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Cost-effective production of bacterial cellulose in static cultures using distillery wastewater
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e7, 2012 www.elsevier.com/locate/jbiosc
Cost-effective production of bacterial cellulose in static cultures using distillery wastewater Jyh-Ming Wu* and Ren-Han Liu Department of Chemical and Materials Engineering, Chinese Culture University, 55 Hwa-Kang Road, Yang-Ming-Shan, Taipei 11114, Taiwan, ROC Received 9 February 2012; accepted 20 September 2012 Available online xxx
Thin stillage (TS), wastewater from rice wine distillery, was used as a cost-free feedstock to replace the costly traditional Hestrin and Schramm (HS) medium for BC production by Gluconacetobacter xylinus. Due to the rich organic acids and amino acids content in TS, BC production was significantly enhanced as 50 (v/v) % of HS medium was replaced with TS. In the 50/50 TSeHS medium, BC concentration of 6.26 g/l could be obtained after 7 days static cultivation which is approximately 50% higher than that could be produced in HS-only medium. The BC produced by TS containing medium had slightly denser reticulated structures and higher crystallinity index values but with lower water holding capacities than that obtained from HS medium. Based on the 50% cost-free TS, the 50/50 TSeHS medium had a BC production feedstock cost about 67% lower than that of traditional HS medium. The employment of cost-free TS to replace a portion of HS medium to achieve a higher BC production not only can reduce the BC production cost but also solve the wastewater disposal problem of winery industry. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Bacterial cellulose; Gluconacetobacter xylinus; Thin stillage; Distillery wastewater; Cost reduction; Static culture]
Bacterial cellulose (BC) can be produced by Gluconacetobacter xylinus (formerly Acetobacter xylinum) as a white leather-like pellicle at the aireliquid interface. Although the molecular structure is identical to that of plant cellulose, BC displays many unique properties including higher purity, higher crystallinity, higher degree of polymerization, higher water absorption and retaining capacity, higher tensile strength, and stronger biological adaptability. These unique properties as well as its purity enable many successful applications in the field of biomedical materials in recent time (1e3). Various sugars such as glucose, sucrose, fructose, molasses, xylose, glycerol, mannitol and arabitol have been used to produce BC by Gluconacetobacter (4e9). Among those sugars, mannitol and fructose are better carbon sources. However, the high cost of mannitol and fructose as well as relative low-yield production with these carbon sources (4,10) limit commercial production and extended applications of BC. Therefore, there are many investigations have been carried out to look for a new and inexpensive carbon source for high-yield BC production. The hydrolyzate of hemicelluloses (11), konjac glucomannan (12), rice bark (13), waste cotton fabrics (14), agricultural wastes (15), fruit juices (16), glycerol (4,17) and organic acids (17) have also been used as carbon sources to culture Gluconacetobacter for the costeffective production of BC. * Corresponding author. Tel.: þ886 2 28610511x33131; fax: þ886 2 28614011. E-mail addresses: [email protected] (J.-M. Wu), hank90189@ hotmail.com (R.-H. Liu).
Thin stillage (TS) or distillery wastewater is the liquid portion of distillery stillage from the fermentation of grain-based feedstock. Its volume is approximately 10 times than that of ethanol produced. Rice wine is mainly used for Chinese cooking and thin stillage from rice wine distilleries is rich in carbon sources and organic acids (18,19). But its low pH and high biological oxygen demand make TS a nuisance to distillery industries. Although TS can be effectively treated by active sludge or anaerobic digestion, from the resource recovery point of view, TS is better to be considered as nutrients rather than wastewater. Thus, many studies have been carried out to ferment TS of rice wine distilleries for the production of value-added product such as enzymes, ethanol, biomass and etc. (19e22). Recently, using the waste from wine fermentation broth for BC production has been investigated and the BC yield has been improved (23,24). Since the supplementation of organic acids in G. xylinus culture medium has been reported to be very effective for the enhancement of BC production (17,25,26), in our previous study (24), TS from rice wine distillery which is rich in organic acids was employed to supplement the traditional BC production Hestrin and Schramm (HS) medium for BC production. The result showed that BC production was enhanced 2.5-fold after 7 days cultivation when 100% TS was supplemented to HS medium. In this study, however, TS was used as a cost-free feedstock to replace the costly HS medium for BC production. The replacement of HS medium with TS for BC production not only can reduce production cost but also can properly dispose the wastewater from winery industry. In this work, an effectiveness factor for BC
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.09.014
Please cite this article in press as: Wu, J.-M., and Liu, R.-H., Cost-effective production of bacterial cellulose in static cultures using distillery wastewater, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.09.014
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J. BIOSCI. BIOENG.,
production based on the cost of feedstock was defined to evaluate to which extent the amount of HS medium can be replaced with the cost-free TS. Additionally, the effect of TS on the structural characteristics of BC was also studied.
WHC ðg water=g BCÞ ¼ ðWh Wd Þ=Wd
(1)
where Wh and Wd are the weights of hydrate and dry BC, respectively.
RESULTS AND DISCUSSION
MATERIALS AND METHODS Bacterial strain G. xylinus (BCRC 12334) obtained from the Bioresource Collection and Research center (Hsinchu, Taiwan) was used as a bacterial cellulose producing strain. Medium and cultivation The Hestrin and Schramm (HS) medium used for growing G. xylinus contains 20 g/l glucose, 20 g/l peptone, 10 g/l yeast extract, 1.15 g/l citric acid, 6.8 g/l Na2HPO4 with pH adjusted to 6.0. Thin stillage (TS) from a rice wine distillery (Taichung Distillery of Taiwan Tobacco & Liquor Corporation, Taichung, Taiwan) was filtrated using a 1.2 mm membrane to remove particulates in the solution. The filtrate was stored at 4 C for further usage. The composition of this TS is shown in Table 1 (18,19,24). TSeHS medium with different TS volume fractions were employed to grow G. xylinus. TS and HS medium were separately adjusted to pH 6.0 and autoclaved at 120 C for 20 min. After cooling, TS and HS medium were mixed with different portions to prepare TSeHS medium. Inoculum culture was prepared by transferring G. xylinus cell suspension stored at 80 C into HS medium and statically cultivated at 30 C for 3 days. The statically grown culture was then shaken vigorously to release the attached cells from the cellulose pellicle. The resulting cell suspension of 5 ml was inoculated into a 250 ml Erlenmeyer flask containing 45 ml of TSeHS medium and then statically incubated at 30 C for 7 days in triplicate experiments. Analytical methods The reducing sugar concentration of the culture medium was determined using the dinitrosalicylic acid (DNS) method. The cellulose pellicle produced in the static cultures was purified by heating the pellicle in 1 N NaOH at 95 C for 3 h to eliminate the entrapped cells, followed by thoroughly washing with deionized water to neutral pH. The purified cellulose pellicle was dried at 90 C to a constant weight. The dry cell weight in the culture was determined by collecting the BC pellicle with culture broth by centrifugation at 12,000 g for 10 min. The pellet obtained was suspended in pH 4.8, 0.1 M citrate buffer and 1% (v/v) cellulase (2500 U/ml, ACCELLERASE 1500, Danisco Inc., USA) was added to degrade the cellulose pellicle. After 200 rpm shaking for 9 h at 50 C to reach a complete hydrolysis, the digested suspension was centrifuged and washed twice with triple amount of deionized water then dried at 90 C (27). Scanning electron microscope (SEM) was used to observe the morphology of BC pellicle. The BC samples were dried by Critical Point Dryers (Samdri-PVT-3, Tousimis Inc., USA). A field emission SEM, model JSM-6335F (JEOL Ltd., Japan), with accelerating voltage of 5.0 kV was used. Samples were mounted onto a substrate with carbon tape and coated with a thin layer of gold. X-ray diffraction (XRD) measurement was carried out to analyze the change in crystallinity of the produced BC by an X-ray diffractometer (D2 Phaser, Bruker Inc., Germany) with reflection geometry and CuKa radiation of wavelength 0.154 nm. Operating voltage and current were 30 kV and 10 mA, respectively. The angle of scanning was varied from 5 to 40 with a scanning speed of 5 /min. The crystallinity index of BC was determined as CrI (%) ¼ [(I002 Iam)/I002] 100%, where I002 represents the maximum intensity of the (0 0 2) lattice diffraction at 2q about 22.8 , and Iam is the intensity of the baseline at 2q about 18 (28). The water holding capacity (WHC) of BC was measured according to the method of Seifert et al. (29). The BC samples were immersed in distilled water for 3 h at 30 C to completely swell up, centrifuged at 1500 g for 10 min, and dried at 90 C until a constant weight was obtained. The WHC of BC was calculated by using the following formula (Eq. 1):
