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Use of electrospun threads in immobilized cell reactors for continuous ethanol production
Colloids and Surfaces B: Biointerfaces 181 (2019) 989–993
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Use of electrospun threads in immobilized cell reactors for continuous ethanol production Akira Nordmeiera, Dev Chidambarama,b, a b
T
⁎
Chemical and Materials Engineering, University of Nevada, Reno, 1664 N. Virginia St., Reno, NV 89557-0338, United States Nevada Institute for Sustainability, University of Nevada, Reno, 1664 N. Virginia St., Reno, NV 89557-0338, United States
ARTICLE INFO
ABSTRACT
Keywords: Yeast FDMA Bioethanol
Saccharomyces cerevisiae immobilized in electrospun Pluronic F127 dimethacrylate (FDMA) was successfully employed for the production of ethanol in an immobilized cell reactor. Yeast cells were immobilized into fibers formed through the process of electrospinning and cross-linking. The threads had an average diameter of 0.88 μm and were used in continuous-flow immobilized cell reactors. The immobilized cell reactors were able to maintain a high ethanol yield of > 90% from day 4 through to the end of the time course at day 14. The reactor was able to achieve a maximum ethanol yield of 94.3%. This study shows that the use of electrospinning is a promising method for continuous ethanol production through immobilized cell-based continuous flow reactors.
1. Introduction Every year approximately 11 billion metric tons of fossil fuels are consumed, and it is estimated that all known oil reserves will be depleted by 2052 [1]. The need for alternative renewable liquid transportation fuels has shifted the focus onto biologically generated energy (biofuels) to minimize environmental costs [2,3]. Ethanol produced from microorganisms has been chosen by various governments to be the transportation fuel-of-choice, as so-produced ethanol is considered a biofuel and helps meet the Renewable Fuel Standards (RFS) [4]. It is estimated that the United States produced 15.25 billion gallons of fuelquality ethanol in 2016, which is a 788.2% increase in production from 2000. Production of ethanol is expected to continue to increase as the U.S. congressional mandate requiring the blending of at least 36 billion gallons of ethanol into transportation fuels by the year 2022. This requirement would more than double the current levels of ethanol production. To meet these production goals, significant increases in process efficiency and manufacturing capacity will be required. There have been various techniques designed to improve the production of ethanol, including continuous culturing and cell recycling [5]. The use of immobilized cell reactors (ICR) has been shown to hold great promise for the efficient production of bio-ethanol [6–8]. The most common of studied immobilization methods makes use of alginate beads and has been studied since Thiele first started this work in 1947 [9]. The use of alginate for whole cell entrapment offers high survival rates and has been studied extensively with different alginate
⁎
concentrations, designs, microorganisms, feed stocks, and feed rates [10–13]. However, the substrate/product must diffuse through the alginate to and from the microorganisms; the diffusion rate is often slow and creates microenvironments. This limits the metabolic activity of the microorganisms which lowers production and yield [14,15]. To minimize this effect, new techniques have been studied to increase the porosity of the materials through the use of hybrid materials such as polyvinyl alcohol (PVA), and polyethylene oxide (PEO) [16,17]. An alternative method is to reduce the size of the immobilization, this would reduce the distance needed for the substrate/product to diffuse [18]. Although spheres offer great surface area-to-volume ratios, the rate of diffusion by the substrate will be limited by the radius of the sphere. Furthermore, decreasing the size of the beads poses problems with retention of the beads within the reactor. One method to reduce the size of the immobilized bead without encountering bead retention issues is to form threads. Alginate threads, when compared to beads with similar sizes, have been shown to have higher diffusion rates [19]. Further, reduction in the size of the threads could minimize the time required for diffusion through the polymer as well as polymer/microorganism interaction. Electrospinning is a technique that is being studied for its use in biotechnology as this technique can produce nanometer-sized threads that form highly porous mats [20,21]. This method has been used with various polymer blends and has shown to have high cell viability [22,23]. Previous work by the principle investigator of this work showed that microorganisms could be immobilized in electrospun threads using PEO blended hydrogel Pluronic
Corresponding author at: University of Nevada, Reno, 1664 N. Virginia St., Reno, NV 89557-0338, United States. E-mail address: [email protected] (D. Chidambaram).
https://doi.org/10.1016/j.colsurfb.2019.05.013 Received 1 February 2019; Received in revised form 18 April 2019; Accepted 7 May 2019 Available online 08 May 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
Colloids and Surfaces B: Biointerfaces 181 (2019) 989–993
A. Nordmeier and D. Chidambaram
F127 dimethacrylate (FDMA or PEO99-: polypropylene oxide (PPO)67PEO99 DMA) as the immobilization material [24]. This work focuses on (I) the use of electrospinning to minimize the amount of polymer surrounding the microorganisms and evaluate the effect on the ethanol yield using a model organism Saccharomyces cerevisiae and (II) the applicability of electrospun threads for use in a continuous ICR.
