 300452, People’s Republic of China C. Li : Z. Zhou : J. Wen : Y. Mao : C. Lu : G. Huo : X. Jia Department of Biological Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People’s Republic of China J. Wen : X. Jia Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin 300072, People’s Republic of China J. Wen : X. Jia (*) Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, People’s Republic of China e-mail: [email protected]
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investigations and optimal strategies to alkane hydrocarbons biodegradation were utilized for enhancing crude oil biodegradation. Keywords Biodegradation . Crude oil . Chromatography . Microbial growth . Biosurfactant . Waste treatment
Introduction Oil spills happened constantly, posing critical hazards for ecological system and mankind [9]. It is still a reinforcing challenge to eliminate oil pollution effectively and eco-friendly [21]. Governments, companies, and research organizations have been under an increasing pressure to develop clean and environmental technologies for treatment of the pollutants in the wake of environmental consciousness [15]. The growing environment crisis caused by oil pollution spurs focused efforts into developing effective and environment-friendly technologies for the treatment of the pollutants. Physical treatments cannot remove the pollutants completely, and the main disadvantage is too costly [19], Whereas, chemical treatments were likely to cause a second pollution to the environment due to the unexpected release of artificial toxic materials into the environment [18]. Therefore, the safe and effective biotreatment methods of oil pollution have attracted extensive attention [28]. Many strains growing on crude oil as the sole carbon source have been isolated for the biodegradation of oil [21], such as Rhodococcus [4], Acinetobacter [12], Achromobacter [2], Dietzia [5], Pseudomonas [20], etc. Among them, Acinetobacter has shown high performance for the biodegradation of crude oil [9, 14]. Acinetobacter calcoaceticus could degrade long carbon-chain n-alkane (contained more than 33 carbon atoms) and even the branched chain alkane but failed to grow on cycloalkanes [1]. Acinetobucter calcoaceticus and Alcaligenes odorans cultured in combination were more effective in degrading crude oil than their pure cultures [9]. Moreover, some strains, many of which were Pseudomonas sp., could produce biosurfactant with proper nutrients available [8, 16]. Additionally, biosurfactant, usually including rhamnolipid, saponin, and alkyl polyglucosides could overcome the difficulties in oil–cell surface contact to accelerate the biodegradation of crude oil and its production by decreasing the surface tension of medium [13, 24]. However, most studies focused on the fermentation and purification of biosurfactants with nutrient medium as well as effects of biosurfactants on crude oil or alkane hydrocarbon biodegradation. Little is known about the application and interaction mechanisms of mixed culture of biosurfactant producers and oil degraders [8]. Strategies to enhance biodegradation of crude oil, including amendments of nutrient and addition of biosurfactant, were prohibitive, and adding these exotic substances might produce additional stress, being a restrain of the degraders due to the booming growth of the other strains. Furthermore, utilization of single strain in crude oil biodegradation was often limited by complex realistic conditions. Biosurfactant was of great use in the biodegradation and exploitation of crude oil, meaning it has a dual role in improving the environmental and economic benefits, yet it can easily be washed away by agitation. To solve this problem, a suggestion was proposed that the biosurfactant could be mixed with nutrients to form particulate fertilizers and applied to the pollutant area or the oil well [23]. However, biosurfactants in fertilizer would be of little use in the absence of water or would be dissolved and washed away in aqueous environment. Another brilliant thought was to functionalize composite material combined with Acinetobacter venetianus 2AW (alkane-degrading bacteria), and biodegradation of alkane was facilitated by the immobilized bacterial population [12]. To enhance the effect of the degraders and the biosurfactant-producing strains, a possible alternative method is to immobilize the mixed strains
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containing the degraders and some biosurfactant-producing strains, forming indissolvable tiny particles and fertilized them into polluted areas. Thus, leaked crude oil was easy to be degraded by improving and stabilizing the local microorganism community. As most biosurfactants are secondary metabolites, and their production was activated by depletion of nutrients [3] and could be restrained by crude oil, the importance of producing biosurfactant in situ with no supplemented nutrients but crude oil was obviously revealed. Complete biodegradation of crude oil pollutants commonly depends on the synergies of bacteria in environment [6], and the mixed strains showed a greater capability of oil biodegradation compared with the pure ones [7]. Great efforts have been made in the preference of mixed degraders to the single strains, yet little is available for the mutualism between the degrader and nondegraders (functional bacteria) in oil biotreatment. Particularly, the relationships of the degraders and biosurfactant-producing strains are still a critical issue on crude oil biodegradation, and the mechanism requires a deeper insight. As for future study on in situ crude oil bioremediation, it became more valuable to investigate the interaction among strains with oil degrading and the coexisted ones. In this paper, a method of co-culturing biosurfactant-producing strain and oil degrader was developed to enhance the biodegradation rate of alkane hydrocarbons and crude oil. With investigations on consortium composition changing during biodegradation of alkane hydrocarbons, the possible reasons for these changes and the strategies to improve biodegradation were proposed.
