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The influence of support structures on cell immobilization and acetone–butanol–ethanol (ABE) fermentation performance
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The influence of support structures on cell immobilization and acetone–butanol–ethanol (ABE) fermentation performance Chi-Ruei He a, Ming-Chieh Lee a, Yu-Yuan Kuo a, Tzong-Ming Wu b, Si-Yu Li a,∗ a b
Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan Department of Materials Science and Engineering, National Chung Hsing University, Taichung 402, Taiwan
a r t i c l e
i n f o
Article history: Received 26 January 2017 Revised 9 May 2017 Accepted 15 May 2017 Available online xxx Keywords: Polyhydroxybutyrate (PHB) Wet-jet process Immobilization Clostridium acetobutylicum Acetone–butanol–ethanol (ABE) fermentation
a b s t r a c t Cell immobilization is an efficient technique for achieving high-cell density; it has been shown to benefit acetone–butanol–ethanol (ABE) fermentation. In this study, the biodegradable material, poly(3hydroxybutyrate) (PHB), was fabricated into plate-like structures by solvent casting (SC) and into fibrous structures by wet jet (WJ) and electrospinning (ES). The BET data showed that the specific surface areas of PHBSC , PHBWJ , and PHBES were 0.17, 18.30, and 22.45 m2 /g, respectively. Butanol yields of 0.25 ± 0.04, 0.28 ± 0.01, and 0.20 ± 0.01 g-butanol/g-glucose were obtained for PHBSC , PHBWJ , and PHBES during batch ABE fermentation. OD600 of fermentation broths that contain PHBSC , PHBWJ , and PHBES can be used to evaluate the immobilization ability. It was found that while OD600 of fermentation broths without an immobilization material was 12.0 ± 1.6, OD600 of fermentation broths containing PHBWJ was 4.3 ± 0.3, which was the best for the three PHB materials tested. In summary, a high specific surface area with a pore size up to several hundred microns is recommended. The robustness and stability of PHB as an immobilization material for ABE fermentation was clearly confirmed. © 2017 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Acetone–butanol–ethanol (ABE) fermentation was the major industrial acetone and butanol production process up to the 1970s. After that, the production of acetone and butanol was undertaken by the petrochemical industry because of competitive cost [1,2]. Growing awareness of the need for a sustainable future has resulted in revitalization of ABE fermentation. Several strategies have been extensively studied to reduce the production cost of ABE fermentation, including the development of robust and efficient strains [3,4], low-cost feedstock [5–8], efficient fermentation techniques [9,10], and new separation methods [11,12]. Cell immobilization is a manageable and efficient technique that enables high performance fermentation [10,13–17]. Different types of materials have been studied for cell immobilization [18], such as brick [9,19], zeolite [20], bone char [21], agricultural waste [2,22], and cotton [16,17]. The choice of immobilization supports can vary, but the golden rule is the concern of biocompatibility where no inhibitors or toxic matter would be produced during their use [18,23]. The other important concern is the specific surface area of the immobilization material [24]. Immobilization supports with a high specific surface area not only provide space
∗
Corresponding author. E-mail address: [email protected] (S.-Y. Li).
for bacteria to accumulate, but also provide protection against the stress of pH, temperature, and shear changes [18]. Among these choices, brick and cotton have been shown to be materials that can effectively support ABE performance [25,26]. Poly(3-hydroxybutyrate) (PHB) is a polyester that can be produced by various microorganisms. The physical properties of PHB are similar to those of polypropylene (PP) whereas the biodegradability of PHB makes it a good alternative to PP. PHB has been used in plastic products such as bottles, bags, disposable nappies, and carriers for drug release [27–30]. PHB can be a good immobilization material not only for its sustainability, but also because it is a plastic material that can be formed into a wide range of structures to suit the cultivation of bacteria, fungi, cell lines, and so on. Currently, high specific area is nothing more than a qualitative criterion for choosing immobilization supports for use in ABE fermentation. The reason for this is that the mechanism involved in Clostridium acetobutylicum immobilization on the support material is not fully understood. In this study, PHB was first employed as the raw material to serve as the immobilization support. PHB was fabricated into different geometries including plates, thick and thin fibrous shapes, which were respectively obtained by using solvent casting (SC), wet jet (WJ), and electrospinning (ES). The effects of geometry and morphology of the support on the immobilization capacity and the performance of ABE fermentation are discussed. Furthermore, because PHB is a renewable material, the feasibility
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Please cite this article as: C.-R. He et al., The influence of support structures on cell immobilization and acetone–butanol–ethanol (ABE) fermentation performance, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.05.016
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of PHB as an immobilization support was investigated by testing its stability during ABE fermentation. 2. Materials and methods 2.1. Fabrication of PHB-based supports Solvent casting for fabricating PHBSC was performed by first dissolving PHB (Sigma-Aldrich, US) in chloroform at 80 °C to obtain a 3 wt% PHB solution. The transparent PHB solution was poured into a glass dish to make a uniformly dispersed liquid film. Chloroform was removed from the PHB in a vacuum drying oven at 30 °C under partial vacuum. The PHBWJ with micro-scale fibrous structure was fabricated using the phase separation of PHB in ethanol as described earlier [31]. A syringe with a 23-gauge needle (ID 0.34 mm) was filled with 3 wt% PHB solution in chloroform. The PHB solution was then directly injected into a 99.5% alcohol solution (ECHO CHEMICAL, Taiwan) using a syringe pump (FES-COL, Falco Tech, Taiwan). The injection velocity was controlled at 0.432 L/h and the alcohol phase was stirred with a magnetic bar at 800 rpm to collect the PHB fibers (PHBWJ ). Electrospun PHB (PHBES ) was fabricated as described in a previous study [32]. In brief, to obtain PHB fibers with diameters of 0.5–1.0 μm, TFE (trifluoroethanol) was used as a solvent to dissolve PHB, at a concentration of 10 wt% [32]. Then 2.5 mL of this PHB solution was injected from 5 mL syringe through a 22-gauge (ID 0.42 mm) at a voltage of 12 kV and a velocity of 4 mL/h. The electrospinning was carried out at room temperature with a distance of 20 cm between the capillary tip and the collecting foil. The preparation of PHBES-Fe3O4 was similar to that of PHBES , but 10 wt% PHB in TFE containing 5% Fe3 O4 was used instead [32]. 