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High-level of xylanase production by the thermophilic Paecilomyces themophila J18 on wheat straw in solid-state fermentation
Bioresource Technology 97 (2006) 1794–1800
High-level of xylanase production by the thermophilic Paecilomyces themophila J18 on wheat straw in solid-state fermentation S.Q. Yang a, Q.J. Yan a, Z.Q. Jiang a
a,*
, L.T. Li
a,*
, H.M. Tian a, Y.Z. Wang
b
Department of Biotechnology, College of Food Science and Nutritional Engineering, P.O. Box 294, China Agricultural University, No. 17 Qinghua Donglu, Haidian District, Beijing 100083, China b Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China Received 6 May 2005; received in revised form 29 August 2005; accepted 5 September 2005 Available online 17 October 2005
Abstract The production of extracellular xylanase by a newly isolated thermophilic fungus, Paecilomyces themophila J18, on the lignocellulosic materials was studied in solid-state fermentation (SSF). The strain grew well at 50 C and produced a high-level of xylanase activity using the selected lignocellulosic materials, especially wheat straw. Production of xylanase by P. themophila J18 on wheat straw was enhanced by optimizing the particle size of wheat straw, nitrogen source, initial moisture level, growth temperature and initial pH of the culture medium. Under the optimized conditions, yield as high as 18 580 U g 1 of carbon source of xylanase was achieved. No CMCase activity was observed. The xylanase exhibited remarkable stability and retained more than 50% of its original activity at 70 C for 4 h at pH 7.0– 8.0. Therefore, P. themophila J18 could to be a promising microorganism for thermostable, cellulase-free xylanase production in SSF. 2005 Elsevier Ltd. All rights reserved. Keywords: Cellulase-free; Paecilomyces themophila; Solid-state fermentation; Thermophilic; Wheat straw; Xylanase
1. Introduction Xylanase (EC 3.2.1.8) catalyzes the hydrolysis of the xylopyranosyl linkages of b-1,4-xylan, a plant polysaccharide next only to cellulose in abundance. Owing to the increasing biotechnological importance of thermostable xylanases, many thermophilic fungi have been examined for xylanases production (Maheshwari et al., 2000; Singh et al., 2003). These strains include Thermoascus auranticus, Talaromyces emersonii, Thermomyces lanuginosus, Melanocarpus albomyces and Sporotrichum thermophile (Jain, 1995; Haltrich et al., 1996; Topakas et al., 2003).
* Corresponding authors. Tel.: +86 10 62737689; fax: +86 10 82388508 (Z.Q. Jiang). E-mail addresses: [email protected] (Z.Q. Jiang), [email protected] (L.T. Li).
0960-8524/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.09.007
To date, the production of thermostable xylanases has been widely studied in submerged culture processes, but the relatively high cost of enzyme production has hindered the industrial application of thermostable xylanases (Haltrich et al., 1996; Beg et al., 2000; Virupakshi et al., 2005). Solid-state fermentation (SSF) is an attractive method for xylanase production, especially for fungal cultivations, because it presents many advantages, such as the higher productivity per reactor volume as well as the lower operation and capital cost (Purkarthofer et al., 1993; Pandey et al., 1999). The cost of carbon source plays another major role in the economics of xylanase production. Hence, another approach to reduce the cost of xylanase production is the use of lignocellulosic materials as substrates rather than opting for the expensive pure xylans (Haltrich et al., 1996; Beg et al., 2000; Senthilkumar et al., 2005). Solid-state fermentation can be performed on a variety of lignocellulosic materials, such as wheat straw, wheat
S.Q. Yang et al. / Bioresource Technology 97 (2006) 1794–1800
bran and corncob, and has been shown to be an efficient technique in the production of xylanase (Alam et al., 1994; Hoq and Deckwer, 1995; Haltrich et al., 1996; Gawande and Kamat, 1999; Kang et al., 2004; Sonia et al., 2005). Xylanase production has been described for many fungal species, including the thermophilic fungi, but only a few reports are available on the production of xylanase from Paecilomyces sp. strains (Kelly et al., 1989; Haltrich et al., 1996; Ferreira et al., 1999; Maheshwari et al., 2000). Until now, no study on the xylanases production of any Paecilomyces species using solid-state fermentation has ever appeared in the literature. P. themophila J18, a thermophilic fungus newly isolated from Sinkiang Province, was proved to be a good producer of xylanase (data not shown). Therefore, the purpose of this study was to investigate the ability of P. themophila J18 to produce xylanase in SSF. We optimized some of the critical factors affecting xylanase production by this strain in SSF. To our knowledge, this is the first paper that reports the production of xylanases by the thermophilic fungi Paecilomyces sp. strains in SSF.