Methods Chemicals and Reagents The oil-contaminated soil was sampled from the Boxi Offshore Oil Field, Tianjin, China. No. 0 diesel oil was purchased from a local service station of Sinopec Group and the crude oil was donated by Boxi Offshore Oil Field of China National Offshore Oil Corporation (CNOOC). Alkane hydrocarbons were a mixture of crude oil and No. 0 diesel with a volume ratio of 1:4. All other chemicals used in this study were of analytical grade. Isolation and Identification of the Strains The two bacterial strains were enriched and isolated from the soil samples. The enrichment medium comprised NaNO3, 2 g; K2HPO4·3H2O, 0.5 g; KH2PO4, 1 g; NaCl, 0.5 g; MgSO4, 0.1 g; and 10 mL trace element solution in 1 L distilled water. The trace element solution contained 1.2 % Na2EDTA·H2O, 0.2 % NaOH, 0.04 % MnSO4 4H2O, 0.04 % ZnSO4 7H2O, 0.05 % concentrated H2SO4, 0.01 % Na2M0O4·2H2O, 0.2 % FeSO4·7H2O, 0.01 % CuSO4·5H2O, 0.1 % CaCl2, and 1 % Na2SO4. Adjusting the pH value to 7.2, 100 mL of the screening media was fractionally fed into 250-mL shake flasks. Five grams soil samples and 0.5 mL alkane hydrocarbons were amended into the sterilized screening media, which were afterwards cultivated at 30 °C in a shaker with agitation of 200 rpm for 5 days. Onemilliliter culture solution was transferred into another fresh culture solution with 0.5 mL alkane hydrocarbons, and five repeats were carried out. Then, the strains were purified on agar plates with enrichment media and 1.8 % (mass ratio) agar powder. One strain designated as XM-02 was selected for its good biodegradation ability of alkane hydrocarbons. Meanwhile, biosurfactant-producing strains were isolated on blue agar plate as described previously [22], and one strain named XM-01 was selected as its greatest performance in surface tension reduction in cultivation.
