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Biodiesel production using magnetic whole-cell biocatalysts by immobilization of Pseudomonas mendocina on Fe3O4-chitosan microspheres
Accepted Manuscript Title: Biodiesel production using magnetic whole-cell biocatalysts by immobilization of Pseudomonas mendocina on Fe3 O4 -chitosan microspheres Author: Guanyi Chen Jing Liu Yun Qi Jingang Yao Beibei Yan PII: DOI: Reference:
S1369-703X(16)30156-5 http://dx.doi.org/doi:10.1016/j.bej.2016.06.003 BEJ 6483
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
19-2-2016 29-5-2016 4-6-2016
Please cite this article as: Guanyi Chen, Jing Liu, Yun Qi, Jingang Yao, Beibei Yan, Biodiesel production using magnetic whole-cell biocatalysts by immobilization of Pseudomonas mendocina on Fe3O4-chitosan microspheres, Biochemical Engineering Journal http://dx.doi.org/10.1016/j.bej.2016.06.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Biodiesel production using magnetic whole-cell biocatalysts by immobilization of Pseudomonas mendocina on Fe3O4-chitosan microspheres
Guanyi Chena, b, c, 1, Jing Liua, 1, Yun Qia,*, Jingang Yaoa, *, Beibei Yana, d
a
School of Environmental Science and Engineering/State Key Lab of Engines, Tianjin
University, Tianjin 300072, China b
School of Science, Tibet University, Lhasa 850012, China
c
Tianjin Engineering Center of Biomass-derived Gas/Oil Technology, Tianjin 300072,
China d
Key Laboratory of Efficient Utilization of Low and Medium Grade Energy (Tianjin
University), Ministry of Education, Tianjin 300072, China
*
Corresponding author. Tel.: +86 22 27891921. E-mail address: [email protected],cn (Y. Qi), [email protected] (J. Yao).
1
Both authors contributed equally to this work.
1
Graphical abstract
Highlights 1. First report on Pseudomonas mendocina containing MWCBs for lipid conversion. 2. A biodiesel yield of 87.32% was achieved at optimized conditions within 48 h. 3. MWCBs prepared can be reused for 10 cycles with retaining 83.57% yield. 4. Kinetic data followed Michaelis-Menten model with methanol competitive inhibition.
2
Abstract Magnetic whole-cell biocatalysts (MWCBs) for enzymatic biodiesel production from soybean oil were constructed by immobilizing Pseudomonas mendocina cells into Fe3O4-chitosan microspheres. The effects of the MWCBs concentration, temperature, the molar ratio of methanol to oil and water content on biodiesel yield were studied. For the MWCBs, a biodiesel yield of 87.32% was obtained under the optimum operating condition (MWCBs concentration of 10 wt.%, water content of 10 wt.%, 35 oC, methanol to oil molar ratio of 4:1 and a four-step addition of methanol) for 48 h. The kinetic data followed Michaelis-Menten model (rmax, 2.1801 mg/min; Km, 0.4272 mg/mL; KI, 0.2308 mg/mL) with competitive inhibition by methanol. MWCBs had an excellent reusability and still gave a biodiesel yield of 83.57% after 10 cycles, which was higher than that of Fe3O4-uncontained whole cell biocatalysts (74.06%). Moreover, MWCBs could be separated and be recycled easily due to their superparamagnetism. Therefore, MWCBs would be promising for large-scale biodiesel industry. Keywords: Biodiesel; Pseudomonas mendocina; Magnetic whole-cell biocatalyst; Transesterification; Immobilization
1. Introduction The depletion of petroleum and global warming issues have triggered a growing interest in developing alternative liquid fuel. Biodiesel is deemed to be an ideal substitute for being renewable, nontoxic and biodegradable [1]. Currently, biodiesel is 3
mainly produced through the conventional chemical method. However, there are several inherent drawbacks for chemical-catalyzed process, such as high requirement of temperature and pressure, unwanted side reactions, expensive downstream processes and costly recovery of the catalyst. Moreover, it has problems of glycerol recovery and wastewater treatment [2-5]. Enzymatic transesterification using lipase is becoming more attractive due to its easier recovery of glycerol, mild reaction conditions, minimal requirement of wastewater treatment and wide adaptability for feedstock [6-11]. However, extracellular lipases as biocatalysts require complicated purification, recovery, and immobilization. Therefore, whole-cell biocatalysts which could directly use lipase-producing microorganism have been of prime interest in the past decade. Some microorganisms can be used as whole-cell biocatalysts for biodiesel production [12-14]. As reported, using Aspergillus sp. (RBD01) cells as whole-cell biocatalysts could give methyl ester yields of 87.5% from oleic, 71% from palmitic and 41% from stearic acid, respectively [15]. Hama et al. reported that Rhizopus oryzae cells immobilized within BSP achieved a high biodiesel content of over 90% even after 5 cycles in a packed bed reactor [16]. Risa et al. reported that the immobilized whole cell (wild-type R. oryzae) successfully catalyzed the ethanolysis of rapeseed oil and achieved a biodiesel yield of 79% [17]. However, whole-cell biocatalysts also suffer the problems of separation, low mass transport, complicated operation and long period of catalytic reaction. Magnetic whole-cell biocatalysts (MWCBs) constructed by immobilizing cells into magnetic particles offer an 4
approach to solve these problems. Owning to their superparamagnetism, MWCBs can be easily separated from the reaction system with an external magnetic field. Moreover, the MWCBs can disperse uniformly and keep stability in the reaction system by slight shaking without the presence of the external magnetic field [18]. The use of MWCBs in magnetic fluidized bed reactor was also reported to have obvious advantages such as elimination of solid mixing, ease of solid transportation and the possibility of operation at increased fluid velocities (even in countercurrent flotation process) [19]. Therefore, transesterification catalyzed by MWCBs makes it possible for biodiesel production on a large scale. Chitosan has become attractive as a carrier for immobilization due to its characteristics of nontoxicity, physiological inertness, hydrophilicity and biocompatibility. Introducing Fe3O4 in chitosan microspheres contributes to the formation of microspheres with superparamagnetism. In this work, MWCBs, which were constructed by immobilizing P. mendocina cells into Fe3O4-chitosan microspheres, were characterized and applied for biodiesel production from soybean oil. Factors affecting the biodiesel yield during transesterification reaction, such as MWCBs concentration, temperature, the molar ratio of methanol to oil, water content, were investigated. Furthermore, the reusability of MWCBs was also studied to indicate its potential in biodiesel production. 2. Materials and methods 2.1. Strains and chemicals P. mendocina CGMCC 7644 was obtained from China General Microbiological 5