by the ratio between the cell dry weight and polymer weight. The cell dry weight of 10 ml at 2.5 O.D. was 70 mg. The media was fed to the ICR column from a feedstock reservoir located beside the column. The feedstock consisted of (g/l) glucose, 100; yeast extract, 10 in DiH2O. A Thermo Scientific FH100 M peristaltic pump was used to transfer the feed medium from the reservoir to the ICR. The effluent from the column was collected in a 1 l conical flask which served as the product reservoir. The feed media was pumped in an upward-flow manner. The retention time (RT) of continuous culture was maintained at 9.8 h and 24 h. Aliquots of 0.5 ml were collected at various time intervals and centrifuged for 5 min at 4 °C for ethanol and glucose analysis as described elsewhere by the authors [17]. Experiments between 1 sheet and 3 sheets of immobilized yeast were conducted in triplicate and averaged.
2. Methods 2.1. Culture media Fermentation was conducted using dry activated distillers yeast (DADY) (Saccharomyces cerevisiae) purchased from Red Star. The organism was cultured in sterilized media containing (g/l) glucose, 50; peptone, 10; yeast extract, 10; MgSO4, 1; and K2HPO4, 1 in deionized water (DiH2O). The culture was maintained in an shaking incubator (125 rpm, 28 ± 1 °C). Culture viability and growth was monitored by the (O.D.) of the culture at 600 nm.
3. Results & discussion In order to ensure the proper formation of FDMA, the FDMA was analyzed using FTIR for the functional groups and was compared to the unmodified Pluronic F-127. The methacrylate group that was added to the polymer can be determined by the appearance of peaks at 1713 and 1635 cm−1 (Fig. 1A). These peaks correspond to the carbonyl vibration of the ester group and to the vinyl double bond, respectively [25]. DSC analysis showed that the melting point of F-127 was 62 °C and the FDMA was 54 °C, which is comparable to the results shown by Cohn et al. [26]. Also, the melting point of the FDMA/PEO blend was between those of pure PEO powder and that of FDMA powder, suggesting proper blending of the polymers (Fig. 1B). Using the FDMA/PEO blended polymer individual yeast cells were encapsulated in threads, as shown in Fig. 2A. The distribution of fiber sizes after electrospinning was analyzed using ImageJ image analysis tool, the size distribution is shown in Fig. 2B. The size distribution of 200 fibers, at spots not containing yeast, showed a narrow distribution of fiber diameter, in which, ∼80% of the fibers were between 0.5 and 1 μm in diameter. As these threads are smaller than the yeast cell this ensures that there is a minimal amount of polymer immobilizing the yeast cell. This size distribution is also similar to those obtained by similar methods [22,27]. The cross-linked FDMA sheets were then placed into the ICR. Production of ethanol is often discussed in terms of yield, which was calculated as follows:
2.2. F127 DMA synthesis and FDMA/PEO blend F127 dimethacrylate (FDMA) was synthesized using the method described in Sosnik et al. [25]. As discussed in Liu et al. 2009, FDMA is not optimal for immobilization and was blended with PEO to facilitate a porous mat formation during the electrospinning process [24]. FDMA/ PEO solution was made using 13 wt% FDMA and 3 wt% PEO in DiH2O. For the immobilization of yeast, the culture was transferred to a new media vial and incubated at 28 °C for 24 h. Cultures were collected when the O.D. reached 2.5 in 50 ml of media. The cultures were collected by centrifugation in 10 ml increments and were re-suspended in 10 ml of FDMA/PEO. 2.3. Characterization FTIR analysis was performed using a Thermo Nicolet 6700 FTIR spectrometer to confirm the synthesis of FDMA. Differential scanning calorimetry (DSC) of F-127, FDMA, FDMA/PEO-blend, and PEO was performed using a Netzsch Jupiter STA 449 F3. DSC measurements were conducted in a nitrogen environment at a heating rate of 20 °C/ min in the temperature range between 25 °C and 500 °C using 15 mg samples loaded in alumina crucibles under dry conditions.