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The morphological, physiological, and phylogenetic properties of the strains were assayed based on 16S rDNA analysis, demonstrated as foregoing documents [17]. Analysis of 16S rDNA sequences was performed under standard reaction conditions with Taq DNA polymerase. Both primers synthesis (27F: 5′-AGA GTT TGA TCC TGG CTC AG-3′ and 1492R: 5′GGT TAC CTT GTT ACG ACT T-3′) and gene sequencing were performed by Beijing Genomics Institute (China). Identification and homology search were carried out with BLAST program at the National Center for Biotechnology Information (NCBI) Website (http://blast. ncbi.nlm.nih.gov). GenBank accession numbers for the 16S rDNA sequences were obtained, and phylogenetic trees of the strains were established by MEGA 5.05 with NJ method. Physiological and biochemical appraisals of the strains were conducted with biochemical appraisal kits (nonfermentative bacteria biochemical coding assessor GYZ-15n, Microbial Reagent Co., Ltd., Hangzhou, China). Analysis of Composition of Mixed Culture During Hydrocarbon Degradation The crude oil was heterogeneous, hardly dissoluble, and unevenly dispersed [14]. The crude oil was diluted by a fourfold volume of diesel and then used as the sole carbon source for screening the degrader and the strains’ synergy analysis. The culture media was the same as the screening media, and cell growth was determined spectrophotometrically and valued in OD600. Four groups of experiments were performed: group 1, a control group without strains inoculated; group 2, Pseudomonas sp. XM-01 with initial OD600 value of 0.01 was inoculated; group 3, the culture media was inoculated by Acinetobacter sp. XM-02 with an identical initial OD600; and group 4, Pseudomonas sp. XM-01 and Acinetobacter sp. XM-02 were both inoculated with OD600 value of 0.1 separately; all the screening media were added with 0.5 % (v/v) alkane hydrocarbons as the sole carbon source and energy supply. Two milliliters of the media was fetched in the process of cultivation, and cell growth was conducted by colony forming unit (CFU) detected every 8 h. The culture was diluted to its 10−6, 10−7, 10−8, and 10−9 and 100 μL of the diluted solution was plated on LB agar plates which were incubated at 30 °C for 3 days. The two strains in mixed strains were distinguished by the different colonial morphology. All the plates were in quintuplicate. GC-MS Analysis of the Substrates Five samples of groups 3 and 4 were taken in the 5-day experiment. In particular, the culture media were internally marked by 10 % octane and then sampled, extracted, and analyzed by gas chromatography and mass spectrometer (GC-MS) system Agilent 6890 (Agilent Technologies Co. Ltd., USA) with HP-5MS capillary column (maximum temperature, 325 °C; rated length, 30.0 m; nominal inside diameter, 250.00 μm; nominal thickness of the film, 0.25 μm, the initial flow, 1.0 mL min−1; rated initial pressure, 7.63 psi; and an average flow speed, 36 cm s−1), the carrier gas was helium with a total flow of 24.0 mL min−1. The initial oven temperature was set as 50 °C for 0.5 min as the balance time and then heated up till 310 °C with a linear rate of 10 °C min−1. Mixed with 1,000 times the volume of n-hexane, a uniform and flowing phase was formed and 1 μL of the miscible liquids were injected. Characterization of the Biosurfactants Produced in the Mixed Cultures Surface tension of the four groups described above was measured every 8 h by the surface tension measurement with a full-automatic surface tensiometer JK99B (Zhongchen Digital Technology Equipment Co., Ltd., Shanghai, China). The biosurfactant produced after
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biodegradation by the mixed strains (group 4) was extracted as [16] described, identified, and analyzed by thin chromatography (TLC) and LC-MS spectrum [11]. Biodegradation of Crude Oil by the Single and Mixed Strains The crude oil biodegradation media were the same as the screening media, but the carbon source was replaced by 5 % (v/v) crude oil. The single strains were purified in tube, collected, and diluted by sterilized water to be dispersed as concentrated bacterial suspension. Group 1, Acinetobacter sp. XM-02 with an initial OD600 of 0.1 was inoculated into 100 mL media in 250 mL flasks; group 2, Acinetobacter sp. XM-02 and Pseudomonas sp. XM-01 with initial OD600 of 0.1 separately were both inoculated into the media. After being cultivated under a rotatation speed of 200 rpm at 30 °C for 10 days, the residual crude oil was extracted by methods described before [26]. All the experiments were carried out in quintuplicate. To validate the effect of the mixed strains, repeat crude oil biodegradation was carried out for a new round. The mixed strains after the first biodegradation were harvested by centrifugation and washed twice with sterile mineral culture. Cells were resuspended, and three groups of experiments for identifying the components transformation were redone. Group 1, cells harvested from crude oil biodegradation by the mixed strains in round one were inoculated into 100 mL fresh sterile mineral culture; group 2, cells from round one and washed Pseudomonas sp. XM-01 were inoculated into 100 mL fresh sterile mineral culture; group 3, cells from round one and washed Acinetobacter sp. XM-02 were inoculated into 100 mL fresh sterile mineral culture. Five milliliters of crude oil were added into these three cultures, final cell concentrations were all quantified to OD600 of 0.1, and re-cultivation was undertaken under a rotation speed of 200 rpm at 30 °C for 10 days. GC-MS for Crude Oil Crude oil in control and the residual oil after biodegradation of the pure and mixed strains were extracted and analyzed by GC-MS, as described above.
