TWI592488B - Poly-l-lactic acid (plla) microtube array membrane-immobilized yeast cells for bioethanol fermentation - Google Patents

Description

Poly-L-lactic acid (PLLA) microtubule array membrane for bioethanol fermentation - immobilized yeast cells

The present invention relates to immobilized yeast cells for fermentation. In particular, the present invention provides a yeast cell immobilized on a regenerable poly-1-lactic acid (PLLA) microtubule array membrane (MTAM) and its use in bioethanol fermentation.

Fossil fuel prices and environmental issues are driving search for renewable energy. Bioethanol is a type of biofuel that can be produced by fermentation of biological quality. The first generation of bioethanol production utilizes sugar and starch from crops as raw materials. Challenges such as rising food prices and scarcity of arable land have led to an urgent need for alternative solutions. Third generation bioethanol production uses algae and requires biomass to be pretreated and hydrolyzed for saccharification and to produce fermentable sugars for use by other microorganisms to produce bioethanol via fermentation. However, pretreatment and hydrolysis of biomass is often accompanied by the formation of inhibitors that severely hamper the subsequent microbial fermentation of bioethanol production. Therefore, the key challenges associated with this energy source relate to the ability to produce bioethanol more efficiently without competing with food crop supplies and arable land.

Fixed to a method of isolating or localizing intact cells in a space and maintaining their catalytic activity. The fixed cells can effectively reduce the negative effects of the inhibitor and the processing cost of the inoculum preparation for continuous or batch fed fermentation of the microorganisms. Fixation techniques can be classified as follows: (1) immobilization on the surface of a solid support, (2) capture in a porous matrix, (3) mechanical containment behind the barrier and (4) cell flocculation (assembly). Bioethanol fermentation using fixed cells increases cell density, shortens fermentation time, increases ethanol and inhibitor tolerance, and improves the feasibility of using continuous fermentation, resulting in more efficient bioethanol production. When sodium alginate beads were used for Saccharomyces cerevisiae immobilization for bioethanol fermentation, it was found that immobilized yeast cells were more efficiently converted to alcohol than free yeast cells during batch fermentation, and the beads were fixed in yeast cells. Can be reused for 5 consecutive batch operations. In addition, the use of a porous sterile loofah sponge or mash for Kluyveromyces marxianus fixation promotes ethanol fermentation compared to the use of free cells. In addition, Saccharomyces cerevisiae VS3 immobilized on rice straw enhances bioethanol fermentation and can be reused for 8 consecutive batch operations (AKChandel, MLNarasu, G. Chandrasekhar, A. Manikyam, LVRao, Use of Saccharum spontaneum (wild sugarcane) as Biomaterial for cell immobilization and modulated ethanol production by thermotolerant Saccharomyces cerevisiae VS3, Bioresour. Technol. 100 (2009) 2404-2410).

Current techniques for immobilizing cells have several drawbacks. Surface attachment of cells using chemical bonding agents such as glutaraldehyde may not be suitable for the production of ethanol or beverages. In addition, cell detachment and relocation can contaminate the product because there are no barriers between the solution and the cells. Another method of cell fixation is to use a porous gel matrix, such as calcium alginate, to capture cells and obtain a high biomass mass loading for fermentation; however, the bead structure can be in acid or diffusion-limited gases such as ethanol The presence of CO ₂ in the fermentation is stabilized, resulting in cracking of the beads. Cell flocculation is considered a relatively low cost method; however, some yeast flocculation is inhibited by the presence of sugars such as glucose and ethanol. When a cell-free product is desired, a method such as a barrier behind the barrier for a microporous membrane filter for cell fixation is most suitable. However, there are inherent problems such as possible membrane fouling, high cost and container recycling problems (DJO'Brien, LHRoth, AJ McAloon, Ethanol production by continuous fermentation-pervaporation: a preliminary economic analysis, J. Membr. Sci. 166 ( 2000) 105-111).

One mole of sucrose is converted to two moles of ethanol and two moles of carbon dioxide by yeast alcohol fermentation. Due to the CO ₂ production during the alcohol fermentation, the gas produced can destroy the gel structure of the gel-fixed yeast and thus cause problems with the stability of the gel-fixed yeast. The present invention uses MTAM to immobilize yeast, and the fixed yeast for alcohol fermentation has high stability and yield. The present invention provides a combination of a yeast cell and a porous microtubule array membrane (MTAM), wherein the yeast cells are fixed in MTAM, and wherein the yeast encapsulation efficiency exceeds 60%. In some embodiments, the yeast encapsulation efficiency exceeds 65%. In some embodiments, the yeast encapsulation efficiency is from about 60% to about 75%, from about 65% to about 75%, or from about 65% to about 70%.

