Degumming of ramie bers by alkalophilic bacteria and their polysaccharide-degrading enzymes

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1 Bioresource Technology 78 (2001) 89±94 Degumming of ramie bers by alkalophilic bacteria and their polysaccharide-degrading enzymes Lianshuang Zheng, Yumin Du *, Jiayao Zhang College of Chemistry and Environmental Science, Wuhan University, Wuhan , People's Republic of China Received 7 January 2000; received in revised form 8 September 2000; accepted 25 September 2000 Abstract Three strains of alkalophilic bacteria, Bacillus sp. NT-39, NT-53 and NT-76, were selected for the degumming of ramie bers and production of polysaccharide-degrading enzymes. After 48 h of incubation with the strains, the loss of the gum might amount to 5.0% or more of the bers and a number of polysaccharide-degrading enzymes were secreted to the culture supernatants. The residual gum of the bers decreased to 9.4% after 5 h of enzymatic degumming. Analysis of gum contents and enzyme activities revealed that pectate lyase and xylanase played an important role in the degradation of residual gum. Enzymatic degumming resulted in an increment of 5.4 ISO units in ber brightness, whereas the reduction in bundle breaking tenacity of the bers was less than 5.0%. The results con rmed that degumming of ramie bers by alkalophilic bacteria and their enzymes had substantial advantages. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: Degumming; Alkalophilic bacteria; Pectate lyase; Xylanase; Ramie bers 1. Introduction Ramie (Bohesmeria nivea) bers are considered the longest, strongest and silkiest of plant bers and an excellent natural textile material. However, decorticated ramie bers contain 20±35% gum consisting mainly of pectin and hemicellulose. This gum should be removed as fully as possible to ful l the textile requirement (1.5± 2.5% residual gum). As the conventional degumming process with a solution of NaOH not only has high consumption of chemicals and energy but also results in serious environmental pollution, it is imperative to nd out substitutive techniques. The degumming of ramie bers by microorganisms or their enzymes has attracted wide attention, and several reports are available (Gurucharanam and Deshpande, 1986; Nasser et al., 1993; Bruhlman et al., 1994). In our previous study (Cao et al., 1992), several strains of alkalophilic bacteria were selected based on the activity of alkaline pectinase and their abilities to degum the bers were determined. The preliminary results showed that alkalophilic bacteria had potential value in the degumming of ramie bers. * Corresponding author. Tel.: ; fax: address: lszheng@whu.edu.cn (Y. Du). The objective of this work was to select the alkalophilic bacteria capable of utilizing alkali-soluble gum and removing residual gum from ramie bers. The polysaccharide-degrading enzymes related to degumming are also investigated. The e ect of enzymatic degumming on ber properties and advantages of degumming by alkalophilic bacteria are discussed. 2. Methods 2.1. Bacterial strains Eighty strains of alkalophilic bacteria, coded from NT-1 to NT-80, were previously isolated from soil and maintained at 4 C on agar slops. Most of them belong to the genus Bacillus and their alkalophilic characters have been ascertained (Cao et al., 1992) Preparation of alkali-soluble gum from ramie bers 100 g of decorticated ramie bers (purchased from Xingshan, Hubei, China) was extracted with 1 dm 3 solution of 1% Na 2 CO 3 at 100 C for 20 min. The extract was precipitated with acetone, then the precipitate was washed with 95% ethanol and anhydrous ethanol, /01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S ( 0 0 )