Culture Collection Center. The standard fatty acid methyl esters (FAMEs) were purchased from Sigma-Aldrich Co. (USA). Peptone, yeast extract and agar powder were derived from Aoboxing Bio-Tech Co., Ltd. (Beijing, China). Soybean oil was purchased from Luhua Group Co., Ltd. (Shandong, China). FeSO4·7H2O, FeCl3·6H2O, NaOH, NH3·H2O, polyethylene glycol, chitosan, tetraethoxysilane, glutaraldehyde, olive oil, polyvinyl alcohol, methanol and ethanol were analytical grade (Yuanli Chemical Co., Ltd., Tianjin, China). 2.2. Synthesis of magnetic nanoparticle (Fe3O4) FeSO4·7H2O and FeCl3·6H2O (the molar ratio of Fe2+/Fe3+ was 1:2) were dissolved in 1 wt.% polyethylene glycol solution. Then 25 wt.% NH3·H2O was added into the solution, stirring at 60 oC for 1 h. The magnetic precipitates were subsequently removed by magnetic separation. Finally, tetraethoxysilane (10 mL), NH3·H2O (5 mL), ethanol (150 mL) and the magnetic precipitates (4 g) were mixed, stirring vigorously for 5 h. The SiO2-coated magnetic nanoparticles (Fe3O4) were rinsed thoroughly with water and ethanol and then dried in a vacuum freeze dryer. The magnetic nanoparticles obtained owned a black color and showed a strong response to an external magnetic field. 2.3. P. mendocina whole-cell biocatalysts preparation P. mendocina CGMCC 7644 was cultivated in LB liquid medium at 35 °C and with 160 rpm for 24 h. Suspended cells (>107 cells/mL) and chitosan solution (2.5%, w/v) containing 4% (w/v) SiO2-coated magnetic nanoparticle (Fe3O4) were mixed (1:12, v/v) thoroughly using ultrasonic wave for 10 min. The mixture was injected dropwise 6
into a sterilized NaOH solution by a needle. Thereafter, glutaraldehyde (25 wt.%) was added to the solution, stirring for 2 h. Finally, these magnetic particles with whole-cell biocatalysts were separated by a magnet and washed with water until neutral pH. For the preparation of Fe3O4-uncontanied whole cell biocatalysts, all the steps were identical with the exception of the need for introducing SiO2-coated Fe3O4. The immobilized cell numbers entrapped in particles were determined by microscopic counting [19,20]. 2.4. Transesterification catalyzed by MWCBs The reaction using soybean oils as substrate was carried out in a cylindrical glass bottle (50 mL) by batch operation. Bottles containing soybean oil, MWCBs, water and methanol were stirred in a reciprocal shaker (160 rpm). The reaction was initiated at 35 oC with a MWCBs concentration of 10 wt.% and a water content of 10 wt.%. For the optimization of transesterification, MWCBs with a concentration range of 2-18 wt.%, temperature with a range of 15-55 oC, methanol to oil in the molar ratio with a range of 1-6:1 and water content with a range of 0-40 wt.% were investigated. 2.5. Recycling MWCBs for transesterification MWCBs were separated from the reaction mixtures by magnet after each cycle and were directly reused into new reaction solutions for the next cycle. Samples for the measurement of biodiesel yield were collected at designated intervals. 2.6 Analytical methods 2.6.1 Characterization analysis of MWCBs The SEM images of MWCBs particles were obtained using SU-8010 equipment 7
(Hitachi, Japan). XRD patterns were recorded by D/MAX-2500 diffractometer (Rigaku, Japan). The magnetic property of MWCBs was observed by LDJ-9600 VSM (LDJ Electronics，USA). 2.6.2 Lipase activity 4 mL emulsion with an olive oil to polyvinyl alcohol ratio of 1:3 (v/v), 0.5 g MWCBs and 5 mL 50 mM phosphate buffer (pH 7) were mixed, stirring at 35 oC for 20 min. It was subsequently terminated by adding 10 mL 95% (v/v) ethanol. Non-catalytic reaction was performed as a control. One-unit lipase activity of MWCBs was defined as the amount of lipase that released 1.0 μmol fatty acids per minute under the assay condition. The released fatty acids were quantified by titration with NaOH (5 mM) solution [18]. 2.6.3 FAMEs analysis Samples of FAMEs obtained from the reaction mixtures were centrifuged at 9000 rpm for 10 min. The supernatants were analyzed using Agilent 7890A GC (INNOWAX, Agilent, 30 m × 0.25 mm × 0.25 µm) equipped with FID. The carrier gas was nitrogen. The temperatures of injector and detector were 330 and 360 o
C, respectively. The temperature of column was started at 100 oC for 2 min, raised to
350 oC at the speed of 10 oC/min, then raised to 380 oC at the speed of 6 oC/min and maintained at 380 oC for 10 min. The concentration of biodiesel could be obtained by a standard curve using a series of external standards. All reported data were averages of experiments performed at least in triplicate. The yield of biodiesel was calculated by Eq. (1): 8
Yield (%)
weight of biodiesel produced 100% theoretical maximum weight of biodiesel
(1)
3. Results and discussion 3.1. Characterization of MWCBs P. mendocina cells were entrapped in the chitosan microspheres and cross-linked with chitosan using glutaraldehyde. The morphology of MWCBs is shown in Fig. 1a. These MWCBs are spherical and have average diameters ranging from 2 to 5 mm. Micro-structural features of MWCBs are observed by SEM (Fig. 1b). SEM analysis confirms a good immobilization of P. mendocina cells on the surface or/and in the internal gaps of magnetic particles owning to chitosan’s flocculating ability and cross-linkable property. Moreover, a porous surface and an interconnected porous structure are observed for MWCBs, suggesting their excellent immobilization performances. Crystalline structure of MWCBs was examined by XRD (Fig. 1c). XRD spectrum of MWCBs was identified by standard spectrum of Fe3O4. As observed, component of particles shows all characteristic peaks of magnetite (standard Fe3O4) and maintain a cubic structure of Fe3O4 (JCPDS card 19-0629). The mean diameter of P. mendocina cells immobilized Fe3O4 derived from Scherrer equation is about 12.4 nm, which is below the superparamagnetic critical size (20 nm) [21]. These results reveal that MWCBs have properties of superparamagnetism. To evaluate the superparamagnetism of MWCBs, the hysteresis of the magnetization was analyzed by VSM at room temperature. As shown in Fig. 1d, a magnetization curve with S-shape is observed for MWCBs. The MWCBs own a saturation magnetization of 47.3 emu/g, 9
