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Xanthan gum production from waste sugar beet pulp
Bioresource Technology 70 (1999) 105±109
Short communication
Xanthan gum production from waste sugar beet pulp Seong D. Yoo1, Sarah W. Harcum* Department of Chemical Engineering, New Mexico State University, P.O. Box 30001, MSC 3805, Las Cruces, NM 88003, USA Received 17 April 1998; revised 6 January 1999; accepted 31 January 1999
Abstract The feasibility of using waste sugar beet pulp (WSBP) as a supplemental substrate for xanthan gum production from Xanthomonas campestris was investigated. For the range of incubation periods and contact times investigated (1 to 5 days), there were no dierences in the mean WSBP degradation. The mean WSBP degradation was signi®cantly greater for incubation temperatures of 28°C as compared to incubation temperatures of 32°C. WSBP degradation was insensitive to the contact temperatures evaluated. These results indicate that optimal cell growth might optimize WSBP degradation. Xanthan gum production from the WSBP supplemented cultures was signi®cantly greater than the unsupplemented production medium. Based on a preliminary analysis, the use of WSBP for xanthan gum production has the potential to be a cost-eective supplemental substrate to produce non-food grade xanthan gum. Ó 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Xanthan gum is a water-soluble hetero-polysaccharide that is produced industrially from sucrose or glucose by fermentation using the gram-negative bacterium X. campestris. The X. campestris cultures produce large mucoid colonies on agar and highly viscous broths in culture (Tait et al., 1986). The excellent rheological properties of xanthan gum contribute to its wide-range of applications as a suspending, stabilizing, and/or thickening agent in the food industry and its use as an emulsi®er, lubricant, thickening agent, and/or mobilitycontrol agent to enhance oil recovery (Margaritis and Pace, 1985; Katzbauer, 1998). Currently, the worldwide consumption of xanthan gum is approximately 23 million kg/y, approximately 5 million kg/y are used as a drilling ¯uid viscosi®er in the oil industry (Yang and Silva, 1995; Katzbauer, 1998). Xanthan gum consumption in the United States has an estimated annual growth rate between 5 and 10% (Glazer and Nikaido, 1994). The petrochemical industry uses other plant-derived polysaccharides and synthetic polymers instead of xanthan gum based on the relative costs of xanthan gum to the other polymers (Cottrell and Kang, 1978; Shu and Yang, 1990). In the United States, the only commercially1 Present address: Michigan State University, Department of Chemical Engineering, East Lansing, MI 48824. * Corresponding author. Tel.: 001 505 646 4145; fax: 001 505 646 7706; e-mail: [email protected]
available xanthan gum is food grade. Commerciallyavailable xanthan gum is relatively expensive due to glucose or sucrose being used as the sole carbon source and the very stringent purity standards of the Food and Drug Administration for foods. For food-grade xanthan gum, up to 50% of the production costs are related to downstream puri®cation steps, many of which would not be necessary for non-food applications. Another cost reduction could be achieved by using less expensive substrates, such as waste agricultural products. Several researchers have investigated using less expensive carbon sources to produce xanthan gum (Roseiro et al., 1992; Bilanovic et al., 1994; Green et al., 1994; Jana and Ghosh, 1995; Yang and Silva, 1995; Lopez and Ramos-Cormenzana, 1996). Roseiro et al. (1992) demonstrated that carob extract could be used to produce xanthan gum. Lopez and Ramos-Cormenzana (1996) used olive-mill wastewaters to produce xanthan gum. Green et al. (1994) and Bilanovic et al. (1994) investigated the use of citrus waste as a low cost substrate for xanthan gum production. By fractionating the citrus waste into pectin, hemicellulose, and cellulose fractions, the researchers determined that pectin was converted to xanthan gum. The xanthan gum yield from the pectin fraction was similar to that of the whole citrus waste. They concluded that the pectin was the carbon and energy source for xanthan gum production and the whole citrus waste did not inhibit the xanthan gum synthesis. Jana and Ghosh (1995) reported that citric acid could be used as both the carbon and energy source for xanthan
