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Production of bacterial cellulose membranes in a modified airlift bioreactor by Gluconacetobacter xylinus
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e6, 2015 www.elsevier.com/locate/jbiosc
Production of bacterial cellulose membranes in a modified airlift bioreactor by Gluconacetobacter xylinus Sheng-Chi Wu* and Meng-Hsun Li Department of Biotechnology, Fooyin University, 151 Jinxue Road, Daliao Dist., Kaohsiung City 83102, Taiwan, ROC Received 14 August 2014; accepted 23 February 2015 Available online xxx
In this study, a novel bioreactor for producing bacterial cellulose (BC) is proposed. Traditional BC production uses static culture conditions and produces a gelatinous membrane. The potential for using various types of bioreactor, including a stirred tank, conventional airlift, and modified airlift with a rectangular wire-mesh draft tube, in large-scale production has been investigated. The BC obtained from these bioreactors is fibrous or in pellet form. Our proposed airlift bioreactor produces a membrane-type BC from Gluconacetobacter xylinus, the water-holding capacity of which is greater than that of cellulose types produced using static cultivation methods. The Young’s modulus of the product can be manipulated by varying the number of net plates in the modified airlift bioreactor. The BC membrane produced using the proposed bioreactor exhibits potential for practical application. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Airlift bioreactor; Bacterial cellulose; Water-holding capacity; Young’s modulus; Bacterial cellulose membrane; Gluconacetobacter xylinus]
Bacterial cellulose (BC) is a food indigenous to Southeast Asia. The material possesses unique physical properties that differ from those of plant cellulose, and it has received considerable attention because of its potential applications as a functional material. Bacteria can be used to produce ultrafine cellulose fibrils that are 50e80 nm across and 3e8 nm thick (1). These fibrils form 3dimensional network structures on a micrometer to nanometer scale. The unique structural and physical properties, including excellent mechanical strength, ultrafine fibers, biodegradability, and high crystallinity, have received a considerable amount of research interest throughout the past decade (2e4). BC has been applied in the food industry (5,6), high-strength paper and electronic paper production (7e9), enzyme immobilization (10), biosensing (11), and in medical applications (3,12,13), such as wound dressings (14), artificial skin (15), artificial blood vessels (16), and drug delivery (17). Traditional BC production use static culture conditions, in which a thick, gelatinous membrane is accumulated on the surface of a culture medium (18). However, despite the labor-intensiveness and low productivity involved in this method, it remains in use now. Stirred-tank bioreactors, which are the most widely used bioreactor in the fermentation industry, are believed to be a reasonable alternative. Moreover, previous studies have screened and obtained BC-producing bacteria that are suitable for agitated cultures (19e23). However, the morphological and structural characteristics of the produced BC differ according to the production method used. Static culture produces cellulose in pellicular form, whereas stirred-
* Corresponding author. Tel.: þ886 7 7811151x5792; fax: þ886 7 7862707. E-mail address: [email protected] (S.-C. Wu).
tank reactors yield fibrous cellulose. In a previous study, the crystallinity index, Young’s modulus, and degree of polymerization were lower in fibrous BC than in pellicular cellulose (21). During BC production, an enriched oxygen supply is an important factor. In stirred tank bioreactor, the suspension of fibrous BC with high cell density generated a highly viscous fluid. This caused oxygen transfer limit and a higher agitation power acquired, resulting in increasing energy consumption (24). Another common type of fermentation reactor is airlift bioreactor which is more energy efficient and less shear stress than stirred-tank reactors. When the airlift bioreactor was used in BC production, oxygen transfer rate became more crucial because oxygen-enriched air was essential for increasing the DO content in the broth (25,26). The modified airlift bioreactor with wire-mesh draft tube was identical to that of a traditional airlift reactor in the geometry, except that it features a rectangular wire-mesh draft tube in place of a simple draft tube (27). The modified airlift bioreactor mitigates this problems arising from limited oxygen supply during the production process in BC (28). Both airlift reactor types produce BC with elliptical pellet morphologies. Most applications involve using a membrane-type of BC (29). We proposed a novel bioreactor that directly produces BC as a membrane. Few reports have addressed this type of bioreactor, although some recent reports have described a rotating disk system (30,31) in which a rotating disk containing bacteria was immersed in a growth medium exposed to the atmosphere. Factors affecting the BC yield in a rotating disc reactor include the volume of the medium, rotation speed, and number of disks (32). The bundling and aggregation of thin layers and filaments cause the formation of nonuniform BC (33). Kralisch et al. (33) proposed a horizontal lift reactor that semicontinuously generated a BC membrane, the
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.02.018
Please cite this article in press as: Wu, S.-C., and Li, M.-H., Production of bacterial cellulose membranes in a modified airlift bioreactor by Gluconacetobacter xylinus, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.02.018
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length and height of which were adjustable. The material properties of the produced membrane are comparable to those produced using static cultivation methods. In this study, we used a modified airlift bioreactor with a series of simple net plates to produce BC membranes. Gluconacetobacter xylinus (formerly Acetobacter xylinum) BPR2001 was selected because of its high cellulose yield in stirred-tank systems (19). We discuss the bioreactor parameters and report the physical and mechanical properties of the product.