2 90 L. Zheng et al. / Bioresource Technology 78 (2001) 89±94 respectively. After ltration, the alkali-soluble gum was dried in a vacuum desiccator Screening of strains utilizing alkali-soluble gum The selective medium containing 30 g of alkali-soluble gum, 1 g of K 2 HPO 4, 3 g of NaNO 3, 18 g of agar in 900 cm 3 of H 2 O was autoclaved at 121 C for 20 min, and adjusted to ph 10.0 with a sterilized solution of 10% Na 2 CO 3 at 60 C before pouring into plates. 80 strains of alkalophilic bacteria were applied to the plates. After incubation at 37 C for 24 h, the strains that grew vigorously were picked and transferred to the plates again, and incubated under the above conditions. The strains forming colonies of >1.0 cm were selected for the degumming test Bacterial degumming Bacterial degumming was conducted as follows: a loop of each selected strain was inoculated into a 250 cm 3 Erlenmeyer ask containing 5 g of the ramie bers pretreated with 50 cm 3 mineral salt solution (K 2 HPO 4, 1 g; NaNO 3, 3 g; Na 2 CO 3,10gdm 3 H 2 O, ph 10.0) at 100 C for 20 min. The asks were incubated at 37 C for 24±48 h with shaking at 180 rpm. The ramie bers were then washed thoroughly with tap water on a 120-mesh screen and dried at 105 C. The degumming liquor could be used as inoculum if necessary. The experiments were performed in triplicate and the means are reported Enzyme assays Bacterial cells and ramie ber debris were removed by centrifugation (10; 000 g), and the supernatant was used as crude enzyme solution. Substrate solutions were all prepared with 0:05 mol dm 3 glycine±naoh bu er (ph 10.0), and enzyme activities were all determined at 40 C. Pectate lyase (EC , PAL) activity was determined spectrophotometrically by measuring the increment in A 235. Crude enzyme solution was mixed with 0.1% sodium polygalacturonate (Sigma) solution (containing 1 mmol dm 3 CaCl 2 ). One unit (U) of activity was de ned as the amount of enzyme that catalyzed the formation of 1 lmol of unsaturated uronide product min 1 with a molar extinction coe cient of 4600 mol 1 cm 1 (Whitaker, 1989). Polygalacturonase (EC , PGase) activity was determined according to the method outlined by Horikoshi (1972). One unit (U) of the enzyme activity was de ned as the amount of enzyme releasing 1 lmol galacturonic acid equivalent min 1. Xylanase (EC ) activity was determined by the DNS-stopping method described by Bailey et al. (1992). Crude enzyme solution was mixed with 0.5% oat spelt xylan (Sigma). One unit (U) of xylanase activity was de ned as the amount of enzyme that produced 1 lmol of xylose equivalent min 1. Cellulase (EC ) activity was detected by measuring the release of reducing sugar from 0.5% carboxymethylcellulose (sodium salt) or cellulose power of nished ramie bers with DNS reagent (Horikoshi et al., 1984). One unit (U) of the enzyme activity was de ned as the amount of enzyme which liberated 1 lmol of glucose equivalent min 1. Enzyme assays were all performed in triplicate and mean values are reported Enzymatic degumming Enzymatic degumming was conducted in 150 cm 3 Erlenmeyer asks with cell-free supernatants (adjusted to ph 10.0 with a solution of Na 2 CO 3 ) from 48 h-old degumming liquors. Each ask contained 50 cm 3 of the supernatant and 5 g of the ramie bers (pretreated with a solution of 1% Na 2 CO 3 at 100 C for 20 min). After incubation at 40 C for 5 h, the bers were washed and dried as described in bacterial degumming Analysis of ber properties The chemical components, brightness and bundle breaking tenacity of ramie bers were separately determined by the standard methods (Cao et al., 1992). The gum loss of the bers was calculated as follows: Gum loss % ˆ G o G r =W Š100; where G o is the original gum weight of ramie bers before degumming, G r the residual gum weight of the - bers after degumming and W is the weight of the bers before degumming. 3. Results 3.1. Growth of strains on selective medium Eighty strains of alkalophilic bacteria were tested for the capability of utilizing alkali-soluble gum. On the selective agar plates, 25 strains showed vigorous growth and formed colonies of >1.0 cm on the plates after incubation of 24 h. Further, 37 strains showed visible growth after incubation for 48 h. It indicated that most of the test strains (77.5%) could utilize the gum as sole organic nutrient. In view of the fact that degumming time may be shortened with the strains growing rapidly, only the 25 strains showing vigorous growth were selected for the degumming test.