Experimental amount of ethanol amount of ethanol theoretically
× 100
2.4. Electrospinning
Maximum
The FDMA/PEO solution containing yeast was placed into a 10 ml syringe with a 22 G needle. The syringe was attached to a syringe pump (New Era Pump Systems) which provided a steady solution flow rate of 20 μl/min. A high-voltage power supply (Gamma High Voltage Research, Ormond Beach, FL) was employed to apply a potential of 12 kV between the 22 G needle and the metal target which was horizontally placed 18 cm away from the tip of the needle. The metal target was a 9 × 9 cm Al foil. Electrospinning was conducted for 1 h and approximately 0.1 g was collected. After collection, the threads were cross-linked using the process described in Liu et al. and allowed to incubate at 4 °C for 12 h [24].
Wherein, the experimental amount of ethanol was measured using HPLC and the theoretical amount was calculated using stoichiometry per the following equation:
possible
C6H12O6 → 2 C2H5OH + 2 CO2 For example, if 80 g/l of glucose is consumed, the theoretical maximum of ethanol will be 40.88 g/l. Fig. 3 shows a comparison between the ICR containing 1 sheet and 3 sheets of the electrospun threads with immobilized yeast. Each reactor was operated in triplicate and with an RT of 9.8 h to ensure repeatability of the reactors as well as the immobilized yeast. The reactors with 1 sheet were able to obtain the highest glucose conversion during the time course, reaching 38.18 g/l of ethanol in 72 h. Because 19.5 g/l of glucose remained at the end of the period, a total of 80.5 g/l of glucose were consumed. Based on this glucose consumption, the theoretical amount of ethanol that can be produced is 41.13 g/l. Thus, the reactor was able to achieve 92.8% yield. This is a notable result as most strains of yeast are capable of an efficiency of 90–92% in batch reactors [28,29]; however, in slow fermenters, DADY has been shown to achieve up to 95% yield [30]. However, the reactor was not stable, and there was a decrease in ethanol production thereafter. This decrease in ethanol production could be due to the loose packing of the reactor,
2.5. Experimental reactor system and analysis The ICR was built using polyvinyl chloride sheet with a 3.2 cm (height) × 2.4 cm (width) × 0.5 cm (depth) with inlet/outlet ports on the top and bottom of the reactor. The volume of the reactor prior to packing was 3.7 ml. The reactors were placed on a bench top at ambient temperature and prefilled with yeast media containing 10 g/l yeast extract in DiH2O. A control reactor was also conducted with 1.75 ml of a yeast culture, as this was determined to contain approximately the same amount of yeast as the immobilized reactors. This was determined 990
Colloids and Surfaces B: Biointerfaces 181 (2019) 989–993
A. Nordmeier and D. Chidambaram
Fig. 2. SEM images of yeast immobilized through electrospinning FDMA/yeast solution A) 400x magnification of dense threads with an inset providing a perspective of the fiber and the cell B) Size distribution of the diameter of 200 fibers with an average of 0.88 μm.
Fig. 1. (A) FTIR spectra of F127 (top spectrum) and F127 dimethacrylate (FDMA) (bottom spectrum) showing the addition of 2 peaks at 1714 and 1636 cm−1, corresponding to the addition of the methacrylate group to the F127 base material. (B) DSC analyses of different polymer samples F-127, FDMA, FDMA/ PEO blend, and PEO.
which caused significant bubble formation to occur. These bubbles formed around the sheet and limited the interaction of the yeast with the media. The ICRs with 3 sheets required a longer startup period but showed less variation between reactors and continued to have an increased production of ethanol throughout the time course reaching a maximum of 86.7% yield. Although bubble formation did occur, the bubbles were significantly smaller and often dissipated. This is likely due to the packing of the reactor which limits the space available. Neither the 1 sheet nor the 3 sheet reactors were able to completely reduce the glucose concentration with the RT of 9.8 h therefore, a longer RT is needed. A study with a longer time course of 14 days and longer RT of 24 h was performed with the ICRs with 3 sheets. This reactor was then compared to a control reactor which had 1.75 ml of yeast culture inoculated into the reactor with no other additions. Fig. 4 shows that the immobilized yeast was able to obtain a maximum ethanol concentration of 46.35 g/l or 94.3% yield, which is close to the 95% ethanol yield from DADY as discussed earlier [30]. The control reactor was also able to obtain similar yields throughout the time course; however, the ethanol production and glucose consumption did not stabilize. Both the control the immobilized yeast reactors were not able to attain complete
Fig. 3. The comparison of the concentration of glucose to the production yield of ethanol with an RT of 9.6 h for ICR containing 1 sheet and 3 sheets of FDMA electrospun threads immobilizing yeast.
glucose reduction. Similar to the previous experiment the ICR with 3 sheets of immobilized yeast often had bubbles in the reactor. However, these bubbles did not dissipate which reduce the volume of the reactor and resulted in a faster retention time. 991
Colloids and Surfaces B: Biointerfaces 181 (2019) 989–993
A. Nordmeier and D. Chidambaram
Fig. 4. The comparison of the concentration of glucose to the production yield of ethanol with an RT of 24 h for ICR containing 3 sheets of FDMA electrospun threads immobilizing yeast and a control reactor inoculated with 1.75 ml of yeast culture.