TABLE 1. The composition of thin stillage from a rice wine distillery in this study. pH Sugar (mg/l) Glucose Reducing sugar Total nitrogen (mg/l) Acid (mg/l) Total organic acid Succinic acid Gluconic acid Acetic acid Citric acid Malic acid Total amino acid BOD (ppm) COD (ppm)
3.5 35 1120 1258 3833 2254 1018 243 161 157 760 25,000 50,920
Effect of TS in TSeHS medium for BC production In order to reduce the feedstock cost for BC production, the cost-free TS of a rice winery was employed to replace the commonly used but costly HS medium. Since ethanol is a very important factor for enhancing BC production (30,31), the residual concentration of ethanol in TSeHS medium has to be determined. After analysis by HPLC, no residual ethanol can be measured in TSeHS medium. Therefore, the factor of ethanol from TS for improving BC production can be completely ruled out. The effect of TS volume fraction in TSeHS medium on BC production was first studied. As shown in Fig. 1A, in a 7 days static cultivation, the cell concentration as well as BC concentration increased with TS volume fraction up to 50% but declined abruptly afterward. This indicates that the highest amount of BC (6.31 g/l) can be obtained in a 50/50 TSeHS medium in which HS medium was diluted to 50% by TS. The BC concentration obtained in 50/50 TSeHS medium was about 50% and 100% higher than that obtained in 100% HS medium and 100% TS medium, respectively. The residual reducing sugar concentration in the culture medium was also analyzed to determine the reducing sugar to BC conversion in TSeHS medium. As shown in Fig. 1B, the total reducing sugar consumed declined steadily from 1.01 g to 0.12 g as TS volume fraction increased to 87.5%. Because, as shown in Table 1, there is only an appreciable amount of reducing sugars in TS (approximately 0.1%), the initial 20 g/l glucose in 100% HS medium was diluted as the amount of TS employed to replace HS medium increased. The percentage of reducing sugar consumed was nearly constant (approximately 96%) in TSeHS medium with TS less than 50%. When TS content was higher than 50%, the percentage of reducing sugar consumed decreased significantly from 96% to 73% as TS increased from 50% to 87.5%. Evidently, as the nutrients of TS became richer in TSeHS medium, G. xylinus switched to utilize TS nutrients rather than the appreciable glucose brought in from HS medium. The less efficient utilization of the easily digestible glucose leads to an apparent drop of cell growth and BC production (Fig. 1A). Based on the amount of reducing sugar consumed, the reducing sugar to BC conversion yield can be determined as shown in Fig. 1B that 120% can be obtained at 87.5% TS volume fraction. The higher than 100% reducing sugar to BC yield indicates that nutrients other than reducing sugars, probably the rich organic acids and amino acids in TS, were metabolized by G. xylinus to synthesize BC. Time courses of BC production The time courses of BC production by G. xylinus in static cultures using 100% HS, 50% HS, 50/50 TSeHS, 50% TS and 100% TS medium were studied. As shown in Fig. 2A, the reducing sugar concentrations decreased to a very low level after first 3 days in all medium containing HS medium. This is because the glucose dehydogenase (GDH) located in cell membrane of G. xylinus is very active and can easily convert the glucose from HS medium into gluconic acid (32) that also resulted in apparent pH decrease (Fig. 2B). In contrast, very low but slightly increased reducing sugar concentrations were detected in 100% and 50% TS medium. The slightly increased reducing sugar concentrations observed is probably due to the reductive metabolites produced from G. xylinus while utilizes the organic acids and amino acids in TS-only medium for cell growth and BC synthesis. As shown in Fig. 2C, the 50% diluted TS medium exhibited a poorest BC production that only 1 g/l BC was obtained after 7 days static cultivation. The 100% TS medium (without dilution), however, can sustain the cell growth and BC
Please cite this article in press as: Wu, J.-M., and Liu, R.-H., Cost-effective production of bacterial cellulose in static cultures using distillery wastewater, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.09.014
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FIG. 1. Effect of different percentage of TS employed to replace HS in TSeHS medium after 7 days static cultivation. (A) Initial reducing sugar concentration in 50 ml culture medium, DCW and BC concentration; (B) reducing sugar consumed and BC/reducing sugar conversion yield. Data are the means of three independent replicates. Error bars indicate standard deviations.
FIG. 2. Time courses of DCW (closed symbols) and reducing sugar concentration (open symbols) (A), pH (B), BC concentration (C) produced by G. xylinus in 50 ml static cultivation of different TSeHS medium. Each value represents the average value of three independent experiments. Error bars represent standard deviations.
Please cite this article in press as: Wu, J.-M., and Liu, R.-H., Cost-effective production of bacterial cellulose in static cultures using distillery wastewater, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.09.014
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production to a level slightly higher than that obtained in 50% HS medium which is half strength of traditional BC production medium. When 1:1 volume ratio of TS to HS medium (50/50 TSeHS medium) was employed, the cell growth rate (Fig. 2A) was slower than that of 100% HS medium during first 3 days probably because the amount of easily digestible glucose is about half of 100% HS medium. A second stage with a faster cell growth was observed after the 3rd day and the BC production nearly maintained at a constant rate and no apparent decrease as in 100% HS medium was observed. Evidently, the nutrients in 50% TS medium although cannot sustain a good growth and BC production but can significantly enhance BC production when supplemented to a 50% HS medium. The synergetic effect of TS on enhancing BC production could be clearly observed from the 6.26 g/l BC produced in 50/50 TSeHS medium which is about 3and 6-fold higher than that produced by 50% HS and 50% TS medium, respectively. The significant enhancement of BC production probably resulted from the rich organic acids and amino acids content in TS (Table 1). In 50/50 TSeHS medium, the organic acids and amino acids not only can provide the enough nutrients to support the cell growth of G. xylinus but also has some extent of inhibition effect on converting glucose into gluconic acid probably due to the second rich gluconic acid existed in TS so that the amount of glucose included in 50% HS medium can be efficiently utilized as building blocks for BC biosynthesis. This has been confirmed in our previous study (24). Moreover, it is worth mentioning that the richest organic acid in TS is succinic acid (approximately 60%), which is probably helpful to BC production. To verify this possibility, the supplementation of succinic acid in 100% HS and 50% HS medium were performed to culture G. xylinus for producing BC. As shown in Fig. 3, in a 7 days static cultivation, the BC production increased with succinic acid concentration up to 0.4% (w/v) but declined abruptly afterward in 100% HS medium as well as 50% HS medium. The highest amounts of BC were 6.17 and 4.18 (g/l), respectively. This indicates that succinic acid is a very effective supplement for BC production. Although the concentration of 0.1% (w/v) succinic acid is not high, which is around the same level of succinic acid in 50/50 TSeHS medium, its BC production reached a comparable
FIG. 3. Effect of succinic acid concentrations on BC production by G. xylinus in 50 ml 100% HS and 50% HS medium after 7 days static cultivation. Data are the means of three independent replicates. Error bars indicate standard deviations.
J. BIOSCI. BIOENG., level to that in higher succinic acid concentrations supplemented to 50% HS medium. While 50% TS medium supplemented to 50% HS medium (50/50 TSeHS medium) produced 6.26 g/l BC yield. This reveals that succinic acid in TS can greatly enhance BC production in 50/50 TSeHS medium. The significant effect of succinic acid on BC production enhancement has been reported previously (17,25,33,34). In addition to succinic acid, other organic acids in TS such as gluconic acid, acetic acid, citric acid and malic acid are also beneficial for BC production enhancement (17,25,33). These organic acids are all located in the pathway of TCA cycle. Effect of TS on the structural characteristics of BC The effect of TS on the morphology of BC pellicle produced was also studied. As shown in Fig. 4A, light brownish pellicle was obtained from the culture medium after thoroughly washing with DI water. Probably due to the original darker color of TS wastewater, the color intensities of pellicles produced from TS containing medium were slightly higher than that produced from HS medium. The pellicle obtained in 50/50 TSeHS medium was obviously thicker than that produced in other medium. After the pellicles were treated in 1 N NaOH to remove the biomass of G. xylinus, purified white BC pellicles were obtained (Fig. 4B). All the purified BC pellicles showed the reticulated structure consisting of ultrafine cellulose nanofibrils and no significant difference in the size of cellulose fibrils as illustrated in the scanning electron microscope (SEM) images (Fig. 5). TS did not significantly change the major ultrafine nanofibril structure, however, the BC nanofibrils produced by TS containing medium resulted in a slightly denser reticulated structure than that
FIG. 4. Digital images of BC pellicles produced from various 50 ml of TSeHS medium after 7 days static cultivation before (A), and after (B) NaOH treatment to remove the biomass of G. xylinus.