In some embodiments, the yeast cell is K. marxianus, Kluyveromyces lactis , Kluyveromyces lipolytica Saccharomyces cerevisiae or Schizosaccharomyces pombe .

In some embodiments, the MTAM is a highly aligned and closely packed fiber assembly wherein the fibers are filled together to form a single layer and the fibers are oriented no more than +/- 5°. In one embodiment, the fibers can be hollow or solid. In some embodiments, the diameter of the pore size of the MTAM ranges from about 20 nm to about 40 nm, from about 25 nm to about 40 nm, from about 30 nm to about 40 nm, from about 20 nm to about 35 nm, from about 20 nm to about 30 nm, or from about 28 nm to about 32 nm. In some embodiments, the MTAM consists of a biodegradable and/or bioabsorbable polymer.

The present invention also provides a process for bioethanol fermentation, comprising the steps of: (a) preparing or number from about ¹⁰⁷ to about ¹⁰⁹ cells in a fixed MTAM (CFU / mL) prepared by siphon of yeast cells in situ The yeast cells are encapsulated into the MTAM with an encapsulation efficiency of more than 60%; and (b) the resulting MTAM is used to ferment sugar by batch fermentation to produce ethanol.

In some embodiments, the number of cells of the yeast cells is about 10 ⁸ CFU/mL; the sugar is glucose, fructose or sucrose; and the batch fermentation is repeated batch fermentation.

Figure 1 (A) to (C) show (A) SEM partial image of MTAM and (B) Free K. marxianus and (C) Light microscopy of immobilized Marker's yeast using MTAM prepared by in situ method image.

Figures 2 (A) and (B) show (A) the amount of sugar and (B) bioethanol production in a batch fermentation using YPD free and fixed K. marxianus. Batch fermentation of YPD medium containing 5% (w/v) glucose was incubated at 42 ° C with shaking at 200 rpm. Data are expressed as mean ± SD (n = 3).

Figures 3 (A) and (B) show (A) the amount of sugar and (B) bioethanol production in repeated batch fermentation of YPD using immobilized K. marxianus. Repeated batch fermentation of YPD medium containing 5% (w/v) glucose was incubated at 42 ° C with shaking at 200 rpm. Data are expressed as mean ± SD (n = 3).

The present invention uses a highly porous microtubule array membrane (MTAM) to immobilize yeast cells for bioethanol fermentation. MTAM acts as an excellent substrate for immobilizing yeast for fermentation processes on an industrial scale.

Although many of the words, terms, and headings used herein are commonly used and conventionally understood within traditional medical and scientific content, some of the terms and descriptions and definitions of specific names, naming, designations, or designations are provided as follows. These descriptions and definitions are used to assist in the identification and appreciation of the correct variations and ranges of applications that are intended to be included within the scope of the method of the invention.

In the specification and patent application of this specification, the words "comprise" and other forms of the word (such as "comprising" and "including" (comprises)") is meant to include, but is not limited to, and is not intended to exclude such additional additives, components, integers or steps.

As used herein, the singular forms "a", "the" and "the"

Ranges may be expressed herein as "about" a particular value and/or to "about" another particular value. When the range is expressed, another aspect includes from a particular value and/or to another particular value. Similarly, when values are expressed as approximations by using the prefix "about", it should be understood that the particular values form another aspect. It is further understood that the endpoints of each of the ranges are important relative to the other endpoints and independent of the other endpoints. It should also be understood that a number of values are disclosed herein, and that values are also disclosed herein as "about" a particular value other than the value itself. For example, if the value "5" is revealed, "about 5" is also revealed. It should also be understood that when a value is disclosed, as is well understood by those skilled in the art, "less than or equal to the value", "greater than or equal to the value" and the possible range between the values are also disclosed. For example, if the value "5" is revealed, "less than or equal to 5" and "greater than or equal to 5" are also revealed. It is also to be understood that in the present application, information is provided in a number of different forms and such information represents the endpoints and starting points and the scope of any combination of such data points. For example, if a specific data point "5" and a specific data point "10" are disclosed, it should be understood that the disclosure is greater than, greater than or equal to, less than, less than or equal to, and equal to 5 and 10 and between 5 and 10. It should also be understood that each unit between two specific units is also disclosed. For example, if 5 and 10 are disclosed, 6, 7, 8, and 9 are also disclosed.