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MATERIALS AND METHODS Materials Glucose and peptone were purchased from Merck Co. Ltd., Germany, and yeast extract was purchased from Sigma Chemical Co. All reagents were of analytical grade. Microorganism and cultivation conditions The microorganism used in this study, G. xylinus subsp. Sucrofermentans BPR2001, was purchased from the Japan Collection of Microorganisms (RIKEN, Saitama, Japan). The culture medium contained 2.5% w/v mannitol, 0.5% w/v yeast extract, 0.3% w/v peptone, and 1.5% w/v agar. Cultures were incubated twice at 30 C for 24 h and preserved at 70 C. For the seed culture, we used the Hestrin and Schramn (HS) medium, which was composed of 2.0% w/v glucose, 0.5% w/v yeast extract, 0.5% w/v peptone, 0.27% w/v Na2HPO4,12H2O, and 0.115% w/v citric acid monohydrate (28). Prior to sterilization at 121 C, the pH value of the medium was adjusted to 5.0. The preserved organisms were transferred into 500-mL shaking flasks containing 300 mL of a medium at 30 C and stirred at 130 rpm for 48 h. For seed cultivation, cell suspension samples (30 mL) were inoculated in a 500-mL shaking flask containing 270 mL of fresh HS medium, and then cultivated in the medium at 30 C and stirred at 130 rpm for 24 h.
3.9cm 2.7cm 2.7cm 3.9cm
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Modified airlift bioreactor A modified airlift bioreactor with a working volume of 5 L was used to produce the BC membrane in this study. The reactor was described in a previous report (27) as featuring a rectangular wire-mesh draft tube. However, in the modified reactor, these tubes were replaced by a series of simple net plates. Fig. 1 shows a schematic diagram of the modified bioreactor equipped with 4 net plates. Prior to sterilization at 121 C, the HS medium was adjusted to a pH value of 5.0. Throughout the experiments, the temperature was maintained at 30 C, whereas the pH was not controlled. The air was used as the oxygen supply, and the aeration rate was 2 volumes of air per liquid volume per minute (vvm) unless otherwise indicated. Cellulose production and purification To measure the cellulose yield, the membrane was removed from the bioreactor when cultivation was completed. The crude membrane was cleaned by immersing it in 0.1 M NaOH at 105 C for 1 h and then washing it several times with deionized water until its pH was neutral. The purified cellulose was dried at 60 C for 24 h and then weighed. Residual glucose measurement The residual glucose in the broth was determined colorimetrically after performing a series of enzyme reactions by using a glucose reagent (ASK, Tonyar Biotech. Inc., Taiwan) that contained glucose oxidase and peroxidase. Water-holding capacity measurement We determined the water-holding capacity (WHC) by centrifuging the cellulose samples for 15 min at various speeds (32) and then measuring the volume of the extracted fluid. We defined the WHC as the ratio of the mass of the extracted fluid to that of the dry weight of the cellulose yield. Mechanical properties The tensile strength and Young’s modulus of the dried BC samples were determined using a tensile-test machine (Yang Yi Technology Co., Ltd, Taiwan). The edges of the purified cellulose samples were trimmed to avoid end effects, and the trimmed samples were cut into 10 2.5 cm strips. The strips were fixed in the clamps of the tensile-test machine and stretched at a rate of 50 mm/min until failure occurred. The tests were conducted in duplicate.
RESULTS AND DISCUSSION Comparison of bioreactors equipped with different numbers of plates The proposed airlift bioreactor used in this study featured rectangular net plates in place of the simple draft tube used in general airlift bioreactors. We investigated how the number of plates affected the flow within the reactor. We observed that an even number of plates must be used to maintain a uniform flow pattern. Therefore, the experiments were performed using 4, 6, 8, or 10 net plates. Fig. 2 shows the time courses for the production of BC for the various net-plate configurations. Glucose concentrations exhibited little change from the initial concentration for approximately the first 30 h of fermentation (Fig. 2A). The glucose concentrations decreased substantially to nearly half of
Air distributor
Air in FIG. 1. Schematic diagram of the modified airlift bioreactor.
the initial concentration at 66 h, regardless of net-plate configuration. Thus, the number of net plates used did not influence the glucose consumption, and glucose availability was not a limiting factor in the production of the cellulose. Gluconacetobacter xylinus is a highly aerobic bacteria; thus, adequate oxygen supply is crucial for cultivation. The time courses of the DO experiments in Fig. 2B shows that culturing BC by using 4 net plates resulted in a substantial decrease in the DO concentration after 20 h of fermentation, and most of the DO was consumed after 28 h. Thus, DO was a limiting factor in our proposed bioreactor. However, a previous study using a similarly modified airlift bioreactor, but with a rectangular wire-mesh draft tube, did not report this effect (28). Installing 6 or 8 net plates in the airlift bioreactor resulted in delays of 50 h and 43 h, respectively, for the DO concentrations to fall below 10% of the initial DO concentration (Fig. 2B). Using 10 net plates did not produce any oxygen-limiting effects. The DO concentration in the broth was maintained above 70% throughout the fermentation procedure, indicating that the oxygen supply in the modified airlift bioreactor was sufficient.