2. Methods 2.1. Materials Tryptone and yeast extract were products from Oxoid (England). Birchwood xylan and carboxymethylcellulose (CMC, low viscosity) were purchased from Sigma Chemical Company, St. Louis, USA. The lignocellulosic materials, namely corncobs, corn straw, rice husk, rice straw, sugar cane bagasse, wheat bran and wheat straw, were obtained locally and were chopped by a chopper into small pieces, dried and ground in a hammer mill. These ground materials were then separated by sieves into particles of different sizes and the fraction that passed through the 0.45 mm (40 mesh) sieve were used in media. Wheat straw particles were divided into six fragments by sieving. The 0.45–0.9 mm fragment consisted of particles that passed through the 0.9 mm sieve, but not through the 0.45 mm sieve. The same procedure was used to sort out the <0.18, 0.18–0.3, 0.3–0.45, 0.45–0.9, and 0.9–2 mm fragments. Dry weight and moisture content of the substrates were determined gravimetrically after drying the samples at 60 C. All other chemicals used were analytical grade reagents unless otherwise stated. 2.2. Strain P. themophila J18 was isolated from the soil samples under decaying tree fibers layer at Tianchi Lake (heavenly lake) in Urumqi of Sinkiang Province, and was identified by the Institute of Microbiology of Chinese Academy of Sciences (IMCAS). This strain was deposited (under the number AS3.6885) at the center for culture collection of
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microorganisms of China. Stock cultures were maintained on potato dextrose agar (PDA) at 4 C and were transferred every 6–7 weeks. The strain was maintained on potato dextrose agar at 50 C for 5 days for spore production. The conidial suspensions were prepared by adding 5 ml of 20% glycerol solution to slant cultures and the surface was gently rubbed with a sterilized wire loop. Spore suspension was counted at 105–106 spores/ml by a haemacytometer. An inoculum size of 2.0 ml/5 g substrate was used for xylanase production in every experiment. 2.3. Xylanase production by solid-state fermentation (SSF) and enzyme extraction Xylanase production was carried out using wheat straw as the basic solid substrate unless otherwise stated. P. themophila J18 was grown in 300 ml Erlenmeyer flasks containing 5 g of each substrate (coarser than 0.45 mm) and 0.2 g yeast extract, and deionized water was added to adjust the moisture content to the desired levels. The initial pH of the growth media was pH 6.9 before sterilization. All flasks were sterilized at 121 C for 20 min. To each flask 2.0 ml of spore suspension was inoculated. The cultures were incubated statically at 50 C for 7 days. After suitable periods of growth time, the xylanases were extracted from the fermented carbon source with 10-fold (v/w) distilled water by shaking (200 rpm) at 30 C for 60 min. The suspended materials and fungal biomass were separated by centrifugation (10 000 · g for 15 min) and the clarified supernatant was used as the source of crude enzyme. 2.4. Enzyme assays and protein determination Xylanase activity was assayed according to the method of Bailey et al. (1992). The reaction mixture containing 0.9 ml of 1.0 (w/v) birchwood xylan (preincubated at 50 C for 3 min) in 50 mM, pH 6.5 MOPS [3-(N-morpholino)-propane sulphonic acid] buffer and 0.1 ml of a suitably diluted enzyme solution was incubated at 50 C for 10 min. The reaction was stopped by adding 1 ml of 1.0% (w/v) DNS (dinitrosalicylic acid). Amount of reducing sugar liberated was determined by DNS method using xylose (Sigma) as the standard (Miller, 1959). One unit of xylanase activity was defined as the amount of enzyme that produced 1 lmol of xylose equivalent per minute of reaction and per milliliter of enzyme solution, in the assay condition. A similar method was used to assay CMCase by using 1.0% (w/v) carboxymethylcellulose as the substrate and D-glucose as the standard. Results were expressed as the mean of at least three different experiments. All assays results were expressed units per gram of initial dry carbon source. Protein concentrations were measured by the Lowry method (Lowry et al., 1951) with BSA (bovine serum albumin) as the standard.
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2.5. Effect of various substrates on xylanase production To investigate the effect of various substrates on xylanase production, P. themophila J18 was cultivated for 7 days on the medium containing each lignocellulosic material as the sole substrate. Initial moisture content was adjusted to 80%. Wheat straw of different particle sizes was tested in order to determine their effects on xylanase production. 2.6. Effect of moisture level on xylanase production The effect of moisture level on xylanase production was tested by varying the moisture content in the range of 75– 88%. All liquid added to the flask was taken into consideration in calculating the moisture content. 2.7. Effect of different nitrogen sources on xylanase production Various organic and inorganic nitrogen sources were used with a fixed concentration of nitrogen at 19.6 mg of N/5 g of wheat straw. The pH was adjusted to 6.0 before sterilization. 2.8. Effect of the initial culture pH and temperature on xylanase production To evaluate the effects of initial culture pH of solid substrate on xylanase production, the initial pH values were adjusted to 4.0, 5.0 6.0, 7.0, 8.0 and 9.0. The production of xylanase by P. themophila J18 was studied by incubating the flasks at 30–55 C for 7 days. 2.9. Stability of the partially purified xylanase The crude xylanase was precipitated with ammonium sulfate (20–60% saturation). The precipitate was recovered by centrifugation at 10 000 · g for 15 min and dissolved in distilled water. The enzyme solution was desalted on a Sephadex G-25 column. Stability of the partially purified xylanase (930 U mg 1) was determined at pH 5.0–10.0 and at 65–70 C. A 1.0 ml xylanase (10 lg ml 1) was incubated at varying pH using the above buffers at different temperatures. A 50 ll aliquot was withdrawn after 15, 30, 60, 120, 180, 240, 300, 360, 420 and 480 min and assayed for residual xylanase activities.
Smith, 1957; Samson, 1974). But significant differences still existed between the two organisms. J18 was thermophilic and grew well at 50 C, while, P. javanicus growth is restricted to 30 C. The colonies grew to 70 mm in diameter after 5 days of incubation and mature colonies appeared to be dull dark white to pink or wine color (Fig. 1(a)).1 The ellipsoidal conidia of J18 (Fig. 1(b)) were shorter and broader (2.5–6.5 · 2.4–3.2 lm) than that of P. javanicus, which were cylindrical to fusiform in shape (5–7.4 · 1.4– 1.7 lm). Because J18 does not match any of the Paecilomyces species, we identify it as a new species, Paecilomyces themophila sp. nov. 3.2. Effect of carbon source on xylanase production Among the lignocellulosic materials tested as carbon sources, wheat straw was far more effective for xylanase production (7745 U g 1). Wheat bran exhibited moderate activity (4517 U g 1), whereas the other carbon sources resulted in significantly lower activities (data not shown). Since the xylanase activity observed during P. themophila J18 growth on wheat straw was much higher than those observed on the other substrates, wheat straw was used as the only carbon source in subsequent experiments. Xylan is costly for large-scale production of xylanases, lignocellulosic materials can be used as cost-effective substrates for xylanase production (Haltrich et al., 1996; Beg et al., 2000). Various lignocellulosic materials and microbial cultures have been used successfully in solid-state fermentation for xylanase production (Topakas et al., 2003; Sonia et al., 2005). The significant difference in xylanase titers, when wheat straw was used as the carbon source may be attributed to its hemicellulose nature and favorable degradability, the presence of some nutrients in the carbon source (Sonia et al., 2005). Wheat straw has been known for being ideally suitable for xylanase production in T. aurantiacus and Penicillium canescens cultures (Kalogeris et al., 1998; Bakri et al., 2003). Wheat straws of different particle sizes were tested in order to determine their effects on xylanase production. It was apparent that particle size affected the enzyme production (data not shown). The highest titre of 9868 U g 1 xylanase was produced by the wheat straw of particle size 0.3–0.45 mm whereas lower activities were produced on the wheat straw of other sizes. These findings confirmed that the size of carbon source was an important factor in xylanase production (Kalogeris et al., 1998).