Acknowledgment
This study provides promising short-term results in the use of immobilized yeast for continuous ethanol production. The ability to achieve a high (94%) yield and maintain it over short period of several days is most promising. Longer-term studies on the order of 4–8 weeks are needed prior to implementation in the industry so as to allow downtime only once a month or every two months. An aspect that needs to be improved is the yield, as batch processes provide higher (˜98%) yield. Yield can be increased by optimizing the flow rates and retention time. The vast majority of commercial ethanol is currently produced using batch processes, which have significant startup time requirements. Using continuous flow will require less capital as the reactor sizes for equivalent production will be much smaller. Since these are continuous flow reactors where in the glucose solution needs to be in contact with the immobilized cells, the reactor volume and production volumes per reactor are expected to be small; however, connecting several small reactors to scale up production, as is often conducted in biotechnology, chemical and pharmaceutical industries, will be simple. Thus, successful implementation in the industry will need significantly lower capital than current batch processing plants. Finally, while this study focused on production of ethanol, this process can be used for production of other bioproducts as well.
This material is based upon work supported by the National Science Foundation under Grant No. 1159772. References [1] K. Aleklett, M. Höök, K. Jakobsson, M. Lardelli, S. Snowden, B. Söderbergh, The peak of the oil age–analyzing the world oil production reference scenario in world energy outlook 2008, Energy Policy 38 (2010) 1398–1414. [2] John Houghton, Sharlene Weatherwax, A.J. Ferrell, Breaking the Biological Barriers to Cellulosic Ethanol: A Joint Research Agenda: A Research Roadmap Resulting from the Biomass to Biofuels Workshop, December 7–9, 2005, Department of Energy Office of the Biomass Program, Rockville, Maryland, United States, 2006. [3] A.E. Farrell, R.J. Plevin, B.T. Turner, A.D. Jones, M. O’Hare, D.M. Kammen, Ethanol can contribute to energy and environmental goals, Science 311 (2006) 506–508. [4] X. Tang, H. Feng, W.N. Chen, Metabolic engineering for enhanced fatty acids synthesis in Saccharomyces cerevisiae, Metab. Eng. 16 (2013) 95–102. [5] G.R. Cysewski, C.R. Wilke, Process design and economic studies of alternative fermentation methods for the production of ethanol, Biotechnol. Bioeng. 20 (1978) 1421–1444. [6] G.D. Najafpour, Chapter 8 – immobilisation of microbial cells for the production of organic acid and ethanol, in: G.D. Najafpour (Ed.), Biochemical Engineering and Biotechnology, Elsevier, Amsterdam, 2007, pp. 199–227. [7] T. Iida, H. Izumida, Y. Akagi, M. Sakamoto, Continuous ethanol fermentation in molasses medium using Zymomonas mobilis immobilized in photo-crosslinkable resin gels, J. Ferment. Bioeng. 75 (1993) 32–35. [8] P. Verbelen, D. De Schutter, F. Delvaux, K. Verstrepen, F. Delvaux, Immobilized yeast cell systems for continuous fermentation applications, Biotechnol. Lett. 28 (2006) 1515–1525. [9] F. Cassiola, H.S. Santos, I. Joekes, Saccharomyces cerevisiae entrapped in chrysotile increases life-span for up to 3 years, Colloids Surf. B: Biointerfaces 30 (2003) 283–289. [10] L. Peijun, W. Xin, F. Stagnitti, L. Ling, G. Zongqiang, Z. Hairong, X. Xianzhe, A. Chris, Degradation of phenanthrene and pyrene in soil slurry reactors with immobilized bacteria Zoogloea sp, Environ. Eng. Sci. 22 (2005) 390–399. [11] P. Bangrak, S. Limtong, M. Phisalaphong, Continuous ethanol production using immobilized yeast cells entrapped in loofa-reinforced alginate carriers, Braz. J. Microbiol. 42 (2011) 676–684. [12] I. Eş, J.D.G. Vieira, A.C. Amaral, Principles, techniques, and applications of biocatalyst immobilization for industrial application, Appl. Microbiol. Biotechnol. 99 (2015) 2065–2082. [13] D.-H. Kim, S.-B. Lee, H.-D. Park, Effect of air-blast drying and the presence of protectants on the viability of yeast entrapped in calcium alginate beads with an aim to improve the survival rate, Appl. Microbiol. Biotechnol. 101 (2017) 93–102. [14] B. Mattiasson, B. Hahn-Hägerdal, Microenvironmental effects on metabolic behaviour of immobilized cells a hypothesis, Eur. J. Appl. Microbiol. Biotechnol. 16 (1982) 52–55. [15] B. Mattiasson, M. Larsson, B. HAHn-hägerdal, Metabolic behavior of immobilized cells—effects of some microenvironmental factorsa, Ann. N. Y. Acad. Sci. 434 (1984) 475–478.