Results and Discussion Isolation and Characterization of the Strains Strains XM-01 and XM-02 were isolated from soil samples polluted by crude oil for years. XM-01 was gram negative, different in size, club shaped, with one apical seta on one end, and nonsporeformer by micro-examination; colonies cultivated for 3 days were brown, round, smooth, semi-humid, and serrated edges; this strain was arginine, citrate, urea, and oxidase positive, indicating the possibility of belonging to Pseudomonas sp. It was verified that XM-01 could produce rhamnolipid (a biosurfactant) with glycerol as sole carbon source. Strain XM-02 was proved to be a good degrader for alkane hydrocarbons as well as crude oil. It was gram negative, but the crystal violet was not easy to fade from its cell wall, with ability to make capsule, nonflagellum, nonsporeformer, arranged in pairs, small, and slender; colonies on agar media cultivated for 3 days were generally pale yellow, round, semitransparent, moist, and glossy with neat edge. The strain is urea positive and nitrate-reducing negative (Table 1), suggesting that it might be one of Acinetobacter (by checking on the nonfermentative bacteria biochemical appraisal code book).
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Table 1 Physiology and biochemistry appraisals of the strain XM-01 and XM-02
“+” positive, “−” negative
Item
XM-01
XM-02
Arginine
+
−
Ornithine Lysine
− −
− −
Citrate
+
−
Nitrate reducing
−
−
Urea
+
+
Aesculin
−
−
Imdole
−
−
ONPG
−
−
Sucrose Maltose
− −
− −
Xylose
−
−
Hydrogen sulfide
−
−
Glucose
+
−
Oxidase
+
−
16S rDNA sequences analysis showed XM-01 (with the GenBank accession number being JX093569) belong to Pseudomonas sp and XM-02 (GenBank accession number, JX093571) belongs to Acinetobacter sp (as shown in Fig. 1) Alkane Hydrocarbons Biodegradation Changes in Bacterial Species Composition in Mixed Culture The four groups of experiments were carried out during 5 days cultivation in alkane hydrocarbons; cell concentrations and the strains’ composition of the mixed strains were measured every 8 h (as shown in Fig. 2). For group 1, there was no bacteria inoculated and the OD600 remained zero till the end; for group 2, cells grew a little bit and then the growth was inhibited at the 16th hour. The culture remained clear but turned a bit light green, and the viable cells stayed invariable till the end, showing that the Pseudomonas sp. XM-01 entered a rapid growth period by utilizing the biodegradable components in alkane hydrocarbons; for group 3, cells entered the logarithmic period and grew rapidly after being delayed for 16 h. The maximum OD600 value was above 5.0 at the 80th hour, proving its effective biodegradation of the alkane hydrocarbons. The culture solution turned light brown since the 32th hour and then became increasingly darker. For group 4, cell grew slowly in the first 16 h, which was almost the same as group 3. Afterward, cell concentration of group 4 increased more sharply than group 3, and the color turned light brown at the 24th hour, 12 h earlier than Acinetobacter sp alone. At the 40th hour, cells began to grow gently with a mild increase till the 64th hour, and the color turned darker in the process. Another rapid cell growth period was observed after the 64th hour, and the color of the media turned greener, and bubbles started to appear. Based on these results, a model was proposed: Acinetobacter sp. XM-02 in group 3 grew well and degraded alkane hydrocarbons effectively, whereas the growth pattern of Pseudomonas sp. XM-01 and Acinetobacter sp. XM-02 in group 4 was shown to be more interesting: Acinetobacter sp. XM-02 (the degrader of alkane hydrocarbons) grew rapidly with sufficient alkane hydrocarbons at the beginning, and Pseudomonas sp. XM-01 (the biosurfactant
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Fig. 1 Phylogenetic trees of Pseudomonas sp. XM-01 and Acinetobacter sp. XM-02