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gum production. In addition to olive-mill, carob, and citrus waste, Yang and Silva (1995) and Konicek et al. (1993) have suggested using waste whey to produce xanthan gum. The objective of this work was to determine the feasibility of producing a lower cost non-food-grade xanthan gum alternative from WSBP when used as a supplemental substrate for the X. campestris fermentations. Yang and Silva (1995) reported that the culture conditions that optimize growth and xanthan gum production were not identical for cultures grown on glucose. They observed optimal growth in glucose at 28°C and optimal xanthan gum production from glucose at 32°C. This study focused on the incubation period prior to the WSBP addition, the contact duration with the WSBP, the incubation temperature, and the contact temperature. The incubation and contact parameters were varied to determine optimal conditions for WSBP degradation and subsequent xanthan gum production. The quality of the xanthan gum produced was beyond the scope of this preliminary study. 2. Methods 2.1. Microorganism X. campestris NRRL B-1459 was obtained from Northern Regional Research Laboratory, US Department of Agriculture. The bacteria were maintained on sucrose agar plates (10 g/l K2 HPO4 , 2 g/l yeast extract, 0.5 g/l MgSO4 á 7H2 O, 35 g/l sucrose, 15 g/l agar). Cultures were transferred at 2-week intervals. Plates were incubated at room temperature (Cadmus and Knutson, 1983). 2.2. Media The production medium was sucrose-based and the same as the sucrose agar except agar was omitted. The inoculum growth medium was YM Broth (5 g/l K2 HPO4 , 4 g/l yeast extract, 0.5 g/l MgSO4 á 7H2 O, 2 g/l malt extract, 10 g/l glucose). The pH of the media was adjusted to between 6.5 and 7.5. Tap water was used to provide trace minerals (Cadmus and Knutson, 1983). 2.3. Inoculum preparation One loop of cells grown on agar plates was used to inoculate a 500 ml ¯ask containing 50 ml of liquid YM Broth. The shake ¯asks were incubated for 48 h at 28°C and 200 rpm. 2.4. Waste sugar beet pulp fermentations Thirteen ml of the inoculum culture was added to 200 ml production medium in a 1000 ml shake ¯ask.
Shake ¯ask cultures were incubated at 28 or 32°C and at 250 rpm. The incubation period, de®ned as the time allowed for cell growth in the production medium prior to the WSBP addition, varied from 1 to 5 days for different shake ¯ask cultures. After incubation, 50 g wet weight (7.47 g dry weight) of sterile WSBP (obtained from Holly Sugar, Browley, CA) was added to each shake ¯ask culture. Shake ¯ask cultures were contacted with the WSBP at 28 or 32°C and 250 rpm. The contact duration, de®ned as the time the cells are in contact with the WSBP following incubation, was also varied from 1 to 5 days for dierent shake ¯ask cultures. The incubation and contact temperatures were selected based on preliminary experiments and the work of Shu and Yang (1990). Dissolved oxygen and pH were not controlled during incubation or contact. Following WSBP contact, the entire shake ¯ask culture was harvested, and the insoluble content and xanthan gum concentration were determined. 2.5. Analytical methods The insoluble mass in the culture was determined after washing, ®ltering, and drying the entire shake ¯ask contents. Filter pore size was 5 lm, allowing cells and very small insolubles to wash through. The ®lter cake was dried for 72 h at 52°C. As a control, autoclaved WSBP (not contacted with cells) was also washed, ®ltered, and dried con®rming that WSBP was insoluble in water and did not contain residual soluble sugars. 2.6. Xanthan gum quanti®cation For the sucrose production medium fermentations, the amount of xanthan gum produced was determined by precipitating the entire fermentation broth with three volumes of 95% ethanol (Cadmus and Knutson, 1983). The dried mass was the amount of xanthan gum. For the sucrose production medium plus WSBP, the amount of xanthan gum produced was determined by precipitating the entire fermentation broth with three volumes of 95% ethanol. The precipitate contained xanthan gum and insoluble WSBP. Since xanthan gum is water soluble, the precipitate was washed with copious amounts of water to remove the xanthan gum, and dried to determine the non-degraded (insoluble) WSBP mass. 3. Results and discussion 3.1. Incubation period and contact duration studies The mean WSBP degradations (with 95% con®dence intervals) for a variety of incubation periods and contact duration experiments are shown in Fig. 1. These
S.D. Yoo, S.W. Harcum / Bioresource Technology 70 (1999) 105±109
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3.2. Eect of temperature on WSBP degradation
Fig. 1. Mean WSBP degradation (with 95% con®dence intervals) for the following conditions: autoclaved WSBP (not contacted with cells) was washed, ®ltered, and dried (Control); incubation and contact temperatures held constant at 28°C, incubation period varied from 1±5 days, and contact duration held constant at 3 days (Incubation at 28°C); incubation and contact temperatures held constant at 28°C, incubation period held constant at 4 days, and contact duration varied from 1±5 days (Contact at 28°C); and incubation and contact temperatures held constant at 32°C, incubation period held constant at 4 days, and contact duration varied from 1±5 days (Contact at 32°C).