Please cite this article in press as: Wu, S.-C., and Li, M.-H., Production of bacterial cellulose membranes in a modified airlift bioreactor by Gluconacetobacter xylinus, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.02.018
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TABLE 1. Comparison of the BC yield and Young’s modulus based on the number of plates used in the modified airlift bioreactor.
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Culture condition
Cellulose concentration (g/L)
Young’s modulus (Gpa)
1.3 1.8 1.6 1.6
7.1 1.2 4.7 0.5 2.1 0.0 ea
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The plates were too close, causing the membrane to coagulate.
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(B) FIG. 2. Effects of varying the number of plates in the modified airlift bioreactor on (A) glucose and (B) DO.
Table 1 lists the concentrations and mechanical properties of BC yield according to the number of net plates used. A peak cellulose concentration of 1.8 g/L was obtained using 6 net plates. We attributed the relatively low BC yield of the 4-plate configuration to the oxygen-supply-limiting factor discussed in relation to Fig. 2B. Young’s modulus values decreased when the plate number increased. The 4-net-plate configuration produced BC at a peak Young’s modulus of 7.1 GPa, which was similar to that obtained when producing membranes under static culture conditions (7.2 0.8 GPa). Thus, the strength of the BC membranes produced using the proposed airlift bioreactor was identical to that of the membranes grown using traditional methods. In the rotating disk system, the membrane strength (0.3 GPa) was considerably lower than that produced using static culture conditions (2.7 GPa), indicating that the high membrane strength obtained using the traditional static culture can also be maintained using the bioreactor. WHC is a crucial characteristic in biomedical applications (34,35). We measured the WHC of the BC yield of the bacteria grown in an airlift reactor with various net-plate configurations (Fig. 3). The WHC of the static cultivation was 23 g of water per gram of cellulose at 300 g. Increasing the centrifugal force substantially reduced the WHC values. Comparing the WHC of the BC
from the culture grown in the airlift reactor fitted with various netplate configurations revealed considerable differences in WHC at 300 g, and the maximal WHC of 185 g of water per gram of cellulose was obtained when using the 6-net-plate configuration. The BC cultured in the 6-net-plate airlift bioreactor could hold nearly 8fold more water compared with that obtained under static conditions. Effects of aeration rate Maximal cellulose membrane production was achieved by using the 6-net-plate configuration in the airlift bioreactor; thus, we adopted this design in the following experiments. Substantial oxygen limitation occurred 50 h after initiating cultivation. Therefore, the oxygen supply was another crucial operational factor. We selected 3 aeration rates (i.e., 1.5, 2.0, and 3.0 vvm) to investigate the effects of a limited oxygen supply on glucose and DO consumption, the results of which are shown in Fig. 4. Fig. 4A shows that the trends in the residual glucose under the 3 sets of conditions were similar. The glucose concentrations exhibited little change until approximately 30 h, after which they decreased steadily. The 3-vvm aeration sample produced the greatest rate of glucose depletion; however, the glucose levels remained higher than 8 g/L, even after 72 h of cultivation. Therefore, glucose was not a limiting nutrient. Fig. 4B shows the DO time under various aeration rates. The 1.5vvm sample exhibited a considerable decrease in DO at 10 h, decreasing to below 60% after 20 h of cultivation. No additional data were obtained because the sensor became covered by cellulose; however, assuming that DO limitation occurred would be reasonable. The figure shows that DO limitation occurred after 50 h of cultivation, even at aeration rates of 2 and 3 vvm. Therefore, varying the aeration rate did not eliminate the oxygen limitation in the production of BC when using the 6-net-plate configuration. This conclusion is consistent with that of Chao et al. (21), who produced BC in a conventional airlift bioreactor and reported that oxygen was a limiting factor. To overcome the oxygen limitation, that study used oxygen-enriched gas instead of air. 200
WHC (g water/g cellulose)
glucose (g/L)
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centrifugation (g) FIG. 3. WHC obtained using different numbers of plates in the modified airlift bioreactor.
Please cite this article in press as: Wu, S.-C., and Li, M.-H., Production of bacterial cellulose membranes in a modified airlift bioreactor by Gluconacetobacter xylinus, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.02.018
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FIG. 5. WHC at various aeration rates in the modified airlift bioreactor.
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(B) FIG. 4. Effects of varying the aeration rate in the modified airlift bioreactor on (A) glucose and (B) DO.