Please cite this article in press as: Wu, J.-M., and Liu, R.-H., Cost-effective production of bacterial cellulose in static cultures using distillery wastewater, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.09.014
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FIG. 5. SEM images of purified BC pellicles produced from various 50 ml of TSeHS medium after 7 days static cultivation. (A) 100% HS, (B) 50% HS, (C) 50/50 TSeHS, (D) 50% TS, and (E) 100% TS medium.
produced from HS medium. The morphological changes in BC can influence various properties and microstructures such as crystallinity. In order to evaluate the crystalline structure and the change in crystallinity of BC produced by TS containing medium, X-ray diffraction was used. The diffraction diagrams of BC produced by all TS containing medium showed three main peaks at 2q 14.4 , 16.7 and 22.8 (Fig. 6CeE), corresponding to the crystallographic planes of (1 0 1), (101) and (0 0 2), respectively (35). These peaks demonstrate that BC possesses the typical crystalline forms of cellulose I (36). The diffraction angles 2q of the peaks for BC obtained in HS medium (Fig. 6A and B) were the same as those in TS containing medium, suggesting that TS did not alter the crystalline structure of BC. The crystallinity index values of BC are listed in Table 2. The crystallinity index values of BC from TS containing medium (approximately 83%) were slightly higher than that of BC from HS medium (approximately 79%).
This reveals that TS can affect the aggregation and crystallization of BC microfibrils and increase the crystallinity index. The water holding capacity (WHC) represents the weight of water held per unit weight of cellulose nanofibrils forming a reticulated structure. The BC pellicles exhibited high retention of water (Table 2) due to the BC composed of ultrafine nanofibrils in ultrafine network as illustrated in Fig. 5. However, the WHC of BC produced by all TS containing medium were approximately 20% lower than that of BC obtained from HS medium. The water molecules are caught physically in the reticulated nanofibrils of BC (37). When the BC nanofibrils are loosely arranged, resulting in more vacant space to trap more water molecules and thus the WHC is high. The BC obtained from TS containing medium showed a more crowded cellulose network with less free space to trap water than that from HS medium (Fig. 5). This could explain the decreased WHC of BC from TS containing medium (29).
Please cite this article in press as: Wu, J.-M., and Liu, R.-H., Cost-effective production of bacterial cellulose in static cultures using distillery wastewater, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.09.014
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J. BIOSCI. BIOENG., TABLE 3. Cost factor of BC production based on HS medium according to different volume fraction of TS in TSeHS medium after 7 days static cultivation at 30 C. Volume fraction of TS in TSeHS (%) (f) 0 12.5 25.0 37.5 50.0 62.5 75.0 87.5 100
FIG. 6. XRD patterns of purified BC pellicles produced from various 50 ml of TSeHS medium after 7 days static cultivation. (A) 100% HS, (B) 50% HS, (C) 50/50 TSeHS, (D) 50% TS, and (E) 100% TS medium.
Cost effectiveness for BC production The BC production cost-down based on a cheaper medium by replacing a fraction of HS medium with cost-free TS is shown in Table 3. The BC production based on a 7 days static cultivation was enhanced with the increase of TS in TSeHS medium up to 50%. A 1.5-fold enhancement (relative to 100% HS medium) was achieved when 50% of HS medium was replaced by TS in a 50/50 TSeHS medium. In other words, the nutrient cost not only can be reduced to half but also with a 50% enhancement of BC production. In order to evaluate the effectiveness of nutrient cost reduction for BC production in TSeHS medium, a cost reduction factor (h) is defined as the ratio of volume fraction of HS medium in the TSeHS medium to BC production enhancement (Eq. 2).
h ¼
1f E
(2)
where E is the BC concentration enhancement relative to 100% HS medium, and f is the volume fraction of TS in TSeHS medium. The lower the cost reduction factor is, the more effective for reducing the nutrient cost will be to produce a unit weight of BC based on a unit cost of HS medium. For example, the cost of 50/50 TSeHS medium is about 67% lower than 100% HS medium to produce a same amount of BC. The cost factor can be reduced to 0.17 and nil as the amount of HS medium employed in TSeHS medium decreased to 12.5% and 0% (100% TS), respectively. This indicates that the nutrient cost approached to nil when 100% TS was employed to produce 3 g/l of BC after 7 days static cultivation. Although there will be no nutrient cost for employing 100% TS to produce BC, the BC concentration is only about half of that obtained in 50/50 TSeHS medium. Taking BC productivity into account in a 7 days static culture, 50/50 TSeHS medium will be the ultimate choice. In our previous study, when TS was employed to replace distilled water for preparing HS medium (100/100 TSeHS TABLE 2. Crystallinity index and water holding capacity of BC produced from various medium after 7 days static cultivation. 100% HS
50% HS
Crystallinity index (%) 79.5 78.7 98.5 4.2 103.1 4.8 Water holding capacity (g water/g BC)
50/50 TSeHS 82.4 83.0 3.6
50% TS
100% TS
83.5 83.9 80.8 2.9 79.1 3.3
BC concentration (g/l)
BC enhancement fold (E)
Cost reduction factor (h)
1.00 1.16 1.33 1.40 1.50 0.92 0.77 0.76 0.74
1.00 0.76 0.56 0.45 0.33 0.41 0.32 0.17 0
4.17 4.82 5.54 5.85 6.26 3.85 3.21 3.15 3.08
0.29 0.22 0.25 0.17 0.23 0.16 0.20 0.14 0.16
medium), BC production in this 100/100 TSeHS medium was enhanced 2.5-fold to a concentration of 10.38 g/l after 7 days static cultivation (24). In this study, however, TS was used to replace the costly HS medium for BC production. In the 50/50 TSeHS medium, BC concentration of 6.26 g/l can be achieved after 7 days static cultivation. This indicates that when the 100/100 TSeHS medium is diluted to 50% by distilled water to become two volumes of the 50/ 50 TSeHS medium, 12.5 g/l of BC could be obtained after 7 days static cultivation which is higher than that produced by 100/100 TSeHS medium. Therefore, the utilization of TS in this work is a good strategy for BC production by G. xylinus. TS from rice winery industry can be used as a very effective feedstock to support the growth of G. xylinus for BC production. Up to 50% traditional BC producing HS medium was replaced with the cost-free TS, the amount of BC produced could be enhanced 50% as compared with that of HS medium. The presence of TS in excess of 50% in TSeHS medium significantly reduced the reducing sugar consumption by G. xylinus that leads to a reduced BC production. The BC production enhancement along with reduced feedstock cost achieved in 50/50 TSeHS medium resulted in a 67% feedstock cost reduction for BC production. The BC produced by TS containing medium had slightly denser reticulated structures and higher crystallinity index values but with lower WHC than that obtained from HS medium. The employment of cost-free TS to replace a portion of HS medium to achieve a higher BC concentration not only can reduce the BC production cost but also solve the wastewater disposal problem of winery industry. ACKNOWLEDGMENTS This research study was financially supported by the Chinese Culture University and the National Science Council of Taiwan (NSC 99-2621-M-034-002). References 1. Petersen, N. and Gatenholm, P.: Bacterial cellulose-based materials and medical devices: current state and perspectives, Appl. Microbiol. Biotechnol., 91, 1277e1286 (2011). 2. Klemm, D., Kramer, F., Moritz, S., Lindström, T., Ankerfors, M., Gray, D., and Dorris, A.: Nanocelluloses: a new family of nature-based materials, Angew. Chem. Int. Ed., 50, 5438e5466 (2011). 3. Czaja, W. K., Young, D. J., Kawecki, M., and Brown, R. M., Jr.: The future prospects of microbial cellulose in biomedical applications, Biomacromolecules, 8, 1e12 (2007). 4. Mikkelsen, D., Flanagan, B. M., Dykes, G. A., and Gidley, M. J.: Influence of different carbon sources on bacterial cellulose production by Gluconacetobacter xylinus strain ATCC 53524, J. Appl. Microbiol., 107, 576e583 (2009). 5. Keshk, S. and Sameshima, K.: Evaluation of different carbon sources for bacterial cellulose production, Afr. J. Biotechnol., 4, 478e482 (2005). 6. Keshk, S. and Sameshima, K.: The utilization of sugar cane molasses with/ without the presence of lignosulfonate for the production of bacterial cellulose, Appl. Microbiol. Biotechnol., 72, 291e296 (2006). 7. Bae, S. and Shoda, M.: Bacterial cellulose production by fed-batch fermentation in molasses medium, Biotechnol. Prog., 20, 1366e1371 (2004).