4. Conclusions Short-term continuous ethanol production in an ICR was successfully demonstrated using yeast immobilized in FDMA via electrospinning. The ICR was able to achieve 94% yield and maintained high ethanol yields after a 4-day startup period with an average of 90.3% yield and a standard deviation of 3.96. To reduce the RT of the reactors and increase the stability, multiple reactors should be run in series. Although proof of principle, this study focused on the short-term analysis to evaluate the ability of electrospun fibers to be used in a continuous flow reactor, the results are promising and warrant further investigation. Conflict of interest The authors declare that they have no conflict of interest.
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Colloids and Surfaces B: Biointerfaces 181 (2019) 989–993
A. Nordmeier and D. Chidambaram [16] R. Jovanovic-Malinovska, M. Cvetkovska, S. Kuzmanova, C. Tsvetanov, E. Winkelhausen, Immobilization of Saccharomyces cerevisiae in novel hydrogels based on hybrid networks of poly(ethylene oxide), alginate and chitosan for ethanol production, Maced. J. Chem. Chem. Eng. 29 (2010) 169–179. [17] A. Nordmeier, D. Chidambaram, The influence of dopants on the effectiveness of alginate beads in immobilized cell reactors, Appl. Biochem. Biotechnol. 178 (2016) 1503–1509. [18] M. Núñez, J. Lema, Cell immobilization: application to alcohol production, Enzyme Microb. Technol. 9 (1987) 642–651. [19] F. Que, Using a thread type of alginate gel particles as cell-immobilised support and some concept of packed bed fermenter design, Biotechnol. Technol. 7 (1993) 755–760. [20] J. Venugopal, S. Ramakrishna, Applications of polymer nanofibers in biomedicine and biotechnology, Appl. Biochem. Biotechnol. 125 (2005) 147–157. [21] M. Amina, T. Amna, N. Al-Musayeib, S.A. Zabin, M.S. Hassan, M.-S. Khil, Encapsulation of β-sitosterol in polyurethane by sol–gel electrospinning, Appl. Biochem. Biotechnol. 182 (2017) 624–634. [22] O.F. Sarioglu, N.O. San Keskin, A. Celebioglu, T. Tekinay, T. Uyar, Bacteria encapsulated electrospun nanofibrous webs for remediation of methylene blue dye in water, Colloids Surf. B: Biointerfaces 152 (2017) 245–251. [23] B. Chaudhuri, B. Mondal, S. Ray, S. Sarkar, A novel biocompatible conducting polyvinyl alcohol (PVA)-polyvinylpyrrolidone (PVP)-hydroxyapatite (HAP)
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Use of electrospun threads in immobilized cell reactors for continuous ethanol production Akira Nordmeiera, Dev Chidambarama,b, a b
T
⁎
Chemical and Materials Engineering, University of Nevada, Reno, 1664 N. Virginia St., Reno, NV 89557-0338, United States Nevada Institute for Sustainability, University of Nevada, Reno, 1664 N. Virginia St., Reno, NV 89557-0338, United States
ARTICLE INFO
ABSTRACT
Keywords: Yeast FDMA Bioethanol
Saccharomyces cerevisiae immobilized in electrospun Pluronic F127 dimethacrylate (FDMA) was successfully employed for the production of ethanol in an immobilized cell reactor. Yeast cells were immobilized into fibers formed through the process of electrospinning and cross-linking. The threads had an average diameter of 0.88 μm and were used in continuous-flow immobilized cell reactors. The immobilized cell reactors were able to maintain a high ethanol yield of > 90% from day 4 through to the end of the time course at day 14. The reactor was able to achieve a maximum ethanol yield of 94.3%. This study shows that the use of electrospinning is a promising method for continuous ethanol production through immobilized cell-based continuous flow reactors.