Although the DO curves differed according to the number of net plates used in the bioreactors, the glucose consumption was similar throughout the cultivation period. A previous study also observed this unusual phenomenon in the production of BC where a bubble column and modified airlift with rectangular net plates were used (28). The principal effects of producing BC might be the fermentation time, carbon source concentration, and surfaceevolume ratio, or relative surface area of the fermentation system (3). Therefore, based on our results, we speculated that cellulose formation might depend on oxygen uptake rather than the medium. In other words, the bacteria growth was related to medium, whereas the cellulose formation was markedly affected by oxygen. However,
TABLE 2. Comparison of the BC yield and Young’s modulus based on the aeration rate in the modified airlift bioreactor. Culture condition Six plates in 1.5 vvm Six plates in 2 vvm Six plates in 3 vvm
Effect of cultivation time From a production perspective, cultivation time is a crucial factor. Fig. 6 shows the cellulose concentrations after 2, 3, and 4 days of growth. The cells exhibited slight growth by 2 days postinoculum, followed by an exponential growth phase on Days 3 and 4. The BC membrane
3.0
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Cellulose concentration (g/L)
Young’s modulus (Gpa)
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0.9 1.8 1.0
10.3 0.0 4.7 0.5 ea
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production rate (g/L/day)
DO
80
cellulose concentration (g/L)
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this conjecture requires further scientific experimentation to explain this phenomenon. Table 2 shows the cellulose concentrations and mechanical properties of the BC membranes cultivated at various aeration rates. A maximal BC concentration of 1.8 g/L was obtained at an aeration rate of 2 vvm, and the yield at 1.5 and 3 vvm was approximately half of that. We assumed that the production was low at 1.5 vvm because of the oxygen limitation, and the poor yield at 3 vvm was attributed to the high shear rates at the net plates, which rendered it difficult for the bacteria to attach to the net plate to form a membrane. In addition to the difference in BC yield, the Young’s modulus of samples produced at 1.5 vvm was greater than that of samples produced at 2 vvm. The membrane produced at 3 vvm was not fully integrated. The peak Young’s modulus of the sample obtained at 1.5 vvm when using the 6-net-plate configuration was 10.3 GPa. Fig. 5 shows the WHC obtained at the various aeration rates. Significant decreases in the WHC occurred at 300 g in all 3 sets of conditions, and the highest WHC was obtained at an aeration rate of 2 vvm. Thus, the aeration conditions affected the BC structure.
0.0
two days
three days
four days
a
The membrane was too thin to collect; therefore, the membrane was not integrated.
FIG. 6. Effects of varying the cultivation time in the modified airlift bioreactor.
Please cite this article in press as: Wu, S.-C., and Li, M.-H., Production of bacterial cellulose membranes in a modified airlift bioreactor by Gluconacetobacter xylinus, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.02.018
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concentration reached 2.6 g/L after 4 days of culturing, yielding a mean BC production rate of 0.027 g/L h. The growth rate was 0.02 g/L h on Day 2, 0.054 g/L h on Day 3, and 0.033 g/L h on Day 4. Therefore, the maximal growth rate occurred on Day 3 before decreasing on Day 4. The average production rate was slightly greater than the static culture 7-day average rate of 0.02 g/L h. Our proposed modified airlift bioreactor, which was fitted with net plates in place of the rectangular wire-mesh draft tube employed in the original design, was used to produce BC membranes. A previous study reported that a modified airlift bioreactor fitted with a rectangular wire-mesh draft tube eliminated the DO limitation of BC growth that occurs in unmodified airlift bioreactors (27). Cheng et al. (28) reported that using an airlift bioreactor that was modified with a rectangular wire-mesh draft tube maintained a DO concentration above 35% during BC production. In that study, 3 wire-mesh draft tubes were installed in the bioreactor; however, the authors did not discuss the effects of the number of plates. Our results indicated that using more net plates supplies more DO during cultivation. This finding is consistent with a report by Wu and Hsiun (36), who used an airlift bioreactor modified with multiple axial-net draft tubes to generate a greater oxygen transfer rate than using a single axial-net draft tube would. In this study, we proved that the amount of DO correlates with the number of net plates in the bioreactor, where using more net plates increases the oxygen transfer rate. We optimized the proposed bioreactor to maximize the WHC values of BC, and determined that using 6 net plates yielded BC with the most favorable WHC. Specifically, this configuration produced BC that can hold nearly 8-fold more fluid than that produced under static fermentation conditions could. Furthermore, the WHC is greater than that of BC produced using a rotating disc bioreactor, which can retain 5-fold more fluid than BC grown under static conditions does (29). We observed that varying the number of net plates and improving broth aeration affected the Young’s modulus of the BC produced. The peak Young’s modulus was obtained using at an aeration rate of 1.5 vvm when using the 6-net-plate configuration. In previous studies, the basic concept was that the process of cellulose formation should occur at the medium/pellicle interface, and the membrane-producing bacteria should be near this interface (37) because no cellulose formation occurs elsewhere in the medium (38). However, this article is the first to report a successful case of producing cellulose in the medium. The BC membrane produced using the proposed bioreactor has potential for various practical applications. ACKNOWLEDGMENTS We are very grateful for the financial support provided by the National Science Council, Taiwan through grants NSC 94-2214-E242 -001 and NSC 95-2622-E-242-005-CC3. References 1. Yoshinaga, F., Tonouchi, J., and Watanabe, K.: Research progress in production of bacterial cellulose by aeration and agitation culture and its application as a new industrial material, Biosci. Biotechnol. Biochem., 61, 219e224 (1997). 2. Ross, P., Mayer, R., and Benziman, M.: Cellulose biosynthesis and function in bacteria, Microbiol. Mol. Biol. Rev., 55, 35e58 (1991). 3. Jonas, R. and Farah, L. F.: Production and application of microbial cellulose, Polym. Degrad. Stab., 59, 101e106 (1998). 4. Iguchi, M., Yamanaka, S., and Budhiono, A.: Bacterial cellulose-a masterpiece of nature’s arts, J. Mater. Sci., 35, 261e270 (2000). 5. Okiyama, A., Motoki, M., and Yamanaka, S.: Bacterial cellulose II. Proceeding of the gelatinous cellulose for food materials, Food Hydrocolloid., 6, 479e487 (1992).