3 L. Zheng et al. / Bioresource Technology 78 (2001) 89±94 91 Fig. 1. Gum loss of ramie bers after incubation with the strains. Ramie bers were pretreated with mineral salt solution, then inoculated with a loop of each strain and incubated at 37 C for 48 h. The values given are the means of three replicates standard deviation. The loss of gum (15.5%) by pretreatment and incubation has been subtracted Degumming abilities of strains The abilities of the twenty- ve strains to degum ramie bers were individually investigated by shake ask test. Although the strains all showed vigorous growth on the selective medium, their abilities to degrade residual gum on the bers were considerably di erent (Fig. 1). After 48 h of incubation, the gum loss was 5.0% or more of the bers for strains NT-39, NT-53 and NT-76, but less than 0.5% for strains NT-9, NT-31, NT-59 and NT-76. Degumming abilities of the strains may be also determined by comparing their degumming courses. Although strains NT-39, NT-53 and NT-76 all showed high activity for degumming, the times required to achieve similar degumming levels were di erent (Fig. 2(a)). The gum loss was more than 5.0% of the bers within 28 h of incubation for strain NT-39, however, removing a similar amount of the gum required 48 h for strain NT-76. Furthermore, the degumming abilities of the strains were also related to the inoculum. For each of the three strains, if 10% degumming liquor was retained at the end of degumming and used as the inoculum for the next process, the degumming course might be shortened to less than 24 h, and prolonging incubation could not improve the degumming e ect (Fig. 2(b)) E ect of pretreatment on bacterial degumming In order to know what was the e ect of pretreatment on the bacterial degumming, the untreated bers were incubated with the degumming liquor of strain NT-39. The results showed that a long degumming course (>96 h) was required to achieve a desirable level of residual gum (about 12%). It indicated that the pretreatment of the bers could accelerate the course of bacterial degumming. Fig. 2. (a) Time-dependent degumming of the strains. A loop of each strain was incubated with ramie bers at 37 C with shaking at 180 rpm. The loss of gum (15.5%) by pretreatment and incubation has been subtracted. Strain: : NT-39; M: NT-53; : NT-76. (b) E ect of inoculum on degumming time. A 10% degumming liquor was retained at the end of degumming and used as inoculum. The cultivation conditions and symbols are the same as (a). The raw ramie bers tested contained 33% of the gum, of which about 10% was water soluble. The pretreatment could remove the water solubles and a part of other components of the gum from the bers (Table 1). These soluble gum components (up to 15.3% of the - bers) might be providing for the growth of the strains and the production of polysaccharide-degrading enzymes. Furthermore, the pretreatment might result in the swelling of ramie ber tissues mainly due to higher temperature and alkalinity, therefore, it would increase the accessibility of the residual gum to the enzymes. After the pretreatment, 24 h of incubation with the degumming liquor of strain NT-39 could reduce the residual gum of the bers from 17.7% to 11.9%. This content of residual gum is slightly higher than that of the residual gum achieved by chemical degumming, but lower than that of the residual gum achieved by mixed bacterial degumming (Paul and Bhattacharyya, 1979).

4 92 L. Zheng et al. / Bioresource Technology 78 (2001) 89±94 Table 1 Changes in the gum contents of the bers treated by di erent degumming processes a Sample no Degumming process Water-soluble (%) Pectin (%) Hemicellulose (%) Gum(%) 1 Untreated 9:8 0:2 5:6 0:3 16:8 0:3 33:0 0:3 2 PT b ± 3:5 0:2 14:6 0:3 17:7 0:4 3 PT-BD c ± 1:0 0:2 10:9 0:4 11:9 0:3 4 CD d ± 0:9 0:2 9:1 0:3 9:6 0:3 a Values are means of three replicates standard deviation. b Pretreatment: 10 cm 3 of mineral salt solution/g bers, 100 C, 20 min. c Bacterial degumming: 10% degumming liquor of strain NT-39, 37 C, 24 h. d Chemical degumming: 1.5% NaOH, 135 C, 2 h Enzymes related to degumming Pectate lyase (PAL), polygalacturonase (PGase), xylanase and cellulase in the 48 h-old culture supernatants of four selected strains were detected (Table 2). The activity of PAL could be detected in the supernatants of three strains (NT-39, NT-53, NT-76) with high degumming ability, but was absent in the supernatant of a strain (NT-80) with low degumming ability. PGase could be detected in all the supernatants, and the activities ranged from 0.52 to 1:80 U cm 3. High xylanolytic activity (2:24 U cm 3 ) was observed in the supernatant of strain NT-39, but strain NT-80 exhibited very low xylanolytic activity. Since pectin and xylan are both major components of ramie gum, it could be deduced