Please cite this article in press as: Wu, J.-M., and Liu, R.-H., Cost-effective production of bacterial cellulose in static cultures using distillery wastewater, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.09.014
VOL. xx, 2012 8. Ishihara, M., Matsunaga, M., Hayashi, N., and Tisler, V.: Utilization of D-xylose as carbon source for production of bacterial cellulose, Enzyme Microb. Technol., 31, 986e991 (2002). 9. Oikawa, T., Morino, T., and Ameyama, M.: Production of cellulose from Darabitol by Acetobacter xylinum KU-1, Biosci. Biotechnol. Biochem., 59, 1564e1565 (1995). 10. Bae, S., Sugano, Y., and Shoda, M.: Improvement of bacterial cellulose production by addition of agar in a jar fermentor, J. Biosci. Bioeng., 97, 33e38 (2004). 11. Hong, F., Zhu, Y. X., Yang, G., and Yang, X. X.: Wheat straw acid hydrolysate as a potential cost-effective feedstock for production of bacterial cellulose, J. Chem. Technol. Biotechnol., 86, 675e680 (2011). 12. Hong, F. and Qiu, K.: An alternative carbon source from konjac powder for enhancing production of bacterial cellulose in static cultures by a model strain Acetobacter aceti subsp. xylinus ATCC 23770, Carbohydr. Polym., 72, 545e549 (2008). 13. Goelzer, F. D. E., Faria-Tischer, P. C. S., Vitorino, J. C., Sierakowski, M. R., and Tischer, C. A.: Production and characterization of nanospheres of bacterial cellulose from Acetobacter xylinum from processed rice bark, Mater. Sci. Eng. C, 29, 546e551 (2009). 14. Kuo, C. H., Lin, P. J., and Lee, C. K.: Enzymatic saccharification of dissolution pretreated waste cellulosic fabrics for bacterial cellulose production by Gluconacetobacter xylinus, J. Chem. Technol. Biotechnol., 85, 1346e1352 (2010). 15. Kongruang, S.: Bacterial cellulose production by Acetobacter xylinum strains from agricultural waste products, Appl. Biochem. Biotechnol., 148, 245e256 (2008). 16. Kurosumi, A., Sasaki, C., Yamashita, Y., and Nakamura, Y.: Utilization of various fruit juices as carbon source for production of bacterial cellulose by Acetobacter xylinum NBRC 13693, Carbohydr. Polym., 76, 333e335 (2009). 17. Jung, H. I., Jeong, J. H., Lee, O. M., Park, G. T., Kim, K. K., Park, H. C., Lee, S. M., Kim, Y. G., and Son, H. J.: Influence of glycerol on production and structuralphysical properties of cellulose from Acetobacter sp. V6 cultured in shake flasks, Bioresour. Technol., 101, 3602e3608 (2010). 18. Hsieh, C., Hsu, T. H., and Yang, F. C.: Production of polysaccharides of Ganoderma lucidum (CCRC36021) by reusing thin stillage, Process Biochem., 40, 909e916 (2005). 19. Yang, F. C. and Lin, I. H.: Production of acid protease using thin stillage from a rice-spirit distillery by Aspergillus niger, Enzyme Microb. Technol., 23, 397e402 (1998). 20. Ahn, J. H., Sang, B. I., and Um, Y.: Butanol production from thin stillage using Clostridium pasteurianum, Bioresour. Technol., 102, 4934e4937 (2011). 21. Gonzalez, R., Campbell, P., and Wong, M.: Production of ethanol from thin stillage by metabolically engineered Escherichia coli, Biotechnol. Lett., 32, 405e411 (2010). 22. Yang, F. C. and Tung, H. L.: Reuse of thin stillage from rice spirit for the culture of the yeast Saccharomyces cerevisiae, Process Biochem., 31, 617e620 (1996). 23. Ha, J. H., Shehzad, O., Khan, S., Lee, S. Y., Park, J. W., Khan, T., and Park, J. K.: Production of bacterial cellulose by a static cultivation using the waste from beer culture broth, Korean J. Chem. Eng., 25, 812e815 (2008).
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24. Wu, J. M. and Liu, R. H.: Thin stillage supplementation greatly enhances bacterial cellulose production by Gluconacetobacter xylinus, Carbohydr. Polym., 90, 116e121 (2012). 25. Dudman, W. F.: Cellulose production by Acetobacter acetigenum in defined medium, J. Gen. Microbiol., 21, 327e337 (1959). 26. Wang, Z. G., Wei, Y. L., Wei, M. S., Wang, X. B., and Xiang, D.: Effect of organic acids on bacterial cellulose production in defined medium by Acetobacter xylinum, Food Sci., 29, 295e297 (2008). 27. Hu, Y. and Catchmark, J. M.: Influence of 1-methylcyclopropene (1-MCP) on the production of bacterial cellulose biosynthesized by Acetobacter xylinum under the agitated culture, Lett. Appl. Microbiol., 51, 109e113 (2010). 28. Segal, L., Creely, J., Martin, A., and Conrad, C.: An empirical method for estimating the degree of crystallinity of native cellulose using the X-ray diffractometer, Text. Res. J., 29, 786e794 (1959). 29. Seifert, M., Hesse, S., Kabrelian, V., and Klemm, D.: Controlling the water content of never dried and reswollen bacterial cellulose by the addition of water-soluble polymers to the culture medium, J. Polym. Sci., 42, 463e470 (2004). 30. Naritomi, T., Kouda, T., Yano, H., and Yoshinaga, F.: Effect of ethanol on bacterial cellulose production from fructose in continuous culture, J. Ferment. Bioeng., 85, 598e603 (1998). 31. Park, J. K., Jung, J. Y., and Park, Y. H.: Cellulose production by Gluconacetobacter hansenii in a medium containing ethanol, Biotechnol. Lett., 25, 2055e2059 (2003). 32. Shigematsu, T., Takamine, K., Kitazato, M., Morita, T., Naritomi, T., Morimura, S., and Kida, K.: Cellulose production from glucose using a glucose dehydrogenase gene (gdh)-deficient mutant of Gluconacetobacter xylinus and its use for bioconversion of sweet potato pulp, J. Biosci. Bioeng., 99, 415e422 (2005). 33. Kim, J. D., Jung, H. I., Jeong, J. H., Park, K. H., Jeon, Y. D., Hwang, D. Y., Lee, C. Y., and Son, H. J.: Production and structural analysis of cellulose by Acetobacter sp. V6 using static culture, Korean J. Microbiol., 45, 275e280 (2009). 34. Benziman, M. and Burger-Rachamimov, H.: Synthesis of cellulose from pyruvate by succinate-grown cells of Acetobacter xylinum, J. Bacteriol., 84, 625e630 (1962). 35. Castro, C., Zuluag, R., Putaux, J., Caro, G., Mondragon, I., and Ganan, P.: Structural characterization of bacterial cellulose produced by Gluconacetobacter swingsii sp. from Colombian agroindustrial wastes, Carbohydr. Polym., 84, 96e102 (2011). 36. Oh, S. Y., Yoo, D. I., Shin, Y., and Kim, H. C.: Crystalline structure analysis of cellulose treated with sodium hydroxide and carbon dioxide by means of X-ray diffraction and FTIR spectroscopy, Carbohydr. Res., 340, 2376e2391 (2005). 37. Watanabe, K., Tabuchi, M., Morinaga, Y., and Yoshinaga, F.: Structural features and properties of bacterial cellulose produced in agitated culture, Cellulose, 5, 187e200 (1998).
