Xanthan Gum Production from Waste Sugar Beet Pulp Seong D

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 Xantho- monas 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 available xanthan gum is food grade. Commercially- available xanthan gum is relatively expensive due to Xanthan gum is a water-soluble hetero-polysaccha- glucose or sucrose being used as the sole carbon source ride that is produced industrially from sucrose or glucose and the very stringent purity standards of the Food and by fermentation using the gram-negative bacterium X. Drug Administration for foods. For food-grade xanthan campestris. The X. campestris cultures produce large gum, up to 50% of the production costs are related to mucoid colonies on agar and highly viscous broths in downstream puri®cation steps, many of which would not culture (Tait et al., 1986). The excellent rheological be necessary for non-food applications. Another cost properties of xanthan gum contribute to its wide-range reduction could be achieved by using less expensive of applications as a suspending, stabilizing, and/or substrates, such as waste agricultural products. thickening agent in the food industry and its use as an Several researchers have investigated using less ex- emulsi®er, lubricant, thickening agent, and/or mobility- pensive carbon sources to produce xanthan gum (Ro- control agent to enhance oil recovery (Margaritis and seiro et al., 1992; Bilanovic et al., 1994; Green et al., Pace, 1985; Katzbauer, 1998). Currently, the worldwide 1994; Jana and Ghosh, 1995; Yang and Silva, 1995; consumption of xanthan gum is approximately 23 mil- Lopez and Ramos-Cormenzana, 1996). Roseiro et al. lion kg/y, approximately 5 million kg/y are used as a (1992) demonstrated that carob extract could be used to drilling ¯uid viscosi®er in the oil industry (Yang and produce xanthan gum. Lopez and Ramos-Cormenzana Silva, 1995; Katzbauer, 1998). Xanthan gum consump- (1996) used olive-mill wastewaters to produce xanthan tion in the United States has an estimated annual growth gum. Green et al. (1994) and Bilanovic et al. (1994) in- rate between 5 and 10% (Glazer and Nikaido, 1994). The vestigated the use of citrus waste as a low cost substrate petrochemical industry uses other plant-derived poly- for xanthan gum production. By fractionating the citrus saccharides and synthetic polymers instead of xanthan waste into pectin, hemicellulose, and cellulose fractions, gum based on the relative costs of xanthan gum to the the researchers determined that pectin was converted to other polymers (Cottrell and Kang, 1978; Shu and Yang, xanthan gum. The xanthan gum yield from the pectin 1990). In the United States, the only commercially- 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 1 Present address: Michigan State University, Department of Chem- citrus waste did not inhibit the xanthan gum synthesis. ical Engineering, East Lansing, MI 48824. * Corresponding author. Tel.: 001 505 646 4145; fax: 001 505 646 Jana and Ghosh (1995) reported that citric acid could be 7706; e-mail: [email protected] used as both the carbon and energy source for xanthan

0960-8524/99/$ ± see front matter Ó 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 9 9 ) 0 0 0 1 3 - 9 106 S.D. Yoo, S.W. Harcum / Bioresource Technology 70 (1999) 105±109 gum production. In addition to olive-mill, carob, and Shake ¯ask cultures were incubated at 28 or 32°C and at citrus waste, Yang and Silva (1995) and Konicek et al. 250 rpm. The incubation period, de®ned as the time (1993) have suggested using waste whey to produce allowed for cell growth in the production medium prior xanthan gum. to the WSBP addition, varied from 1 to 5 days for dif- The objective of this work was to determine the fea- ferent shake ¯ask cultures. After incubation, 50 g wet sibility of producing a lower cost non-food-grade xan- weight (7.47 g dry weight) of sterile WSBP (obtained than gum alternative from WSBP when used as a from Holly Sugar, Browley, CA) was added to each supplemental substrate for the X. campestris fermenta- shake ¯ask culture. Shake ¯ask cultures were contacted tions. Yang and Silva (1995) reported that the culture with the WSBP at 28 or 32°C and 250 rpm. The contact conditions that optimize growth and xanthan gum duration, de®ned as the time the cells are in contact with production were not identical for cultures grown on the WSBP following incubation, was also varied from glucose. They observed optimal growth in glucose at 1 to 5 days for dierent shake ¯ask cultures. The incu- 28°C and optimal xanthan gum production from glu- bation and contact temperatures were selected based on cose at 32°C. This study focused on the incubation pe- preliminary experiments and the work of Shu and Yang riod prior to the WSBP addition, the contact duration (1990). Dissolved oxygen and pH were not controlled with the WSBP, the incubation temperature, and the during incubation or contact. Following WSBP contact, contact temperature. The incubation and contact pa- the entire shake ¯ask culture was harvested, and the rameters were varied to determine optimal conditions insoluble content and xanthan gum concentration were for WSBP degradation and subsequent xanthan gum determined. production. The quality of the xanthan gum produced was beyond the scope of this preliminary study. 2.5. Analytical methods