a remnant magnetization intensity of 0.0053 emu/g and a coercivity of 0.0107 emu/g, respectively, indicating good superparamagnetism of MWCBs [22]. As presented in Fig. 2, enzyme activity of MWCBs increases significantly with incubation time. Highest enzyme activity of 2810 U/g was achieved at 8 h. This result confirms that lipases produced by MWCBs are secreted outside the cells. In the previous studies, enzyme activities of 560 U/g from Aspergillus niger [23], 1233 U/g from Thermomyces lanuginosus [24] and 1785 U/g from recombinant Pichia pastoris [25] were obtained, respectively, which are all lower than that of MWCBs. 3.2. Effects of methanol addition strategy on transesterification catalyzed by MWCBs Fig. 3 shows the time course of biodiesel yield obtained at 35 oC under the conditions of 10 wt.% MWCBs and 10 wt.% of water. It is obvious that methanol-to-oil ratio has an important influence on MWCBs catalytic transesterification. The biodiesel yield increases at higher methanol concentration. A biodiesel yield of 73.49% was obtained at a methanol/oil ratio of 4. However, excessive methanol concentration beyond 4 leads to a decrease in biodiesel yield owning to irreversible inactivation of MWCBs [26]. Similar phenomena have been reported in other literatures [27]. To decrease the deactivation of MWCBs, stepwise addition of methanol was used for transesterification of soybean oils [28]. One mole equivalent of methanol was added stepwise into reaction system at 12 h intervals. As indicated in Fig. 3, the biodiesel yield was improved significantly with the highest biodiesel yield of 90.48% obtained after 72 h. However, the fifth addition of methanol only gives an increase of 10
3.13% in biodiesel yield. The possible reason for this variation is that excess addition of methanol could result in a lower concentration of lipids, which leads to a negative effect to enzymatic activity [29]. These results reveal that the deactivation of MWCBs is avoided by the stepwise addition of methanol. Based on these results, a four-step addition of methanol is deemed to be an appropriately way to increase the biodiesel yield and to reduce the cost of biodiesel production. Given that, all subsequent investigations were carried out at a four-step addition of methanol. 3.3. Effect of biocatalysts concentration on transesterification As seen in Fig. 4, the biodiesel yield increases significantly by increasing the MWCBs concentration from 2 wt.% to 10 wt.%. Highest biodiesel yield of 84.52% was attained at MWCBs concentration of 10 wt.%. However, no significant increase in biodiesel yield was observed by further increasing the MWCBs concentration. These changes are due to the fact that overmuch biocatalyst particles could lead to higher solid content and viscosity in the transesterification, causing difficulties in mixing and mass transfer [30]. But in consideration of the costs, 10 wt.% MWCBs were chosen as the preferential concentration for transesterification. Therefore, the optimal concentration of MWCBs is deemed to be 10 wt.% in this work. Control experiment was performed with Fe3O4-uncontained whole cell biocatalysts under the same conditions. As observed, there is no significant difference in biodiesel yield between MWCBs and Fe3O4-uncontained whole cell biocatalysts. This indicates that the magnetic materials of MWCBs have no apparent impact on 11
transesterification. 3.4. Effect of temperature on transesterification catalyzed by MWCBs As indicated in Fig. 5a, increasing temperature from 15 oC to 35 oC improves the process. This is an expected result because the growth rate of cells and the molecular diffusion are accelerated by higher temperature, leading to higher reaction rate. However, the biodiesel yield reduces remarkably by further increasing temperature beyond 35 oC, which is mainly due to the partial inactivation of biocatalyst in organic solvent at high temperature. These results are in agreement to previous studies [31,32]. The highest biodiesel yield of 85.36% was obtained at 35 oC in this part. Based on these results, the optimal temperature for MWCBs is concluded to be 35 oC. 3.5. Effect of water content on transesterification catalyzed by MWCBs Optimum water content is required to maintain transesterification activity and lipase stability [33]. Fig. 5b shows biodiesel yields obtained at various amounts of water. As observed, biodiesel yield increases by increasing water content from 0 to 10 wt.%. A maximum yield of 83.04% was obtained at a water content of 10 wt.%. This may be due to the fact that an appropriate higher water content increases the flexibility and the mobility of the enzyme and its expressed activity [34,35]. However, a decrease in biodiesel yield was observed by further increasing water content beyond 10 wt.%. The possible reason for the decrease of biodiesel yield in the range is due to the hydrolytic activity enhanced by excess water content [36]. 3.6. Kinetic analysis of MWCBs-catalyzed transesterification The mechanism of MWCBs-catalyzed transesterification is assumed to follow the 12
Michaelis-Menten and Lineweaver-Burk model [37-39]. Some assumptions are made to simplify model development: (1) The mass transfer limitations in the reaction are ignored. (2) Methanol is regarded as the main inhibitor in the transesterification and other inhibitors are negligible. (3) The methanol inhibits the enzyme by a competitive inhibition mechanism. With these assumptions, the rate equation of transesterification is expressed by Eq. (2):
r
rmax Cs CI Km (1 ) Cs KI
(2)
where r, rmax, CS and CI are transesterification rate, maximum transesterification rate, concentration of substrate (triglyceride) and concentration of inhibitor (methanol), respectively. Km and KI are Michaelis constant and the constant of inhibition, respectively. The double-reciprocal form of the Michaelis-Menten equation is defined by: K 1 1 CI 1 m (1 ) r rmax rmax KI Cs
(3)
Kinetic parameters in Eq. (3) are obtained from reactions with varying methanol concentrations (20%, 25%, 30%, 35%, 40%, v/v) under the optimized conditions (Fig. 6). The values obtained for rmax, Km and KI are 2.1801 mg/min, 0.4272 mg/mL and 0.2308 mg/mL, respectively. As seen from Fig. 6, higher methanol concentration gives higher slope of lines, but an invariant rmax, indicating that methanol was a competitive inhibitor of enzyme activity [40]. These observations conform the above assumption and clearly support the results described in section 3.2.