3. Results and discussion 3.3. Effect of initial moisture on xylanase production 3.1. Isolation and identification of P. themophila J18 The newly isolated thermophilic fungus J18 from the soil samples of Sinkiang Province in China was identified as P. themophila (Fig. 1). After carefully comparing the features of fungus J18 with the characteristics of other Paecilomyces species, it was observed that the species was somewhat similar to Paecilomyces javanicus (Brown and
Initial moisture content is one of the key factors influencing xylanase production. The highest xylanase activity (13 230 U g 1) was obtained with initial moisture content
1 For interpretation of the references in colour in this figure, the reader is referred to the web version of this article.
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Fig. 1. (a) Colony morphology of Paecilomyces themophila J18 after growth at 50 C for 5 days on potato dextrose agar (PDA). (b) Electron micrograph showing mature ellipsoidal spores of Paecilomyces themophila J18.
of 83% (data not shown). The moisture content of the medium has critical importance to SSF. Many researchers have reported the similar effect of moisture content on xylanases production (Ferreira et al., 1999; Bakri et al., 2003). This could be attributed to the faster growth of microorganism at higher moisture content and the subsequent early initiation of the enzyme production (Kalogeris et al., 1998). As reported elsewhere, high moisture enhanced fungal growth and xylanases production when lignocellulosic substrates were the carbon sources in SSF (Purkarthofer et al., 1993; Alam et al., 1994; Narang et al., 2001; Bakri et al., 2003). 3.4. Effect of nitrogen source on xylanase production The effect of different nitrogen sources was also studied. When a number of nitrogen sources were tested, the results showed that xylanase activities were higher with organic nitrogen sources (data not shown). Maximum xylanase activity (13 440 U g 1) was evident when yeast extract was added. These results are in agreement with those reported in the literature where fungi were found to produce higher xylanase activities on organic nitrogen sources (Purkarthofer et al., 1993; Lemos et al., 2001; Bakri et al., 2003). 3.5. Effects of initial culture pH and growth temperature on xylanase production The effect of initial culture pH on the xylanase production was investigated (data not shown). It was found that maximum activity for xylanase (15 574 U g 1) was obtained when the initial pH was adjusted to 7.0. High-level
xylanase production (more than 10 000 U g 1) of this strain was observed in pH 5.0–8.0. To examine the effect of temperature on xylanase production, growth of P. themophila J18 between 30 C and 55 C were studied. Maximum xylanase production (15 582 U g 1) occurred at 50 C. At 55 C, a significant decline (27.5%) in xylanase activity was evident (data not shown). The optimum temperature for xylanase production by P. themophila J18 was similar to some thermophilic fungi, such as T. lanuginosus (Purkarthofer et al., 1993), T. aurantiacus (Kalogeris et al., 1998), and Sporotrichum thermophile (Topakas et al., 2003) grow under SSF. The results clearly indicated the thermophilic nature of the fungus. 3.6. Time course of xylanase production by P. themophila J18 in SSF The time course of xylanase production was further investigated (data not shown). Maximum production (18 580 U g 1) was observed after 8 days. Further incubation after this time did not show any increment in the level of enzyme production. The volumetric productivity (U l 1 h 1) is an important parameter to assess the effectiveness of the process (Haltrich et al., 1996). The productivity of xylanase (16 451 U l 1 h 1) in SSF was compared with those reported for other xylanase-producing microorganisms. Furthermore, no CMCase was produced along with the xylanase in this study. In some fungi, high xylanase production has been shown to be linked strictly to cellulase production (Haltrich et al., 1996; Kang et al., 2004), but P. themophila J18 like T. lanuginosus did not produce cellulases despite of the use of cellulose-rich substrate,
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i.e., wheat straw (Singh et al., 2003). This is one of the attractive features of this strain. 3.7. Comparisons of xylanase production using SSF Quantitative comparison of xylanase activities reported in literature is not always possible as no standard enzyme substrate has yet been adopted. As shown in Table 1, xylanase productivity of P themophila J18 observed in this work was only lower to T. lanuginosus D2W3 and DSM 5826 but was much higher than the optimum productivities reported for some fungi grown in SSF. Filamentous fungi have been widely used to produce xylanases for industrial applications, of which xylanase levels are generally much higher than those produced by yeast or bacteria (Haltrich et al., 1996). The production of xylanase by T. lanuginosus DSM 5826 in solid-state cultivation using corncobs as carbon source was studied with an initial water content of 70% (w/w) at 50 C. An activity of 336 700 nkat/g dry solid (20 161.7 U g 1) was obtained after 9 days of cultivation (Purkarthofer et al., 1993). Under the optimized condition, yield as high as 6193 U g 1 of carbon source (wheat straw) was obtained for T. aurantiacus under solid-state culture (SSC) (Kalogeris et al., 1998). Melanocarpus albomyces IIS-68 produced the maximum xylanase (7760 U g 1) when growth on wheat straw after 4 days of cultivation at 45 C (Narang et al., 2001). Xylanase activity of 5071 U g 1 was obtained in solid-state fermentation of rice straw by Aspergillus niger mutant under the optimized conditions (Park et al., 2002). Addition of the purified xylan in SSF increased xylanase production considerably in some fungi, however this practice also leads to the higher production cost. Xylanase production (9632 U g 1) of