As used herein, the term "encapsulated" is used to include one thing in another, and thus the things included are not obvious. In the present invention, "encapsulated" means that the yeast cells are included in the porous microtubule array membrane.

As used herein, the term "fixed" refers to a substance that is attached to an inert, insoluble carrier.

As used herein, the term "electrospun" refers to the use of fluid power and charged surfaces. The interaction between the two polymers of electrospun fibers is known from the solution. In general, the formation of electrospun fibers involves providing a solution to a hole in an object in electrical communication with a voltage source, wherein the electrical power assists in the formation of fine fibers deposited on the surface that can be grounded or at a voltage below the object. . In electrospinning, a polymerization solution or melt provided from one or more needles, slots or other holes is fed into a high voltage relative to the collection grid. The power overcomes the surface tension and causes a fine spray of the polymerization solution or melt toward the ground or the oppositely charged accumulation grid.

As used herein, the term "polymer" means and generally includes, but is not limited to, homopolymers, copolymers such as block, graft, random and alternating copolymers, terpolymers, and the like, and blends thereof. And variants. Preferably, it may include, but is not limited to, polylactide, polylactic acid, polyolefin, polyacrylonitrile, polyurethane, polycarbonate, polycaprolactone, polyvinyl alcohol (PVA), Cellulose, polyglucosamine (for example, nylon 6, nylon 406, nylon 6-6, etc.), polystyrene, proteins, and the like, or combinations thereof. Unless specifically limited otherwise, the term "polymer" is intended to include all possible geometric configurations of the material. Such configurations include, but are not limited to, isotactic, syndiotactic, and random symmetry. Suitable solvents for each polymer may be selected from solvents known to those skilled in the art including, but not limited to, sulfuric acid, formic acid, chloroform, tetrahydrofuran, dimethylformamide, water, acetone, and the like. combination.

As used herein, the term "nano-sized fibers" or "nanofibers" refers to very small diameter fibers having an average diameter of no more than about 1500 nanometers (nm). Nanofibers are generally understood to have a fiber diameter in the range of from about 10 to about 1500 nm, more specifically from about 10 to about 1000 nm, still more specifically from about 20 to about 500 nm, and most specifically from about 20 to about 400 nm. Other exemplary ranges include from about 50 to about 500 nm, from about 100 to 500 nm, or from about 40 to about 200 nm. In a case where particles are present and non-uniformly distributed on the nanofibers, the average diameter of the nanofibers can be measured using known techniques (for example, image analysis tools combined with electromicroscopy), but excluding A portion of the fiber that is substantially enlarged by the addition of particles relative to the free portion of the particles of the fiber.

As used herein, the term "oriented fiber" indicates that substantially all of the fibers in a particular structure or array are arranged in parallel with one another in a longitudinal direction ("unidirectional orientation") or in a well-defined three-dimensional network ("three-dimensional orientation"). In other words, the fibers are spatially non-randomly aligned with respect to each other. In most instances, the fibers described herein are grown in a substantially vertical direction relative to the surface of the support substrate, and if present, there are few individual branched fiber strands.

As used herein, the term "single layer of material" or "single layer material" means a material consisting of a single layer of varying thickness.

As used herein, the term "plurality of layers" or "multilayer material" refers to a "stack" of a single layer of material.

In one aspect, the invention provides a combination of a yeast cell and a porous microtubule array membrane (MTAM), wherein the yeast cells are immobilized in the MTAM, and wherein the yeast encapsulation efficiency exceeds 60%. Preferably, the yeast encapsulation efficiency exceeds 65%. More preferably, the yeast encapsulation efficiency is from about 60% to about 75%, from about 65% to about 75%, or from about 65% to about 70%.

In some embodiments, the yeast cell is K. marxianus, Kluyveromyces lactis, Kluyveromyces cerevisiae, Saccharomyces cerevisiae or Schizosaccharomyces pombe. Preferably, the yeast cell is K. marxianus.

In some embodiments, the MTAM is a highly aligned and closely packed fiber assembly in which the fibers are filled together to form a single layer and the fibers are oriented no more than +/- 5°. In one embodiment, the fibers can be hollow or solid. Preferably, the MTAM is hollow. More preferably, the MTAM is a single layer having a hollow tube. Preferably, the diameter of the tube ranges from about 30 pm to about 50 pm, from about 35 pm to about 50 pm, from about 40 pm to about 50 pm, from about 30 pm to about 45 pm, from about 30 pm to about 40 pm, from about 35 pm to about 45 pm, or from about 38 pm to about 42 pm. Inside. More preferably, the tube has a diameter of about 40 pm.