Please cite this article in press as: Wu, J.-M., and Liu, R.-H., Cost-effective production of bacterial cellulose in static cultures using distillery wastewater, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.09.014
Cost-effective production of bacterial cellulose in static cultures using distillery wastewater Jyh-Ming Wu* and Ren-Han Liu Department of Chemical and Materials Engineering, Chinese Culture University, 55 Hwa-Kang Road, Yang-Ming-Shan, Taipei 11114, Taiwan, ROC Received 9 February 2012; accepted 20 September 2012 Available online xxx
Thin stillage (TS), wastewater from rice wine distillery, was used as a cost-free feedstock to replace the costly traditional Hestrin and Schramm (HS) medium for BC production by Gluconacetobacter xylinus. Due to the rich organic acids and amino acids content in TS, BC production was significantly enhanced as 50 (v/v) % of HS medium was replaced with TS. In the 50/50 TSeHS medium, BC concentration of 6.26 g/l could be obtained after 7 days static cultivation which is approximately 50% higher than that could be produced in HS-only medium. The BC produced by TS containing medium had slightly denser reticulated structures and higher crystallinity index values but with lower water holding capacities than that obtained from HS medium. Based on the 50% cost-free TS, the 50/50 TSeHS medium had a BC production feedstock cost about 67% lower than that of traditional HS medium. The employment of cost-free TS to replace a portion of HS medium to achieve a higher BC production not only can reduce the BC production cost but also solve the wastewater disposal problem of winery industry. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Bacterial cellulose; Gluconacetobacter xylinus; Thin stillage; Distillery wastewater; Cost reduction; Static culture]
Bacterial cellulose (BC) can be produced by Gluconacetobacter xylinus (formerly Acetobacter xylinum) as a white leather-like pellicle at the aireliquid interface. Although the molecular structure is identical to that of plant cellulose, BC displays many unique properties including higher purity, higher crystallinity, higher degree of polymerization, higher water absorption and retaining capacity, higher tensile strength, and stronger biological adaptability. These unique properties as well as its purity enable many successful applications in the field of biomedical materials in recent time (1e3). Various sugars such as glucose, sucrose, fructose, molasses, xylose, glycerol, mannitol and arabitol have been used to produce BC by Gluconacetobacter (4e9). Among those sugars, mannitol and fructose are better carbon sources. However, the high cost of mannitol and fructose as well as relative low-yield production with these carbon sources (4,10) limit commercial production and extended applications of BC. Therefore, there are many investigations have been carried out to look for a new and inexpensive carbon source for high-yield BC production. The hydrolyzate of hemicelluloses (11), konjac glucomannan (12), rice bark (13), waste cotton fabrics (14), agricultural wastes (15), fruit juices (16), glycerol (4,17) and organic acids (17) have also been used as carbon sources to culture Gluconacetobacter for the costeffective production of BC. * Corresponding author. Tel.: þ886 2 28610511x33131; fax: þ886 2 28614011. E-mail addresses: [email protected] (J.-M. Wu), hank90189@ hotmail.com (R.-H. Liu).
Thin stillage (TS) or distillery wastewater is the liquid portion of distillery stillage from the fermentation of grain-based feedstock. Its volume is approximately 10 times than that of ethanol produced. Rice wine is mainly used for Chinese cooking and thin stillage from rice wine distilleries is rich in carbon sources and organic acids (18,19). But its low pH and high biological oxygen demand make TS a nuisance to distillery industries. Although TS can be effectively treated by active sludge or anaerobic digestion, from the resource recovery point of view, TS is better to be considered as nutrients rather than wastewater. Thus, many studies have been carried out to ferment TS of rice wine distilleries for the production of value-added product such as enzymes, ethanol, biomass and etc. (19e22). Recently, using the waste from wine fermentation broth for BC production has been investigated and the BC yield has been improved (23,24). Since the supplementation of organic acids in G. xylinus culture medium has been reported to be very effective for the enhancement of BC production (17,25,26), in our previous study (24), TS from rice wine distillery which is rich in organic acids was employed to supplement the traditional BC production Hestrin and Schramm (HS) medium for BC production. The result showed that BC production was enhanced 2.5-fold after 7 days cultivation when 100% TS was supplemented to HS medium. In this study, however, TS was used as a cost-free feedstock to replace the costly HS medium for BC production. The replacement of HS medium with TS for BC production not only can reduce production cost but also can properly dispose the wastewater from winery industry. In this work, an effectiveness factor for BC
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.09.014
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J. BIOSCI. BIOENG.,
production based on the cost of feedstock was defined to evaluate to which extent the amount of HS medium can be replaced with the cost-free TS. Additionally, the effect of TS on the structural characteristics of BC was also studied.
WHC ðg water=g BCÞ ¼ ðWh Wd Þ=Wd
(1)
where Wh and Wd are the weights of hydrate and dry BC, respectively.
RESULTS AND DISCUSSION
MATERIALS AND METHODS Bacterial strain G. xylinus (BCRC 12334) obtained from the Bioresource Collection and Research center (Hsinchu, Taiwan) was used as a bacterial cellulose producing strain. Medium and cultivation The Hestrin and Schramm (HS) medium used for growing G. xylinus contains 20 g/l glucose, 20 g/l peptone, 10 g/l yeast extract, 1.15 g/l citric acid, 6.8 g/l Na2HPO4 with pH adjusted to 6.0. Thin stillage (TS) from a rice wine distillery (Taichung Distillery of Taiwan Tobacco & Liquor Corporation, Taichung, Taiwan) was filtrated using a 1.2 mm membrane to remove particulates in the solution. The filtrate was stored at 4 C for further usage. The composition of this TS is shown in Table 1 (18,19,24). TSeHS medium with different TS volume fractions were employed to grow G. xylinus. TS and HS medium were separately adjusted to pH 6.0 and autoclaved at 120 C for 20 min. After cooling, TS and HS medium were mixed with different portions to prepare TSeHS medium. Inoculum culture was prepared by transferring G. xylinus cell suspension stored at 80 C into HS medium and statically cultivated at 30 C for 3 days. The statically grown culture was then shaken vigorously to release the attached cells from the cellulose pellicle. The resulting cell suspension of 5 ml was inoculated into a 250 ml Erlenmeyer flask containing 45 ml of TSeHS medium and then statically incubated at 30 C for 7 days in triplicate experiments. Analytical methods The reducing sugar concentration of the culture medium was determined using the dinitrosalicylic acid (DNS) method. The cellulose pellicle produced in the static cultures was purified by heating the pellicle in 1 N NaOH at 95 C for 3 h to eliminate the entrapped cells, followed by thoroughly washing with deionized water to neutral pH. The purified cellulose pellicle was dried at 90 C to a constant weight. The dry cell weight in the culture was determined by collecting the BC pellicle with culture broth by centrifugation at 12,000 g for 10 min. The pellet obtained was suspended in pH 4.8, 0.1 M citrate buffer and 1% (v/v) cellulase (2500 U/ml, ACCELLERASE 1500, Danisco Inc., USA) was added to degrade the cellulose pellicle. After 200 rpm shaking for 9 h at 50 C to reach a complete hydrolysis, the digested suspension was centrifuged and washed twice with triple amount of deionized water then dried at 90 C (27). Scanning electron microscope (SEM) was used to observe the morphology of BC pellicle. The BC samples were dried by Critical Point Dryers (Samdri-PVT-3, Tousimis Inc., USA). A field emission SEM, model JSM-6335F (JEOL Ltd., Japan), with accelerating voltage of 5.0 kV was used. Samples were mounted onto a substrate with carbon tape and coated with a thin layer of gold. X-ray diffraction (XRD) measurement was carried out to analyze the change in crystallinity of the produced BC by an X-ray diffractometer (D2 Phaser, Bruker Inc., Germany) with reflection geometry and CuKa radiation of wavelength 0.154 nm. Operating voltage and current were 30 kV and 10 mA, respectively. The angle of scanning was varied from 5 to 40 with a scanning speed of 5 /min. The crystallinity index of BC was determined as CrI (%) ¼ [(I002 Iam)/I002] 100%, where I002 represents the maximum intensity of the (0 0 2) lattice diffraction at 2q about 22.8 , and Iam is the intensity of the baseline at 2q about 18 (28). The water holding capacity (WHC) of BC was measured according to the method of Seifert et al. (29). The BC samples were immersed in distilled water for 3 h at 30 C to completely swell up, centrifuged at 1500 g for 10 min, and dried at 90 C until a constant weight was obtained. The WHC of BC was calculated by using the following formula (Eq. 1):