Penicillium canescens 10-10c in SSF was obtained by using the mixture of wheat straw and xylan as substrate (Bakri et al., 2003). Under an optimized condition, T. lanuginosus D2W3 produced xylanase activity as high as 48 000 U g 1 of carbon source (sorghum straw) in SSF (Sonia et al., 2005), which was the highest titre of xylanase activity ever reported. There are some reports where bacterial strains have been used successfully for the production of xylanase by using SSF, but their xylanase levels are generally lower than those in fungi. A thermostable xylanase has been produced from Streptomyces sp. GG-11-3 in SSF (Beg et al., 2000). The maximum xylanase yield obtained using wheat bran and eucalyptus kraft pulp as the prime solid substrates were 2360 U g 1 and 1200 U g 1, respectively. Maximum production of xylanase (3644 U g 1) by Bacillus sp. JB-99 in SSF was obtained using rice bran as the prime substrate (Virupakshi et al., 2005). 3.8. Stability of the partially purified xylanase Ammonium sulphate fraction (20–60% saturation) of crude xylanase yielded 74% of the enzyme with 3.6-fold purification (data not shown). The stability of the partially purified xylanase at 65 C and 70 C at different pH (pH 5.0–10.0) is shown in Fig. 2. Xylanase exhibited remarkable stability and was completely stable at temperature up to 65 C for 60 min at pH 6.0–9.0, while at 70 C it showed complete stability at pH 7.0–8.0 for 30 min. Nevertheless, xylanase retained over 70% of its original activity for 2 h at pH 7.0–9.0 at 70 C while retained over 50% of its original activity for 4 h at pH 7.0–8.0. Similar stability has been reported for xylanases from thermophilic fungi, Melanocarpus albomyces, Talaromyces byssochlamydoides,
Table 1 Comparisons of xylanase production from different fungi grown on lignocellulosic materials Organism
Aspergillus niger KK2 mutant Melanocarpus albomyces IIS-68 Paecilomyces themophila J18 Penicillium canescens Sporotrichum thermophile ATCC 34628 Schizophyllum commune Streptomyces sp. Thermomyces lanuginosus DSM 5826 Thermomyces lanuginosus RT9 Thermomyces lanuginosus D2W3 Thermoascus aurantiacus BJT 190 Thermoascus aurantiacus IMI 216529 Thermoascus aurantiacus ATCC 204492 a b
Substrate
Initial moisture content (%)
Cultivation
28 C, 45 C, 50 C, 30 C,
Xylanase Productivity (U l 1 h 1)b
5071 7760 18 580 9632
14 790 11 317 16 451 5551
Park et al. (2002) Narang et al. (2001) This work Bakri et al. (2003)
Rice straw Wheat straw Wheat straw Wheat straw + xylan Wheat straw
65 86 83 83
Static, Static, Static, Static,
80
Static, 50 C, 6 days
320
444
Topakas et al. (2003)
Unbleached pulp Wheat bran Corncobs
75 75 70
Static, 30 C, 16 days Static, 37 C, 7 days Static, 50 C, 9 days
14 800
Wheat bran Sorghum straw Wheat bran
50 80 50
Static, 55 C, 7 days Static, 50 C, 6 days Static, 55 C, 7 days
22 700 2360 337 000 nkat/g (20179.6 U g 1) 1900 U ml 1 48 000 706 U ml 1
5650 33 333 2100
Haltrich et al. (1996) Beg et al. (2000) Purkarthofer et al. (1993) Alam et al. (1994) Sonia et al. (2005) Alam et al. (1994)
Wheat straw
80
Static, 50 C, 5 days
6193
10 322
Kalogeris et al. (1998)
Bagasse
81
Static, 45 C, 10 days
2700
2318
Souza et al. (1999)
Maximum activity. Volumetric productivity; calculation is based on the initial moisture content.
5 days 4 days 8 days 12 days
References
Activitya (U g 1)
28 100
100 90 80 70 60 50 40 30 20 10 0 0
(a)
60
120
180
240
300
360
Time (min)
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100 90 80 70 60 50 40 30 20 10 0
Relative residual xylanase (%)
Relative residual xylanase (%)
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0
(b)
60
120
180
240
300
360
Time (min)
Fig. 2. Stability of the partially purified xylanase at pH 5.0–10.0 and at 65 C (a) and 70 C (b) as a function time. The crude xylanase was produced by Paecilomyces themophila J18 in SSF for 8 days. Different pH values used: pH 5.0 (), pH 6.0 (j), pH 7.0 (m), pH 8.0 (), pH 9.0 (h), pH 10.0 (n).
Thermomyces lanuginosus (Jain, 1995; Maheshwari et al., 2000; Singh et al., 2003; Li et al., 2005). 4. Conclusions The present work has established the potential of the newly isolated thermophilic P. themophila J18 for xylanase production in SSF. High-level xylanase production by this strain in SSF suggests that P. themophila J18 is an interesting source of newer thermostable, cellulase-free xylanase and will have important economic advantages. The stability of xylanase at 65–70 C could be of considerable commercial interest for its potential applications. Further studies on purification and characterization of the xylanase from P. themophila J18 are in progress. Acknowledgements This work was financially supported by The National High Technology Research and Development Program of China (863 Program, No. 2003AA214020). The authors thank Dr. Szesze Tan for the critical editing of the manuscript. References Alam, M., Gomes, I., Mohiuddin, G., Hoq, M.M., 1994. Production and characterization of thermostable xylanases by Thermomyces lanuginosus and Thermoascus aurantiacus grown on lignocelluloses. Enzyme Microb. Technol. 16, 298–302. Bailey, M.J., Biely, P., Poutanen, K., 1992. Interlaboratory testing of methods for assay of xylanase activity. J. Biotechnol. 23, 257– 270. Bakri, Y., Jacques, P., Thonart, P., 2003. Xylanase production by Penicillium canescens 10-10c in solid-state fermentation. Appl. Biochem. Biotechnol. 105–108, 737–747. Beg, Q.K., Bhushan, B., Kapoor, M., Hoondal, G.S., 2000. Enhanced production of a thermostable xylanase from Streptomyces sp. QG-11-3 and its application in biobleaching of eucalyptus kraft pulp. Enzyme Microb. Technol. 27, 459–466. Brown, A.H.S., Smith, G., 1957. The genus Paecilomyces Bainier and its perfect stages Byssochlamys Westling. Trans. Br. Mycol. Soc. 40, 17– 89.