The insoluble mass in the culture was determined 2. Methods after washing, ®ltering, and drying the entire shake ¯ask contents. Filter pore size was 5 lm, allowing cells 2.1. Microorganism and very small insolubles to wash through. The ®lter cake was dried for 72 h at 52°C. As a control, auto- X. campestris NRRL B-1459 was obtained from claved WSBP (not contacted with cells) was also wa- Northern Regional Research Laboratory, US Depart- shed, ®ltered, and dried con®rming that WSBP was ment of Agriculture. The bacteria were maintained on insoluble in water and did not contain residual soluble sucrose agar plates (10 g/l K HPO , 2 g/l yeast extract, 2 4 sugars. 0.5 g/l MgSO4 á 7H2O, 35 g/l sucrose, 15 g/l agar). Cultures were transferred at 2-week intervals. Plates 2.6. Xanthan gum quanti®cation were incubated at room temperature (Cadmus and Knutson, 1983). For the sucrose production medium fermentations, the amount of xanthan gum produced was determined 2.2. Media by precipitating the entire fermentation broth with three volumes of 95% ethanol (Cadmus and Knutson, 1983). The production medium was sucrose-based and the The dried mass was the amount of xanthan gum. For same as the sucrose agar except agar was omitted. The the sucrose production medium plus WSBP, the amount inoculum growth medium was YM Broth (5 g/l of xanthan gum produced was determined by precipi- K HPO , 4 g/l yeast extract, 0.5 g/l MgSO á 7H O, 2 g/l 2 4 4 2 tating the entire fermentation broth with three volumes malt extract, 10 g/l glucose). The pH of the media was of 95% ethanol. The precipitate contained xanthan gum adjusted to between 6.5 and 7.5. Tap water was used to and insoluble WSBP. Since xanthan gum is water solu- provide trace minerals (Cadmus and Knutson, 1983). ble, the precipitate was washed with copious amounts of 2.3. Inoculum preparation water to remove the xanthan gum, and dried to determine the non-degraded (insoluble) WSBP mass. 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 3. Results and discussion and 200 rpm. 3.1. Incubation period and contact duration studies 2.4. Waste sugar beet pulp fermentations The mean WSBP degradations (with 95% con®dence Thirteen ml of the inoculum culture was added to intervals) for a variety of incubation periods and contact 200 ml production medium in a 1000 ml shake ¯ask. duration experiments are shown in Fig. 1. These S.D. Yoo, S.W. Harcum / Bioresource Technology 70 (1999) 105±109 107 3.2. Eect of temperature on 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 3- day 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 Fig. 1. Mean WSBP degradation (with 95% con®dence intervals) for considered: incubation at 28°C/contact at 28°C (28/28), the following conditions: autoclaved WSBP (not contacted with cells) incubation at 28°C/contact at 32°C (28/32), incubation was washed, ®ltered, and dried (Control); incubation and contact at 32°C/contact at 28°C (32/28), and incubation at 32°C/ temperatures held constant at 28°C, incubation period varied from 1±5 contact at 32°C (32/32). The fermentations were run in days, and contact duration held constant at 3 days (Incubation at triplicate from three dierent inocula. The mean WSBP 28°C); incubation and contact temperatures held constant at 28°C, incubation period held constant at 4 days, and contact duration varied degradation for the four treatments were statistically from 1±5 days (Contact at 28°C); and incubation and contact tem- dierent (p < 0.0005) based on a Bonferroni (Dunn) t- peratures held constant at 32°C, incubation period held constant at test for multiple comparisons. Using the two-sample t- 4 days, and contact duration varied from 1±5 days (Contact at 32°C). test 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 experiments demonstrate the impact of incubation pe- the fermentations incubated at 32°C (32/28 vs. 32/32) riod and contact duration on WSBP degradation. As were not statistically dierent. This indicates that for the described earlier, autoclaved WSBP (not contacted with contact temperatures evaluated, WSBP degradation was cells) was washed, ®ltered, and dried con®rming that not aected. However, the incubation temperature does WSBP was insoluble and no degradation occurred result in a statistically dierent WSBP degradation. (Control). The conditions for the ®rst treatment were Therefore incubation temperature is an important pa- incubation and contact temperatures held constant at rameter in¯uencing WSBP degradation. This further 28°C, incubation period varied from 1 to 5 days, and supports the hypothesis that optimal cell growth might contact duration held constant at 3 days (Incubation at optimize WSBP degradation. 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 corre- sponds to Shu and Yang's observation (Shu and Yang, 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 1990) of optimal cell growth at 28°C, which supports the temperature conditions: incubation at 28°C/contact at 28°C (28/28), hypothesis that optimal cell growth might optimize incubation at 28°C/contact at 32°C (28/32), incubation at 32°C/contact WSBP degradation. at 28°C (32/28), and incubation at 32°C/contact at 32°C (32/32). 108 S.D. Yoo, S.W. Harcum / Bioresource Technology 70 (1999) 105±109