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3.7. Reusability of MWCBs MWCBs were separated from the reaction system by using a magnet and reused into the fresh reactants. Fig. 7 shows the time course of biodiesel yield obtained at 35 o
C with stepwise addition of methanol (biocatalysts concentration of 10 wt.%,
methanol to oil molar ratio of 4:1, water content of 10 wt.%). As seen, a biodiesel yield of about 90% is maintained over reused MWCBs. No remarkable decline in biodiesel yield is observed after the sixth reuse. A biodiesel yield of 83.57% is retained even after the tenth reuse. However, the yield obtained over Fe3O4-uncontained whole cell biocatalysts decreased gradually after each reuse, and only maintained 74.06% yield after tenth cycle. The decreasing biodiesel yields in the case of control batch were due to the fact that collisions between particles and collisions with container wall occurred during processes of separating Fe3O4-uncontanied whole-cell biocatalysts from the reaction mixture, leading to damages of microspheres and cell leakage from these microspheres. Owning to their superparamagnetic property, MWCBs were easily and quickly separated from the reaction system by applying an external magnetic field [18,19,41]. The damage caused by collision and the cell leakage were decreased significantly since MWCBs moved rapidly along the magnetic field lines. These results indicate that MWCBs are promising for industrial biodiesel production.
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4. Conclusions Magnetic whole-cell biocatalysts were constructed, characterized and applied for biodiesel production from soybean oil. A maximum biodiesel yield of 87.32% was obtained under the optimum operating condition (MWCBs concentration of 10 wt.%, water content of 10 wt.%, 35 oC, methanol to oil molar ratio of 4:1 and a four-step addition of methanol) for 48 h. The kinetic data followed Michaelis-Menten model (rmax, 2.1801 mg/min; Km, 0.4272 mg/mL; KI, 0.2308 mg/mL) with competitive inhibition by methanol. MWCBs had an excellent reusability and still gave a biodiesel yield of 83.57% after 10 cycles, which was higher than that of Fe3O4-uncontained whole cell biocatalysts (74.06%). Therefore, MWCBs would be promising for large-scale biodiesel industry. Acknowledgments The authors gratefully acknowledge financial support from Natural Science Foundation of Tianjin, China (16JCYBJC20700) and the National Energy Administration of China (NY2013040302).
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Appl. Energ. 113 (2014) 1614-1631. [38] S. Liu, K. Nie, X. Zhang, M. Wang, L. Deng, X. Ye, F. Wang, T. Tan, Kinetic study on lipase-catalyzed biodiesel production from waste cooking oil, J. Mol. Catal. B: Enzym. 99 (2014) 43-50. [39] B. Bharathiraja, J. Jayamuthunagai, R. Praveenkumar, M. Jayakumar, S. Palani, The Kinetics of interesterfication on waste cooking oil (sunflower oil) for the production of fatty acid alkyl esters using a whole cell biocatalyst (Rhizopus oryzae) and pure lipase enzyme, Int. J. Green. Energy. 12 (2015) 1012-1017. [40] L.C. Meher, D. Vidya Sagar, S.N. Naik, Technical aspects of biodiesel production by transesterification-a review, Renew. Sust. Energ. Rev. 10 (2006) 248-268. [41] G. Zhou, G. Chen, B. Yan, Biodiesel production in a magnetically-stabilized, fluidized bed reactor with an immobilized lipase in magnetic chitosan microspheres, Biotechnol. Lett. 36 (2014) 63-68.
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Figure Captions Fig. 1. a, Magnetic particles of MWCBs. b, Surface features of magnetic particles observed with SEM. c, XRD spectrum of MWCBs comparing with standard spectrum of Fe3O4. d, Magnetization curves of MWCBs at 300 K. Fig. 2. Time course of enzyme activity of MWCBs. Fig. 3. Time course of MWCBs catalyzed transesterification. Reaction conditions: MWCBs concentration of 10 wt.%, water content of 10 wt.%, 35 oC, 160 rpm. Fig. 4. Effect of biocatalysts concentration on transesterification catalyzed by MWCBs. Reaction conditions: methanol to oil molar ratio of 4:1, water content of 10 wt.%, 35 oC, 160 rpm, 48h. Fig. 5. Effects of temperature and water contents on transesterification catalyzed by MWCBs. Reaction conditions: MWCBs concentration of 10 wt.%, methanol to oil molar ratio of 4:1, 160 rpm, 48 h. Fig. 6. Lineweaver-Burk plot of MWCBs-catalyzed transesterification under different methanol concentrations. Reaction conditions: MWCBs concentration of 10 wt.%, water content of 10 wt.%, 35 oC, 160 rpm. Fig. 7. Reusability of biocatalysts for repeated batch transesterification. Reaction conditions: biocatalysts concentration of 10 wt.%, water content of 10 wt.%, methanol to oil molar ratio of 4:1, 35 oC, 160 rpm.
22
Fig. 1. a, Magnetic particles of MWCBs. b, Surface features of magnetic particles observed with SEM. c, XRD spectrum of MWCBs comparing with standard spectrum of Fe3O4. d, Magnetization curves of MWCBs at 300 K.
23
Fig. 2. Time course of enzyme activity of MWCBs.
24
Fig. 3. Time course of MWCBs catalyzed transesterification. Reaction conditions: MWCBs concentration of 10 wt.%, water content of 10 wt.%, 35 ℃, 160 rpm.
25
Fig. 4. Effect of biocatalysts concentration on transesterification catalyzed by MWCBs. Reaction conditions: methanol to oil molar ratio of 4:1, water content of 10 wt.%, 35 ℃, 160 rpm, 48h.