In some embodiments, the diameter of the pore size of the MTAM ranges from about 20 nm to about 40 nm, from about 25 nm to about 40 nm, from about 30 nm to about 40 nm, from about 20 nm to about 35 nm, from about 20 nm to about 30 nm, or from about 28 nm to about 32 nm. Preferably, the aperture of the MTAM is straight The diameter is about 30 nm.

In some embodiments, the MTAM consists of a biodegradable and/or bioabsorbable polymer selected from the group consisting of ethylene oxide, polyethylene oxide (PEO), ethylene glycol, polyethylene glycol (PEG) ), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(ethylene oxide) (PEO), nylon, polyester, polyamide , poly(etheramine acid), polyimine, polyether, polyketone, polyurethane, polycaprolactone, polyacrylonitrile, aromatic polyamine, conjugated polymer such as electroluminescent polymerization , poly(2-methoxy, 5 ethyl(2'hexyloxy)p-phenylene vinyl) (MEH-PPV), polyphenylenevinylene, poly(arylene), polythiophenevinyl, Polypyrrole-ethylene, poly(arylene), polyaniline, polyphenylene, poly(arylene) compound, polythiophene, polypyrrole, polyheteroaryl compound, polyphenylene-acetylene, poly(arylene)-acetylene , polythiophene-acetylene, polyheteroaryl-acetylene and mixtures thereof. Preferably, the polymer is PLA.

The preparation of MTAM refers to KLOu, CSChen, LHLin, JCLu, YCShu, WCTseng, JCYang, SYLee, CCChen, Membranes of epitaxial-like packed, super aligned electrospun micron hollow poly (L-lactic acid (PLLA)fibers, Eur. Polym. J. 47 (2011) 882-892; JSJang, Y.Cho, GTJeong, SKKim, Optimization of saccharification and ethanol production by simultaneous saccharification and fermentation (SSF) from seaweed, Saccharina japonica, Bioprocess Biosyst.Eng.35(2012)11-18.;JCYang,SYLee,WCTseng,YCShu,JCLu,HSShie,CCChen,Formation of highly aigned,single-layered,hollow fibrous assemblies And the fabrication of large pieces of PLLA Membranes, Macromol. Mater. Eng. 297 (2012) 115-122.; and LCLin, YCShu, JCYang, HSShie, SYLee, CCChen, Nano-porous poly-L -lactic acid microtube array membranes, Curr. Nanosci. 10 (2014) 227-234.

The present invention uses highly porous MTAM to immobilize yeast cells for bioethanol fermentation, for the first time for bioethanol production. MTAM has a similar but superior structure to hollow fibers and was developed using coaxial electrospinning. Compared to typical hollow fibers, the diameter of the MTAM is reduced by 1-2 orders of magnitude and the wall thickness is >2 orders of magnitude; most surprisingly it can be self-assembled into a single diaphragm. The MTAM is further functionalized by establishing a nanopore using a pore former on the surface and then leaching out the process. Along with these structural features, the MTAM provides a large surface area per unit volume and a short diffusion distance, and has a self-supporting barrier function. It is easy to handle and low in cost. MTAM acts as an excellent substrate for immobilizing yeast for fermentation processes on an industrial scale.

In one aspect, the invention provides a method for bioethanol fermentation comprising the steps of: (a) immobilizing from about 10 ⁷ to about ¹⁰⁹ cells in MTAM by in situ preparation or siphon preparation (CFU/ The yeast cells of mL) are encapsulated into the MTAM with an encapsulation efficiency of more than 60%; and (b) the resulting MTAM is used to ferment sugar by batch fermentation to produce ethanol.

Preferably, the number of cells of the yeast cell is about 10 ⁸ CFU/mL.

Preferably, the sugar is glucose, fructose or sucrose. More preferably, the sugar is glucose having a concentration ranging from about 40 g/L to about 60 g/L. More preferably, the concentration of glucose is about 50 g/L.

Preferably, the batch fermentation is a repeated batch fermentation. More preferably, the batch fermentation is repeated in 2 to 7 or 2 to 6 batches. More preferably, the batch fermentation is repeated in 3 to 5 batches.

Embodiments of encapsulation efficiency and MTAM are described herein.

The present invention also optimizes the conditions for preparing MTAM-fixed cells and evaluates their potential for batch bioethanol fermentation.