2.2. Physical characterization of PHB-based supports The macro morphology of the PHBSC , PHBWJ, PHBES , and PHBES-Fe3O4 supports was recorded using a digital camera. A fieldemission Scanning Electron Microscope (FESEM, JSM-6700F, JEOL Ltd., Tokyo, Japan) was used to characterize the surface morphology before and after ABE fermentation. Newly fabricated PHB supports and PHB supports after ABE fermentation were dried at 40–50 °C to remove residual moisture. Samples were sputtercoated with gold for 90 s using a Jeol JFC-1600, auto Fine Coater. The specific surface areas of PHB-based supports were measured by N2 adsorption using a Micromeritics ASAP 2010 porosimeter and the de-gassed condition was controlled at 80 °C. 2.3. Bacterial strain, culture media, and growth conditions C. acetobutylicum ATCC 824 was used in this study. Bacteria were stored in spore form at room temperature until needed [33]. Batch fermentation was started as follows: test tubes were filled with 9 mL of the Reinforced Clostridial Medium (RCM, OXOID, UK) and then capped with rubber septa and sealed with alumina seals. The head space of the test tubes was flushed for 10 min with nitrogen filtered through a 0.2 μm syringe filter. The test tubes were then sterilized by autoclaving at 121 °C for 20 min. After cooling to room temperature, a mineral stock solution (60 g/L MgSO4 ·7H2 O, 11 g/L FeSO4 ·7H2 O, 0.8 g/L CaCl2 ) and the glucose solution of 600 g/L were added to create the RCM-s medium containing (per liter) 38 g RCM, 0.6 g MgSO4 ·7H2 O, 0.008 g CaCl2 , 0.1 g FeSO4 ·7H2 O, and 60 g glucose. 1.0 mL of spore stock was aseptically transferred to a test tube and cultivated at 37 °C and 200 rpm. Note that the heat-shock germination process was avoided as discussed earlier [12].
In the experiments to test the efficiency of the PHB-based supports on the performances of ABE fermentation and cell immobilization: 0.05 g of PHBCS , 0.05 g of PHBWJ , 0.01 g of PHBES , and 0.011 g of PHBES-Fe3O4 supports were added to individual test tube before autoclaving. 2.4. Sampling and analytical methods The concentration of suspended cells was detected using a UV– vis Spectrophotometer (GENESYS 10S, Thermo Scientific, USA) to measure the optical density (OD) at 600 nm. The glucose concentration was measured by the DNS method [34]. In brief, a 1 mL reagent containing 10 g/L of 3,5-dinitrosalicylic acid, 16 g/L sodium hydroxide, and 300 g/L potassium sodium tartrate was reacted for 10 min in a sealed test tube with 10-μL samples containing reducing sugars at 100 °C. After the reaction, the mixture was cooled off and the absorbance at 550 nm was measured. Acetone, butanol, ethanol, acetic acid, and butyric acids were measured using gas chromatography (Hewlett Packard HP 5890 Series II) as described elsewhere [35]. 2.5. The degradability and durability of PHB by C. acetobutylicum For the degradability and durability test of PHB, approximately 50 mg PHBSC as prepared in this study were rinsed with distilled water followed by 70% ethanol. The PHBSC film was left to dry under UV irradiation and then transferred into the serum bottle that contained the sterilized RCM-s’ medium (RCM-s medium without the supplement of additional glucose; in this manner, the initial glucose concentration of around 5 g/L was derived from RCM). The head-space of the serum bottle was flushed with N2 for 5 min and then inoculation was carried out using the pre-culture of C. acetobutylicum at 10 v/v%. After fermentation at 37 °C and 200 rpm for 6 days, the fermentation broth was subjected to HPLC analysis while the film was vigorously rinsed with distilled water, followed by 70% ethanol. After drying, the weight of the films was recorded. The degradability and durability of PHBSC was evaluated by the weight loss [36,37]. 3. Results and discussion 3.1. The physical geometries and the specific surface area of the PHB supports PHB supports fabricated by solvent casting (SC), wet jet (WJ), and electrospinning (ES) were employed in this study as the cell immobilization matrices for ABE fermentation. Images made by a digital camera and a scanning electron microscope were used to investigate the appearance of the PHB supports. It can be seen in Fig. 1(a) and (e) that the PHB support fabricated by the solvent casting process (PHBSC ) was a plate-like support with no welldefined 3-D structure at 250 × magnification. This result is reasonable since PHB molecules were expected to deposit one by one during the evaporation of the solvent to form a relatively intensive packing geometry. Huag et al. have used the solvent casting to fabricate a PHBSC material which they called dense PHB membrane [38]. On the other hand, the fibrous structure can be obtained by the wet-jet (PHBWJ ) and the electrospinning (PHBES ) processes (see Fig. 1(b), (c), (f), and (g)). The differences in the fibrous structure between the PHBWJ and the PHBES were in the uniformity and the diameter of fibers. While the diameters of PHBES fibers were uniformly within a range of 0.5–1 μm as described in our previous study [32], the diameter of the fibers generated by the wet jet process were widely distributed between 5 and 50 μm. Unlike PHBSC , PHB molecules in PHBWJ and PHBES were packed in a loosely tangled fibrous structure with sponge-like morphology that provided
Please cite this article as: C.-R. He et al., The influence of support structures on cell immobilization and acetone–butanol–ethanol (ABE) fermentation performance, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.05.016
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Fig. 1. Digital camera images of (a) PHBSC (b) PHBWJ (c) PHBES (d) PHBES_ 5% Fe3O4 and field-emission scanning electron microscopic (FESEM) images of (e) PHBSC (f) PHBWJ (g) PHBES and (h) PHBES_ 5% Fe3O4. The subscripts of SC, WJ, and ES represent solvent casting, wet-jet, and electrospinning.