experiments demonstrate the impact of incubation period and contact duration on WSBP degradation. As described earlier, autoclaved WSBP (not contacted with cells) was washed, ®ltered, and dried con®rming that WSBP was insoluble and no degradation occurred (Control). The conditions for the ®rst treatment were incubation and contact temperatures held constant at 28°C, incubation period varied from 1 to 5 days, and contact duration held constant at 3 days (Incubation at 28°C). The conditions for the second treatment were incubation and contact temperatures held constant at 28°C, incubation period held constant at 4 days, and contact duration varied from 1 to 5 days (Contact at 28°C). The conditions for the ®nal treatment were incubation and contact temperatures held constant at 32°C, incubation period held constant at 4 days, and contact duration varied from 1 to 5 days (Contact at 32°C). Using a two-sampled t-test, the mean WSBP degradation from the ®rst two treatments are not statistically dierent. This indicates that for 28°C and the range of incubation periods and contact durations selected, incubation period and contact duration do not impact WSBP degradation. The mean WSBP degradation for the last two treatments are statistically dierent (p < 0.015). This indicates that signi®cantly more WSBP was degraded in cultures maintained at 28°C than in cultures maintained at 32°C. Note that the highest WSBP degradation in these experiments corresponds to Shu and Yang's observation (Shu and Yang, 1990) of optimal cell growth at 28°C, which supports the hypothesis that optimal cell growth might optimize WSBP degradation.
Since Shu and Yang (1990) reported optimal xanthan gum production at 32°C, the eect of an incubation period at 28°C for optimal growth, followed by a 32°C contact temperature was examined. Based on the results presented in the previous section demonstrating that WSBP degradation was not sensitive to incubation period and contact duration, these experiments used a 3day incubation period and a 4-day contact duration. The mean WSBP degradation (with 95% con®dence intervals) for selected incubation and contact temperatures are shown in Fig. 2. Four treatments were considered: incubation at 28°C/contact at 28°C (28/28), incubation at 28°C/contact at 32°C (28/32), incubation at 32°C/contact at 28°C (32/28), and incubation at 32°C/ contact at 32°C (32/32). The fermentations were run in triplicate from three dierent inocula. The mean WSBP degradation for the four treatments were statistically dierent (p < 0.0005) based on a Bonferroni (Dunn) ttest for multiple comparisons. Using the two-sample ttest for pairwise comparisons, it was determined that the mean WSBP degradation for fermentations incubated at 28°C (28/28 vs. 28/32) were not statistically dierent and the fermentations incubated at 32°C (32/28 vs. 32/32) were not statistically dierent. This indicates that for the contact temperatures evaluated, WSBP degradation was not aected. However, the incubation temperature does result in a statistically dierent WSBP degradation. Therefore incubation temperature is an important parameter in¯uencing WSBP degradation. This further supports the hypothesis that optimal cell growth might optimize WSBP degradation.
Fig. 2. Mean WSBP degradation (with 95% con®dence intervals) for a 3-day incubation period and a 4-day contact duration for the following temperature conditions: incubation at 28°C/contact at 28°C (28/28), incubation at 28°C/contact at 32°C (28/32), incubation at 32°C/contact at 28°C (32/28), and incubation at 32°C/contact at 32°C (32/32).
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Table 1 A comparison of xanthan gum (XG) production from sucrose production medium (Sucrose) and sucrose production medium plus WSBP (Sucrose + WSBP) fermentations for an incubation period of 3 days and a contact duration of 4 days. XG production from the production medium, the WSBP supplemented medium, and the dierence between the cultures are given. The amount of sucrose in the shake ¯asks was 7.0 g. The amount of WSBP added was 7.47 g dry weight. The xanthan gum yields for the degraded WSBP and sucrose (S) are given on a gram per gram basis Incubation/Contact Temperature (°C)
XG Production from (g)a Sucrose
Sucrose + WSBP
28/28 28/32 32/28 32/32 Mean
3.49 3.45 2.12 2.04 2.78
5.95 5.51 3.82 4.05 4.83
a
Delta XG from WSBP
WSBP Degraded (g)
YieldXG=S
YieldXG=WSBP
2.46 2.06 1.70 2.01
3.20 3.06 1.91 2.29
0.50 0.49 0.30 0.29
0.77 0.67 0.89 0.88
Sucrose plus WSBP XG production was signi®cantly greater (p < 0.001) than sucrose XG production using a two-sided paired t-test.