producer) began to increase with the intermediates metabolized by the biodegradation of alkane hydrocarbons (after the 16th hour). With the prosperity of Pseudomonas sp. XM-01, the degrader was restrained gradually and the color of the media was changed by the rapid growth of Pseudomonas sp. XM-01 (from lighter brown to darker green). After the 64th hour,
Fig. 2 Cell growth of the four groups in alkane hydrocarbon biodegradation
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faster growing Pseudomonas sp. XM-01 began to dominate the mixed strains and a secondary growth came into being. With the changing culture condition, it became more suitable for Pseudomonas sp. XM-01 and more restraining for Acinetobacter sp. XM-02, leading to the decline of the degrader and the booming of the biosurfactant producer. As the growth of Pseudomonas sp. XM-01 stabilized, biosurfactant (identified as glycolipids, results are shown in “Identification of Biosurfactant Produced”) began to synthetize, causing the reduction in surface tension and decrease in content of heavy components in alkane hydrocarbons. The conclusion could be verified with the appearance of adding alkane hydrocarbons into the media of group 4 after 120 h, and there was no biodegradation of alkane hydrocarbons observed (meaning the available cells were biosurfactant producer and no degrader left). While with a supply of Acinetobacter sp. XM-02, alkane hydrocarbons was once again degradable and the color of the mixed culture (green present) turned brown and then green again, the same as the appearance of group 4 previously. This verification consolidated the conclusion of the interaction between the two strains in alkane hydrocarbon biodegradation. As a speculative conclusion on the changing pattern of the mixed strains in biodegradation was drawn, it is necessary to study the transformation of cell composition in the mixed strains. The two strains behaved dissimilarly in the process of alkane hydrocarbon co-treating based on CFU counts shown in Fig. 3. The development of Acinetobacter sp. XM-02 presents a common microorganism growth curve with four roughly legible growth periods. Whereas, Pseudomonas sp. XM-01 keeps booming since the 24th hour and peaks at the 104th hour. As shown in Fig. 3, the study of the two strains’ cell growth pattern was discovered: Acinetobacter sp. XM-02 began to boom while Pseudomonas sp. XM-01 grew next to nothing. The culture was still clear with few cells and intermediates in the first 16 h. With more alkane hydrocarbons biodegraded, Pseudomonas sp. XM-01 started to play a more important role in the biodegradation process, as metabolic activity of Pseudomonas sp. XM-01 gets stronger, the secondary metabolism of Pseudomonas sp. XM-01 would commence and the secondary metabolite, rhamnolipid, was produced. Rhamnolipid, a kind of glycolipida biosurfactant, could make the alkane
Fig. 3 Growth curve of available cells in the mixed cultures
Appl Biochem Biotechnol
hydrocarbons more biodegradable. Pseudomonas sp. XM-01 and its productions favor the biodegradation of alkane hydrocarbons in this stage. With the decline of alkane hydrocarbons and fast-growing Pseudomonas sp. XM-01, the predominance of Acinetobacter sp. XM-02 was challenged by Pseudomonas sp. XM-01 which kept flourishing with the intermediates as its substrates, resulting in more biosurfactant produced; however, the available cells of Acinetobacter sp. XM-02 were of low metabolic activity and declined gradually, and the color of the media was changing from brown to green (the characteristic color of Pseudomonas sp. XM-01 in culture). Finally, the available cells of Acinetobacter sp. XM-02 decreased to zero and Pseudomonas sp. XM-01 began to slow down, at this phase, the media color was green and the surface tension of the media was reduced to 28.6×10−3 N/m, generating a lot of bubbles. To verify the consortium composition changing above, 0.5 mL of fresh alkane hydrocarbons was added into the culture media of group 4 after 120 h, and it showed a negative degradation of the substrates. However, when the media was further added with a batch of fresh Acinetobacter sp. XM-02, growing observation of cells and biodegradation of the alkane hydrocarbons showed up again. This