TABLE 1. The composition of thin stillage from a rice wine distillery in this study. pH Sugar (mg/l) Glucose Reducing sugar Total nitrogen (mg/l) Acid (mg/l) Total organic acid Succinic acid Gluconic acid Acetic acid Citric acid Malic acid Total amino acid BOD (ppm) COD (ppm)
3.5 35 1120 1258 3833 2254 1018 243 161 157 760 25,000 50,920
Effect of TS in TSeHS medium for BC production In order to reduce the feedstock cost for BC production, the cost-free TS of a rice winery was employed to replace the commonly used but costly HS medium. Since ethanol is a very important factor for enhancing BC production (30,31), the residual concentration of ethanol in TSeHS medium has to be determined. After analysis by HPLC, no residual ethanol can be measured in TSeHS medium. Therefore, the factor of ethanol from TS for improving BC production can be completely ruled out. The effect of TS volume fraction in TSeHS medium on BC production was first studied. As shown in Fig. 1A, in a 7 days static cultivation, the cell concentration as well as BC concentration increased with TS volume fraction up to 50% but declined abruptly afterward. This indicates that the highest amount of BC (6.31 g/l) can be obtained in a 50/50 TSeHS medium in which HS medium was diluted to 50% by TS. The BC concentration obtained in 50/50 TSeHS medium was about 50% and 100% higher than that obtained in 100% HS medium and 100% TS medium, respectively. The residual reducing sugar concentration in the culture medium was also analyzed to determine the reducing sugar to BC conversion in TSeHS medium. As shown in Fig. 1B, the total reducing sugar consumed declined steadily from 1.01 g to 0.12 g as TS volume fraction increased to 87.5%. Because, as shown in Table 1, there is only an appreciable amount of reducing sugars in TS (approximately 0.1%), the initial 20 g/l glucose in 100% HS medium was diluted as the amount of TS employed to replace HS medium increased. The percentage of reducing sugar consumed was nearly constant (approximately 96%) in TSeHS medium with TS less than 50%. When TS content was higher than 50%, the percentage of reducing sugar consumed decreased significantly from 96% to 73% as TS increased from 50% to 87.5%. Evidently, as the nutrients of TS became richer in TSeHS medium, G. xylinus switched to utilize TS nutrients rather than the appreciable glucose brought in from HS medium. The less efficient utilization of the easily digestible glucose leads to an apparent drop of cell growth and BC production (Fig. 1A). Based on the amount of reducing sugar consumed, the reducing sugar to BC conversion yield can be determined as shown in Fig. 1B that 120% can be obtained at 87.5% TS volume fraction. The higher than 100% reducing sugar to BC yield indicates that nutrients other than reducing sugars, probably the rich organic acids and amino acids in TS, were metabolized by G. xylinus to synthesize BC. Time courses of BC production The time courses of BC production by G. xylinus in static cultures using 100% HS, 50% HS, 50/50 TSeHS, 50% TS and 100% TS medium were studied. As shown in Fig. 2A, the reducing sugar concentrations decreased to a very low level after first 3 days in all medium containing HS medium. This is because the glucose dehydogenase (GDH) located in cell membrane of G. xylinus is very active and can easily convert the glucose from HS medium into gluconic acid (32) that also resulted in apparent pH decrease (Fig. 2B). In contrast, very low but slightly increased reducing sugar concentrations were detected in 100% and 50% TS medium. The slightly increased reducing sugar concentrations observed is probably due to the reductive metabolites produced from G. xylinus while utilizes the organic acids and amino acids in TS-only medium for cell growth and BC synthesis. As shown in Fig. 2C, the 50% diluted TS medium exhibited a poorest BC production that only 1 g/l BC was obtained after 7 days static cultivation. The 100% TS medium (without dilution), however, can sustain the cell growth and BC
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FIG. 1. Effect of different percentage of TS employed to replace HS in TSeHS medium after 7 days static cultivation. (A) Initial reducing sugar concentration in 50 ml culture medium, DCW and BC concentration; (B) reducing sugar consumed and BC/reducing sugar conversion yield. Data are the means of three independent replicates. Error bars indicate standard deviations.
FIG. 2. Time courses of DCW (closed symbols) and reducing sugar concentration (open symbols) (A), pH (B), BC concentration (C) produced by G. xylinus in 50 ml static cultivation of different TSeHS medium. Each value represents the average value of three independent experiments. Error bars represent standard deviations.
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production to a level slightly higher than that obtained in 50% HS medium which is half strength of traditional BC production medium. When 1:1 volume ratio of TS to HS medium (50/50 TSeHS medium) was employed, the cell growth rate (Fig. 2A) was slower than that of 100% HS medium during first 3 days probably because the amount of easily digestible glucose is about half of 100% HS medium. A second stage with a faster cell growth was observed after the 3rd day and the BC production nearly maintained at a constant rate and no apparent decrease as in 100% HS medium was observed. Evidently, the nutrients in 50% TS medium although cannot sustain a good growth and BC production but can significantly enhance BC production when supplemented to a 50% HS medium. The synergetic effect of TS on enhancing BC production could be clearly observed from the 6.26 g/l BC produced in 50/50 TSeHS medium which is about 3and 6-fold higher than that produced by 50% HS and 50% TS medium, respectively. The significant enhancement of BC production probably resulted from the rich organic acids and amino acids content in TS (Table 1). In 50/50 TSeHS medium, the organic acids and amino acids not only can provide the enough nutrients to support the cell growth of G. xylinus but also has some extent of inhibition effect on converting glucose into gluconic acid probably due to the second rich gluconic acid existed in TS so that the amount of glucose included in 50% HS medium can be efficiently utilized as building blocks for BC biosynthesis. This has been confirmed in our previous study (24). Moreover, it is worth mentioning that the richest organic acid in TS is succinic acid (approximately 60%), which is probably helpful to BC production. To verify this possibility, the supplementation of succinic acid in 100% HS and 50% HS medium were performed to culture G. xylinus for producing BC. As shown in Fig. 3, in a 7 days static cultivation, the BC production increased with succinic acid concentration up to 0.4% (w/v) but declined abruptly afterward in 100% HS medium as well as 50% HS medium. The highest amounts of BC were 6.17 and 4.18 (g/l), respectively. This indicates that succinic acid is a very effective supplement for BC production. Although the concentration of 0.1% (w/v) succinic acid is not high, which is around the same level of succinic acid in 50/50 TSeHS medium, its BC production reached a comparable
FIG. 3. Effect of succinic acid concentrations on BC production by G. xylinus in 50 ml 100% HS and 50% HS medium after 7 days static cultivation. Data are the means of three independent replicates. Error bars indicate standard deviations.
J. BIOSCI. BIOENG., level to that in higher succinic acid concentrations supplemented to 50% HS medium. While 50% TS medium supplemented to 50% HS medium (50/50 TSeHS medium) produced 6.26 g/l BC yield. This reveals that succinic acid in TS can greatly enhance BC production in 50/50 TSeHS medium. The significant effect of succinic acid on BC production enhancement has been reported previously (17,25,33,34). In addition to succinic acid, other organic acids in TS such as gluconic acid, acetic acid, citric acid and malic acid are also beneficial for BC production enhancement (17,25,33). These organic acids are all located in the pathway of TCA cycle. Effect of TS on the structural characteristics of BC The effect of TS on the morphology of BC pellicle produced was also studied. As shown in Fig. 4A, light brownish pellicle was obtained from the culture medium after thoroughly washing with DI water. Probably due to the original darker color of TS wastewater, the color intensities of pellicles produced from TS containing medium were slightly higher than that produced from HS medium. The pellicle obtained in 50/50 TSeHS medium was obviously thicker than that produced in other medium. After the pellicles were treated in 1 N NaOH to remove the biomass of G. xylinus, purified white BC pellicles were obtained (Fig. 4B). All the purified BC pellicles showed the reticulated structure consisting of ultrafine cellulose nanofibrils and no significant difference in the size of cellulose fibrils as illustrated in the scanning electron microscope (SEM) images (Fig. 5). TS did not significantly change the major ultrafine nanofibril structure, however, the BC nanofibrils produced by TS containing medium resulted in a slightly denser reticulated structure than that
FIG. 4. Digital images of BC pellicles produced from various 50 ml of TSeHS medium after 7 days static cultivation before (A), and after (B) NaOH treatment to remove the biomass of G. xylinus.