Fig. 3. The comparison of OD600 and the butanol concentration using different adsorbents at fermentation time of 48 h.
Fig. 2. Specific surface areas of PHBSC , PHBWJ , and PHBES .
a sophisticated porosity with a high specific surface area (Fig. 1(f)– (h)) [39]. To quantify the specific surface areas of PHB supports, the BETnitrogen adsorption method was employed. In Fig. 2, the specific surface areas of PHBSC , PHBWJ , and PHBES were 0.17, 18.30, and 22.45 m2 /g, respectively. This is in agreement with previous results that electrospun materials typically have high surface area [40]. This demonstrated that the PHB-based support with the fibrous structure has a specific surface area two orders of magnitude higher than that of the plate-like PHBSC . This is consistent with Fig. 1 and the above observations. 3.2. Immobilization of C. acetobutylicum on PHB supports for ABE fermentation The competences of PHBSC , PHBWJ , and PHBES as immobilization matrices for ABE fermentation were investigated, with each PHB material being individually added during the cultivation of C. acetobutylicum in the batch mode. It can be seen from Fig. 3 that the free cell condition reached an OD600 of 12.0 ± 1.6 with a butanol concentration of 9.2 ± 1.0 g/L after 48 h of cultivation. When PHBSC was employed as the immobilization support during the batch cultivation, both the OD600 of supernatant and the butanol concentration went down to 4.3 ± 0.3 and 5.4 ± 0.3 g/L, respectively. The low OD600 with low butanol concentration indicates poor
growing conditions, which may be attributed to a significant resistance in macroscopic mass transfer between the bacteria and nutrients. Given the same weight of PHB supports that were used in this study, the plate-like PHBSC has the widest extension in two dimensions compared to fibrous PHB supports. Owing to the dense, hydrophobic, and impermeable nature of aqueous solutes of PHBSC , mixing during batch cultivation may be encumbered to restrict the uptake of glucose and nutrients. Although macroscopic mass transfer hindrance was observed, the immobilization of C. acetobutylicum can be observed by the SEM image, as shown in Fig. 4(a). It can be clearly seen that C. acetobutylicum can be well immobilized on the hydrophobic surface of PHBSC , indicating the suitability of PHB as a green material for bacterial immobilization. Nevertheless, the low specific surface area of PHBSC is a drawback. On the other hand, when fibrous PHBWJ was used as a support, a low OD600 of 3.0 ± 0.7 and a high butanol concentration of 9.7 ± 0.2 g/L were simultaneously achieved. The production of butanol with a high glucose consumption, which is comparable to the free cell culture, indicates a thriving bacteria population. The low OD600 of 3.0 ± 0.7 indicates PHBWJ as a competent immobilization support, where most of the bacteria are efficiently immobilized. The immobilization of bacteria on PHBSC and PHBWJ can be observed in the SEM images in Fig. 4(b). A comparable butanol concentration of 9.1 ± 0.4 g/L was reported using PHBES as the immobilization support for ABE. Nevertheless, a curtailment of immobilization efficiency was observed where a significant increase in OD600 from 3.0 ± 0.7 to 9.1 ± 3.4 occurred, compared to the use of PHBWJ .
Please cite this article as: C.-R. He et al., The influence of support structures on cell immobilization and acetone–butanol–ethanol (ABE) fermentation performance, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.05.016
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Fig. 6. The evaluation of degradability and durability of PHB to C. acetobutylicum by the weight loss. Fig. 4. Field-emission scanning electron microscopic (FESEM) images of (a) PHBSC (b) PHBWJ (c) PHBES and (d) PHBES_ 5% Fe3O4 after 48 h ABE fermentation. The subscripts of SC, WJ, and ES represent solvent casting, wet-jet, and electrospinning.
Fig. 5. The comparison of the butanol yield using different adsorbents at fermentation time of 48 h.
It is suggested that the fibrous structure providing the high specific surface area is essential for fabricating the PHB-based immobilization supports, considering the performances of PHBES and PHBWJ . On the other hand, the pore size of the support should also be taken into consideration. It can be estimated (see Fig. 1(f)) that the pore size of PHBWJ can increase to a few hundred microns while the pore size of PHBES is one order of magnitude less than that of PHBWJ . The decrease in the immobilization efficiency can be attributed to increased mass transfer where immobilized bacteria with biofilm-like structure clog the pores on the PHBES support matrix (see Fig. 4(c)). This hindered the mass transfer of nutrients within the support matrix and prevented bacterial growth inside. Thus, it would seem that the capacity of PHB support for bacteria immobilization is determined not only by specific surface area but also by pore size. Moreover, the formation of biofilm-like materials suggests that entrapment plays an important role in the immobilization of C. acetobutylicum within PHBWJ . In summary, a high specific surface area with a pore size up to several hundred microns is recommended. In Fig. 5, the highest butanol yield of 0.28 ± 0.03 g/g was reported for PHBWJ , while the butanol yield for the free cell was 0.18 ± 0.03 g/g. Interestingly, with appropriate immobilization of C. acetobutylicum, butanol yields of over 0.2 g/g were observed during continuous operation [41,42]. Compared to chemostat operations, where butanol yields range from 0.15 to 0.21 g/g [41] the immobilized C. acetobutylicum were presumed to be in a phase that favored solvent production rather than acid production. This