indicates: (1) the only one that was left behind of group 4 was Pseudomonas sp. XM01 at the end of the experiment; (2) the tolerance nature but not the degradation ability to alkane hydrocarbons by Pseudomonas sp. XM-01; and (3) the importance of alkane hydrocarbons to the degrader. Thus, a hypothetical model could be made: the main factor for the decrease even extinction of available Acinetobacter sp. XM-02 was the lack of substrates: alkane hydrocarbons, but not the toxicity of mass-growing Pseudomonas sp. XM-01. Identification of Biosurfactant Produced To state the biosurfactant production in the biodegradation of alkane hydrocarbons, surface tensions of the four groups were measured (as shown in Fig. 4). No changing of surface tension was observed in group 1. As for group 2, the surface tension dropped a little to 64.7× 10−3 N/m till the 56th hour and remained steady afterwards, meaning a slight growth of Pseudomonas sp. XM-01 (see Fig. 2). Acinetobacter sp. XM-02 could degrade the vast majority of alkane hydrocarbons rapidly in 5 days; the surface tension of the media dropped slowly and smoothly to an indistinctive terminal value of 51.4×10−3 N/m. All the three groups produced no bubbles in the surface of the media. In group 4, surface tension of the media dropped dramatically since the 8th hour and became stable at the 96th hour. It dropped to 28.6×10−3 N/m, which was rarely found in biodegradation of alkane hydrocarbons. Crude biosurfactant of group 4 was collected and identified as an affiliation to glycolipids. Further investigation of the glycolipids by TLC and LC-MS spectrum confirmed it was rhamnolipid. Previously reported substrates used for biosurfactant fermentations by Pseudomonas sp. were mannitol [16], soybean oil, peanut oil [23], or glucose [3]. There were few reports on the production of biosurfactant by cultivation in the media with diesel and the crude oil as the sole carbon source [16, 23]. Generation of rhamnolipids with alkane hydrocarbons as substrates was uncommon to Pseudomonas sp. and a great part was taken by the degrader at this process. One possible reason could be that some metabolic intermediate substances was utilized by Pseudomonas sp. XM-01, and the mixed oil–water phases promoted the composition changes in cell wall and the biosynthesis of rhamnolipid as its stress reaction to oil. Alkane Hydrocarbons Biodegradation Analysis Alkane hydrocarbons degraded by the pure and the mixed strains, namely groups 3 and 4 were sampled and extracted by n-hexane, analyzed by GC-MS. Examination of alkane hydrocarbons was carried out by the integral area comparison out of gas chromatogram. The
Appl Biochem Biotechnol
Fig. 4 Surface tension of the four groups in alkane hydrocarbon biodegradation
degradation rate of alkane hydrocarbons were 89.35 and 97.41 % in groups 3 and 4, respectively. To investigate the biodegradation kinetically, cultures were sampled when cell concentration changed greatly was chosen as its potential transformation and indication of the tremendous changing in substrate. As shown in Fig. 5, GC-MS analysis of the extractions at each sampling point indicated the reducing tendency of alkane hydrocarbons, as well as its variations in different components. These results show that biodegradation of alkane hydrocarbons by Acinetobacter sp. XM-02 was carried out steadily; however, the biodegradation by the mixed strains was different, more than 80 % of the substrates were biodegraded by the 80th hour, and the process went slow thereafter. This phenomenon corresponded with the consortium composition changing of the mixed strains when the available cells of Acinetobacter sp. XM-02 began to decrease dramatically and Pseudomonas sp. XM-01 kept soaring and producing biosurfactants. Another conclusion which had been reported repeatedly could be confirmed by the GC-MS results: the biodegradation of short-chain alkane hydrocarbons was prioritized and the heavy ones remained gradually decomposable [25]. Compositional variation of the two degradation patterns at some time points was studied. At the 