26
Fig. 5. Effects of temperature and water contents on transesterification catalyzed by MWCBs. Reaction conditions: MWCBs concentration of 10 wt.%, methanol to oil molar ratio of 4:1, 160 rpm, 48 h.
27
Fig. 6. Lineweaver-Burk plot of MWCBs-catalyzed transesterification under different methanol concentrations. Reaction conditions: MWCBs concentration of 10 wt.%, water content of 10 wt.%, 35 ℃, 160 rpm.
28
Fig. 7. Reusability of biocatalysts for repeated batch transesterification. Reaction conditions: biocatalysts concentration of 10 wt.%, water content of 10 wt.%, methanol to oil molar ratio of 4:1, 35 ℃, 160 rpm.
29
S1369-703X(16)30156-5 http://dx.doi.org/doi:10.1016/j.bej.2016.06.003 BEJ 6483
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
19-2-2016 29-5-2016 4-6-2016
Please cite this article as: Guanyi Chen, Jing Liu, Yun Qi, Jingang Yao, Beibei Yan, Biodiesel production using magnetic whole-cell biocatalysts by immobilization of Pseudomonas mendocina on Fe3O4-chitosan microspheres, Biochemical Engineering Journal http://dx.doi.org/10.1016/j.bej.2016.06.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Biodiesel production using magnetic whole-cell biocatalysts by immobilization of Pseudomonas mendocina on Fe3O4-chitosan microspheres
Guanyi Chena, b, c, 1, Jing Liua, 1, Yun Qia,*, Jingang Yaoa, *, Beibei Yana, d
a
School of Environmental Science and Engineering/State Key Lab of Engines, Tianjin
University, Tianjin 300072, China b
School of Science, Tibet University, Lhasa 850012, China
c
Tianjin Engineering Center of Biomass-derived Gas/Oil Technology, Tianjin 300072,
China d
Key Laboratory of Efficient Utilization of Low and Medium Grade Energy (Tianjin
University), Ministry of Education, Tianjin 300072, China
*
Corresponding author. Tel.: +86 22 27891921. E-mail address: [email protected],cn (Y. Qi), [email protected] (J. Yao).
1
Both authors contributed equally to this work.
1
Graphical abstract
Highlights 1. First report on Pseudomonas mendocina containing MWCBs for lipid conversion. 2. A biodiesel yield of 87.32% was achieved at optimized conditions within 48 h. 3. MWCBs prepared can be reused for 10 cycles with retaining 83.57% yield. 4. Kinetic data followed Michaelis-Menten model with methanol competitive inhibition.
2
Abstract Magnetic whole-cell biocatalysts (MWCBs) for enzymatic biodiesel production from soybean oil were constructed by immobilizing Pseudomonas mendocina cells into Fe3O4-chitosan microspheres. The effects of the MWCBs concentration, temperature, the molar ratio of methanol to oil and water content on biodiesel yield were studied. For the MWCBs, a biodiesel yield of 87.32% was obtained under the optimum operating condition (MWCBs concentration of 10 wt.%, water content of 10 wt.%, 35 oC, methanol to oil molar ratio of 4:1 and a four-step addition of methanol) for 48 h. The kinetic data followed Michaelis-Menten model (rmax, 2.1801 mg/min; Km, 0.4272 mg/mL; KI, 0.2308 mg/mL) with competitive inhibition by methanol. MWCBs had an excellent reusability and still gave a biodiesel yield of 83.57% after 10 cycles, which was higher than that of Fe3O4-uncontained whole cell biocatalysts (74.06%). Moreover, MWCBs could be separated and be recycled easily due to their superparamagnetism. Therefore, MWCBs would be promising for large-scale biodiesel industry. Keywords: Biodiesel; Pseudomonas mendocina; Magnetic whole-cell biocatalyst; Transesterification; Immobilization
1. Introduction The depletion of petroleum and global warming issues have triggered a growing interest in developing alternative liquid fuel. Biodiesel is deemed to be an ideal substitute for being renewable, nontoxic and biodegradable [1]. Currently, biodiesel is 3
mainly produced through the conventional chemical method. However, there are several inherent drawbacks for chemical-catalyzed process, such as high requirement of temperature and pressure, unwanted side reactions, expensive downstream processes and costly recovery of the catalyst. Moreover, it has problems of glycerol recovery and wastewater treatment [2-5]. Enzymatic transesterification using lipase is becoming more attractive due to its easier recovery of glycerol, mild reaction conditions, minimal requirement of wastewater treatment and wide adaptability for feedstock [6-11]. However, extracellular lipases as biocatalysts require complicated purification, recovery, and immobilization. Therefore, whole-cell biocatalysts which could directly use lipase-producing microorganism have been of prime interest in the past decade. Some microorganisms can be used as whole-cell biocatalysts for biodiesel production [12-14]. As reported, using Aspergillus sp. (RBD01) cells as whole-cell biocatalysts could give methyl ester yields of 87.5% from oleic, 71% from palmitic and 41% from stearic acid, respectively [15]. Hama et al. reported that Rhizopus oryzae cells immobilized within BSP achieved a high biodiesel content of over 90% even after 5 cycles in a packed bed reactor [16]. Risa et al. reported that the immobilized whole cell (wild-type R. oryzae) successfully catalyzed the ethanolysis of rapeseed oil and achieved a biodiesel yield of 79% [17]. However, whole-cell biocatalysts also suffer the problems of separation, low mass transport, complicated operation and long period of catalytic reaction. Magnetic whole-cell biocatalysts (MWCBs) constructed by immobilizing cells into magnetic particles offer an 4
approach to solve these problems. Owning to their superparamagnetism, MWCBs can be easily separated from the reaction system with an external magnetic field. Moreover, the MWCBs can disperse uniformly and keep stability in the reaction system by slight shaking without the presence of the external magnetic field [18]. The use of MWCBs in magnetic fluidized bed reactor was also reported to have obvious advantages such as elimination of solid mixing, ease of solid transportation and the possibility of operation at increased fluid velocities (even in countercurrent flotation process) [19]. Therefore, transesterification catalyzed by MWCBs makes it possible for biodiesel production on a large scale. Chitosan has become attractive as a carrier for immobilization due to its characteristics of nontoxicity, physiological inertness, hydrophilicity and biocompatibility. Introducing Fe3O4 in chitosan microspheres contributes to the formation of microspheres with superparamagnetism. In this work, MWCBs, which were constructed by immobilizing P. mendocina cells into Fe3O4-chitosan microspheres, were characterized and applied for biodiesel production from soybean oil. Factors affecting the biodiesel yield during transesterification reaction, such as MWCBs concentration, temperature, the molar ratio of methanol to oil, water content, were investigated. Furthermore, the reusability of MWCBs was also studied to indicate its potential in biodiesel production. 2. Materials and methods 2.1. Strains and chemicals P. mendocina CGMCC 7644 was obtained from China General Microbiological 5