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Please cite this article in press as: Wu, S.-C., and Li, M.-H., Production of bacterial cellulose membranes in a modified airlift bioreactor by Gluconacetobacter xylinus, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.02.018
Production of bacterial cellulose membranes in a modified airlift bioreactor by Gluconacetobacter xylinus Sheng-Chi Wu* and Meng-Hsun Li Department of Biotechnology, Fooyin University, 151 Jinxue Road, Daliao Dist., Kaohsiung City 83102, Taiwan, ROC Received 14 August 2014; accepted 23 February 2015 Available online xxx
In this study, a novel bioreactor for producing bacterial cellulose (BC) is proposed. Traditional BC production uses static culture conditions and produces a gelatinous membrane. The potential for using various types of bioreactor, including a stirred tank, conventional airlift, and modified airlift with a rectangular wire-mesh draft tube, in large-scale production has been investigated. The BC obtained from these bioreactors is fibrous or in pellet form. Our proposed airlift bioreactor produces a membrane-type BC from Gluconacetobacter xylinus, the water-holding capacity of which is greater than that of cellulose types produced using static cultivation methods. The Young’s modulus of the product can be manipulated by varying the number of net plates in the modified airlift bioreactor. The BC membrane produced using the proposed bioreactor exhibits potential for practical application. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Airlift bioreactor; Bacterial cellulose; Water-holding capacity; Young’s modulus; Bacterial cellulose membrane; Gluconacetobacter xylinus]
Bacterial cellulose (BC) is a food indigenous to Southeast Asia. The material possesses unique physical properties that differ from those of plant cellulose, and it has received considerable attention because of its potential applications as a functional material. Bacteria can be used to produce ultrafine cellulose fibrils that are 50e80 nm across and 3e8 nm thick (1). These fibrils form 3dimensional network structures on a micrometer to nanometer scale. The unique structural and physical properties, including excellent mechanical strength, ultrafine fibers, biodegradability, and high crystallinity, have received a considerable amount of research interest throughout the past decade (2e4). BC has been applied in the food industry (5,6), high-strength paper and electronic paper production (7e9), enzyme immobilization (10), biosensing (11), and in medical applications (3,12,13), such as wound dressings (14), artificial skin (15), artificial blood vessels (16), and drug delivery (17). Traditional BC production use static culture conditions, in which a thick, gelatinous membrane is accumulated on the surface of a culture medium (18). However, despite the labor-intensiveness and low productivity involved in this method, it remains in use now. Stirred-tank bioreactors, which are the most widely used bioreactor in the fermentation industry, are believed to be a reasonable alternative. Moreover, previous studies have screened and obtained BC-producing bacteria that are suitable for agitated cultures (19e23). However, the morphological and structural characteristics of the produced BC differ according to the production method used. Static culture produces cellulose in pellicular form, whereas stirred-
* Corresponding author. Tel.: þ886 7 7811151x5792; fax: þ886 7 7862707. E-mail address: [email protected] (S.-C. Wu).
tank reactors yield fibrous cellulose. In a previous study, the crystallinity index, Young’s modulus, and degree of polymerization were lower in fibrous BC than in pellicular cellulose (21). During BC production, an enriched oxygen supply is an important factor. In stirred tank bioreactor, the suspension of fibrous BC with high cell density generated a highly viscous fluid. This caused oxygen transfer limit and a higher agitation power acquired, resulting in increasing energy consumption (24). Another common type of fermentation reactor is airlift bioreactor which is more energy efficient and less shear stress than stirred-tank reactors. When the airlift bioreactor was used in BC production, oxygen transfer rate became more crucial because oxygen-enriched air was essential for increasing the DO content in the broth (25,26). The modified airlift bioreactor with wire-mesh draft tube was identical to that of a traditional airlift reactor in the geometry, except that it features a rectangular wire-mesh draft tube in place of a simple draft tube (27). The modified airlift bioreactor mitigates this problems arising from limited oxygen supply during the production process in BC (28). Both airlift reactor types produce BC with elliptical pellet morphologies. Most applications involve using a membrane-type of BC (29). We proposed a novel bioreactor that directly produces BC as a membrane. Few reports have addressed this type of bioreactor, although some recent reports have described a rotating disk system (30,31) in which a rotating disk containing bacteria was immersed in a growth medium exposed to the atmosphere. Factors affecting the BC yield in a rotating disc reactor include the volume of the medium, rotation speed, and number of disks (32). The bundling and aggregation of thin layers and filaments cause the formation of nonuniform BC (33). Kralisch et al. (33) proposed a horizontal lift reactor that semicontinuously generated a BC membrane, the
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.02.018
Please cite this article in press as: Wu, S.-C., and Li, M.-H., Production of bacterial cellulose membranes in a modified airlift bioreactor by Gluconacetobacter xylinus, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.02.018
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length and height of which were adjustable. The material properties of the produced membrane are comparable to those produced using static cultivation methods. In this study, we used a modified airlift bioreactor with a series of simple net plates to produce BC membranes. Gluconacetobacter xylinus (formerly Acetobacter xylinum) BPR2001 was selected because of its high cellulose yield in stirred-tank systems (19). We discuss the bioreactor parameters and report the physical and mechanical properties of the product.