Instance Materials and methods Source of Mark Skrvest's yeast

Kluyveromyces cerevisiae (BCRC 21363) was purchased from the Bioresources Collection and Research Center (BCRC) in Hsinchu, Taiwan.

Preparation of PLLA MTAM-fixed K. marxianus

K. marxianus was cultured in YPD medium (1% yeast extract, 2% bacterial peptone and 2% dextrose) at 42 ° C to achieve a cell density of 10 ⁸ and 10 ⁹ cfu/mL for immobilization. The materials used were PLLA (BioTech One, Taiwan), polyethylene glycol/polyethylene oxide (PEG/PEO; Sigma-Aldrich), N,N-dimethylformamide (DMF; Tedia, USA) and dichloromethane ( DCM; Mallinckrodt, USA). To prepare an electrospun spinning dope, PLLA was dissolved in mixed DCM/DMF (8/2) at room temperature to obtain a 10% solution. PEG was added to the PLLA solution to obtain a PEG/PLLA (30/70) solution as previously described.

An electrostatic charger (ChargeMaster, Simco-Ion, Alameda, CA, USA) or a power supply unit (You-Shang Co., Fongshan, Taiwan) was used as a source of static electricity. Typically, electrospinning is delivered by a syringe pump (KDS-100, KD Scientific, Holliston, MA, USA) at a rate of 4-9 mL/h, a voltage of 5-7 kV, and a distance of 3-5 cm from the drum collector. The PLLA (shell) and PEG (core) solutions were carried out through a co-axial spindle spinning head made in the chamber. All electrospinning procedures were carried out indoors at a relative humidity of 50 ± 5% and a temperature of 25 ± 1 °C. The nanoporous PLLA MTAM is obtained by washing to remove the core component PEG, followed by drying. In situ preparation of yeast-containing PLLA MTAM by using a typical MTAM electrospinning method having parameters similar to those described above, using yeast cells (cell density of 10 ⁸ mL ^-1 and ¹⁰⁹ mL ^-1 ) medium solution and The internal solution of the mixed solution of the PEG solution (1:9 volume) was completed. The resulting MTAM sample was then cut to a size of 3 cm x 0.5 cm x 0.0004 cm and both ends were sealed by a heated coating metal. PLLA MTAM containing siphon yeast cells was prepared by placing only one open end of the dried PLLA-MTAM into a fixed volume (10 μL) of yeast cell medium solution (cell density of 10 ⁸ mL ^-1 and 10 ⁹ mL ^-1 ) . After the capillary force up the siphon solution throughout the MTAM, both ends are sealed by a heated coating metal.

Stability testing of PLLA MTAM at various rpm and ethanol concentrations

PLLA MTAM was prepared as previously described and stored in 0.1% (w/v) peptone water at 4 °C. For the stability test of PLLA MTAM at various ethanol concentrations, the prepared MTAM was placed at room temperature to ethanol solutions at concentrations of 3%, 6%, 9%, 12%, and 15% (v/v), respectively. The PLLA MTAM structure at each ethanol concentration was observed for any fracture by naked eye and microscope. For the stability test of PLLA MTAM, the prepared PLLA MTAM was incubated in 0.1% (w/v) peptone water at 100, 200, 300, 400 and 500 rpm, respectively, at room temperature. The PLLA MTAM structure at each ethanol concentration was observed for any break by naked eye and microscope.

Analysis of encapsulation efficiency

PLLA MTAM-fixed K. marxianus was torn by using scissors and immersed in 0.1% (w/v) peptone. The mixture is vortexed sufficiently to release the fixed cells into the peptone. The mixture was then diluted and applied to a YPD pan and the plates were incubated at 42 °C for 24 h. The community is calculated and the actual cell count of the fixed yeast is obtained. The actual cell count was divided by the theoretical cell count of the fixed yeast to obtain encapsulation efficiency.