Culture Collection Center. The standard fatty acid methyl esters (FAMEs) were purchased from Sigma-Aldrich Co. (USA). Peptone, yeast extract and agar powder were derived from Aoboxing Bio-Tech Co., Ltd. (Beijing, China). Soybean oil was purchased from Luhua Group Co., Ltd. (Shandong, China). FeSO4·7H2O, FeCl3·6H2O, NaOH, NH3·H2O, polyethylene glycol, chitosan, tetraethoxysilane, glutaraldehyde, olive oil, polyvinyl alcohol, methanol and ethanol were analytical grade (Yuanli Chemical Co., Ltd., Tianjin, China). 2.2. Synthesis of magnetic nanoparticle (Fe3O4) FeSO4·7H2O and FeCl3·6H2O (the molar ratio of Fe2+/Fe3+ was 1:2) were dissolved in 1 wt.% polyethylene glycol solution. Then 25 wt.% NH3·H2O was added into the solution, stirring at 60 oC for 1 h. The magnetic precipitates were subsequently removed by magnetic separation. Finally, tetraethoxysilane (10 mL), NH3·H2O (5 mL), ethanol (150 mL) and the magnetic precipitates (4 g) were mixed, stirring vigorously for 5 h. The SiO2-coated magnetic nanoparticles (Fe3O4) were rinsed thoroughly with water and ethanol and then dried in a vacuum freeze dryer. The magnetic nanoparticles obtained owned a black color and showed a strong response to an external magnetic field. 2.3. P. mendocina whole-cell biocatalysts preparation P. mendocina CGMCC 7644 was cultivated in LB liquid medium at 35 °C and with 160 rpm for 24 h. Suspended cells (>107 cells/mL) and chitosan solution (2.5%, w/v) containing 4% (w/v) SiO2-coated magnetic nanoparticle (Fe3O4) were mixed (1:12, v/v) thoroughly using ultrasonic wave for 10 min. The mixture was injected dropwise 6
into a sterilized NaOH solution by a needle. Thereafter, glutaraldehyde (25 wt.%) was added to the solution, stirring for 2 h. Finally, these magnetic particles with whole-cell biocatalysts were separated by a magnet and washed with water until neutral pH. For the preparation of Fe3O4-uncontanied whole cell biocatalysts, all the steps were identical with the exception of the need for introducing SiO2-coated Fe3O4. The immobilized cell numbers entrapped in particles were determined by microscopic counting [19,20]. 2.4. Transesterification catalyzed by MWCBs The reaction using soybean oils as substrate was carried out in a cylindrical glass bottle (50 mL) by batch operation. Bottles containing soybean oil, MWCBs, water and methanol were stirred in a reciprocal shaker (160 rpm). The reaction was initiated at 35 oC with a MWCBs concentration of 10 wt.% and a water content of 10 wt.%. For the optimization of transesterification, MWCBs with a concentration range of 2-18 wt.%, temperature with a range of 15-55 oC, methanol to oil in the molar ratio with a range of 1-6:1 and water content with a range of 0-40 wt.% were investigated. 2.5. Recycling MWCBs for transesterification MWCBs were separated from the reaction mixtures by magnet after each cycle and were directly reused into new reaction solutions for the next cycle. Samples for the measurement of biodiesel yield were collected at designated intervals. 2.6 Analytical methods 2.6.1 Characterization analysis of MWCBs The SEM images of MWCBs particles were obtained using SU-8010 equipment 7
(Hitachi, Japan). XRD patterns were recorded by D/MAX-2500 diffractometer (Rigaku, Japan). The magnetic property of MWCBs was observed by LDJ-9600 VSM (LDJ Electronics，USA). 2.6.2 Lipase activity 4 mL emulsion with an olive oil to polyvinyl alcohol ratio of 1:3 (v/v), 0.5 g MWCBs and 5 mL 50 mM phosphate buffer (pH 7) were mixed, stirring at 35 oC for 20 min. It was subsequently terminated by adding 10 mL 95% (v/v) ethanol. Non-catalytic reaction was performed as a control. One-unit lipase activity of MWCBs was defined as the amount of lipase that released 1.0 μmol fatty acids per minute under the assay condition. The released fatty acids were quantified by titration with NaOH (5 mM) solution [18]. 2.6.3 FAMEs analysis Samples of FAMEs obtained from the reaction mixtures were centrifuged at 9000 rpm for 10 min. The supernatants were analyzed using Agilent 7890A GC (INNOWAX, Agilent, 30 m × 0.25 mm × 0.25 µm) equipped with FID. The carrier gas was nitrogen. The temperatures of injector and detector were 330 and 360 o
C, respectively. The temperature of column was started at 100 oC for 2 min, raised to
350 oC at the speed of 10 oC/min, then raised to 380 oC at the speed of 6 oC/min and maintained at 380 oC for 10 min. The concentration of biodiesel could be obtained by a standard curve using a series of external standards. All reported data were averages of experiments performed at least in triplicate. The yield of biodiesel was calculated by Eq. (1): 8
Yield (%)
weight of biodiesel produced 100% theoretical maximum weight of biodiesel
(1)
3. Results and discussion 3.1. Characterization of MWCBs P. mendocina cells were entrapped in the chitosan microspheres and cross-linked with chitosan using glutaraldehyde. The morphology of MWCBs is shown in Fig. 1a. These MWCBs are spherical and have average diameters ranging from 2 to 5 mm. Micro-structural features of MWCBs are observed by SEM (Fig. 1b). SEM analysis confirms a good immobilization of P. mendocina cells on the surface or/and in the internal gaps of magnetic particles owning to chitosan’s flocculating ability and cross-linkable property. Moreover, a porous surface and an interconnected porous structure are observed for MWCBs, suggesting their excellent immobilization performances. Crystalline structure of MWCBs was examined by XRD (Fig. 1c). XRD spectrum of MWCBs was identified by standard spectrum of Fe3O4. As observed, component of particles shows all characteristic peaks of magnetite (standard Fe3O4) and maintain a cubic structure of Fe3O4 (JCPDS card 19-0629). The mean diameter of P. mendocina cells immobilized Fe3O4 derived from Scherrer equation is about 12.4 nm, which is below the superparamagnetic critical size (20 nm) [21]. These results reveal that MWCBs have properties of superparamagnetism. To evaluate the superparamagnetism of MWCBs, the hysteresis of the magnetization was analyzed by VSM at room temperature. As shown in Fig. 1d, a magnetization curve with S-shape is observed for MWCBs. The MWCBs own a saturation magnetization of 47.3 emu/g, 9