demonstrated that the culture using PHBWJ has the highest solvent production with very low acid concentrations (data not shown) and this implies that the high density of bacteria clustered within the support matrix may be in a condition microscopically different from that of the freely suspended C. acetobutylicum. Magnetic nanoparticles may stimulate the regeneration of bone tissue [43] and to examine the influence of magnetic Fe3 O4 nanoparticles on the activity of immobilized C. acetobutylicum, PHBES containing 5% of Fe3 O4 , PHBES-Fe3O4 , was fabricated and used as the immobilization support during batch ABE fermentation. It can be seen in Fig. 1(d) that the exterior of the PHBES-Fe3O4 support is pale yellow, differing from that of PHBES . This indicates that 5% of Fe3 O4 is high enough to affect the exterior color on a macro scale. There was no significant difference in the fiber size or structure in the SEM images (Fig. 1(h)). Furthermore, there was no significant difference in butanol yield or butanol productivity when PHBES-Fe3O4 was used (Figs. 3 and 5) and the magnetic nanoparticles may play no role in the enhancement of cell growth. On the other hand, the magnetic nanoparticles also exhibited no toxicity to bacteria during batch ABE fermentation. 3.3. The degradability and durability of PHB by C. acetobutylicum It can be seen in Fig. 6 that the weight loss of PHBSC was negative after a 24-day degradation test, dropping from 0 to −5 ± 5%. This indicated that C. acetobutylicum does not degrade PHB in 24 days and that PHBSC can endure all the conditions for regular C. acetobutylicum cultivation. On the other hand, the steady negative weight loss presumably suggests that C. acetobutylicum may attach so firmly to the surface of the film, that neither water nor 70% ethanol removed it. Note that the RSM-s’ medium (low initial glucose concentration of 5 g/L) for the degradation test was used because it is known that the expression of PHB depolymerase is repressed by the presence of glucose [44] The study demonstrated clearly that PHB is a robust immobilization material that can be reused. Unlike other fibrous materials [17], PHB can be 3D-printed into a uniform structure that is best suited for ABE fermentation and other fermentation processes as well [45]. 4. Conclusion ABE fermentation using immobilized C. acetobutylicum is a promising technique for the achievement of high cell density fermentation, to give high butanol productivity and a reasonable
Please cite this article as: C.-R. He et al., The influence of support structures on cell immobilization and acetone–butanol–ethanol (ABE) fermentation performance, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.05.016
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yield. PHBES-Fe3O4 has no significant influence on the butanol yield or productivity and the choice of support matrices for cell immobilization depends on specific surface area and porosity. It is suggested that specific surface area is important for an immobilization material because PHBWJ has a better performance than PHBSC in bacterial immobilization. It is further suggested that porosity is equally important. Although PHBES has a similar specific surface area to that of PHBWJ , it has inferior C. acetobutylicum immobilization. This can be attributed to the natural production of a biofilmlike material by C. acetobutylicum. This may clog the pores of PHBES during fermentation, hinder bacterial growth inside PHBES and interfere with the mass transfer of substrate. The formation of a biofilm material suggests that entrapment plays an important role in the immobilization of the bacteria inside the PHBWJ . A high specific surface area with a pore size up to several hundred microns is recommended. The robustness and stability of PHB materials as immobilization materials for ABE fermentation is confirmed. Acknowledgments This work was funded by the Ministry of Science and Technology Taiwan, MOST-103-2221-E-005-072-MY3 and MOST-104-2621M-0 05-0 04-MY3. References [1] Green EM. Fermentative production of butanol—the industrial perspective. Curr Opin Biotechnol 2011;22:337–43. [2] Cai D, Chang Z, Gao L, Chen C, Niu Y, Qin P, et al. Acetone–butanol–ethanol (ABE) fermentation integrated with simplified gas stripping using sweet sorghum bagasse as immobilized carrier. Chem Eng J 2015;277:176–85. [3] Kumar M, Gayen K. Developments in biobutanol production: new insights. Appl Energy 2011;88:1999–2012. [4] Lu C, Zhao J, Yang S-T, Wei D. Fed-batch fermentation for n-butanol production from cassava bagasse hydrolysate in a fibrous bed bioreactor with continuous gas stripping. Bioresour Technol 2012;104:380–7. [5] Jesse T, Ezeji T, Qureshi N, Blaschek H. Production of butanol from starch-based waste packing peanuts and agricultural waste. J Ind Microbiol Biotechnol. 2002;29:117–23. [6] Qureshi N, Saha BC, Hector RE, Hughes SR, Cotta MA. Butanol production from wheat straw by simultaneous saccharification and fermentation using Clostridium beijerinckii. Part I—batch fermentation. Biomass Bioenergy 2008;32:168–75. [7] He C-R, Huang C-L, Lai Y-C, Li S-Y. The utilization of sweet potato vines as carbon sources for fermenting bio-butanol. J Taiwan Inst Chem Eng. https:// doi.org/10.1016/j.jtice.2017.02.022. [8] He C-R, Kuo Y-Y, Li S-Y. Lignocellulosic butanol production from Napier grass using semi-simultaneous saccharification fermentation. Bioresour Technol 2017;231:101–8. [9] Yen H-W, Li R-J, Ma T-W. The development process for a continuous acetone–butanol–ethanol (ABE) fermentation by immobilized Clostridium acetobutylicum. J Taiwan Inst Chem Eng 2011;42:902–7. [10] Tripathi A, Sami H, Jain SR, Viloria-Cols M, Zhuravleva N, Nilsson G, et al. Improved bio-catalytic conversion by novel immobilization process using cryogel beads to increase solvent production. Enzyme Microb Technol 2010;47:44–51. [11] Lu C, Dong J, Yang S-T. Butanol production from wood pulping hydrolysate in an integrated fermentation-gas stripping process. Bioresour Technol 2013;143:467–75. [12] Lu K-M, Li S-Y. An integrated in situ extraction-gas stripping process for acetone–butanol–ethanol (ABE) fermentation. J Taiwan Inst Chem Eng 2014;45:2106–10. [13] Pilkington P, Margaritis A, Mensour N, Russell I. Fundamentals of immobilised yeast cells for continuous beer fermentation: a review. J Inst Brew 1998;104:19–31. [14] Börner RA, Zaushitsyna O, Berillo D, Scaccia N, Mattiasson B, Kirsebom H. Immobilization of Clostridium acetobutylicum DSM 792 as macroporous aggregates through cryogelation for butanol production. Process Biochem 2014;49:10–18. [15] Yen H-W, Li R-J. The effects of dilution rate and glucose concentration on continuous acetone–butanol–ethanol fermentation by Clostridium acetobutylicum immobilized on bricks. J Chem Technol Biotechnol 2011;86:1399–404. [16] Chen Y, Zhou T, Liu D, Li A, Xu S, Liu Q, et al. Production of butanol from glucose and xylose with immobilized cells of Clostridium acetobutylicum. Biotechnol Bioproc E 2013;18:234–41.