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MATERIALS AND METHODS Materials Glucose and peptone were purchased from Merck Co. Ltd., Germany, and yeast extract was purchased from Sigma Chemical Co. All reagents were of analytical grade. Microorganism and cultivation conditions The microorganism used in this study, G. xylinus subsp. Sucrofermentans BPR2001, was purchased from the Japan Collection of Microorganisms (RIKEN, Saitama, Japan). The culture medium contained 2.5% w/v mannitol, 0.5% w/v yeast extract, 0.3% w/v peptone, and 1.5% w/v agar. Cultures were incubated twice at 30 C for 24 h and preserved at 70 C. For the seed culture, we used the Hestrin and Schramn (HS) medium, which was composed of 2.0% w/v glucose, 0.5% w/v yeast extract, 0.5% w/v peptone, 0.27% w/v Na2HPO4,12H2O, and 0.115% w/v citric acid monohydrate (28). Prior to sterilization at 121 C, the pH value of the medium was adjusted to 5.0. The preserved organisms were transferred into 500-mL shaking flasks containing 300 mL of a medium at 30 C and stirred at 130 rpm for 48 h. For seed cultivation, cell suspension samples (30 mL) were inoculated in a 500-mL shaking flask containing 270 mL of fresh HS medium, and then cultivated in the medium at 30 C and stirred at 130 rpm for 24 h.
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Modified airlift bioreactor A modified airlift bioreactor with a working volume of 5 L was used to produce the BC membrane in this study. The reactor was described in a previous report (27) as featuring a rectangular wire-mesh draft tube. However, in the modified reactor, these tubes were replaced by a series of simple net plates. Fig. 1 shows a schematic diagram of the modified bioreactor equipped with 4 net plates. Prior to sterilization at 121 C, the HS medium was adjusted to a pH value of 5.0. Throughout the experiments, the temperature was maintained at 30 C, whereas the pH was not controlled. The air was used as the oxygen supply, and the aeration rate was 2 volumes of air per liquid volume per minute (vvm) unless otherwise indicated. Cellulose production and purification To measure the cellulose yield, the membrane was removed from the bioreactor when cultivation was completed. The crude membrane was cleaned by immersing it in 0.1 M NaOH at 105 C for 1 h and then washing it several times with deionized water until its pH was neutral. The purified cellulose was dried at 60 C for 24 h and then weighed. Residual glucose measurement The residual glucose in the broth was determined colorimetrically after performing a series of enzyme reactions by using a glucose reagent (ASK, Tonyar Biotech. Inc., Taiwan) that contained glucose oxidase and peroxidase. Water-holding capacity measurement We determined the water-holding capacity (WHC) by centrifuging the cellulose samples for 15 min at various speeds (32) and then measuring the volume of the extracted fluid. We defined the WHC as the ratio of the mass of the extracted fluid to that of the dry weight of the cellulose yield. Mechanical properties The tensile strength and Young’s modulus of the dried BC samples were determined using a tensile-test machine (Yang Yi Technology Co., Ltd, Taiwan). The edges of the purified cellulose samples were trimmed to avoid end effects, and the trimmed samples were cut into 10 2.5 cm strips. The strips were fixed in the clamps of the tensile-test machine and stretched at a rate of 50 mm/min until failure occurred. The tests were conducted in duplicate.
RESULTS AND DISCUSSION Comparison of bioreactors equipped with different numbers of plates The proposed airlift bioreactor used in this study featured rectangular net plates in place of the simple draft tube used in general airlift bioreactors. We investigated how the number of plates affected the flow within the reactor. We observed that an even number of plates must be used to maintain a uniform flow pattern. Therefore, the experiments were performed using 4, 6, 8, or 10 net plates. Fig. 2 shows the time courses for the production of BC for the various net-plate configurations. Glucose concentrations exhibited little change from the initial concentration for approximately the first 30 h of fermentation (Fig. 2A). The glucose concentrations decreased substantially to nearly half of
Air distributor
Air in FIG. 1. Schematic diagram of the modified airlift bioreactor.
the initial concentration at 66 h, regardless of net-plate configuration. Thus, the number of net plates used did not influence the glucose consumption, and glucose availability was not a limiting factor in the production of the cellulose. Gluconacetobacter xylinus is a highly aerobic bacteria; thus, adequate oxygen supply is crucial for cultivation. The time courses of the DO experiments in Fig. 2B shows that culturing BC by using 4 net plates resulted in a substantial decrease in the DO concentration after 20 h of fermentation, and most of the DO was consumed after 28 h. Thus, DO was a limiting factor in our proposed bioreactor. However, a previous study using a similarly modified airlift bioreactor, but with a rectangular wire-mesh draft tube, did not report this effect (28). Installing 6 or 8 net plates in the airlift bioreactor resulted in delays of 50 h and 43 h, respectively, for the DO concentrations to fall below 10% of the initial DO concentration (Fig. 2B). Using 10 net plates did not produce any oxygen-limiting effects. The DO concentration in the broth was maintained above 70% throughout the fermentation procedure, indicating that the oxygen supply in the modified airlift bioreactor was sufficient.