a remnant magnetization intensity of 0.0053 emu/g and a coercivity of 0.0107 emu/g, respectively, indicating good superparamagnetism of MWCBs [22]. As presented in Fig. 2, enzyme activity of MWCBs increases significantly with incubation time. Highest enzyme activity of 2810 U/g was achieved at 8 h. This result confirms that lipases produced by MWCBs are secreted outside the cells. In the previous studies, enzyme activities of 560 U/g from Aspergillus niger [23], 1233 U/g from Thermomyces lanuginosus [24] and 1785 U/g from recombinant Pichia pastoris [25] were obtained, respectively, which are all lower than that of MWCBs. 3.2. Effects of methanol addition strategy on transesterification catalyzed by MWCBs Fig. 3 shows the time course of biodiesel yield obtained at 35 oC under the conditions of 10 wt.% MWCBs and 10 wt.% of water. It is obvious that methanol-to-oil ratio has an important influence on MWCBs catalytic transesterification. The biodiesel yield increases at higher methanol concentration. A biodiesel yield of 73.49% was obtained at a methanol/oil ratio of 4. However, excessive methanol concentration beyond 4 leads to a decrease in biodiesel yield owning to irreversible inactivation of MWCBs [26]. Similar phenomena have been reported in other literatures [27]. To decrease the deactivation of MWCBs, stepwise addition of methanol was used for transesterification of soybean oils [28]. One mole equivalent of methanol was added stepwise into reaction system at 12 h intervals. As indicated in Fig. 3, the biodiesel yield was improved significantly with the highest biodiesel yield of 90.48% obtained after 72 h. However, the fifth addition of methanol only gives an increase of 10
3.13% in biodiesel yield. The possible reason for this variation is that excess addition of methanol could result in a lower concentration of lipids, which leads to a negative effect to enzymatic activity [29]. These results reveal that the deactivation of MWCBs is avoided by the stepwise addition of methanol. Based on these results, a four-step addition of methanol is deemed to be an appropriately way to increase the biodiesel yield and to reduce the cost of biodiesel production. Given that, all subsequent investigations were carried out at a four-step addition of methanol. 3.3. Effect of biocatalysts concentration on transesterification As seen in Fig. 4, the biodiesel yield increases significantly by increasing the MWCBs concentration from 2 wt.% to 10 wt.%. Highest biodiesel yield of 84.52% was attained at MWCBs concentration of 10 wt.%. However, no significant increase in biodiesel yield was observed by further increasing the MWCBs concentration. These changes are due to the fact that overmuch biocatalyst particles could lead to higher solid content and viscosity in the transesterification, causing difficulties in mixing and mass transfer [30]. But in consideration of the costs, 10 wt.% MWCBs were chosen as the preferential concentration for transesterification. Therefore, the optimal concentration of MWCBs is deemed to be 10 wt.% in this work. Control experiment was performed with Fe3O4-uncontained whole cell biocatalysts under the same conditions. As observed, there is no significant difference in biodiesel yield between MWCBs and Fe3O4-uncontained whole cell biocatalysts. This indicates that the magnetic materials of MWCBs have no apparent impact on 11
transesterification. 3.4. Effect of temperature on transesterification catalyzed by MWCBs As indicated in Fig. 5a, increasing temperature from 15 oC to 35 oC improves the process. This is an expected result because the growth rate of cells and the molecular diffusion are accelerated by higher temperature, leading to higher reaction rate. However, the biodiesel yield reduces remarkably by further increasing temperature beyond 35 oC, which is mainly due to the partial inactivation of biocatalyst in organic solvent at high temperature. These results are in agreement to previous studies [31,32]. The highest biodiesel yield of 85.36% was obtained at 35 oC in this part. Based on these results, the optimal temperature for MWCBs is concluded to be 35 oC. 3.5. Effect of water content on transesterification catalyzed by MWCBs Optimum water content is required to maintain transesterification activity and lipase stability [33]. Fig. 5b shows biodiesel yields obtained at various amounts of water. As observed, biodiesel yield increases by increasing water content from 0 to 10 wt.%. A maximum yield of 83.04% was obtained at a water content of 10 wt.%. This may be due to the fact that an appropriate higher water content increases the flexibility and the mobility of the enzyme and its expressed activity [34,35]. However, a decrease in biodiesel yield was observed by further increasing water content beyond 10 wt.%. The possible reason for the decrease of biodiesel yield in the range is due to the hydrolytic activity enhanced by excess water content [36]. 3.6. Kinetic analysis of MWCBs-catalyzed transesterification The mechanism of MWCBs-catalyzed transesterification is assumed to follow the 12
Michaelis-Menten and Lineweaver-Burk model [37-39]. Some assumptions are made to simplify model development: (1) The mass transfer limitations in the reaction are ignored. (2) Methanol is regarded as the main inhibitor in the transesterification and other inhibitors are negligible. (3) The methanol inhibits the enzyme by a competitive inhibition mechanism. With these assumptions, the rate equation of transesterification is expressed by Eq. (2):
r
rmax Cs CI Km (1 ) Cs KI
(2)
where r, rmax, CS and CI are transesterification rate, maximum transesterification rate, concentration of substrate (triglyceride) and concentration of inhibitor (methanol), respectively. Km and KI are Michaelis constant and the constant of inhibition, respectively. The double-reciprocal form of the Michaelis-Menten equation is defined by: K 1 1 CI 1 m (1 ) r rmax rmax KI Cs
(3)
Kinetic parameters in Eq. (3) are obtained from reactions with varying methanol concentrations (20%, 25%, 30%, 35%, 40%, v/v) under the optimized conditions (Fig. 6). The values obtained for rmax, Km and KI are 2.1801 mg/min, 0.4272 mg/mL and 0.2308 mg/mL, respectively. As seen from Fig. 6, higher methanol concentration gives higher slope of lines, but an invariant rmax, indicating that methanol was a competitive inhibitor of enzyme activity [40]. These observations conform the above assumption and clearly support the results described in section 3.2.