24th hour, the degradation rate of C17 was 31.42 % by Acinetobacter sp. XM-02. However, it turned to 24.01 % when degraded by the mixed strains (as shown in Fig. 6), but this trend was broken as the degradation rate by the mixed strains increased sharper and exceeded the pure strain at the 80th hour. Finally, the degradation rates of the pure and mixed strains were 88.85 and 93.12 %, respectively. Other components exhibited the same regular pattern. It was during the 48th, 104th, and 120th hours when the biodegradation rates of C11, C23, and C30 that group 4 exceeded group 3, respectively. In addition, nearly all components were more extensively degraded by the mixed strains than the pure ones at the end of the experiment. Disintegration effects of the longer-chain hydrocarbons biodegraded by the mixed strains were faster, and the increasing tendency went sharper after the 80th hour. This anastomosis indicated that the availability for alkane hydrocarbons biodegradation was improved when exposed to the rising concentrations of biosurfactant. As shown in Fig. 6, peak
Appl Biochem Biotechnol
Fig. 5 GC-MS analysis of hydrocarbon biodegradation by the single and mixed strains
areas of C11 were degraded by the mixed strains at the 24th hour, and the hours after which were all less than the peak area degraded by the pure strain. Components of alkane hydrocarbons showed the same degradation pattern as the C11. As the components of alkane hydrocarbons got longer, more time was needed for the over-biodegradation by the mixed strains. It indicated that heavier hydrocarbons showed a much greater resistance to the surface-active agent than the light ones. Crude Oil Biodegradation After degradation for 10 days, 5.13±2.05 % crude oil was lost by abiotic factors, 74.32± 4.09 % of crude oil was biodegraded by Acinetobacter sp. XM-02 alone, and 87.29±2.41 % of crude oil was degraded by the mixed strains, 12.83 % higher than the pure strain (as shown in Fig. 7). The degradable ability of crude oil was remarkably enhanced by the synergy between Acinetobacter sp. XM-02 and Pseudomonas sp. XM-01. The promotion of crude oil biodegradation by mixed culture was of great utility value together with nutriment addition. A batch experiment was conducted as described in “Biodegradation of Crude Oil by the Single and Mixed Strains” to study how the two strains in the mixed culture acted with each other. Just as expected, biodegradation rates of crude oil in 10 days showed great divergences for these three groups: 47.31, 49.26, and 88.54 % of crude oil was decomposed in groups 1, 2, and 3, respectively. Biodegradation rates in groups 1 and 2 were much lower than the first round, indicating the constituent of the mixed strains had already changed after 10 days biodegradation in round one; results of groups 1 and 2 were in a little difference which demonstrated little affection on the addition of biosurfactant-producing strain after round one. However, biodegradation rate in group 3 was much higher than groups 1 and 2, which showed
Appl Biochem Biotechnol
Fig. 6 GC-MS analysis of C11/C14/C17/C23 biodegradation by the single and mixed strains
that the degrading strain added in round 2 played a significant role in the mixed culture. These experimental results verified that the composition of the mixed strains after round one had been changed. The amount of the degrader was reduced but not vanished, which is why more
Fig. 7 Crude oil biodegradation by the single and mixed strains
Appl Biochem Biotechnol