1. Introduction Every year approximately 11 billion metric tons of fossil fuels are consumed, and it is estimated that all known oil reserves will be depleted by 2052 [1]. The need for alternative renewable liquid transportation fuels has shifted the focus onto biologically generated energy (biofuels) to minimize environmental costs [2,3]. Ethanol produced from microorganisms has been chosen by various governments to be the transportation fuel-of-choice, as so-produced ethanol is considered a biofuel and helps meet the Renewable Fuel Standards (RFS) [4]. It is estimated that the United States produced 15.25 billion gallons of fuelquality ethanol in 2016, which is a 788.2% increase in production from 2000. Production of ethanol is expected to continue to increase as the U.S. congressional mandate requiring the blending of at least 36 billion gallons of ethanol into transportation fuels by the year 2022. This requirement would more than double the current levels of ethanol production. To meet these production goals, significant increases in process efficiency and manufacturing capacity will be required. There have been various techniques designed to improve the production of ethanol, including continuous culturing and cell recycling [5]. The use of immobilized cell reactors (ICR) has been shown to hold great promise for the efficient production of bio-ethanol [6–8]. The most common of studied immobilization methods makes use of alginate beads and has been studied since Thiele first started this work in 1947 [9]. The use of alginate for whole cell entrapment offers high survival rates and has been studied extensively with different alginate
⁎
concentrations, designs, microorganisms, feed stocks, and feed rates [10–13]. However, the substrate/product must diffuse through the alginate to and from the microorganisms; the diffusion rate is often slow and creates microenvironments. This limits the metabolic activity of the microorganisms which lowers production and yield [14,15]. To minimize this effect, new techniques have been studied to increase the porosity of the materials through the use of hybrid materials such as polyvinyl alcohol (PVA), and polyethylene oxide (PEO) [16,17]. An alternative method is to reduce the size of the immobilization, this would reduce the distance needed for the substrate/product to diffuse [18]. Although spheres offer great surface area-to-volume ratios, the rate of diffusion by the substrate will be limited by the radius of the sphere. Furthermore, decreasing the size of the beads poses problems with retention of the beads within the reactor. One method to reduce the size of the immobilized bead without encountering bead retention issues is to form threads. Alginate threads, when compared to beads with similar sizes, have been shown to have higher diffusion rates [19]. Further, reduction in the size of the threads could minimize the time required for diffusion through the polymer as well as polymer/microorganism interaction. Electrospinning is a technique that is being studied for its use in biotechnology as this technique can produce nanometer-sized threads that form highly porous mats [20,21]. This method has been used with various polymer blends and has shown to have high cell viability [22,23]. Previous work by the principle investigator of this work showed that microorganisms could be immobilized in electrospun threads using PEO blended hydrogel Pluronic
Corresponding author at: University of Nevada, Reno, 1664 N. Virginia St., Reno, NV 89557-0338, United States. E-mail address: [email protected] (D. Chidambaram).
https://doi.org/10.1016/j.colsurfb.2019.05.013 Received 1 February 2019; Received in revised form 18 April 2019; Accepted 7 May 2019 Available online 08 May 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
Colloids and Surfaces B: Biointerfaces 181 (2019) 989–993
A. Nordmeier and D. Chidambaram
F127 dimethacrylate (FDMA or PEO99-: polypropylene oxide (PPO)67PEO99 DMA) as the immobilization material [24]. This work focuses on (I) the use of electrospinning to minimize the amount of polymer surrounding the microorganisms and evaluate the effect on the ethanol yield using a model organism Saccharomyces cerevisiae and (II) the applicability of electrospun threads for use in a continuous ICR.
by the ratio between the cell dry weight and polymer weight. The cell dry weight of 10 ml at 2.5 O.D. was 70 mg. The media was fed to the ICR column from a feedstock reservoir located beside the column. The feedstock consisted of (g/l) glucose, 100; yeast extract, 10 in DiH2O. A Thermo Scientific FH100 M peristaltic pump was used to transfer the feed medium from the reservoir to the ICR. The effluent from the column was collected in a 1 l conical flask which served as the product reservoir. The feed media was pumped in an upward-flow manner. The retention time (RT) of continuous culture was maintained at 9.8 h and 24 h. Aliquots of 0.5 ml were collected at various time intervals and centrifuged for 5 min at 4 °C for ethanol and glucose analysis as described elsewhere by the authors [17]. Experiments between 1 sheet and 3 sheets of immobilized yeast were conducted in triplicate and averaged.
2. Methods 2.1. Culture media Fermentation was conducted using dry activated distillers yeast (DADY) (Saccharomyces cerevisiae) purchased from Red Star. The organism was cultured in sterilized media containing (g/l) glucose, 50; peptone, 10; yeast extract, 10; MgSO4, 1; and K2HPO4, 1 in deionized water (DiH2O). The culture was maintained in an shaking incubator (125 rpm, 28 ± 1 °C). Culture viability and growth was monitored by the (O.D.) of the culture at 600 nm.