3.3. Xanthan gum production from supplemental WSBP Xanthan gum production experiments, using the temperature pro®les described above, were conducted without replication using the sucrose production medium and the sucrose production medium plus WSBP (WSBP added at the beginning of the contact phase). The WSBP degradation and xanthan gum production for these cultures are summarized in Table 1. The amount of xanthan gum produced in excess of the sucrose fermentation was determined by comparing the amount of xanthan gum produced by the sucrose fermentations to that of the sucrose production medium plus WSBP fermentations. For the range of conditions evaluated, the sucrose production medium plus WSBP fermentations resulted in xanthan gum production that was signi®cantly (p < 0.001) greater than the sucrose production medium using a two-sided paired t-test. Table 1 also summarizes the yield of xanthan gum from the degraded WSBP on a gram per gram basis. The xanthan gum yield from the degraded WSBP ranged from 67% to 89%. The observed xanthan gum yield from sucrose ranged from 29% to 50% based on the sucrose production medium fermentations. The yield of xanthan gum from the total amount WSBP added to the fermentations ranged from 22% to 33% with the maximum observed for the 28/28°C and 28/32°C cultures. Reported yields of xanthan gum from glucose and sucrose are between 27% and 86% (Pinches and Pallent, 1986; Amanullah et al., 1998). The observed yields of xanthan gum from WSBP in this study are similar to the reported values obtained for glucose and sucrose. Although a complete economic analysis was not the purpose of this study, WSBP degradation to produce xanthan gum does appear to be economical. Sucrose on the commodity market costs approximately $720 per ton compared to $80 per ton for dried WSBP (sold as cattle feed). The overall yield from WSBP was observed to be 33% (WSBP derived xanthan gum in excess of parallel sucrose culture for the 28/28°C fermentation). The substrate-associated costs for xanthan gum from WSBP are $267 per ton and $900 per ton xanthan gum from
sucrose, using an 80% yield of xanthan gum from sucrose (Pinches and Pallent, 1986). WSBP has a cost advantage, based only on the substrate-associated costs of at least $633 per ton xanthan gum produced. The use of supplemental WSBP to produce xanthan gum has the potential to be less expensive than the standard fermentations where sucrose or glucose are used as the sole carbon and energy source. Further studies are needed to investigate the quality of the xanthan gum produced from WSBP and issues related to the bulk handling of the WSBP. Acknowledgements The authors would like to acknowledge Dr. John Patton and Mr. Richard L. Clampitt for ®nancial assistance to support SDY. The authors thank Mr. Richard L. Clampitt for transporting the WSBP from California to New Mexico. Additionally, the authors thank Dr. Dennis L. Clason for the statistical analysis of the data. References Amanullah, A., Satti, S., Nienow, A.W., 1998. Enhancing xanthan fermentations by dierent modes of glucose feeding. Biotech. Prog. 14, 265±269. Bilanovic, D., Shelef, G., Green, M., 1994. Xanthan fermentation of citrus waste. Bioresource Tech. 48, 169±172. Cadmus, M. C., Knutson, C.A. (1983). Production of high-pyruvate xanthan gum on synthetic medium. US Patent 4, 394, 447. Cottrell, W.I., Kang, S.K., 1978. Xanthan gum, a unique bacterial polysaccharide for food application. Dev. Ind. Microbiol. 19, 177. Green, M., Shelef, G., Bilanovic, D., 1994. The eect of various citrus waste fractions on xanthan fermentation. Chem. Eng. J. 56, B37± B41. Glazer, A.N., Nikaido, H., (1994). Microbial polysaccharides and polyesters. In: Microbial Biotechnol. W.H. Freeman and Company, New York. 266±282. Katzbauer, B., 1998. Properties and applications of xanthan gum. Poly. Degrad. Stabil. 59, 81±84. Konicek, J., Lasik, J., Safar, H., 1993. Xanthan gum produced from whey by a mutant of Xanthomonas campestris. Folia Microbiologica 38, 403±405.
S.D. Yoo, S.W. Harcum / Bioresource Technology 70 (1999) 105±109 Jana, A.K., Ghosh, P., 1995. Xanthan biosynthesis in continuous culture: citric acid as an energy source. J. Ferm. Bioeng. 80, 485± 491. Lopez, M.J., Ramos-Cormenzana, A., 1996. Xanthan gum production from olive-mill wastewaters. Inter. Biodeter. Biodegrad. 38, 263± 270. Margaritis, A., Pace, G.W., (1985). Microbial polysaccharides. In: Moo-Young, M., Blanch, W.H., Drew, S., Wang, D.I.C. (Eds.), Comprehensive Biotechnology. Pergamon Press, Toronto, pp. 1005±1043. Pinches, A., Pallent, L.J., 1986. Rate and yield relationships in the production of xanthan gum by batch fermentations using complex and chemically de®ned growth media. Biotechnol. Bioeng. 28, 1484±1496.