13
3.7. Reusability of MWCBs MWCBs were separated from the reaction system by using a magnet and reused into the fresh reactants. Fig. 7 shows the time course of biodiesel yield obtained at 35 o
C with stepwise addition of methanol (biocatalysts concentration of 10 wt.%,
methanol to oil molar ratio of 4:1, water content of 10 wt.%). As seen, a biodiesel yield of about 90% is maintained over reused MWCBs. No remarkable decline in biodiesel yield is observed after the sixth reuse. A biodiesel yield of 83.57% is retained even after the tenth reuse. However, the yield obtained over Fe3O4-uncontained whole cell biocatalysts decreased gradually after each reuse, and only maintained 74.06% yield after tenth cycle. The decreasing biodiesel yields in the case of control batch were due to the fact that collisions between particles and collisions with container wall occurred during processes of separating Fe3O4-uncontanied whole-cell biocatalysts from the reaction mixture, leading to damages of microspheres and cell leakage from these microspheres. Owning to their superparamagnetic property, MWCBs were easily and quickly separated from the reaction system by applying an external magnetic field [18,19,41]. The damage caused by collision and the cell leakage were decreased significantly since MWCBs moved rapidly along the magnetic field lines. These results indicate that MWCBs are promising for industrial biodiesel production.
14
4. Conclusions Magnetic whole-cell biocatalysts were constructed, characterized and applied for biodiesel production from soybean oil. A maximum biodiesel yield of 87.32% was obtained under the optimum operating condition (MWCBs concentration of 10 wt.%, water content of 10 wt.%, 35 oC, methanol to oil molar ratio of 4:1 and a four-step addition of methanol) for 48 h. The kinetic data followed Michaelis-Menten model (rmax, 2.1801 mg/min; Km, 0.4272 mg/mL; KI, 0.2308 mg/mL) with competitive inhibition by methanol. MWCBs had an excellent reusability and still gave a biodiesel yield of 83.57% after 10 cycles, which was higher than that of Fe3O4-uncontained whole cell biocatalysts (74.06%). Therefore, MWCBs would be promising for large-scale biodiesel industry. Acknowledgments The authors gratefully acknowledge financial support from Natural Science Foundation of Tianjin, China (16JCYBJC20700) and the National Energy Administration of China (NY2013040302).
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Figure Captions Fig. 1. a, Magnetic particles of MWCBs. b, Surface features of magnetic particles observed with SEM. c, XRD spectrum of MWCBs comparing with standard spectrum of Fe3O4. d, Magnetization curves of MWCBs at 300 K. Fig. 2. Time course of enzyme activity of MWCBs. Fig. 3. Time course of MWCBs catalyzed transesterification. Reaction conditions: MWCBs concentration of 10 wt.%, water content of 10 wt.%, 35 oC, 160 rpm. Fig. 4. Effect of biocatalysts concentration on transesterification catalyzed by MWCBs. Reaction conditions: methanol to oil molar ratio of 4:1, water content of 10 wt.%, 35 oC, 160 rpm, 48h. Fig. 5. Effects of temperature and water contents on transesterification catalyzed by MWCBs. Reaction conditions: MWCBs concentration of 10 wt.%, methanol to oil molar ratio of 4:1, 160 rpm, 48 h. Fig. 6. Lineweaver-Burk plot of MWCBs-catalyzed transesterification under different methanol concentrations. Reaction conditions: MWCBs concentration of 10 wt.%, water content of 10 wt.%, 35 oC, 160 rpm. Fig. 7. Reusability of biocatalysts for repeated batch transesterification. Reaction conditions: biocatalysts concentration of 10 wt.%, water content of 10 wt.%, methanol to oil molar ratio of 4:1, 35 oC, 160 rpm.
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Fig. 1. a, Magnetic particles of MWCBs. b, Surface features of magnetic particles observed with SEM. c, XRD spectrum of MWCBs comparing with standard spectrum of Fe3O4. d, Magnetization curves of MWCBs at 300 K.
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Fig. 2. Time course of enzyme activity of MWCBs.
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Fig. 3. Time course of MWCBs catalyzed transesterification. Reaction conditions: MWCBs concentration of 10 wt.%, water content of 10 wt.%, 35 ℃, 160 rpm.
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Fig. 4. Effect of biocatalysts concentration on transesterification catalyzed by MWCBs. Reaction conditions: methanol to oil molar ratio of 4:1, water content of 10 wt.%, 35 ℃, 160 rpm, 48h.
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Fig. 5. Effects of temperature and water contents on transesterification catalyzed by MWCBs. Reaction conditions: MWCBs concentration of 10 wt.%, methanol to oil molar ratio of 4:1, 160 rpm, 48 h.
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Fig. 6. Lineweaver-Burk plot of MWCBs-catalyzed transesterification under different methanol concentrations. Reaction conditions: MWCBs concentration of 10 wt.%, water content of 10 wt.%, 35 ℃, 160 rpm.
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Fig. 7. Reusability of biocatalysts for repeated batch transesterification. Reaction conditions: biocatalysts concentration of 10 wt.%, water content of 10 wt.%, methanol to oil molar ratio of 4:1, 35 ℃, 160 rpm.
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