than 45 % of the crude oil was still degraded in group 1. However, the biomass and activity of the degrader were inhibited by Pseudomonas sp. XM-01 at the end of the 10-day experiment, so the degradation rate was far lower in the second round than the first round. This reduction was due to the recession of the degrader but not the biosurfactant-producing strain, which was also confirmed by the low degradation rate in group 2, whose composition was similar to group 1 and the significant enhancement of group 3 (group 1 amended with Acinetobacter sp. XM-02), whose colony structure was reconstructed to its similarity with the first-round experiment. The experiments above confirmed that the degrader strain declined but the biosurfactant-producing strain flourished in the process of crude oil biodegradation, which was the same as the findings drawn in “Changes in Bacterial Species Composition in Mixed Culture.” The actual system is more complex; this article only has carried on the preliminary work in laboratory. However, the research provided us with a meaningful idea for enhancing biodegradation of crude oil and its derivatives: by designing the program rationally and controlling the local consortium composition dynamically with nutriment addition to increase the degradability of crude oil, where more research needs to be done in the future. There are plenty of different hydrocarbon compounds in crude oil, leading different biodegradation patterns for different compounds. Basically, the biodegradable order of components in crude oil: n-alkanes>i-alkanes>low molecular weight aromatics>high molecular weight aromatics and cyclic alkanes [10, 27]. Crude oil before and after biodegradation were sampled and analyzed by GC-MS (the result is shown in Fig. 8). Short-chain hydrocarbons were nearly all degraded, and the long-chain ones remained hard to decompose. Interestingly, less heavy components of crude oil biodegraded by the mixed strains were retained than those biodegraded by Acinetobacter sp. XM-02 alone. This condition could be explained by the function of Pseudomonas sp. XM-01 and the biosurfactant produced by it, which was also a confirmation to the conclusion drawn in Fig. 5.
Conclusions Two strains isolated from areas polluted by crude oil were identified and nominated as Pseudomonas sp. XM-01 and Acinetobacter sp. XM-02. Pseudomonas sp. XM-01 with a biosurafactant-producing ability (identified as rhamnolipid) could tolerate alkane hydrocarbons but could not degrade them. Acinetobacter sp. XM-02 was the degrader and it could reduce 74.32±4.09 % of crude oil in 10 days. Enhancement of crude oil biodegraded was achieved by the mixed strains at a biodegradation rate of
Fig. 8 GC-MS analysis of crude oil biodegradation by the single and mixed strains
Appl Biochem Biotechnol
87.29±2.41 %. Moreover, it was found that almost all of the components in alkane hydrocarbons obtained higher degradation rates by the mixed strain than by the single strain. The effect of the biosurfactant produced on the enhancement of short-chain alkane hydrocarbons biodegradation was more remarkable. Studies on the composition changing of the mixed strains in alkane hydrocarbons biodegradation were carried out, indicating that growth of Pseudomonas sp. XM-01 was firstly inhibited and then boosted by the metabolites degraded by Acinetobacter sp. XM-02; meanwhile, biosurfactant identified as rhamnolipid produced by Pseudomonas sp. XM-01 increased the degradation of alkane hydrocarbons. Finally, with the fast growth of Pseudomonas sp. XM-01 and the decrease of available substrates, the degrader was restrained. These observations were in accordance with the crude oil biodegradation in the two round experiments. These results indicated the enhancing ability of the biosurfactant-producing strain in crude oil biodegradation. In conclusion, with the mixed bacterial cultures isolated and new strategies proposed in this study, promising enhancement of crude oil biodegradation was achieved, which would be a potential way for bioremediation of crude oilcontaminated soil and water. Acknowledgments The authors wish to acknowledge the financial support provided by the National Key Basic Research Program of China (No. 2014CB745100), the Natural Science Foundation of Tianjin, the Seed Foundation of Tianjin University, the Program of Introducing Talents of Discipline to Universities (No. B06006), the Science and Technology Project from China National Offshore Oil Corporation (CNOOC-KJ 125 ZDXM 25JAB NFCY 2013-01) and the Science and Technology Project of Tianjin Binhai New Area (2012-XJR23017).
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