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The influence of support structures on cell immobilization and acetone–butanol–ethanol (ABE) fermentation performance Chi-Ruei He a, Ming-Chieh Lee a, Yu-Yuan Kuo a, Tzong-Ming Wu b, Si-Yu Li a,∗ a b
Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan Department of Materials Science and Engineering, National Chung Hsing University, Taichung 402, Taiwan
a r t i c l e
i n f o
Article history: Received 26 January 2017 Revised 9 May 2017 Accepted 15 May 2017 Available online xxx Keywords: Polyhydroxybutyrate (PHB) Wet-jet process Immobilization Clostridium acetobutylicum Acetone–butanol–ethanol (ABE) fermentation
a b s t r a c t Cell immobilization is an efficient technique for achieving high-cell density; it has been shown to benefit acetone–butanol–ethanol (ABE) fermentation. In this study, the biodegradable material, poly(3hydroxybutyrate) (PHB), was fabricated into plate-like structures by solvent casting (SC) and into fibrous structures by wet jet (WJ) and electrospinning (ES). The BET data showed that the specific surface areas of PHBSC , PHBWJ , and PHBES were 0.17, 18.30, and 22.45 m2 /g, respectively. Butanol yields of 0.25 ± 0.04, 0.28 ± 0.01, and 0.20 ± 0.01 g-butanol/g-glucose were obtained for PHBSC , PHBWJ , and PHBES during batch ABE fermentation. OD600 of fermentation broths that contain PHBSC , PHBWJ , and PHBES can be used to evaluate the immobilization ability. It was found that while OD600 of fermentation broths without an immobilization material was 12.0 ± 1.6, OD600 of fermentation broths containing PHBWJ was 4.3 ± 0.3, which was the best for the three PHB materials tested. In summary, a high specific surface area with a pore size up to several hundred microns is recommended. The robustness and stability of PHB as an immobilization material for ABE fermentation was clearly confirmed. © 2017 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Acetone–butanol–ethanol (ABE) fermentation was the major industrial acetone and butanol production process up to the 1970s. After that, the production of acetone and butanol was undertaken by the petrochemical industry because of competitive cost [1,2]. Growing awareness of the need for a sustainable future has resulted in revitalization of ABE fermentation. Several strategies have been extensively studied to reduce the production cost of ABE fermentation, including the development of robust and efficient strains [3,4], low-cost feedstock [5–8], efficient fermentation techniques [9,10], and new separation methods [11,12]. Cell immobilization is a manageable and efficient technique that enables high performance fermentation [10,13–17]. Different types of materials have been studied for cell immobilization [18], such as brick [9,19], zeolite [20], bone char [21], agricultural waste [2,22], and cotton [16,17]. The choice of immobilization supports can vary, but the golden rule is the concern of biocompatibility where no inhibitors or toxic matter would be produced during their use [18,23]. The other important concern is the specific surface area of the immobilization material [24]. Immobilization supports with a high specific surface area not only provide space
∗
Corresponding author. E-mail address: [email protected] (S.-Y. Li).
for bacteria to accumulate, but also provide protection against the stress of pH, temperature, and shear changes [18]. Among these choices, brick and cotton have been shown to be materials that can effectively support ABE performance [25,26]. Poly(3-hydroxybutyrate) (PHB) is a polyester that can be produced by various microorganisms. The physical properties of PHB are similar to those of polypropylene (PP) whereas the biodegradability of PHB makes it a good alternative to PP. PHB has been used in plastic products such as bottles, bags, disposable nappies, and carriers for drug release [27–30]. PHB can be a good immobilization material not only for its sustainability, but also because it is a plastic material that can be formed into a wide range of structures to suit the cultivation of bacteria, fungi, cell lines, and so on. Currently, high specific area is nothing more than a qualitative criterion for choosing immobilization supports for use in ABE fermentation. The reason for this is that the mechanism involved in Clostridium acetobutylicum immobilization on the support material is not fully understood. In this study, PHB was first employed as the raw material to serve as the immobilization support. PHB was fabricated into different geometries including plates, thick and thin fibrous shapes, which were respectively obtained by using solvent casting (SC), wet jet (WJ), and electrospinning (ES). The effects of geometry and morphology of the support on the immobilization capacity and the performance of ABE fermentation are discussed. Furthermore, because PHB is a renewable material, the feasibility
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Please cite this article as: C.-R. He et al., The influence of support structures on cell immobilization and acetone–butanol–ethanol (ABE) fermentation performance, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.05.016
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of PHB as an immobilization support was investigated by testing its stability during ABE fermentation. 2. Materials and methods 2.1. Fabrication of PHB-based supports Solvent casting for fabricating PHBSC was performed by first dissolving PHB (Sigma-Aldrich, US) in chloroform at 80 °C to obtain a 3 wt% PHB solution. The transparent PHB solution was poured into a glass dish to make a uniformly dispersed liquid film. Chloroform was removed from the PHB in a vacuum drying oven at 30 °C under partial vacuum. The PHBWJ with micro-scale fibrous structure was fabricated using the phase separation of PHB in ethanol as described earlier [31]. A syringe with a 23-gauge needle (ID 0.34 mm) was filled with 3 wt% PHB solution in chloroform. The PHB solution was then directly injected into a 99.5% alcohol solution (ECHO CHEMICAL, Taiwan) using a syringe pump (FES-COL, Falco Tech, Taiwan). The injection velocity was controlled at 0.432 L/h and the alcohol phase was stirred with a magnetic bar at 800 rpm to collect the PHB fibers (PHBWJ ). Electrospun PHB (PHBES ) was fabricated as described in a previous study [32]. In brief, to obtain PHB fibers with diameters of 0.5–1.0 μm, TFE (trifluoroethanol) was used as a solvent to dissolve PHB, at a concentration of 10 wt% [32]. Then 2.5 mL of this PHB solution was injected from 5 mL syringe through a 22-gauge (ID 0.42 mm) at a voltage of 12 kV and a velocity of 4 mL/h. The electrospinning was carried out at room temperature with a distance of 20 cm between the capillary tip and the collecting foil. The preparation of PHBES-Fe3O4 was similar to that of PHBES , but 10 wt% PHB in TFE containing 5% Fe3 O4 was used instead [32]. 