Batch and repeated batch bioethanol fermentation using fixed K. marxianus

For bioethanol fermentation, PLLA MTAM-fixed K. marxianus was placed in a 100 mL 5% glucose-containing YPD medium (1% yeast extract, 2% bacterial peptone, and 5% dextrose) at 200 °C with shaking at 200 rpm. The amount of glucose used in the batch fermentation and ethanol production were monitored at 0, 6, 9, 12, 15 and 24 h by HPLC analysis. Observe the optimal batch fermentation time for maximum ethanol production and apply it to repeated batch fermentations. Subsequent repeated batches of bioethanol fermentation were completed using the same growth conditions as batch fermentation. At the end of each fermentation cycle, PLLA MTAM-fixed K. marxianus cells were washed with sterile peptone water and then transferred to the same volume of 5% glucose-containing fresh YPD medium for subsequent fermentation cycles. The cycle is repeated until the production of bioethanol is no longer effective. Glucose and bioethanol fermentation were measured at 0, 3, 6, 9, 12 h during each cycle of fermentation. The parameters used for bioethanol fermentation are shown as C _EtOH (ethanol concentration), r P _EtOH (initial ethanol production rate), and Max. Y _P/S (maximum ethanol production) [5]. C _EtOH (g/L) is the maximum ethanol concentration that should be achieved during fermentation; r P _EtOH (g/L h) is C _EtOH divided by the time (h) taken to reach C _EtOH ; Max.Y _P/S ( g/g) is obtained by dividing by the total ethanol produced during the fermentation.

Analysis of glucose and ethanol using HPLC

Glucose (g) and ethanol (g) were flowed at a flow rate of 0.6 mL/min with a 5 mM H ₂ SO ₄ eliminator using an Aminex HPX-87H column (Bio-Rad, Sunnyvale, CA, USA) and a refractive index detector. All were analyzed at 60 °C. All test samples were filtered through a 0.22 [mu]m membrane filter prior to HPLC analysis.

Statistical Analysis

Data were analyzed statistically using SPSS version 12.0 (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) was used to determine statistical differences between sample components, with a significance level set at P < 0.05. Multiple comparisons of components were done by Tukey testing. All data are expressed as mean ± SD.

Example 1 Encapsulation of Kluyveromyces cerevisiae in PLLA MTAM

A partial scanning electron microscope (SEM) image of MTAM is shown in Figure 1 (A). The MTAM consists of a single-layer hollow tube having a diameter of about 40 μm and a hollow tube wall pore diameter of about 30 nm. Free K. marxianus cells and MTAM-fixed K. marxianus cells are shown in Figures 1 (B) and (C). PLLA-MTAM captures yeast cells for immobilization to achieve high biomass mass loading in fermentation. In terms of enantioselectivity, pure PLLA is a semicrystalline polymer having a glass transition temperature of about 55 ° C and a melting point of about 180 ° C ( Prog. Polym. Sci. 27 (2002) 1123-1163) , indicating PLLA-MTAM It is thermally stable at all known bioethanol fermentation temperatures. In addition, PLLA is insoluble in water, and its solubility in a solvent is highly dependent on the molar mass and crystallinity ( Prog. Polym. Sci. 27 (2002) 1123-1163).

Example 2 Effect of Ethanol and Stirring on the Stability of MTAM

To determine the effect of agitation and ethanol, MTAM-fixed K. marxianus was incubated at different rpm settings and ethanol concentrations. As shown in Table 1, MTAM maintained constant stability over 5%, 6%, 9%, 12%, and 15% (v/v) ethanol for more than 5 weeks (840 h) (data not shown). Our data indicate that MTAM is stable for more than 840 hours at 15% (v/v) ethanol, and cell fixation is suitable for repeated batch and continuous fermentation. The effects of agitation (100, 200, 300, 400 and 500 rpm) on the MTAM were determined using two different methods, a rotary shaker and a fermenter with an impeller. As shown in Table 1, the MTAM began to tear at 200 rpm at a incubation time of 72-96 h on a rotary shaker. As the rpm decreases, the time required for any tearing that occurs on the MTAM increases. At 50 and 100 rpm settings, MTAM did not show tears within 600h (25 days). The MTAM did not exhibit an apparent tear at 100 rpm for up to 60 h and at 200 rpm for up to 36 h. The data indicates that the current PLLA-MTAM is suitable for anaerobic and aerobic fermentation under shaking.

Example 3 encapsulation method and the effect of cell number on encapsulation efficiency

We evaluated the encapsulation efficiency of K. marxianus for two encapsulation methods, in situ preparation and siphon preparation. As shown in Table 2, the method of in situ and siphon 10 ⁸ yeast cells were showing encapsulation of 70.0 ± 2.0% and 67.0 ± 15.0% of the encapsulation efficiency. Two methods show similar efficiency in a sealed capsule at a density of 10 ⁸ cells. Our encapsulation efficiency data indicates that the K. marquee yeast cells survived the electrospinning method.

Table 2

Data are expressed as mean ± SD (n = 3).