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Roseiro, J.C., Costa, D.C., Collaco, M.T.A., 1992. Batch and fedcultivation of Xanthomonas campestris in carob extracts. Food Sci. Tech. 25, 289±293. Shu, C.H., Yang, S.T., 1990. Eect of temperature on cell growth and xanthan production in batch culture of Xanthomonas campestris. Biotechnol. Bioeng. 35, 454±468. Tait, M.I., Sutherland, W.I., Clarke-Sturman, A.J., 1986. Eect of growth conditions on the production composition and viscosity of Xanthomonas campestris exopolysaccharide. J. Gen. Microbiol. 132, 1483±1492. Yang, S.T., Silva, E.M., 1995. Novel products and new technologies for use of a familiar carbohydrate, milk lactose. J. Dairy Sci. 78, 2541±2562.
Short communication
Xanthan gum production from waste sugar beet pulp Seong D. Yoo1, Sarah W. Harcum* Department of Chemical Engineering, New Mexico State University, P.O. Box 30001, MSC 3805, Las Cruces, NM 88003, USA Received 17 April 1998; revised 6 January 1999; accepted 31 January 1999
Abstract The feasibility of using waste sugar beet pulp (WSBP) as a supplemental substrate for xanthan gum production from Xanthomonas campestris was investigated. For the range of incubation periods and contact times investigated (1 to 5 days), there were no dierences in the mean WSBP degradation. The mean WSBP degradation was signi®cantly greater for incubation temperatures of 28°C as compared to incubation temperatures of 32°C. WSBP degradation was insensitive to the contact temperatures evaluated. These results indicate that optimal cell growth might optimize WSBP degradation. Xanthan gum production from the WSBP supplemented cultures was signi®cantly greater than the unsupplemented production medium. Based on a preliminary analysis, the use of WSBP for xanthan gum production has the potential to be a cost-eective supplemental substrate to produce non-food grade xanthan gum. Ó 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Xanthan gum is a water-soluble hetero-polysaccharide that is produced industrially from sucrose or glucose by fermentation using the gram-negative bacterium X. campestris. The X. campestris cultures produce large mucoid colonies on agar and highly viscous broths in culture (Tait et al., 1986). The excellent rheological properties of xanthan gum contribute to its wide-range of applications as a suspending, stabilizing, and/or thickening agent in the food industry and its use as an emulsi®er, lubricant, thickening agent, and/or mobilitycontrol agent to enhance oil recovery (Margaritis and Pace, 1985; Katzbauer, 1998). Currently, the worldwide consumption of xanthan gum is approximately 23 million kg/y, approximately 5 million kg/y are used as a drilling ¯uid viscosi®er in the oil industry (Yang and Silva, 1995; Katzbauer, 1998). Xanthan gum consumption in the United States has an estimated annual growth rate between 5 and 10% (Glazer and Nikaido, 1994). The petrochemical industry uses other plant-derived polysaccharides and synthetic polymers instead of xanthan gum based on the relative costs of xanthan gum to the other polymers (Cottrell and Kang, 1978; Shu and Yang, 1990). In the United States, the only commercially1 Present address: Michigan State University, Department of Chemical Engineering, East Lansing, MI 48824. * Corresponding author. Tel.: 001 505 646 4145; fax: 001 505 646 7706; e-mail: [email protected]
available xanthan gum is food grade. Commerciallyavailable xanthan gum is relatively expensive due to glucose or sucrose being used as the sole carbon source and the very stringent purity standards of the Food and Drug Administration for foods. For food-grade xanthan gum, up to 50% of the production costs are related to downstream puri®cation steps, many of which would not be necessary for non-food applications. Another cost reduction could be achieved by using less expensive substrates, such as waste agricultural products. Several researchers have investigated using less expensive carbon sources to produce xanthan gum (Roseiro et al., 1992; Bilanovic et al., 1994; Green et al., 1994; Jana and Ghosh, 1995; Yang and Silva, 1995; Lopez and Ramos-Cormenzana, 1996). Roseiro et al. (1992) demonstrated that carob extract could be used to produce xanthan gum. Lopez and Ramos-Cormenzana (1996) used olive-mill wastewaters to produce xanthan gum. Green et al. (1994) and Bilanovic et al. (1994) investigated the use of citrus waste as a low cost substrate for xanthan gum production. By fractionating the citrus waste into pectin, hemicellulose, and cellulose fractions, the researchers determined that pectin was converted to xanthan gum. The xanthan gum yield from the pectin fraction was similar to that of the whole citrus waste. They concluded that the pectin was the carbon and energy source for xanthan gum production and the whole citrus waste did not inhibit the xanthan gum synthesis. Jana and Ghosh (1995) reported that citric acid could be used as both the carbon and energy source for xanthan