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High-level of xylanase production by the thermophilic Paecilomyces themophila J18 on wheat straw in solid-state fermentation S.Q. Yang a, Q.J. Yan a, Z.Q. Jiang a
a,*
, L.T. Li
a,*
, H.M. Tian a, Y.Z. Wang
b
Department of Biotechnology, College of Food Science and Nutritional Engineering, P.O. Box 294, China Agricultural University, No. 17 Qinghua Donglu, Haidian District, Beijing 100083, China b Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China Received 6 May 2005; received in revised form 29 August 2005; accepted 5 September 2005 Available online 17 October 2005
Abstract The production of extracellular xylanase by a newly isolated thermophilic fungus, Paecilomyces themophila J18, on the lignocellulosic materials was studied in solid-state fermentation (SSF). The strain grew well at 50 C and produced a high-level of xylanase activity using the selected lignocellulosic materials, especially wheat straw. Production of xylanase by P. themophila J18 on wheat straw was enhanced by optimizing the particle size of wheat straw, nitrogen source, initial moisture level, growth temperature and initial pH of the culture medium. Under the optimized conditions, yield as high as 18 580 U g 1 of carbon source of xylanase was achieved. No CMCase activity was observed. The xylanase exhibited remarkable stability and retained more than 50% of its original activity at 70 C for 4 h at pH 7.0– 8.0. Therefore, P. themophila J18 could to be a promising microorganism for thermostable, cellulase-free xylanase production in SSF. 2005 Elsevier Ltd. All rights reserved. Keywords: Cellulase-free; Paecilomyces themophila; Solid-state fermentation; Thermophilic; Wheat straw; Xylanase
1. Introduction Xylanase (EC 3.2.1.8) catalyzes the hydrolysis of the xylopyranosyl linkages of b-1,4-xylan, a plant polysaccharide next only to cellulose in abundance. Owing to the increasing biotechnological importance of thermostable xylanases, many thermophilic fungi have been examined for xylanases production (Maheshwari et al., 2000; Singh et al., 2003). These strains include Thermoascus auranticus, Talaromyces emersonii, Thermomyces lanuginosus, Melanocarpus albomyces and Sporotrichum thermophile (Jain, 1995; Haltrich et al., 1996; Topakas et al., 2003).
* Corresponding authors. Tel.: +86 10 62737689; fax: +86 10 82388508 (Z.Q. Jiang). E-mail addresses: [email protected] (Z.Q. Jiang), [email protected] (L.T. Li).
0960-8524/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.09.007
To date, the production of thermostable xylanases has been widely studied in submerged culture processes, but the relatively high cost of enzyme production has hindered the industrial application of thermostable xylanases (Haltrich et al., 1996; Beg et al., 2000; Virupakshi et al., 2005). Solid-state fermentation (SSF) is an attractive method for xylanase production, especially for fungal cultivations, because it presents many advantages, such as the higher productivity per reactor volume as well as the lower operation and capital cost (Purkarthofer et al., 1993; Pandey et al., 1999). The cost of carbon source plays another major role in the economics of xylanase production. Hence, another approach to reduce the cost of xylanase production is the use of lignocellulosic materials as substrates rather than opting for the expensive pure xylans (Haltrich et al., 1996; Beg et al., 2000; Senthilkumar et al., 2005). Solid-state fermentation can be performed on a variety of lignocellulosic materials, such as wheat straw, wheat
S.Q. Yang et al. / Bioresource Technology 97 (2006) 1794–1800
bran and corncob, and has been shown to be an efficient technique in the production of xylanase (Alam et al., 1994; Hoq and Deckwer, 1995; Haltrich et al., 1996; Gawande and Kamat, 1999; Kang et al., 2004; Sonia et al., 2005). Xylanase production has been described for many fungal species, including the thermophilic fungi, but only a few reports are available on the production of xylanase from Paecilomyces sp. strains (Kelly et al., 1989; Haltrich et al., 1996; Ferreira et al., 1999; Maheshwari et al., 2000). Until now, no study on the xylanases production of any Paecilomyces species using solid-state fermentation has ever appeared in the literature. P. themophila J18, a thermophilic fungus newly isolated from Sinkiang Province, was proved to be a good producer of xylanase (data not shown). Therefore, the purpose of this study was to investigate the ability of P. themophila J18 to produce xylanase in SSF. We optimized some of the critical factors affecting xylanase production by this strain in SSF. To our knowledge, this is the first paper that reports the production of xylanases by the thermophilic fungi Paecilomyces sp. strains in SSF.