2.2. Physical characterization of PHB-based supports The macro morphology of the PHBSC , PHBWJ, PHBES , and PHBES-Fe3O4 supports was recorded using a digital camera. A fieldemission Scanning Electron Microscope (FESEM, JSM-6700F, JEOL Ltd., Tokyo, Japan) was used to characterize the surface morphology before and after ABE fermentation. Newly fabricated PHB supports and PHB supports after ABE fermentation were dried at 40–50 °C to remove residual moisture. Samples were sputtercoated with gold for 90 s using a Jeol JFC-1600, auto Fine Coater. The specific surface areas of PHB-based supports were measured by N2 adsorption using a Micromeritics ASAP 2010 porosimeter and the de-gassed condition was controlled at 80 °C. 2.3. Bacterial strain, culture media, and growth conditions C. acetobutylicum ATCC 824 was used in this study. Bacteria were stored in spore form at room temperature until needed [33]. Batch fermentation was started as follows: test tubes were filled with 9 mL of the Reinforced Clostridial Medium (RCM, OXOID, UK) and then capped with rubber septa and sealed with alumina seals. The head space of the test tubes was flushed for 10 min with nitrogen filtered through a 0.2 μm syringe filter. The test tubes were then sterilized by autoclaving at 121 °C for 20 min. After cooling to room temperature, a mineral stock solution (60 g/L MgSO4 ·7H2 O, 11 g/L FeSO4 ·7H2 O, 0.8 g/L CaCl2 ) and the glucose solution of 600 g/L were added to create the RCM-s medium containing (per liter) 38 g RCM, 0.6 g MgSO4 ·7H2 O, 0.008 g CaCl2 , 0.1 g FeSO4 ·7H2 O, and 60 g glucose. 1.0 mL of spore stock was aseptically transferred to a test tube and cultivated at 37 °C and 200 rpm. Note that the heat-shock germination process was avoided as discussed earlier [12].
In the experiments to test the efficiency of the PHB-based supports on the performances of ABE fermentation and cell immobilization: 0.05 g of PHBCS , 0.05 g of PHBWJ , 0.01 g of PHBES , and 0.011 g of PHBES-Fe3O4 supports were added to individual test tube before autoclaving. 2.4. Sampling and analytical methods The concentration of suspended cells was detected using a UV– vis Spectrophotometer (GENESYS 10S, Thermo Scientific, USA) to measure the optical density (OD) at 600 nm. The glucose concentration was measured by the DNS method [34]. In brief, a 1 mL reagent containing 10 g/L of 3,5-dinitrosalicylic acid, 16 g/L sodium hydroxide, and 300 g/L potassium sodium tartrate was reacted for 10 min in a sealed test tube with 10-μL samples containing reducing sugars at 100 °C. After the reaction, the mixture was cooled off and the absorbance at 550 nm was measured. Acetone, butanol, ethanol, acetic acid, and butyric acids were measured using gas chromatography (Hewlett Packard HP 5890 Series II) as described elsewhere [35]. 2.5. The degradability and durability of PHB by C. acetobutylicum For the degradability and durability test of PHB, approximately 50 mg PHBSC as prepared in this study were rinsed with distilled water followed by 70% ethanol. The PHBSC film was left to dry under UV irradiation and then transferred into the serum bottle that contained the sterilized RCM-s’ medium (RCM-s medium without the supplement of additional glucose; in this manner, the initial glucose concentration of around 5 g/L was derived from RCM). The head-space of the serum bottle was flushed with N2 for 5 min and then inoculation was carried out using the pre-culture of C. acetobutylicum at 10 v/v%. After fermentation at 37 °C and 200 rpm for 6 days, the fermentation broth was subjected to HPLC analysis while the film was vigorously rinsed with distilled water, followed by 70% ethanol. After drying, the weight of the films was recorded. The degradability and durability of PHBSC was evaluated by the weight loss [36,37]. 3. Results and discussion 3.1. The physical geometries and the specific surface area of the PHB supports PHB supports fabricated by solvent casting (SC), wet jet (WJ), and electrospinning (ES) were employed in this study as the cell immobilization matrices for ABE fermentation. Images made by a digital camera and a scanning electron microscope were used to investigate the appearance of the PHB supports. It can be seen in Fig. 1(a) and (e) that the PHB support fabricated by the solvent casting process (PHBSC ) was a plate-like support with no welldefined 3-D structure at 250 × magnification. This result is reasonable since PHB molecules were expected to deposit one by one during the evaporation of the solvent to form a relatively intensive packing geometry. Huag et al. have used the solvent casting to fabricate a PHBSC material which they called dense PHB membrane [38]. On the other hand, the fibrous structure can be obtained by the wet-jet (PHBWJ ) and the electrospinning (PHBES ) processes (see Fig. 1(b), (c), (f), and (g)). The differences in the fibrous structure between the PHBWJ and the PHBES were in the uniformity and the diameter of fibers. While the diameters of PHBES fibers were uniformly within a range of 0.5–1 μm as described in our previous study [32], the diameter of the fibers generated by the wet jet process were widely distributed between 5 and 50 μm. Unlike PHBSC , PHB molecules in PHBWJ and PHBES were packed in a loosely tangled fibrous structure with sponge-like morphology that provided
Please cite this article as: C.-R. He et al., The influence of support structures on cell immobilization and acetone–butanol–ethanol (ABE) fermentation performance, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.05.016
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Fig. 1. Digital camera images of (a) PHBSC (b) PHBWJ (c) PHBES (d) PHBES_ 5% Fe3O4 and field-emission scanning electron microscopic (FESEM) images of (e) PHBSC (f) PHBWJ (g) PHBES and (h) PHBES_ 5% Fe3O4. The subscripts of SC, WJ, and ES represent solvent casting, wet-jet, and electrospinning.