Please cite this article in press as: Wu, S.-C., and Li, M.-H., Production of bacterial cellulose membranes in a modified airlift bioreactor by Gluconacetobacter xylinus, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.02.018
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TABLE 1. Comparison of the BC yield and Young’s modulus based on the number of plates used in the modified airlift bioreactor.
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(B) FIG. 2. Effects of varying the number of plates in the modified airlift bioreactor on (A) glucose and (B) DO.
Table 1 lists the concentrations and mechanical properties of BC yield according to the number of net plates used. A peak cellulose concentration of 1.8 g/L was obtained using 6 net plates. We attributed the relatively low BC yield of the 4-plate configuration to the oxygen-supply-limiting factor discussed in relation to Fig. 2B. Young’s modulus values decreased when the plate number increased. The 4-net-plate configuration produced BC at a peak Young’s modulus of 7.1 GPa, which was similar to that obtained when producing membranes under static culture conditions (7.2 0.8 GPa). Thus, the strength of the BC membranes produced using the proposed airlift bioreactor was identical to that of the membranes grown using traditional methods. In the rotating disk system, the membrane strength (0.3 GPa) was considerably lower than that produced using static culture conditions (2.7 GPa), indicating that the high membrane strength obtained using the traditional static culture can also be maintained using the bioreactor. WHC is a crucial characteristic in biomedical applications (34,35). We measured the WHC of the BC yield of the bacteria grown in an airlift reactor with various net-plate configurations (Fig. 3). The WHC of the static cultivation was 23 g of water per gram of cellulose at 300 g. Increasing the centrifugal force substantially reduced the WHC values. Comparing the WHC of the BC
from the culture grown in the airlift reactor fitted with various netplate configurations revealed considerable differences in WHC at 300 g, and the maximal WHC of 185 g of water per gram of cellulose was obtained when using the 6-net-plate configuration. The BC cultured in the 6-net-plate airlift bioreactor could hold nearly 8fold more water compared with that obtained under static conditions. Effects of aeration rate Maximal cellulose membrane production was achieved by using the 6-net-plate configuration in the airlift bioreactor; thus, we adopted this design in the following experiments. Substantial oxygen limitation occurred 50 h after initiating cultivation. Therefore, the oxygen supply was another crucial operational factor. We selected 3 aeration rates (i.e., 1.5, 2.0, and 3.0 vvm) to investigate the effects of a limited oxygen supply on glucose and DO consumption, the results of which are shown in Fig. 4. Fig. 4A shows that the trends in the residual glucose under the 3 sets of conditions were similar. The glucose concentrations exhibited little change until approximately 30 h, after which they decreased steadily. The 3-vvm aeration sample produced the greatest rate of glucose depletion; however, the glucose levels remained higher than 8 g/L, even after 72 h of cultivation. Therefore, glucose was not a limiting nutrient. Fig. 4B shows the DO time under various aeration rates. The 1.5vvm sample exhibited a considerable decrease in DO at 10 h, decreasing to below 60% after 20 h of cultivation. No additional data were obtained because the sensor became covered by cellulose; however, assuming that DO limitation occurred would be reasonable. The figure shows that DO limitation occurred after 50 h of cultivation, even at aeration rates of 2 and 3 vvm. Therefore, varying the aeration rate did not eliminate the oxygen limitation in the production of BC when using the 6-net-plate configuration. This conclusion is consistent with that of Chao et al. (21), who produced BC in a conventional airlift bioreactor and reported that oxygen was a limiting factor. To overcome the oxygen limitation, that study used oxygen-enriched gas instead of air. 200
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(B) FIG. 4. Effects of varying the aeration rate in the modified airlift bioreactor on (A) glucose and (B) DO.