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gum production. In addition to olive-mill, carob, and citrus waste, Yang and Silva (1995) and Konicek et al. (1993) have suggested using waste whey to produce xanthan gum. The objective of this work was to determine the feasibility of producing a lower cost non-food-grade xanthan gum alternative from WSBP when used as a supplemental substrate for the X. campestris fermentations. Yang and Silva (1995) reported that the culture conditions that optimize growth and xanthan gum production were not identical for cultures grown on glucose. They observed optimal growth in glucose at 28°C and optimal xanthan gum production from glucose at 32°C. This study focused on the incubation period prior to the WSBP addition, the contact duration with the WSBP, the incubation temperature, and the contact temperature. The incubation and contact parameters were varied to determine optimal conditions for WSBP degradation and subsequent xanthan gum production. The quality of the xanthan gum produced was beyond the scope of this preliminary study. 2. Methods 2.1. Microorganism X. campestris NRRL B-1459 was obtained from Northern Regional Research Laboratory, US Department of Agriculture. The bacteria were maintained on sucrose agar plates (10 g/l K2 HPO4 , 2 g/l yeast extract, 0.5 g/l MgSO4 á 7H2 O, 35 g/l sucrose, 15 g/l agar). Cultures were transferred at 2-week intervals. Plates were incubated at room temperature (Cadmus and Knutson, 1983). 2.2. Media The production medium was sucrose-based and the same as the sucrose agar except agar was omitted. The inoculum growth medium was YM Broth (5 g/l K2 HPO4 , 4 g/l yeast extract, 0.5 g/l MgSO4 á 7H2 O, 2 g/l malt extract, 10 g/l glucose). The pH of the media was adjusted to between 6.5 and 7.5. Tap water was used to provide trace minerals (Cadmus and Knutson, 1983). 2.3. Inoculum preparation One loop of cells grown on agar plates was used to inoculate a 500 ml ¯ask containing 50 ml of liquid YM Broth. The shake ¯asks were incubated for 48 h at 28°C and 200 rpm. 2.4. Waste sugar beet pulp fermentations Thirteen ml of the inoculum culture was added to 200 ml production medium in a 1000 ml shake ¯ask.
Shake ¯ask cultures were incubated at 28 or 32°C and at 250 rpm. The incubation period, de®ned as the time allowed for cell growth in the production medium prior to the WSBP addition, varied from 1 to 5 days for different shake ¯ask cultures. After incubation, 50 g wet weight (7.47 g dry weight) of sterile WSBP (obtained from Holly Sugar, Browley, CA) was added to each shake ¯ask culture. Shake ¯ask cultures were contacted with the WSBP at 28 or 32°C and 250 rpm. The contact duration, de®ned as the time the cells are in contact with the WSBP following incubation, was also varied from 1 to 5 days for dierent shake ¯ask cultures. The incubation and contact temperatures were selected based on preliminary experiments and the work of Shu and Yang (1990). Dissolved oxygen and pH were not controlled during incubation or contact. Following WSBP contact, the entire shake ¯ask culture was harvested, and the insoluble content and xanthan gum concentration were determined. 2.5. Analytical methods The insoluble mass in the culture was determined after washing, ®ltering, and drying the entire shake ¯ask contents. Filter pore size was 5 lm, allowing cells and very small insolubles to wash through. The ®lter cake was dried for 72 h at 52°C. As a control, autoclaved WSBP (not contacted with cells) was also washed, ®ltered, and dried con®rming that WSBP was insoluble in water and did not contain residual soluble sugars. 2.6. Xanthan gum quanti®cation For the sucrose production medium fermentations, the amount of xanthan gum produced was determined by precipitating the entire fermentation broth with three volumes of 95% ethanol (Cadmus and Knutson, 1983). The dried mass was the amount of xanthan gum. For the sucrose production medium plus WSBP, the amount of xanthan gum produced was determined by precipitating the entire fermentation broth with three volumes of 95% ethanol. The precipitate contained xanthan gum and insoluble WSBP. Since xanthan gum is water soluble, the precipitate was washed with copious amounts of water to remove the xanthan gum, and dried to determine the non-degraded (insoluble) WSBP mass. 3. Results and discussion 3.1. Incubation period and contact duration studies The mean WSBP degradations (with 95% con®dence intervals) for a variety of incubation periods and contact duration experiments are shown in Fig. 1. These
S.D. Yoo, S.W. Harcum / Bioresource Technology 70 (1999) 105±109
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3.2. Eect of temperature on WSBP degradation
Fig. 1. Mean WSBP degradation (with 95% con®dence intervals) for the following conditions: autoclaved WSBP (not contacted with cells) was washed, ®ltered, and dried (Control); incubation and contact temperatures held constant at 28°C, incubation period varied from 1±5 days, and contact duration held constant at 3 days (Incubation at 28°C); incubation and contact temperatures held constant at 28°C, incubation period held constant at 4 days, and contact duration varied from 1±5 days (Contact at 28°C); and incubation and contact temperatures held constant at 32°C, incubation period held constant at 4 days, and contact duration varied from 1±5 days (Contact at 32°C).