Fig. 3. The comparison of OD600 and the butanol concentration using different adsorbents at fermentation time of 48 h.
Fig. 2. Specific surface areas of PHBSC , PHBWJ , and PHBES .
a sophisticated porosity with a high specific surface area (Fig. 1(f)– (h)) [39]. To quantify the specific surface areas of PHB supports, the BETnitrogen adsorption method was employed. In Fig. 2, the specific surface areas of PHBSC , PHBWJ , and PHBES were 0.17, 18.30, and 22.45 m2 /g, respectively. This is in agreement with previous results that electrospun materials typically have high surface area [40]. This demonstrated that the PHB-based support with the fibrous structure has a specific surface area two orders of magnitude higher than that of the plate-like PHBSC . This is consistent with Fig. 1 and the above observations. 3.2. Immobilization of C. acetobutylicum on PHB supports for ABE fermentation The competences of PHBSC , PHBWJ , and PHBES as immobilization matrices for ABE fermentation were investigated, with each PHB material being individually added during the cultivation of C. acetobutylicum in the batch mode. It can be seen from Fig. 3 that the free cell condition reached an OD600 of 12.0 ± 1.6 with a butanol concentration of 9.2 ± 1.0 g/L after 48 h of cultivation. When PHBSC was employed as the immobilization support during the batch cultivation, both the OD600 of supernatant and the butanol concentration went down to 4.3 ± 0.3 and 5.4 ± 0.3 g/L, respectively. The low OD600 with low butanol concentration indicates poor
growing conditions, which may be attributed to a significant resistance in macroscopic mass transfer between the bacteria and nutrients. Given the same weight of PHB supports that were used in this study, the plate-like PHBSC has the widest extension in two dimensions compared to fibrous PHB supports. Owing to the dense, hydrophobic, and impermeable nature of aqueous solutes of PHBSC , mixing during batch cultivation may be encumbered to restrict the uptake of glucose and nutrients. Although macroscopic mass transfer hindrance was observed, the immobilization of C. acetobutylicum can be observed by the SEM image, as shown in Fig. 4(a). It can be clearly seen that C. acetobutylicum can be well immobilized on the hydrophobic surface of PHBSC , indicating the suitability of PHB as a green material for bacterial immobilization. Nevertheless, the low specific surface area of PHBSC is a drawback. On the other hand, when fibrous PHBWJ was used as a support, a low OD600 of 3.0 ± 0.7 and a high butanol concentration of 9.7 ± 0.2 g/L were simultaneously achieved. The production of butanol with a high glucose consumption, which is comparable to the free cell culture, indicates a thriving bacteria population. The low OD600 of 3.0 ± 0.7 indicates PHBWJ as a competent immobilization support, where most of the bacteria are efficiently immobilized. The immobilization of bacteria on PHBSC and PHBWJ can be observed in the SEM images in Fig. 4(b). A comparable butanol concentration of 9.1 ± 0.4 g/L was reported using PHBES as the immobilization support for ABE. Nevertheless, a curtailment of immobilization efficiency was observed where a significant increase in OD600 from 3.0 ± 0.7 to 9.1 ± 3.4 occurred, compared to the use of PHBWJ .
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Fig. 6. The evaluation of degradability and durability of PHB to C. acetobutylicum by the weight loss. Fig. 4. Field-emission scanning electron microscopic (FESEM) images of (a) PHBSC (b) PHBWJ (c) PHBES and (d) PHBES_ 5% Fe3O4 after 48 h ABE fermentation. The subscripts of SC, WJ, and ES represent solvent casting, wet-jet, and electrospinning.
Fig. 5. The comparison of the butanol yield using different adsorbents at fermentation time of 48 h.
It is suggested that the fibrous structure providing the high specific surface area is essential for fabricating the PHB-based immobilization supports, considering the performances of PHBES and PHBWJ . On the other hand, the pore size of the support should also be taken into consideration. It can be estimated (see Fig. 1(f)) that the pore size of PHBWJ can increase to a few hundred microns while the pore size of PHBES is one order of magnitude less than that of PHBWJ . The decrease in the immobilization efficiency can be attributed to increased mass transfer where immobilized bacteria with biofilm-like structure clog the pores on the PHBES support matrix (see Fig. 4(c)). This hindered the mass transfer of nutrients within the support matrix and prevented bacterial growth inside. Thus, it would seem that the capacity of PHB support for bacteria immobilization is determined not only by specific surface area but also by pore size. Moreover, the formation of biofilm-like materials suggests that entrapment plays an important role in the immobilization of C. acetobutylicum within PHBWJ . In summary, a high specific surface area with a pore size up to several hundred microns is recommended. In Fig. 5, the highest butanol yield of 0.28 ± 0.03 g/g was reported for PHBWJ , while the butanol yield for the free cell was 0.18 ± 0.03 g/g. Interestingly, with appropriate immobilization of C. acetobutylicum, butanol yields of over 0.2 g/g were observed during continuous operation [41,42]. Compared to chemostat operations, where butanol yields range from 0.15 to 0.21 g/g [41] the immobilized C. acetobutylicum were presumed to be in a phase that favored solvent production rather than acid production. This