Although the DO curves differed according to the number of net plates used in the bioreactors, the glucose consumption was similar throughout the cultivation period. A previous study also observed this unusual phenomenon in the production of BC where a bubble column and modified airlift with rectangular net plates were used (28). The principal effects of producing BC might be the fermentation time, carbon source concentration, and surfaceevolume ratio, or relative surface area of the fermentation system (3). Therefore, based on our results, we speculated that cellulose formation might depend on oxygen uptake rather than the medium. In other words, the bacteria growth was related to medium, whereas the cellulose formation was markedly affected by oxygen. However,
TABLE 2. Comparison of the BC yield and Young’s modulus based on the aeration rate in the modified airlift bioreactor. Culture condition Six plates in 1.5 vvm Six plates in 2 vvm Six plates in 3 vvm
Effect of cultivation time From a production perspective, cultivation time is a crucial factor. Fig. 6 shows the cellulose concentrations after 2, 3, and 4 days of growth. The cells exhibited slight growth by 2 days postinoculum, followed by an exponential growth phase on Days 3 and 4. The BC membrane
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this conjecture requires further scientific experimentation to explain this phenomenon. Table 2 shows the cellulose concentrations and mechanical properties of the BC membranes cultivated at various aeration rates. A maximal BC concentration of 1.8 g/L was obtained at an aeration rate of 2 vvm, and the yield at 1.5 and 3 vvm was approximately half of that. We assumed that the production was low at 1.5 vvm because of the oxygen limitation, and the poor yield at 3 vvm was attributed to the high shear rates at the net plates, which rendered it difficult for the bacteria to attach to the net plate to form a membrane. In addition to the difference in BC yield, the Young’s modulus of samples produced at 1.5 vvm was greater than that of samples produced at 2 vvm. The membrane produced at 3 vvm was not fully integrated. The peak Young’s modulus of the sample obtained at 1.5 vvm when using the 6-net-plate configuration was 10.3 GPa. Fig. 5 shows the WHC obtained at the various aeration rates. Significant decreases in the WHC occurred at 300 g in all 3 sets of conditions, and the highest WHC was obtained at an aeration rate of 2 vvm. Thus, the aeration conditions affected the BC structure.
0.0
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The membrane was too thin to collect; therefore, the membrane was not integrated.
FIG. 6. Effects of varying the cultivation time in the modified airlift bioreactor.
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concentration reached 2.6 g/L after 4 days of culturing, yielding a mean BC production rate of 0.027 g/L h. The growth rate was 0.02 g/L h on Day 2, 0.054 g/L h on Day 3, and 0.033 g/L h on Day 4. Therefore, the maximal growth rate occurred on Day 3 before decreasing on Day 4. The average production rate was slightly greater than the static culture 7-day average rate of 0.02 g/L h. Our proposed modified airlift bioreactor, which was fitted with net plates in place of the rectangular wire-mesh draft tube employed in the original design, was used to produce BC membranes. A previous study reported that a modified airlift bioreactor fitted with a rectangular wire-mesh draft tube eliminated the DO limitation of BC growth that occurs in unmodified airlift bioreactors (27). Cheng et al. (28) reported that using an airlift bioreactor that was modified with a rectangular wire-mesh draft tube maintained a DO concentration above 35% during BC production. In that study, 3 wire-mesh draft tubes were installed in the bioreactor; however, the authors did not discuss the effects of the number of plates. Our results indicated that using more net plates supplies more DO during cultivation. This finding is consistent with a report by Wu and Hsiun (36), who used an airlift bioreactor modified with multiple axial-net draft tubes to generate a greater oxygen transfer rate than using a single axial-net draft tube would. In this study, we proved that the amount of DO correlates with the number of net plates in the bioreactor, where using more net plates increases the oxygen transfer rate. We optimized the proposed bioreactor to maximize the WHC values of BC, and determined that using 6 net plates yielded BC with the most favorable WHC. Specifically, this configuration produced BC that can hold nearly 8-fold more fluid than that produced under static fermentation conditions could. Furthermore, the WHC is greater than that of BC produced using a rotating disc bioreactor, which can retain 5-fold more fluid than BC grown under static conditions does (29). We observed that varying the number of net plates and improving broth aeration affected the Young’s modulus of the BC produced. The peak Young’s modulus was obtained using at an aeration rate of 1.5 vvm when using the 6-net-plate configuration. In previous studies, the basic concept was that the process of cellulose formation should occur at the medium/pellicle interface, and the membrane-producing bacteria should be near this interface (37) because no cellulose formation occurs elsewhere in the medium (38). However, this article is the first to report a successful case of producing cellulose in the medium. The BC membrane produced using the proposed bioreactor has potential for various practical applications. ACKNOWLEDGMENTS We are very grateful for the financial support provided by the National Science Council, Taiwan through grants NSC 94-2214-E242 -001 and NSC 95-2622-E-242-005-CC3. References 1. Yoshinaga, F., Tonouchi, J., and Watanabe, K.: Research progress in production of bacterial cellulose by aeration and agitation culture and its application as a new industrial material, Biosci. Biotechnol. Biochem., 61, 219e224 (1997). 2. Ross, P., Mayer, R., and Benziman, M.: Cellulose biosynthesis and function in bacteria, Microbiol. Mol. Biol. Rev., 55, 35e58 (1991). 3. Jonas, R. and Farah, L. F.: Production and application of microbial cellulose, Polym. Degrad. Stab., 59, 101e106 (1998). 4. Iguchi, M., Yamanaka, S., and Budhiono, A.: Bacterial cellulose-a masterpiece of nature’s arts, J. Mater. Sci., 35, 261e270 (2000). 5. Okiyama, A., Motoki, M., and Yamanaka, S.: Bacterial cellulose II. Proceeding of the gelatinous cellulose for food materials, Food Hydrocolloid., 6, 479e487 (1992).
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