experiments demonstrate the impact of incubation period and contact duration on WSBP degradation. As described earlier, autoclaved WSBP (not contacted with cells) was washed, ®ltered, and dried con®rming that WSBP was insoluble and no degradation occurred (Control). The conditions for the ®rst treatment were incubation and contact temperatures held constant at 28°C, incubation period varied from 1 to 5 days, and contact duration held constant at 3 days (Incubation at 28°C). The conditions for the second treatment were incubation and contact temperatures held constant at 28°C, incubation period held constant at 4 days, and contact duration varied from 1 to 5 days (Contact at 28°C). The conditions for the ®nal treatment were incubation and contact temperatures held constant at 32°C, incubation period held constant at 4 days, and contact duration varied from 1 to 5 days (Contact at 32°C). Using a two-sampled t-test, the mean WSBP degradation from the ®rst two treatments are not statistically dierent. This indicates that for 28°C and the range of incubation periods and contact durations selected, incubation period and contact duration do not impact WSBP degradation. The mean WSBP degradation for the last two treatments are statistically dierent (p < 0.015). This indicates that signi®cantly more WSBP was degraded in cultures maintained at 28°C than in cultures maintained at 32°C. Note that the highest WSBP degradation in these experiments corresponds to Shu and Yang's observation (Shu and Yang, 1990) of optimal cell growth at 28°C, which supports the hypothesis that optimal cell growth might optimize WSBP degradation.
Since Shu and Yang (1990) reported optimal xanthan gum production at 32°C, the eect of an incubation period at 28°C for optimal growth, followed by a 32°C contact temperature was examined. Based on the results presented in the previous section demonstrating that WSBP degradation was not sensitive to incubation period and contact duration, these experiments used a 3day incubation period and a 4-day contact duration. The mean WSBP degradation (with 95% con®dence intervals) for selected incubation and contact temperatures are shown in Fig. 2. Four treatments were considered: incubation at 28°C/contact at 28°C (28/28), incubation at 28°C/contact at 32°C (28/32), incubation at 32°C/contact at 28°C (32/28), and incubation at 32°C/ contact at 32°C (32/32). The fermentations were run in triplicate from three dierent inocula. The mean WSBP degradation for the four treatments were statistically dierent (p < 0.0005) based on a Bonferroni (Dunn) ttest for multiple comparisons. Using the two-sample ttest for pairwise comparisons, it was determined that the mean WSBP degradation for fermentations incubated at 28°C (28/28 vs. 28/32) were not statistically dierent and the fermentations incubated at 32°C (32/28 vs. 32/32) were not statistically dierent. This indicates that for the contact temperatures evaluated, WSBP degradation was not aected. However, the incubation temperature does result in a statistically dierent WSBP degradation. Therefore incubation temperature is an important parameter in¯uencing WSBP degradation. This further supports the hypothesis that optimal cell growth might optimize WSBP degradation.
Fig. 2. Mean WSBP degradation (with 95% con®dence intervals) for a 3-day incubation period and a 4-day contact duration for the following temperature conditions: incubation at 28°C/contact at 28°C (28/28), incubation at 28°C/contact at 32°C (28/32), incubation at 32°C/contact at 28°C (32/28), and incubation at 32°C/contact at 32°C (32/32).