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

a Incubation/Contact XG Production from (g) Delta XG from WSBP WSBP Degraded (g) YieldXG=S YieldXG=WSBP Temperature (°C) Sucrose Sucrose + WSBP 28/28 3.49 5.95 2.46 3.20 0.50 0.77 28/32 3.45 5.51 2.06 3.06 0.49 0.67 32/28 2.12 3.82 1.70 1.91 0.30 0.89 32/32 2.04 4.05 2.01 2.29 0.29 0.88 Mean 2.78 4.83 aSucrose 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 sucrose, using an 80% yield of xanthan gum from sucrose (Pinches and Pallent, 1986). WSBP has a cost Xanthan gum production experiments, using the advantage, based only on the substrate-associated costs temperature pro®les described above, were conducted of at least $633 per ton xanthan gum produced. The use without replication using the sucrose production medi- of supplemental WSBP to produce xanthan gum has the um and the sucrose production medium plus WSBP potential to be less expensive than the standard fer- (WSBP added at the beginning of the contact phase). mentations where sucrose or glucose are used as the sole The WSBP degradation and xanthan gum production carbon and energy source. Further studies are needed to for these cultures are summarized in Table 1. The investigate the quality of the xanthan gum produced amount of xanthan gum produced in excess of the su- from WSBP and issues related to the bulk handling of crose fermentation was determined by comparing the the WSBP. amount of xanthan gum produced by the sucrose fermentations to that of the sucrose production medium plus WSBP fermentations. For the range of conditions Acknowledgements evaluated, the sucrose production medium plus WSBP fermentations resulted in xanthan gum production that The authors would like to acknowledge Dr. John was signi®cantly (p < 0.001) greater than the sucrose Patton and Mr. Richard L. Clampitt for ®nancial production medium using a two-sided paired t-test. assistance to support SDY. The authors thank Table 1 also summarizes the yield of xanthan gum from Mr. Richard L. Clampitt for transporting the WSBP the degraded WSBP on a gram per gram basis. The from California to New Mexico. Additionally, the au- xanthan gum yield from the degraded WSBP ranged thors thank Dr. Dennis L. Clason for the statistical from 67% to 89%. The observed xanthan gum yield from analysis of the data. sucrose ranged from 29% to 50% based on the sucrose production medium fermentations. The yield of xanthan References gum from the total amount WSBP added to the fermentations ranged from 22% to 33% with the maximum Amanullah, A., Satti, S., Nienow, A.W., 1998. Enhancing xanthan observed for the 28/28°C and 28/32°C cultures. Re- fermentations by dierent modes of glucose feeding. Biotech. Prog. ported yields of xanthan gum from glucose and sucrose 14, 265±269. are between 27% and 86% (Pinches and Pallent, 1986; Bilanovic, D., Shelef, G., Green, M., 1994. Xanthan fermentation of Amanullah et al., 1998). The observed yields of xanthan citrus waste. Bioresource Tech. 48, 169±172. Cadmus, M. C., Knutson, C.A. (1983). Production of high-pyruvate gum from WSBP in this study are similar to the reported xanthan gum on synthetic medium. US Patent 4, 394, 447. values obtained for glucose and sucrose. Cottrell, W.I., Kang, S.K., 1978. Xanthan gum, a unique bacterial Although a complete economic analysis was not the polysaccharide for food application. Dev. Ind. Microbiol. 19, 177. purpose of this study, WSBP degradation to produce Green, M., Shelef, G., Bilanovic, D., 1994. The eect of various citrus xanthan gum does appear to be economical. Sucrose on waste fractions on xanthan fermentation. Chem. Eng. J. 56, B37± B41. the commodity market costs approximately $720 per ton Glazer, A.N., Nikaido, H., (1994). Microbial polysaccharides and compared to $80 per ton for dried WSBP (sold as cattle polyesters. In: Microbial Biotechnol. W.H. Freeman and Compa- feed). The overall yield from WSBP was observed to be ny, New York. 266±282. 33% (WSBP derived xanthan gum in excess of parallel Katzbauer, B., 1998. Properties and applications of xanthan gum. sucrose culture for the 28/28°C fermentation). The Poly. Degrad. Stabil. 59, 81±84. Konicek, J., Lasik, J., Safar, H., 1993. Xanthan gum produced from substrate-associated costs for xanthan gum from WSBP whey by a mutant of Xanthomonas campestris. Folia Microbiolog- are $267 per ton and $900 per ton xanthan gum from ica 38, 403±405. S.D. Yoo, S.W. Harcum / Bioresource Technology 70 (1999) 105±109 109

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