3. Results & discussion In order to ensure the proper formation of FDMA, the FDMA was analyzed using FTIR for the functional groups and was compared to the unmodified Pluronic F-127. The methacrylate group that was added to the polymer can be determined by the appearance of peaks at 1713 and 1635 cm−1 (Fig. 1A). These peaks correspond to the carbonyl vibration of the ester group and to the vinyl double bond, respectively [25]. DSC analysis showed that the melting point of F-127 was 62 °C and the FDMA was 54 °C, which is comparable to the results shown by Cohn et al. [26]. Also, the melting point of the FDMA/PEO blend was between those of pure PEO powder and that of FDMA powder, suggesting proper blending of the polymers (Fig. 1B). Using the FDMA/PEO blended polymer individual yeast cells were encapsulated in threads, as shown in Fig. 2A. The distribution of fiber sizes after electrospinning was analyzed using ImageJ image analysis tool, the size distribution is shown in Fig. 2B. The size distribution of 200 fibers, at spots not containing yeast, showed a narrow distribution of fiber diameter, in which, ∼80% of the fibers were between 0.5 and 1 μm in diameter. As these threads are smaller than the yeast cell this ensures that there is a minimal amount of polymer immobilizing the yeast cell. This size distribution is also similar to those obtained by similar methods [22,27]. The cross-linked FDMA sheets were then placed into the ICR. Production of ethanol is often discussed in terms of yield, which was calculated as follows:
2.2. F127 DMA synthesis and FDMA/PEO blend F127 dimethacrylate (FDMA) was synthesized using the method described in Sosnik et al. [25]. As discussed in Liu et al. 2009, FDMA is not optimal for immobilization and was blended with PEO to facilitate a porous mat formation during the electrospinning process [24]. FDMA/ PEO solution was made using 13 wt% FDMA and 3 wt% PEO in DiH2O. For the immobilization of yeast, the culture was transferred to a new media vial and incubated at 28 °C for 24 h. Cultures were collected when the O.D. reached 2.5 in 50 ml of media. The cultures were collected by centrifugation in 10 ml increments and were re-suspended in 10 ml of FDMA/PEO. 2.3. Characterization FTIR analysis was performed using a Thermo Nicolet 6700 FTIR spectrometer to confirm the synthesis of FDMA. Differential scanning calorimetry (DSC) of F-127, FDMA, FDMA/PEO-blend, and PEO was performed using a Netzsch Jupiter STA 449 F3. DSC measurements were conducted in a nitrogen environment at a heating rate of 20 °C/ min in the temperature range between 25 °C and 500 °C using 15 mg samples loaded in alumina crucibles under dry conditions.
Experimental amount of ethanol amount of ethanol theoretically
× 100
2.4. Electrospinning
Maximum
The FDMA/PEO solution containing yeast was placed into a 10 ml syringe with a 22 G needle. The syringe was attached to a syringe pump (New Era Pump Systems) which provided a steady solution flow rate of 20 μl/min. A high-voltage power supply (Gamma High Voltage Research, Ormond Beach, FL) was employed to apply a potential of 12 kV between the 22 G needle and the metal target which was horizontally placed 18 cm away from the tip of the needle. The metal target was a 9 × 9 cm Al foil. Electrospinning was conducted for 1 h and approximately 0.1 g was collected. After collection, the threads were cross-linked using the process described in Liu et al. and allowed to incubate at 4 °C for 12 h [24].
Wherein, the experimental amount of ethanol was measured using HPLC and the theoretical amount was calculated using stoichiometry per the following equation:
possible
C6H12O6 → 2 C2H5OH + 2 CO2 For example, if 80 g/l of glucose is consumed, the theoretical maximum of ethanol will be 40.88 g/l. Fig. 3 shows a comparison between the ICR containing 1 sheet and 3 sheets of the electrospun threads with immobilized yeast. Each reactor was operated in triplicate and with an RT of 9.8 h to ensure repeatability of the reactors as well as the immobilized yeast. The reactors with 1 sheet were able to obtain the highest glucose conversion during the time course, reaching 38.18 g/l of ethanol in 72 h. Because 19.5 g/l of glucose remained at the end of the period, a total of 80.5 g/l of glucose were consumed. Based on this glucose consumption, the theoretical amount of ethanol that can be produced is 41.13 g/l. Thus, the reactor was able to achieve 92.8% yield. This is a notable result as most strains of yeast are capable of an efficiency of 90–92% in batch reactors [28,29]; however, in slow fermenters, DADY has been shown to achieve up to 95% yield [30]. However, the reactor was not stable, and there was a decrease in ethanol production thereafter. This decrease in ethanol production could be due to the loose packing of the reactor,
2.5. Experimental reactor system and analysis The ICR was built using polyvinyl chloride sheet with a 3.2 cm (height) × 2.4 cm (width) × 0.5 cm (depth) with inlet/outlet ports on the top and bottom of the reactor. The volume of the reactor prior to packing was 3.7 ml. The reactors were placed on a bench top at ambient temperature and prefilled with yeast media containing 10 g/l yeast extract in DiH2O. A control reactor was also conducted with 1.75 ml of a yeast culture, as this was determined to contain approximately the same amount of yeast as the immobilized reactors. This was determined 990
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Fig. 2. SEM images of yeast immobilized through electrospinning FDMA/yeast solution A) 400x magnification of dense threads with an inset providing a perspective of the fiber and the cell B) Size distribution of the diameter of 200 fibers with an average of 0.88 μm.