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Table 1 A comparison of xanthan gum (XG) production from sucrose production medium (Sucrose) and sucrose production medium plus WSBP (Sucrose + WSBP) fermentations for an incubation period of 3 days and a contact duration of 4 days. XG production from the production medium, the WSBP supplemented medium, and the dierence between the cultures are given. The amount of sucrose in the shake ¯asks was 7.0 g. The amount of WSBP added was 7.47 g dry weight. The xanthan gum yields for the degraded WSBP and sucrose (S) are given on a gram per gram basis Incubation/Contact Temperature (°C)
XG Production from (g)a Sucrose
Sucrose + WSBP
28/28 28/32 32/28 32/32 Mean
3.49 3.45 2.12 2.04 2.78
5.95 5.51 3.82 4.05 4.83
a
Delta XG from WSBP
WSBP Degraded (g)
YieldXG=S
YieldXG=WSBP
2.46 2.06 1.70 2.01
3.20 3.06 1.91 2.29
0.50 0.49 0.30 0.29
0.77 0.67 0.89 0.88
Sucrose plus WSBP XG production was signi®cantly greater (p < 0.001) than sucrose XG production using a two-sided paired t-test.
3.3. Xanthan gum production from supplemental WSBP Xanthan gum production experiments, using the temperature pro®les described above, were conducted without replication using the sucrose production medium and the sucrose production medium plus WSBP (WSBP added at the beginning of the contact phase). The WSBP degradation and xanthan gum production for these cultures are summarized in Table 1. The amount of xanthan gum produced in excess of the sucrose fermentation was determined by comparing the amount of xanthan gum produced by the sucrose fermentations to that of the sucrose production medium plus WSBP fermentations. For the range of conditions evaluated, the sucrose production medium plus WSBP fermentations resulted in xanthan gum production that was signi®cantly (p < 0.001) greater than the sucrose production medium using a two-sided paired t-test. Table 1 also summarizes the yield of xanthan gum from the degraded WSBP on a gram per gram basis. The xanthan gum yield from the degraded WSBP ranged from 67% to 89%. The observed xanthan gum yield from sucrose ranged from 29% to 50% based on the sucrose production medium fermentations. The yield of xanthan gum from the total amount WSBP added to the fermentations ranged from 22% to 33% with the maximum observed for the 28/28°C and 28/32°C cultures. Reported yields of xanthan gum from glucose and sucrose are between 27% and 86% (Pinches and Pallent, 1986; Amanullah et al., 1998). The observed yields of xanthan gum from WSBP in this study are similar to the reported values obtained for glucose and sucrose. Although a complete economic analysis was not the purpose of this study, WSBP degradation to produce xanthan gum does appear to be economical. Sucrose on the commodity market costs approximately $720 per ton compared to $80 per ton for dried WSBP (sold as cattle feed). The overall yield from WSBP was observed to be 33% (WSBP derived xanthan gum in excess of parallel sucrose culture for the 28/28°C fermentation). The substrate-associated costs for xanthan gum from WSBP are $267 per ton and $900 per ton xanthan gum from
sucrose, using an 80% yield of xanthan gum from sucrose (Pinches and Pallent, 1986). WSBP has a cost advantage, based only on the substrate-associated costs of at least $633 per ton xanthan gum produced. The use of supplemental WSBP to produce xanthan gum has the potential to be less expensive than the standard fermentations where sucrose or glucose are used as the sole carbon and energy source. Further studies are needed to investigate the quality of the xanthan gum produced from WSBP and issues related to the bulk handling of the WSBP. Acknowledgements The authors would like to acknowledge Dr. John Patton and Mr. Richard L. Clampitt for ®nancial assistance to support SDY. The authors thank Mr. Richard L. Clampitt for transporting the WSBP from California to New Mexico. Additionally, the authors thank Dr. Dennis L. Clason for the statistical analysis of the data. References Amanullah, A., Satti, S., Nienow, A.W., 1998. Enhancing xanthan fermentations by dierent modes of glucose feeding. Biotech. Prog. 14, 265±269. Bilanovic, D., Shelef, G., Green, M., 1994. Xanthan fermentation of citrus waste. Bioresource Tech. 48, 169±172. Cadmus, M. C., Knutson, C.A. (1983). Production of high-pyruvate xanthan gum on synthetic medium. US Patent 4, 394, 447. Cottrell, W.I., Kang, S.K., 1978. Xanthan gum, a unique bacterial polysaccharide for food application. Dev. Ind. Microbiol. 19, 177. Green, M., Shelef, G., Bilanovic, D., 1994. The eect of various citrus waste fractions on xanthan fermentation. Chem. Eng. J. 56, B37± B41. Glazer, A.N., Nikaido, H., (1994). Microbial polysaccharides and polyesters. In: Microbial Biotechnol. W.H. Freeman and Company, New York. 266±282. Katzbauer, B., 1998. Properties and applications of xanthan gum. Poly. Degrad. Stabil. 59, 81±84. Konicek, J., Lasik, J., Safar, H., 1993. Xanthan gum produced from whey by a mutant of Xanthomonas campestris. Folia Microbiologica 38, 403±405.
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