2. Methods 2.1. Materials Tryptone and yeast extract were products from Oxoid (England). Birchwood xylan and carboxymethylcellulose (CMC, low viscosity) were purchased from Sigma Chemical Company, St. Louis, USA. The lignocellulosic materials, namely corncobs, corn straw, rice husk, rice straw, sugar cane bagasse, wheat bran and wheat straw, were obtained locally and were chopped by a chopper into small pieces, dried and ground in a hammer mill. These ground materials were then separated by sieves into particles of different sizes and the fraction that passed through the 0.45 mm (40 mesh) sieve were used in media. Wheat straw particles were divided into six fragments by sieving. The 0.45–0.9 mm fragment consisted of particles that passed through the 0.9 mm sieve, but not through the 0.45 mm sieve. The same procedure was used to sort out the <0.18, 0.18–0.3, 0.3–0.45, 0.45–0.9, and 0.9–2 mm fragments. Dry weight and moisture content of the substrates were determined gravimetrically after drying the samples at 60 C. All other chemicals used were analytical grade reagents unless otherwise stated. 2.2. Strain P. themophila J18 was isolated from the soil samples under decaying tree fibers layer at Tianchi Lake (heavenly lake) in Urumqi of Sinkiang Province, and was identified by the Institute of Microbiology of Chinese Academy of Sciences (IMCAS). This strain was deposited (under the number AS3.6885) at the center for culture collection of
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microorganisms of China. Stock cultures were maintained on potato dextrose agar (PDA) at 4 C and were transferred every 6–7 weeks. The strain was maintained on potato dextrose agar at 50 C for 5 days for spore production. The conidial suspensions were prepared by adding 5 ml of 20% glycerol solution to slant cultures and the surface was gently rubbed with a sterilized wire loop. Spore suspension was counted at 105–106 spores/ml by a haemacytometer. An inoculum size of 2.0 ml/5 g substrate was used for xylanase production in every experiment. 2.3. Xylanase production by solid-state fermentation (SSF) and enzyme extraction Xylanase production was carried out using wheat straw as the basic solid substrate unless otherwise stated. P. themophila J18 was grown in 300 ml Erlenmeyer flasks containing 5 g of each substrate (coarser than 0.45 mm) and 0.2 g yeast extract, and deionized water was added to adjust the moisture content to the desired levels. The initial pH of the growth media was pH 6.9 before sterilization. All flasks were sterilized at 121 C for 20 min. To each flask 2.0 ml of spore suspension was inoculated. The cultures were incubated statically at 50 C for 7 days. After suitable periods of growth time, the xylanases were extracted from the fermented carbon source with 10-fold (v/w) distilled water by shaking (200 rpm) at 30 C for 60 min. The suspended materials and fungal biomass were separated by centrifugation (10 000 · g for 15 min) and the clarified supernatant was used as the source of crude enzyme. 2.4. Enzyme assays and protein determination Xylanase activity was assayed according to the method of Bailey et al. (1992). The reaction mixture containing 0.9 ml of 1.0 (w/v) birchwood xylan (preincubated at 50 C for 3 min) in 50 mM, pH 6.5 MOPS [3-(N-morpholino)-propane sulphonic acid] buffer and 0.1 ml of a suitably diluted enzyme solution was incubated at 50 C for 10 min. The reaction was stopped by adding 1 ml of 1.0% (w/v) DNS (dinitrosalicylic acid). Amount of reducing sugar liberated was determined by DNS method using xylose (Sigma) as the standard (Miller, 1959). One unit of xylanase activity was defined as the amount of enzyme that produced 1 lmol of xylose equivalent per minute of reaction and per milliliter of enzyme solution, in the assay condition. A similar method was used to assay CMCase by using 1.0% (w/v) carboxymethylcellulose as the substrate and D-glucose as the standard. Results were expressed as the mean of at least three different experiments. All assays results were expressed units per gram of initial dry carbon source. Protein concentrations were measured by the Lowry method (Lowry et al., 1951) with BSA (bovine serum albumin) as the standard.
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2.5. Effect of various substrates on xylanase production To investigate the effect of various substrates on xylanase production, P. themophila J18 was cultivated for 7 days on the medium containing each lignocellulosic material as the sole substrate. Initial moisture content was adjusted to 80%. Wheat straw of different particle sizes was tested in order to determine their effects on xylanase production. 2.6. Effect of moisture level on xylanase production The effect of moisture level on xylanase production was tested by varying the moisture content in the range of 75– 88%. All liquid added to the flask was taken into consideration in calculating the moisture content. 2.7. Effect of different nitrogen sources on xylanase production Various organic and inorganic nitrogen sources were used with a fixed concentration of nitrogen at 19.6 mg of N/5 g of wheat straw. The pH was adjusted to 6.0 before sterilization. 2.8. Effect of the initial culture pH and temperature on xylanase production To evaluate the effects of initial culture pH of solid substrate on xylanase production, the initial pH values were adjusted to 4.0, 5.0 6.0, 7.0, 8.0 and 9.0. The production of xylanase by P. themophila J18 was studied by incubating the flasks at 30–55 C for 7 days. 2.9. Stability of the partially purified xylanase The crude xylanase was precipitated with ammonium sulfate (20–60% saturation). The precipitate was recovered by centrifugation at 10 000 · g for 15 min and dissolved in distilled water. The enzyme solution was desalted on a Sephadex G-25 column. Stability of the partially purified xylanase (930 U mg 1) was determined at pH 5.0–10.0 and at 65–70 C. A 1.0 ml xylanase (10 lg ml 1) was incubated at varying pH using the above buffers at different temperatures. A 50 ll aliquot was withdrawn after 15, 30, 60, 120, 180, 240, 300, 360, 420 and 480 min and assayed for residual xylanase activities.
Smith, 1957; Samson, 1974). But significant differences still existed between the two organisms. J18 was thermophilic and grew well at 50 C, while, P. javanicus growth is restricted to 30 C. The colonies grew to 70 mm in diameter after 5 days of incubation and mature colonies appeared to be dull dark white to pink or wine color (Fig. 1(a)).1 The ellipsoidal conidia of J18 (Fig. 1(b)) were shorter and broader (2.5–6.5 · 2.4–3.2 lm) than that of P. javanicus, which were cylindrical to fusiform in shape (5–7.4 · 1.4– 1.7 lm). Because J18 does not match any of the Paecilomyces species, we identify it as a new species, Paecilomyces themophila sp. nov. 3.2. Effect of carbon source on xylanase production Among the lignocellulosic materials tested as carbon sources, wheat straw was far more effective for xylanase production (7745 U g 1). Wheat bran exhibited moderate activity (4517 U g 1), whereas the other carbon sources resulted in significantly lower activities (data not shown). Since the xylanase activity observed during P. themophila J18 growth on wheat straw was much higher than those observed on the other substrates, wheat straw was used as the only carbon source in subsequent experiments. Xylan is costly for large-scale production of xylanases, lignocellulosic materials can be used as cost-effective substrates for xylanase production (Haltrich et al., 1996; Beg et al., 2000). Various lignocellulosic materials and microbial cultures have been used successfully in solid-state fermentation for xylanase production (Topakas et al., 2003; Sonia et al., 2005). The significant difference in xylanase titers, when wheat straw was used as the carbon source may be attributed to its hemicellulose nature and favorable degradability, the presence of some nutrients in the carbon source (Sonia et al., 2005). Wheat straw has been known for being ideally suitable for xylanase production in T. aurantiacus and Penicillium canescens cultures (Kalogeris et al., 1998; Bakri et al., 2003). Wheat straws of different particle sizes were tested in order to determine their effects on xylanase production. It was apparent that particle size affected the enzyme production (data not shown). The highest titre of 9868 U g 1 xylanase was produced by the wheat straw of particle size 0.3–0.45 mm whereas lower activities were produced on the wheat straw of other sizes. These findings confirmed that the size of carbon source was an important factor in xylanase production (Kalogeris et al., 1998).