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FIG. 5. SEM images of purified BC pellicles produced from various 50 ml of TSeHS medium after 7 days static cultivation. (A) 100% HS, (B) 50% HS, (C) 50/50 TSeHS, (D) 50% TS, and (E) 100% TS medium.
produced from HS medium. The morphological changes in BC can influence various properties and microstructures such as crystallinity. In order to evaluate the crystalline structure and the change in crystallinity of BC produced by TS containing medium, X-ray diffraction was used. The diffraction diagrams of BC produced by all TS containing medium showed three main peaks at 2q 14.4 , 16.7 and 22.8 (Fig. 6CeE), corresponding to the crystallographic planes of (1 0 1), (101) and (0 0 2), respectively (35). These peaks demonstrate that BC possesses the typical crystalline forms of cellulose I (36). The diffraction angles 2q of the peaks for BC obtained in HS medium (Fig. 6A and B) were the same as those in TS containing medium, suggesting that TS did not alter the crystalline structure of BC. The crystallinity index values of BC are listed in Table 2. The crystallinity index values of BC from TS containing medium (approximately 83%) were slightly higher than that of BC from HS medium (approximately 79%).
This reveals that TS can affect the aggregation and crystallization of BC microfibrils and increase the crystallinity index. The water holding capacity (WHC) represents the weight of water held per unit weight of cellulose nanofibrils forming a reticulated structure. The BC pellicles exhibited high retention of water (Table 2) due to the BC composed of ultrafine nanofibrils in ultrafine network as illustrated in Fig. 5. However, the WHC of BC produced by all TS containing medium were approximately 20% lower than that of BC obtained from HS medium. The water molecules are caught physically in the reticulated nanofibrils of BC (37). When the BC nanofibrils are loosely arranged, resulting in more vacant space to trap more water molecules and thus the WHC is high. The BC obtained from TS containing medium showed a more crowded cellulose network with less free space to trap water than that from HS medium (Fig. 5). This could explain the decreased WHC of BC from TS containing medium (29).
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J. BIOSCI. BIOENG., TABLE 3. Cost factor of BC production based on HS medium according to different volume fraction of TS in TSeHS medium after 7 days static cultivation at 30 C. Volume fraction of TS in TSeHS (%) (f) 0 12.5 25.0 37.5 50.0 62.5 75.0 87.5 100
FIG. 6. XRD patterns of purified BC pellicles produced from various 50 ml of TSeHS medium after 7 days static cultivation. (A) 100% HS, (B) 50% HS, (C) 50/50 TSeHS, (D) 50% TS, and (E) 100% TS medium.
Cost effectiveness for BC production The BC production cost-down based on a cheaper medium by replacing a fraction of HS medium with cost-free TS is shown in Table 3. The BC production based on a 7 days static cultivation was enhanced with the increase of TS in TSeHS medium up to 50%. A 1.5-fold enhancement (relative to 100% HS medium) was achieved when 50% of HS medium was replaced by TS in a 50/50 TSeHS medium. In other words, the nutrient cost not only can be reduced to half but also with a 50% enhancement of BC production. In order to evaluate the effectiveness of nutrient cost reduction for BC production in TSeHS medium, a cost reduction factor (h) is defined as the ratio of volume fraction of HS medium in the TSeHS medium to BC production enhancement (Eq. 2).
h ¼
1f E
(2)
where E is the BC concentration enhancement relative to 100% HS medium, and f is the volume fraction of TS in TSeHS medium. The lower the cost reduction factor is, the more effective for reducing the nutrient cost will be to produce a unit weight of BC based on a unit cost of HS medium. For example, the cost of 50/50 TSeHS medium is about 67% lower than 100% HS medium to produce a same amount of BC. The cost factor can be reduced to 0.17 and nil as the amount of HS medium employed in TSeHS medium decreased to 12.5% and 0% (100% TS), respectively. This indicates that the nutrient cost approached to nil when 100% TS was employed to produce 3 g/l of BC after 7 days static cultivation. Although there will be no nutrient cost for employing 100% TS to produce BC, the BC concentration is only about half of that obtained in 50/50 TSeHS medium. Taking BC productivity into account in a 7 days static culture, 50/50 TSeHS medium will be the ultimate choice. In our previous study, when TS was employed to replace distilled water for preparing HS medium (100/100 TSeHS TABLE 2. Crystallinity index and water holding capacity of BC produced from various medium after 7 days static cultivation. 100% HS
50% HS
Crystallinity index (%) 79.5 78.7 98.5 4.2 103.1 4.8 Water holding capacity (g water/g BC)
50/50 TSeHS 82.4 83.0 3.6
50% TS
100% TS
83.5 83.9 80.8 2.9 79.1 3.3
BC concentration (g/l)
BC enhancement fold (E)
Cost reduction factor (h)
1.00 1.16 1.33 1.40 1.50 0.92 0.77 0.76 0.74
1.00 0.76 0.56 0.45 0.33 0.41 0.32 0.17 0
4.17 4.82 5.54 5.85 6.26 3.85 3.21 3.15 3.08
0.29 0.22 0.25 0.17 0.23 0.16 0.20 0.14 0.16
medium), BC production in this 100/100 TSeHS medium was enhanced 2.5-fold to a concentration of 10.38 g/l after 7 days static cultivation (24). In this study, however, TS was used to replace the costly HS medium for BC production. In the 50/50 TSeHS medium, BC concentration of 6.26 g/l can be achieved after 7 days static cultivation. This indicates that when the 100/100 TSeHS medium is diluted to 50% by distilled water to become two volumes of the 50/ 50 TSeHS medium, 12.5 g/l of BC could be obtained after 7 days static cultivation which is higher than that produced by 100/100 TSeHS medium. Therefore, the utilization of TS in this work is a good strategy for BC production by G. xylinus. TS from rice winery industry can be used as a very effective feedstock to support the growth of G. xylinus for BC production. Up to 50% traditional BC producing HS medium was replaced with the cost-free TS, the amount of BC produced could be enhanced 50% as compared with that of HS medium. The presence of TS in excess of 50% in TSeHS medium significantly reduced the reducing sugar consumption by G. xylinus that leads to a reduced BC production. The BC production enhancement along with reduced feedstock cost achieved in 50/50 TSeHS medium resulted in a 67% feedstock cost reduction for BC production. The BC produced by TS containing medium had slightly denser reticulated structures and higher crystallinity index values but with lower WHC than that obtained from HS medium. The employment of cost-free TS to replace a portion of HS medium to achieve a higher BC concentration not only can reduce the BC production cost but also solve the wastewater disposal problem of winery industry. ACKNOWLEDGMENTS This research study was financially supported by the Chinese Culture University and the National Science Council of Taiwan (NSC 99-2621-M-034-002). References 1. Petersen, N. and Gatenholm, P.: Bacterial cellulose-based materials and medical devices: current state and perspectives, Appl. Microbiol. Biotechnol., 91, 1277e1286 (2011). 2. Klemm, D., Kramer, F., Moritz, S., Lindström, T., Ankerfors, M., Gray, D., and Dorris, A.: Nanocelluloses: a new family of nature-based materials, Angew. Chem. Int. Ed., 50, 5438e5466 (2011). 3. Czaja, W. K., Young, D. J., Kawecki, M., and Brown, R. M., Jr.: The future prospects of microbial cellulose in biomedical applications, Biomacromolecules, 8, 1e12 (2007). 4. Mikkelsen, D., Flanagan, B. M., Dykes, G. A., and Gidley, M. J.: Influence of different carbon sources on bacterial cellulose production by Gluconacetobacter xylinus strain ATCC 53524, J. Appl. Microbiol., 107, 576e583 (2009). 5. Keshk, S. and Sameshima, K.: Evaluation of different carbon sources for bacterial cellulose production, Afr. J. Biotechnol., 4, 478e482 (2005). 6. Keshk, S. and Sameshima, K.: The utilization of sugar cane molasses with/ without the presence of lignosulfonate for the production of bacterial cellulose, Appl. Microbiol. Biotechnol., 72, 291e296 (2006). 7. Bae, S. and Shoda, M.: Bacterial cellulose production by fed-batch fermentation in molasses medium, Biotechnol. Prog., 20, 1366e1371 (2004).