Fig. 1. (A) FTIR spectra of F127 (top spectrum) and F127 dimethacrylate (FDMA) (bottom spectrum) showing the addition of 2 peaks at 1714 and 1636 cm−1, corresponding to the addition of the methacrylate group to the F127 base material. (B) DSC analyses of different polymer samples F-127, FDMA, FDMA/ PEO blend, and PEO.
which caused significant bubble formation to occur. These bubbles formed around the sheet and limited the interaction of the yeast with the media. The ICRs with 3 sheets required a longer startup period but showed less variation between reactors and continued to have an increased production of ethanol throughout the time course reaching a maximum of 86.7% yield. Although bubble formation did occur, the bubbles were significantly smaller and often dissipated. This is likely due to the packing of the reactor which limits the space available. Neither the 1 sheet nor the 3 sheet reactors were able to completely reduce the glucose concentration with the RT of 9.8 h therefore, a longer RT is needed. A study with a longer time course of 14 days and longer RT of 24 h was performed with the ICRs with 3 sheets. This reactor was then compared to a control reactor which had 1.75 ml of yeast culture inoculated into the reactor with no other additions. Fig. 4 shows that the immobilized yeast was able to obtain a maximum ethanol concentration of 46.35 g/l or 94.3% yield, which is close to the 95% ethanol yield from DADY as discussed earlier [30]. The control reactor was also able to obtain similar yields throughout the time course; however, the ethanol production and glucose consumption did not stabilize. Both the control the immobilized yeast reactors were not able to attain complete
Fig. 3. The comparison of the concentration of glucose to the production yield of ethanol with an RT of 9.6 h for ICR containing 1 sheet and 3 sheets of FDMA electrospun threads immobilizing yeast.
glucose reduction. Similar to the previous experiment the ICR with 3 sheets of immobilized yeast often had bubbles in the reactor. However, these bubbles did not dissipate which reduce the volume of the reactor and resulted in a faster retention time. 991
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Fig. 4. The comparison of the concentration of glucose to the production yield of ethanol with an RT of 24 h for ICR containing 3 sheets of FDMA electrospun threads immobilizing yeast and a control reactor inoculated with 1.75 ml of yeast culture.
Acknowledgment
This study provides promising short-term results in the use of immobilized yeast for continuous ethanol production. The ability to achieve a high (94%) yield and maintain it over short period of several days is most promising. Longer-term studies on the order of 4–8 weeks are needed prior to implementation in the industry so as to allow downtime only once a month or every two months. An aspect that needs to be improved is the yield, as batch processes provide higher (˜98%) yield. Yield can be increased by optimizing the flow rates and retention time. The vast majority of commercial ethanol is currently produced using batch processes, which have significant startup time requirements. Using continuous flow will require less capital as the reactor sizes for equivalent production will be much smaller. Since these are continuous flow reactors where in the glucose solution needs to be in contact with the immobilized cells, the reactor volume and production volumes per reactor are expected to be small; however, connecting several small reactors to scale up production, as is often conducted in biotechnology, chemical and pharmaceutical industries, will be simple. Thus, successful implementation in the industry will need significantly lower capital than current batch processing plants. Finally, while this study focused on production of ethanol, this process can be used for production of other bioproducts as well.
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4. Conclusions Short-term continuous ethanol production in an ICR was successfully demonstrated using yeast immobilized in FDMA via electrospinning. The ICR was able to achieve 94% yield and maintained high ethanol yields after a 4-day startup period with an average of 90.3% yield and a standard deviation of 3.96. To reduce the RT of the reactors and increase the stability, multiple reactors should be run in series. Although proof of principle, this study focused on the short-term analysis to evaluate the ability of electrospun fibers to be used in a continuous flow reactor, the results are promising and warrant further investigation. Conflict of interest The authors declare that they have no conflict of interest.
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