that the enzymes responsible for breaking down their glycosidic linkages would play a very important role in the ramie degumming though other enzymes may be involved in the process. Four strains, all showed low cellulolytic activities for carboxymethylcellulose but did not show any activity for natural cellulose of ramie - bers. It is suggested that the tenacity of the bers would not be damaged by microbial and enzymatic degumming. When the supernatants were used to degum the ramie bers, a correlation between the activities of polysaccharide-degrading enzymes (Table 2) and the changes in gum contents of the bers (Table 3) could be observed. The results showed that PAL exerted more in uence on the removal of pectin substances from the bers than did PGase, and the xylanase seemed to be responsible for degradation of residual hemicellulose. Otherwise, when the supernatants of strain NT-39 and NT-53 were mixed and used for degumming, the residual gum of the bers could decrease to 9.4%, lower than the amount achieved by using a single supernatant. It is suggested that different polysaccharide-degrading enzymes produced by the two strains had a synergistic action on the degumming of ramie bers E ect of enzymatic degumming on ber properties Enzymatic degumming with the supernatants increased ber brightness and slightly decreased the bun- Table 2 Activities of polysaccharide-degrading enzymes in the supernatants from 48 h-old degumming liquors Strain Enzyme activities (U cm 3 ) Degradation of ramie PAL PGase Xylanase CMCase ber cellulose NT ) a NT ) NT ) NT-80 ) ) a Absence of activity. Table 3 E ect of enzymatic degumming on the gum contents of ramie bers a Sample no. b Supernatant of strain Pectin (%) Hemicellulose (%) Residual gum (%) 1 ) c 3:5 0:1 14:7 0:2 17:6 0:4 2 NT-39 0:8 0:2 10:5 0:3 11:2 0:3 3 NT-53 1:1 0:3 12:3 0:4 12:8 0:2 4 NT-39 NT-53(1:1) 0:9 0:2 8:8 0:3 9:4 0:4 5 NT-76 1:2 0:1 11:0 0:2 12:5 0:3 6 NT-80 3:0 0:2 14:2 0:4 16:6 0:4 a Values are means of three replicates standard deviation. b The ramie bers were pretreated with 1% Na 2 CO 3 solution at 100 C for 20 min, then treated with the supernatants of the strains at 40 C for 5 h. c Blank control was treated with inactivated supernatant (boiling for 10 min) of strain NT-39.

5 L. Zheng et al. / Bioresource Technology 78 (2001) 89±94 93 Table 4 E ect of enzymatic degumming on the physical properties of ramie bers a Sample no. b Supernatant of strain Brightness (ISO) Bundle breaking tenacity (g D 1 ) 1 ± c 32:4 0:2 6:3 0:3 2 NT-39 35:2 0:2 6:1 0:2 3 NT-53 34:7 0:1 6:2 0:2 4 NT-76 35:0 0:1 6:1 0:3 5 NT-39 NT-53(1:1) 37:8 0:2 6:0 0:2 6 ± 33:0 0:2 6:1 0:2 a Values are means of three replicates standard deviation. b Sample 1 to sample 5 were treated as described in Table 3, and sample 6 was only boiled with a solution of 1.5% NaOH at 135 C for 2 h. c Blank control was treated with inactivated supernatant (boiling for 10 min) of strain NT-39. dle breaking tenacity of ramie bers (Table 4). The results showed that the brightness increased with the removal of residual gum from the bers. When a mixed supernatant of strain NT-39 and NT-53 was used for degumming, the increment of ber brightness could amount to 5.4 ISO units. It indicated that enzymatic degumming could result in the removal of chromophoric material linking gum components. Tenkanen et al. (1999) have shown that the residual lignin linking with xylan in birch kraft pulp could be removed by xylanase. On the other hand, enzymatic degummig caused <5.0% reduction in bundle breaking tenacity. It further con- rmed that the enzymes from the selected strains did not damage ramie bers. Although the values of bundle breaking tenacity were similar for the samples of the bers treated by chemical degumming or enzymatic degumming, higher ber brightness could be achieved by enzymatic degumming. It indicated that the consumption of bleaching agent might be reduced in successive bleaching process. 4. Discussion Although some attempts to degum ramie bers with neutrophilic microorganisms and their enzymes have been made, their industrial applications are rather limited (Cao et al., 1992). High production cost and low degumming e ciency are the major problems hindering the application of this biotechnology. It has been reported that the degumming process can be accelerated by pretreating ramie bers with alkali (Deshpande and Gurucharanam, 1985). Because neutrophilic microorganisms are unable to grow under high ph conditions, alkalophilic bacteria were chosen for the degumming of ramie bers in this study. Our ideal was to cultivate the bacteria with ramie gum and to degum the bers with bacterial enzymes. The results of our study have proved the feasibility of this ideal. The selected strains could utilize ramie gum as sole carbon source for