3. Results and discussion 3.3. Effect of initial moisture on xylanase production 3.1. Isolation and identification of P. themophila J18 The newly isolated thermophilic fungus J18 from the soil samples of Sinkiang Province in China was identified as P. themophila (Fig. 1). After carefully comparing the features of fungus J18 with the characteristics of other Paecilomyces species, it was observed that the species was somewhat similar to Paecilomyces javanicus (Brown and
Initial moisture content is one of the key factors influencing xylanase production. The highest xylanase activity (13 230 U g 1) was obtained with initial moisture content
1 For interpretation of the references in colour in this figure, the reader is referred to the web version of this article.
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Fig. 1. (a) Colony morphology of Paecilomyces themophila J18 after growth at 50 C for 5 days on potato dextrose agar (PDA). (b) Electron micrograph showing mature ellipsoidal spores of Paecilomyces themophila J18.
of 83% (data not shown). The moisture content of the medium has critical importance to SSF. Many researchers have reported the similar effect of moisture content on xylanases production (Ferreira et al., 1999; Bakri et al., 2003). This could be attributed to the faster growth of microorganism at higher moisture content and the subsequent early initiation of the enzyme production (Kalogeris et al., 1998). As reported elsewhere, high moisture enhanced fungal growth and xylanases production when lignocellulosic substrates were the carbon sources in SSF (Purkarthofer et al., 1993; Alam et al., 1994; Narang et al., 2001; Bakri et al., 2003). 3.4. Effect of nitrogen source on xylanase production The effect of different nitrogen sources was also studied. When a number of nitrogen sources were tested, the results showed that xylanase activities were higher with organic nitrogen sources (data not shown). Maximum xylanase activity (13 440 U g 1) was evident when yeast extract was added. These results are in agreement with those reported in the literature where fungi were found to produce higher xylanase activities on organic nitrogen sources (Purkarthofer et al., 1993; Lemos et al., 2001; Bakri et al., 2003). 3.5. Effects of initial culture pH and growth temperature on xylanase production The effect of initial culture pH on the xylanase production was investigated (data not shown). It was found that maximum activity for xylanase (15 574 U g 1) was obtained when the initial pH was adjusted to 7.0. High-level
xylanase production (more than 10 000 U g 1) of this strain was observed in pH 5.0–8.0. To examine the effect of temperature on xylanase production, growth of P. themophila J18 between 30 C and 55 C were studied. Maximum xylanase production (15 582 U g 1) occurred at 50 C. At 55 C, a significant decline (27.5%) in xylanase activity was evident (data not shown). The optimum temperature for xylanase production by P. themophila J18 was similar to some thermophilic fungi, such as T. lanuginosus (Purkarthofer et al., 1993), T. aurantiacus (Kalogeris et al., 1998), and Sporotrichum thermophile (Topakas et al., 2003) grow under SSF. The results clearly indicated the thermophilic nature of the fungus. 3.6. Time course of xylanase production by P. themophila J18 in SSF The time course of xylanase production was further investigated (data not shown). Maximum production (18 580 U g 1) was observed after 8 days. Further incubation after this time did not show any increment in the level of enzyme production. The volumetric productivity (U l 1 h 1) is an important parameter to assess the effectiveness of the process (Haltrich et al., 1996). The productivity of xylanase (16 451 U l 1 h 1) in SSF was compared with those reported for other xylanase-producing microorganisms. Furthermore, no CMCase was produced along with the xylanase in this study. In some fungi, high xylanase production has been shown to be linked strictly to cellulase production (Haltrich et al., 1996; Kang et al., 2004), but P. themophila J18 like T. lanuginosus did not produce cellulases despite of the use of cellulose-rich substrate,
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i.e., wheat straw (Singh et al., 2003). This is one of the attractive features of this strain. 3.7. Comparisons of xylanase production using SSF Quantitative comparison of xylanase activities reported in literature is not always possible as no standard enzyme substrate has yet been adopted. As shown in Table 1, xylanase productivity of P themophila J18 observed in this work was only lower to T. lanuginosus D2W3 and DSM 5826 but was much higher than the optimum productivities reported for some fungi grown in SSF. Filamentous fungi have been widely used to produce xylanases for industrial applications, of which xylanase levels are generally much higher than those produced by yeast or bacteria (Haltrich et al., 1996). The production of xylanase by T. lanuginosus DSM 5826 in solid-state cultivation using corncobs as carbon source was studied with an initial water content of 70% (w/w) at 50 C. An activity of 336 700 nkat/g dry solid (20 161.7 U g 1) was obtained after 9 days of cultivation (Purkarthofer et al., 1993). Under the optimized condition, yield as high as 6193 U g 1 of carbon source (wheat straw) was obtained for T. aurantiacus under solid-state culture (SSC) (Kalogeris et al., 1998). Melanocarpus albomyces IIS-68 produced the maximum xylanase (7760 U g 1) when growth on wheat straw after 4 days of cultivation at 45 C (Narang et al., 2001). Xylanase activity of 5071 U g 1 was obtained in solid-state fermentation of rice straw by Aspergillus niger mutant under the optimized conditions (Park et al., 2002). Addition of the purified xylan in SSF increased xylanase production considerably in some fungi, however this practice also leads to the higher production cost. Xylanase production (9632 U g 1) of