Example 4 Encapsulation Method and Effect of MTAM Porosity on Yeast Growth and Batch Bioethanol Fermentation

Figure 1 (A) shows that the MTAM is a single layer in which the diameter of the empty tube is about 40 μm and the diameter of the nanopore on the surface of each hollow tube is about 30 nm, which allows simple diffusion of sugar, alcohol and other small molecules into and spread. Out. In order to determine whether the larger porosity on the surface of the tube will accelerate sugar consumption and ethanol production, possibly due to the large diffusion of sugar and ethanol across the barrier, we used 10% (v/v) in PLLA-MTAM preparation. The pore agent polyethylene glycol produced a large porosity of a pore diameter of 0.31 ± 0.08 μm on the surface of the hollow wall of the MTAM. As shown in Table 3, in situ preparation of MTAM-fixed K. marxianus with large porosity exhibited 8.76 ± 0.07 g / L of C _EtOH , 0.73 ± 0.01 g / Lh of r P _EtoH during bioethanol fermentation and The maximum Y _{P/S of} 0.43 ± 0.01 ^a (g/g) is better than the corresponding value of the original MTAM. Regarding the MTAM-fixed K. marxianus yeast having a large porosity prepared by the siphon method, there is no obvious difference compared with the parameters of the bioethanol fermentation of the original MTAM. The encapsulation efficiency using this method exhibited a relatively high standard deviation indicating a large difference between the number of cells involved in the original MTAM and the MTAM with larger porosity. We used 10% (v/v) polyethylene glycol to establish MTAM-fixed K. marxianus for the rest of the study.

C _EtOH (ethanol concentration); r P _EtOH (initial ethanol production rate); Max. Y _P/S (maximum ethanol production).

Data are expressed as mean ± SD (n = 3). The different letters in the data column show significant differences (p < 0.05).

Example 5 using a free and MTAM-fixed sugar amount of K. martingus and bioethanol fermentation of glucose

The bioethanol fermentation of YPD by free and MTAM immobilized K. marxianus is shown in Figure 2. The initial cell density of the free and MTAM-fixed K. marxianus in the fermentation was continuously maintained at 10 ⁵ CFU/mL. The results show that batch fermentation with yeast cells immobilized in MTAM requires less fermentation time to convert glucose to bioethanol than free cells. Free cells consumed glucose at a concentration of 50 g/L within 24 h. However, MTAM-fixed cells prepared by in situ and siphon method depleted glucose at 9 h and 12 h, respectively, indicating that MTAM immobilized K. marxianus increased efficiency.

The maximum bioethanol concentration of 18.46 g/L was obtained from fermentation after 15 h of free cells. Yeast cells fixed in MTAM prepared from in situ and siphon tubes exhibited maximum bioethanol concentrations of 20.7 g/L (9 h) and 19.9 g/L (12 h), which were 10% and 8% higher, respectively, than free cells. In this study, we used batch-fermented MTAM to immobilize K. marxianus and increased ethanol productivity by 8-10% compared to free cells, depending on the preparation method used.

Example 6 Repeated Batch Bioethanol Fermentation of YPD by Using MTAM to Fix Marker's Yeast

Glucose consumption and ethanol production of M. serrata using MTAM in repeated batch fermentations were studied as a function of time (Fig. 3). The first batch of MTAM-fixed cells consumed 17 g/L of starting 50 g/L glucose (34%) at 6 h. From batches II to VII, the same MTAM was used, and each batch consumed more than 95% glucose at 6 h (Fig. 3(A)). In addition, all batches, including the first batch, consumed all glucose within 9 hours. Thus, the highest ethanol concentration was obtained in all batches at 9 h of fermentation, which began to decrease at 12 h, possibly due to the oxidative metabolism of ethanol when there was no available glucose. The standard deviation of ethanol concentrations for all batches at 6 h was greater than the standard deviation of their ethanol concentrations at any other time point, indicating that some fixed MTAMs could achieve maximum ethanol production by 9 h. We attempted to encapsulate Marker's yeast in MTAM for repeated batch bioethanol fermentation, successfully reducing the cost of processing inoculum preparation, and accelerating glucose consumption and ethanol production. The kinetic parameters of ethanol production using free cells in fermentation and the evaluation of immobilization of K. marxianus by MTAM in repeated batch fermentation are shown in Table 4. As expected, MTAM fixed cells showed improved efficacy in terms of sugar consumption and ethanol production compared to free cells in each batch. Bioethanol fermentation using free K. marxianus cells showed 93% glucose consumption, 18.46 ± 0.38 g/L C _EtOH , 0.40 ± 0.002 g/g maximum Y _P/S and 1.23 ± 0.02 g/ at 15 h. L h of r P _EtOH . For fixed cells, the first batch showed the lowest ethanol productivity at 9 h, with C _EtOH (20.78 ± 0.16 g/L), maximum Y _P/S (0.42 ± 0.003 g/g), and r P _EtOH (2.31 ± 0.02). g/L h). In subsequent batches, ethanol productivity gradually increased until batch VI, and C _EtOH , maximum T _P/S, and r P _EtOH obtained in batches III, IV, V, and VI were higher than the first two batches. The fixed cells prepared by the in-situ method were subjected to 6 cycles of repeated batch fermentation to obtain a maximum C _{EtOH of} 24.23±0.63 g/L, a maximum Y _{P/S of} 0.48±0.012 g/g, and 2.69±0.07 g/ r P _{EtOH of} L h , slightly decreased in the last cycle of fermentation. Ethanol productivity began to decrease in batch VII, possibly due to aging of yeast cells; physical properties, cell encapsulation efficiency and immobilization techniques were performed therein, and our data showed that MTAM-fixed yeast showed great significance in repeated batch and continuous bioethanol fermentations. potential. The present invention introduces this cell fixation technique for the first time into bioethanol fermentation.