growth and production of polysaccharide-degrading enzymes. Because a part of the gum had been assimilated into bacterial biomass or dissimilated to CO 2 and H 2 O by the strains, the amount of organic waste in degumming ef- uent was considerably decreased. In this aspect, the bacterial degumming is superior to any conventional physical/chemical treatment adopted at present. Moreover, the degumming liquor could be used as the inoculum and enzyme source for further degumming. Therefore, this degumming process would be expected to have signi cant economic and environmental bene ts. Paul and Bhattacharyya (1979) reported that ramie bers still retained 15% gum after 7 days of incubation with mixed bacterial cultures, and a similar residual gum content was also determined on the bers incubated with the supernatants of actinomycete strains for 15 h (Bruhlman et al., 1994). The results of this study have con rmed the superiority of alkalopholic bacteria on ramie degumming. Strain NT-39 exhibited higher degumming e ciency than did other microbial strains. When a mixed supernatant of strain NT-39 and NT-53 was incubated with ramie bers, the degumming e ciency reached the level of a chemical degumming process (<10% residual gum content). Moreover, after bacterial degumming, the residual gum could be easily removed from the bers by successive processes (Cao et al., 1992). Therefore, the high consumption of chemicals and energy resulted from conventional degumming process would be avoided to a great extent. The enzymes involved in ramie degumming have been investigated in this study. The results demonstrated that PAL and xylanase are necessary for e ective degumming. Bruhlman et al. (1994) found that PAL was a key enzyme but xylanase did not make a signi cant contribution to ramie degumming when various actinomycetes were used for degumming. In this study, the e ect of xylanase on ramie degumming may be deduced by comparing the residual gum contents of the bers after enzymatic degumming, since additional gum loss resulted almost entirely from the further removal of hemicellulose rather than of pectin. The ber brightness increased after ramie bers were treated with the supernatants. This e ect might have been related to the activity of xylanase, since the biobleaching e ect of xylanase on kraft pulp has been con rmed (Garg et al., 1998). It is desirable that the selected strains are free of cellulolytic activities for ramie bers. Thus, the high

6 94 L. Zheng et al. / Bioresource Technology 78 (2001) 89±94 tenacity of ramie bers will be retained in the degumming process. Acknowledgements This work was supported by the Chinese Nation Science Foundation and research fond of State Key Laboratory of Pulp and Paper Engineering, China. We thank J. Cao and S. Chen, College of Life Science, Wuhan University, for supplying test strains. References Bailey, M.J., Biely, P., Poutanen, K., Imterlaboratory testing of methods for assay of xylanase activity. J. Biotechnol. 23, 257±270. Bruhlman, F., Kim, K.S., Zimmerman, W., Fiechiter, A., Pectinolytic enzymes from actinomycetes for the degumming of ramie bast bers. Appl. Environ. Microbiol. 61, 2107±2112. Cao, J., Zheng, L., Chen, S., Screening of pectinase producer from alkalophilic bacteria and study on its potential application in degumming of ramie. Enzyme Microbiol. Technol. 14, 1013± Deshpande, K.S., Gurucharanam, K., Degumming of ramie bers: role of cell wall degrading enzyme of Aspergillus versicolor. Ind. J. Bot. 8, 79±81. Garg, A.P., Roberts, J.C., McCarthy, A.J., Bleach boosting e ect of cellulase-free xylanase of Streptomyces thermoviolaceus and its comparison with two commercial enzyme preparations on birchwood kraft pulp. Enzyme Microbiol. Technol. 22, 594±598. Gurucharanam, K., Deshpande, K.S., Polysaccharidase of Curvularia lunate use in degumming of ramie bers. Ind. Phytopathol 39, 385±389. Horikoshi, K., Production of alkaline enzymes by alkalophilic microorganisms. Part III. Alkaline pectinase of Bacillus No. P-4-N. Agric. Biol. Chem. 36, 285±293. Horikoshi, K., Nakau, M., Kurono, Y., Sashihara, N., Cellulase of an alkalophilic Bacillus strain isolated from soil. Can. J. Microbiol. 30, 774±779. Nasser, B.H., Coelho, J.L.C., Araujo, E.F., Silva, D.O., Pectin lyase activity of Penicillin griseoroseum related to degumming of ramie. Rev. Microbiol. 24, 175±178. Paul, N.B., Bhattacharyya, S.K., The microbial degumming of ramie ber. J. Textile Inst. 12, 512±517. Tenkanen, M., Tamminen, B., Hortling, B., Investigation of lignin-carbohydrate complexes in kraft pulps by selective enzymatic treatments. Appl. Microbiol. Biotechnol. 51, 241±248. Whitaker, R.J., Microbial pectolytic enzymes. In: Fogarty, W.M., Kell, C.T. (Eds.), Microbial Enzymes and Biotechnology. Elsevier Applied Science, London, pp. 133±175.