Penicillium canescens 10-10c in SSF was obtained by using the mixture of wheat straw and xylan as substrate (Bakri et al., 2003). Under an optimized condition, T. lanuginosus D2W3 produced xylanase activity as high as 48 000 U g 1 of carbon source (sorghum straw) in SSF (Sonia et al., 2005), which was the highest titre of xylanase activity ever reported. There are some reports where bacterial strains have been used successfully for the production of xylanase by using SSF, but their xylanase levels are generally lower than those in fungi. A thermostable xylanase has been produced from Streptomyces sp. GG-11-3 in SSF (Beg et al., 2000). The maximum xylanase yield obtained using wheat bran and eucalyptus kraft pulp as the prime solid substrates were 2360 U g 1 and 1200 U g 1, respectively. Maximum production of xylanase (3644 U g 1) by Bacillus sp. JB-99 in SSF was obtained using rice bran as the prime substrate (Virupakshi et al., 2005). 3.8. Stability of the partially purified xylanase Ammonium sulphate fraction (20–60% saturation) of crude xylanase yielded 74% of the enzyme with 3.6-fold purification (data not shown). The stability of the partially purified xylanase at 65 C and 70 C at different pH (pH 5.0–10.0) is shown in Fig. 2. Xylanase exhibited remarkable stability and was completely stable at temperature up to 65 C for 60 min at pH 6.0–9.0, while at 70 C it showed complete stability at pH 7.0–8.0 for 30 min. Nevertheless, xylanase retained over 70% of its original activity for 2 h at pH 7.0–9.0 at 70 C while retained over 50% of its original activity for 4 h at pH 7.0–8.0. Similar stability has been reported for xylanases from thermophilic fungi, Melanocarpus albomyces, Talaromyces byssochlamydoides,
Table 1 Comparisons of xylanase production from different fungi grown on lignocellulosic materials Organism
Aspergillus niger KK2 mutant Melanocarpus albomyces IIS-68 Paecilomyces themophila J18 Penicillium canescens Sporotrichum thermophile ATCC 34628 Schizophyllum commune Streptomyces sp. Thermomyces lanuginosus DSM 5826 Thermomyces lanuginosus RT9 Thermomyces lanuginosus D2W3 Thermoascus aurantiacus BJT 190 Thermoascus aurantiacus IMI 216529 Thermoascus aurantiacus ATCC 204492 a b
Substrate
Initial moisture content (%)
Cultivation
28 C, 45 C, 50 C, 30 C,
Xylanase Productivity (U l 1 h 1)b
5071 7760 18 580 9632
14 790 11 317 16 451 5551
Park et al. (2002) Narang et al. (2001) This work Bakri et al. (2003)
Rice straw Wheat straw Wheat straw Wheat straw + xylan Wheat straw
65 86 83 83
Static, Static, Static, Static,
80
Static, 50 C, 6 days
320
444
Topakas et al. (2003)
Unbleached pulp Wheat bran Corncobs
75 75 70
Static, 30 C, 16 days Static, 37 C, 7 days Static, 50 C, 9 days
14 800
Wheat bran Sorghum straw Wheat bran
50 80 50
Static, 55 C, 7 days Static, 50 C, 6 days Static, 55 C, 7 days
22 700 2360 337 000 nkat/g (20179.6 U g 1) 1900 U ml 1 48 000 706 U ml 1
5650 33 333 2100
Haltrich et al. (1996) Beg et al. (2000) Purkarthofer et al. (1993) Alam et al. (1994) Sonia et al. (2005) Alam et al. (1994)
Wheat straw
80
Static, 50 C, 5 days
6193
10 322
Kalogeris et al. (1998)
Bagasse
81
Static, 45 C, 10 days
2700
2318
Souza et al. (1999)
Maximum activity. Volumetric productivity; calculation is based on the initial moisture content.
5 days 4 days 8 days 12 days
References
Activitya (U g 1)
28 100
100 90 80 70 60 50 40 30 20 10 0 0
(a)
60
120
180
240
300
360
Time (min)
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100 90 80 70 60 50 40 30 20 10 0
Relative residual xylanase (%)
Relative residual xylanase (%)
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0
(b)
60
120
180
240
300
360
Time (min)
Fig. 2. Stability of the partially purified xylanase at pH 5.0–10.0 and at 65 C (a) and 70 C (b) as a function time. The crude xylanase was produced by Paecilomyces themophila J18 in SSF for 8 days. Different pH values used: pH 5.0 (), pH 6.0 (j), pH 7.0 (m), pH 8.0 (), pH 9.0 (h), pH 10.0 (n).
Thermomyces lanuginosus (Jain, 1995; Maheshwari et al., 2000; Singh et al., 2003; Li et al., 2005). 4. Conclusions The present work has established the potential of the newly isolated thermophilic P. themophila J18 for xylanase production in SSF. High-level xylanase production by this strain in SSF suggests that P. themophila J18 is an interesting source of newer thermostable, cellulase-free xylanase and will have important economic advantages. The stability of xylanase at 65–70 C could be of considerable commercial interest for its potential applications. Further studies on purification and characterization of the xylanase from P. themophila J18 are in progress. Acknowledgements This work was financially supported by The National High Technology Research and Development Program of China (863 Program, No. 2003AA214020). The authors thank Dr. Szesze Tan for the critical editing of the manuscript. References Alam, M., Gomes, I., Mohiuddin, G., Hoq, M.M., 1994. Production and characterization of thermostable xylanases by Thermomyces lanuginosus and Thermoascus aurantiacus grown on lignocelluloses. Enzyme Microb. Technol. 16, 298–302. Bailey, M.J., Biely, P., Poutanen, K., 1992. Interlaboratory testing of methods for assay of xylanase activity. J. Biotechnol. 23, 257– 270. Bakri, Y., Jacques, P., Thonart, P., 2003. Xylanase production by Penicillium canescens 10-10c in solid-state fermentation. Appl. Biochem. Biotechnol. 105–108, 737–747. Beg, Q.K., Bhushan, B., Kapoor, M., Hoondal, G.S., 2000. Enhanced production of a thermostable xylanase from Streptomyces sp. QG-11-3 and its application in biobleaching of eucalyptus kraft pulp. Enzyme Microb. Technol. 27, 459–466. Brown, A.H.S., Smith, G., 1957. The genus Paecilomyces Bainier and its perfect stages Byssochlamys Westling. Trans. Br. Mycol. Soc. 40, 17– 89.
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