Table 4 shows the kinetic parameters of ethanol production by free cells in batch fermentation and the evaluation of MTAM encapsulation of K. marxianus in repeated batch fermentations.

C _EtOH (ethanol concentration); r P _EtOH (initial ethanol production rate); Max. Y _P/S (maximum ethanol production).

Data are expressed as mean ± SD (n = 3). Different letters in the data column showed significant differences (p < 0.05).

Claims (14)

A combination of a yeast cell and a porous microtubule array membrane (MTAM), wherein the yeast cells are immobilized in the MTAM, and wherein the yeast encapsulation efficiency exceeds 60%. The combination of claim 1, wherein the yeast cell is Kluyveromyces marxianus, Kluyveromyces lactis , Kluyveromyces lipolytica , Saccharomyces cerevisiae or Schizosaccharomyces pombe . A combination of claim 1 wherein the MTAM is a highly aligned and closely packed fiber assembly, and wherein the fibers are filled together to form a single layer, and the fibers are oriented no more than +/- 5[deg.]. A combination of claim 1 wherein the MTAM is a single layer having a hollow tube. A combination of claim 4, wherein the tube has a diameter in the range of from about 30 [mu]m to about 50 [mu]m. A combination of claim 1 wherein the MTAM has a pore size in the range of from about 20 nm to about 40 nm. The combination of claim 1, wherein the MTAM is composed of a biodegradable and/or bioabsorbable polymer selected from the group consisting of ethylene oxide, polyethylene oxide (PEO), ethylene glycol, and polyethylene. Alcohol (PEG), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(ethylene oxide) (PEO), nylon, polyester, Polyamide, poly(proline), polyimine, polyether, polyketone, polyurethane, polycaprolactone, polyacrylonitrile, aromatic polyamine, conjugated polymer such as electricity Luminescent polymer, poly(2-methoxy, 5 ethyl(2'hexyloxy)-p-phenylene vinyl) (MEH-PPV), polyphenylenevinylene, poly(arylene), polythiophene Ethylene, polypyrrole-ethylene, poly(arylene), polyaniline, polyphenylene compound, poly-extension Base compounds, polythiophenes, polypyrroles, polyheteroaryl compounds, polyphenylene-acetylenes, poly(arylene-acetylenes), polythiophene-acetylenes, polyhexamethylene-acetylenes, and mixtures thereof. As in the combination of claim 1, wherein the MTAM is a PLA. A method for bioethanol fermentation comprising the steps of: (a) fixing about 10 ⁷ to about 10 ⁹ cell numbers (CFU/mL) of yeast cells in MTAM by in situ or siphon control, such that The yeast cells are encapsulated into the MTAM by more than 60% encapsulation efficiency; and (b) the resulting MTAM fermented sugar is used to produce ethanol by batch fermentation. The method of claim 9, wherein the number of cells of the yeast cells in (a) is about 10 ⁸ CFU/mL. The method of claim 9, wherein the sugar is glucose, fructose or sucrose. The method of claim 9, wherein the sugar is glucose having a concentration ranging from about 40 g/L to about 60 g/L. The method of claim 9, wherein the batch fermentation is repeated batch fermentation. The method of claim 13, wherein the repeated batch fermentation is from 2 to 7 batches.