demonstrated that the culture using PHBWJ has the highest solvent production with very low acid concentrations (data not shown) and this implies that the high density of bacteria clustered within the support matrix may be in a condition microscopically different from that of the freely suspended C. acetobutylicum. Magnetic nanoparticles may stimulate the regeneration of bone tissue [43] and to examine the influence of magnetic Fe3 O4 nanoparticles on the activity of immobilized C. acetobutylicum, PHBES containing 5% of Fe3 O4 , PHBES-Fe3O4 , was fabricated and used as the immobilization support during batch ABE fermentation. It can be seen in Fig. 1(d) that the exterior of the PHBES-Fe3O4 support is pale yellow, differing from that of PHBES . This indicates that 5% of Fe3 O4 is high enough to affect the exterior color on a macro scale. There was no significant difference in the fiber size or structure in the SEM images (Fig. 1(h)). Furthermore, there was no significant difference in butanol yield or butanol productivity when PHBES-Fe3O4 was used (Figs. 3 and 5) and the magnetic nanoparticles may play no role in the enhancement of cell growth. On the other hand, the magnetic nanoparticles also exhibited no toxicity to bacteria during batch ABE fermentation. 3.3. The degradability and durability of PHB by C. acetobutylicum It can be seen in Fig. 6 that the weight loss of PHBSC was negative after a 24-day degradation test, dropping from 0 to −5 ± 5%. This indicated that C. acetobutylicum does not degrade PHB in 24 days and that PHBSC can endure all the conditions for regular C. acetobutylicum cultivation. On the other hand, the steady negative weight loss presumably suggests that C. acetobutylicum may attach so firmly to the surface of the film, that neither water nor 70% ethanol removed it. Note that the RSM-s’ medium (low initial glucose concentration of 5 g/L) for the degradation test was used because it is known that the expression of PHB depolymerase is repressed by the presence of glucose [44] The study demonstrated clearly that PHB is a robust immobilization material that can be reused. Unlike other fibrous materials [17], PHB can be 3D-printed into a uniform structure that is best suited for ABE fermentation and other fermentation processes as well [45]. 4. Conclusion ABE fermentation using immobilized C. acetobutylicum is a promising technique for the achievement of high cell density fermentation, to give high butanol productivity and a reasonable
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yield. PHBES-Fe3O4 has no significant influence on the butanol yield or productivity and the choice of support matrices for cell immobilization depends on specific surface area and porosity. It is suggested that specific surface area is important for an immobilization material because PHBWJ has a better performance than PHBSC in bacterial immobilization. It is further suggested that porosity is equally important. Although PHBES has a similar specific surface area to that of PHBWJ , it has inferior C. acetobutylicum immobilization. This can be attributed to the natural production of a biofilmlike material by C. acetobutylicum. This may clog the pores of PHBES during fermentation, hinder bacterial growth inside PHBES and interfere with the mass transfer of substrate. The formation of a biofilm material suggests that entrapment plays an important role in the immobilization of the bacteria inside the PHBWJ . A high specific surface area with a pore size up to several hundred microns is recommended. The robustness and stability of PHB materials as immobilization materials for ABE fermentation is confirmed. Acknowledgments This work was funded by the Ministry of Science and Technology Taiwan, MOST-103-2221-E-005-072-MY3 and MOST-104-2621M-0 05-0 04-MY3. References [1] Green EM. Fermentative production of butanol—the industrial perspective. Curr Opin Biotechnol 2011;22:337–43. [2] Cai D, Chang Z, Gao L, Chen C, Niu Y, Qin P, et al. Acetone–butanol–ethanol (ABE) fermentation integrated with simplified gas stripping using sweet sorghum bagasse as immobilized carrier. Chem Eng J 2015;277:176–85. [3] Kumar M, Gayen K. Developments in biobutanol production: new insights. Appl Energy 2011;88:1999–2012. [4] Lu C, Zhao J, Yang S-T, Wei D. Fed-batch fermentation for n-butanol production from cassava bagasse hydrolysate in a fibrous bed bioreactor with continuous gas stripping. Bioresour Technol 2012;104:380–7. [5] Jesse T, Ezeji T, Qureshi N, Blaschek H. Production of butanol from starch-based waste packing peanuts and agricultural waste. J Ind Microbiol Biotechnol. 2002;29:117–23. [6] Qureshi N, Saha BC, Hector RE, Hughes SR, Cotta MA. Butanol production from wheat straw by simultaneous saccharification and fermentation using Clostridium beijerinckii. Part I—batch fermentation. Biomass Bioenergy 2008;32:168–75. [7] He C-R, Huang C-L, Lai Y-C, Li S-Y. The utilization of sweet potato vines as carbon sources for fermenting bio-butanol. J Taiwan Inst Chem Eng. https:// doi.org/10.1016/j.jtice.2017.02.022. [8] He C-R, Kuo Y-Y, Li S-Y. Lignocellulosic butanol production from Napier grass using semi-simultaneous saccharification fermentation. Bioresour Technol 2017;231:101–8. [9] Yen H-W, Li R-J, Ma T-W. The development process for a continuous acetone–butanol–ethanol (ABE) fermentation by immobilized Clostridium acetobutylicum. J Taiwan Inst Chem Eng 2011;42:902–7. [10] Tripathi A, Sami H, Jain SR, Viloria-Cols M, Zhuravleva N, Nilsson G, et al. Improved bio-catalytic conversion by novel immobilization process using cryogel beads to increase solvent production. Enzyme Microb Technol 2010;47:44–51. [11] Lu C, Dong J, Yang S-T. Butanol production from wood pulping hydrolysate in an integrated fermentation-gas stripping process. Bioresour Technol 2013;143:467–75. [12] Lu K-M, Li S-Y. An integrated in situ extraction-gas stripping process for acetone–butanol–ethanol (ABE) fermentation. J Taiwan Inst Chem Eng 2014;45:2106–10. [13] Pilkington P, Margaritis A, Mensour N, Russell I. Fundamentals of immobilised yeast cells for continuous beer fermentation: a review. J Inst Brew 1998;104:19–31. [14] Börner RA, Zaushitsyna O, Berillo D, Scaccia N, Mattiasson B, Kirsebom H. Immobilization of Clostridium acetobutylicum DSM 792 as macroporous aggregates through cryogelation for butanol production. Process Biochem 2014;49:10–18. [15] Yen H-W, Li R-J. The effects of dilution rate and glucose concentration on continuous acetone–butanol–ethanol fermentation by Clostridium acetobutylicum immobilized on bricks. J Chem Technol Biotechnol 2011;86:1399–404. [16] Chen Y, Zhou T, Liu D, Li A, Xu S, Liu Q, et al. Production of butanol from glucose and xylose with immobilized cells of Clostridium acetobutylicum. Biotechnol Bioproc E 2013;18:234–41.
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Please cite this article as: C.-R. He et al., The influence of support structures on cell immobilization and acetone–butanol–ethanol (ABE) fermentation performance, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.05.016