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VOL. xx, 2012 8. Ishihara, M., Matsunaga, M., Hayashi, N., and Tisler, V.: Utilization of D-xylose as carbon source for production of bacterial cellulose, Enzyme Microb. Technol., 31, 986e991 (2002). 9. Oikawa, T., Morino, T., and Ameyama, M.: Production of cellulose from Darabitol by Acetobacter xylinum KU-1, Biosci. Biotechnol. Biochem., 59, 1564e1565 (1995). 10. Bae, S., Sugano, Y., and Shoda, M.: Improvement of bacterial cellulose production by addition of agar in a jar fermentor, J. Biosci. Bioeng., 97, 33e38 (2004). 11. Hong, F., Zhu, Y. X., Yang, G., and Yang, X. X.: Wheat straw acid hydrolysate as a potential cost-effective feedstock for production of bacterial cellulose, J. Chem. Technol. Biotechnol., 86, 675e680 (2011). 12. Hong, F. and Qiu, K.: An alternative carbon source from konjac powder for enhancing production of bacterial cellulose in static cultures by a model strain Acetobacter aceti subsp. xylinus ATCC 23770, Carbohydr. Polym., 72, 545e549 (2008). 13. Goelzer, F. D. E., Faria-Tischer, P. C. S., Vitorino, J. C., Sierakowski, M. R., and Tischer, C. A.: Production and characterization of nanospheres of bacterial cellulose from Acetobacter xylinum from processed rice bark, Mater. Sci. Eng. C, 29, 546e551 (2009). 14. Kuo, C. H., Lin, P. J., and Lee, C. K.: Enzymatic saccharification of dissolution pretreated waste cellulosic fabrics for bacterial cellulose production by Gluconacetobacter xylinus, J. Chem. Technol. Biotechnol., 85, 1346e1352 (2010). 15. Kongruang, S.: Bacterial cellulose production by Acetobacter xylinum strains from agricultural waste products, Appl. Biochem. Biotechnol., 148, 245e256 (2008). 16. Kurosumi, A., Sasaki, C., Yamashita, Y., and Nakamura, Y.: Utilization of various fruit juices as carbon source for production of bacterial cellulose by Acetobacter xylinum NBRC 13693, Carbohydr. Polym., 76, 333e335 (2009). 17. Jung, H. I., Jeong, J. H., Lee, O. M., Park, G. T., Kim, K. K., Park, H. C., Lee, S. M., Kim, Y. G., and Son, H. J.: Influence of glycerol on production and structuralphysical properties of cellulose from Acetobacter sp. V6 cultured in shake flasks, Bioresour. Technol., 101, 3602e3608 (2010). 18. Hsieh, C., Hsu, T. H., and Yang, F. C.: Production of polysaccharides of Ganoderma lucidum (CCRC36021) by reusing thin stillage, Process Biochem., 40, 909e916 (2005). 19. Yang, F. C. and Lin, I. H.: Production of acid protease using thin stillage from a rice-spirit distillery by Aspergillus niger, Enzyme Microb. Technol., 23, 397e402 (1998). 20. Ahn, J. H., Sang, B. I., and Um, Y.: Butanol production from thin stillage using Clostridium pasteurianum, Bioresour. Technol., 102, 4934e4937 (2011). 21. Gonzalez, R., Campbell, P., and Wong, M.: Production of ethanol from thin stillage by metabolically engineered Escherichia coli, Biotechnol. Lett., 32, 405e411 (2010). 22. Yang, F. C. and Tung, H. L.: Reuse of thin stillage from rice spirit for the culture of the yeast Saccharomyces cerevisiae, Process Biochem., 31, 617e620 (1996). 23. Ha, J. H., Shehzad, O., Khan, S., Lee, S. Y., Park, J. W., Khan, T., and Park, J. K.: Production of bacterial cellulose by a static cultivation using the waste from beer culture broth, Korean J. Chem. Eng., 25, 812e815 (2008).
COST-EFFECTIVE BC PRODUCTION BY DISTILLERY WASTEWATER
7
24. Wu, J. M. and Liu, R. H.: Thin stillage supplementation greatly enhances bacterial cellulose production by Gluconacetobacter xylinus, Carbohydr. Polym., 90, 116e121 (2012). 25. Dudman, W. F.: Cellulose production by Acetobacter acetigenum in defined medium, J. Gen. Microbiol., 21, 327e337 (1959). 26. Wang, Z. G., Wei, Y. L., Wei, M. S., Wang, X. B., and Xiang, D.: Effect of organic acids on bacterial cellulose production in defined medium by Acetobacter xylinum, Food Sci., 29, 295e297 (2008). 27. Hu, Y. and Catchmark, J. M.: Influence of 1-methylcyclopropene (1-MCP) on the production of bacterial cellulose biosynthesized by Acetobacter xylinum under the agitated culture, Lett. Appl. Microbiol., 51, 109e113 (2010). 28. Segal, L., Creely, J., Martin, A., and Conrad, C.: An empirical method for estimating the degree of crystallinity of native cellulose using the X-ray diffractometer, Text. Res. J., 29, 786e794 (1959). 29. Seifert, M., Hesse, S., Kabrelian, V., and Klemm, D.: Controlling the water content of never dried and reswollen bacterial cellulose by the addition of water-soluble polymers to the culture medium, J. Polym. Sci., 42, 463e470 (2004). 30. Naritomi, T., Kouda, T., Yano, H., and Yoshinaga, F.: Effect of ethanol on bacterial cellulose production from fructose in continuous culture, J. Ferment. Bioeng., 85, 598e603 (1998). 31. Park, J. K., Jung, J. Y., and Park, Y. H.: Cellulose production by Gluconacetobacter hansenii in a medium containing ethanol, Biotechnol. Lett., 25, 2055e2059 (2003). 32. Shigematsu, T., Takamine, K., Kitazato, M., Morita, T., Naritomi, T., Morimura, S., and Kida, K.: Cellulose production from glucose using a glucose dehydrogenase gene (gdh)-deficient mutant of Gluconacetobacter xylinus and its use for bioconversion of sweet potato pulp, J. Biosci. Bioeng., 99, 415e422 (2005). 33. Kim, J. D., Jung, H. I., Jeong, J. H., Park, K. H., Jeon, Y. D., Hwang, D. Y., Lee, C. Y., and Son, H. J.: Production and structural analysis of cellulose by Acetobacter sp. V6 using static culture, Korean J. Microbiol., 45, 275e280 (2009). 34. Benziman, M. and Burger-Rachamimov, H.: Synthesis of cellulose from pyruvate by succinate-grown cells of Acetobacter xylinum, J. Bacteriol., 84, 625e630 (1962). 35. Castro, C., Zuluag, R., Putaux, J., Caro, G., Mondragon, I., and Ganan, P.: Structural characterization of bacterial cellulose produced by Gluconacetobacter swingsii sp. from Colombian agroindustrial wastes, Carbohydr. Polym., 84, 96e102 (2011). 36. Oh, S. Y., Yoo, D. I., Shin, Y., and Kim, H. C.: Crystalline structure analysis of cellulose treated with sodium hydroxide and carbon dioxide by means of X-ray diffraction and FTIR spectroscopy, Carbohydr. Res., 340, 2376e2391 (2005). 37. Watanabe, K., Tabuchi, M., Morinaga, Y., and Yoshinaga, F.: Structural features and properties of bacterial cellulose produced in agitated culture, Cellulose, 5, 187e200 (1998).
Please cite this article in press as: Wu, J.-M., and Liu, R.-H., Cost-effective production of bacterial cellulose in static cultures using distillery wastewater, J. Biosci. Bioeng., (2012), http://dx.doi.org/10.1016/j.jbiosc.2012.09.014