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Hydrolysis of bamboo cellulose and cellulase characteristics by Streptomyces griseoaurantiacus ZQBC691
Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 220–225
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Hydrolysis of bamboo cellulose and cellulase characteristics by Streptomyces griseoaurantiacus ZQBC691 Feng-Jen Chu a, Chi-Wen Lin a,*, Yet-Po I a, Chih-Hung Wu b, Ding-Hsuan Chen c a
Department of Safety, Health and Environmental Engineering, National Yunlin University of Science and Technology, Yunlin 64002, Taiwan Graduate School of Engineering Science and Technology, National Yunlin University of Science and Technology, Yunlin 64002, Taiwan c Capital Engineering Corporation, Kaohsiung 802, Taiwan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 April 2011 Received in revised form 7 August 2011 Accepted 16 August 2011 Available online 19 September 2011
This investigation studies the cellulolytic characteristics of a microbial isolate native to Taiwan. The bacterium was isolated from a compost of bamboo shoots in Taiwan and identified as Streptomyces griseoaurantiacus ZQBC691 using morphological criteria and 16S rRNA sequence. The strain ZQBC691 grew vigorously on amorphous cellulose (carboxymethylcellulose – CMC) and produced endoglucanase. Less endoglucanase was produced on crystalline cellulose (avicel). Under the optimal growth condition with a of 5.3, a shaking speed of 150 rpm, a culture temperature of 30 8C, and a CMC concentration of 15 g/L, the final concentration of reducing sugar reached 1.60 g/L. Additionally, the strain ZQBC691 can hydrolyze purified bamboo fiber to generate glucose, and ferment monosugars to produce products including lactic acid and acetic acid. Moreover, data obtained in an enzyme activity test indicated that at the optimal temperature of 50 8C strain ZQBC691 generated 34.13 IU/ml of endoglucanase on CMC, and 37.38 IU/ml at the optimal pH of 5. ß 2011 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Keywords: Bioenergy Bamboo cellulolytic microbes Cellulose Isolation Reducing sugar
1. Introduction Lignocellulosic biomass, a potential alternative bioenergy resource, is a renewable and globally abundant organic raw material [1–3]. The exact composition of lignocelluloses depends on their sources, but comprises mostly cellulose, hemicelluloses and lignin. Lignocelluloses are produced from a variety of hardwood, softwood, forestry residues, agricultural residues and municipal solid waste [4–6]. Therefore, they are the most promising feedstock for producing energy, food and chemicals, and they may be used in self-sustainable processes and products. Bamboos are useful woody plants that belong to the subfamily Bambusoideae of the family Gramineae. They are known to be the fastest growing woody plant with a growth rate of 30–100 cm per day [7]. Ninety percent of bamboo in Asia is found in Southeast Asia [8]. The largest number of species is found in China (626 species), followed by India (102 species) and Japan (84 species) [9]. Taiwan’s location is ideal for growing bamboo, which distributes in very large quantities throughout the island [10] and is regarded an important forestry product. Some species in Taiwan are fast growers with high yields and high cellulose contents. They are very
* Corresponding author. Tel.: +886 5 534 2601x4425; fax: +886 5 531 2069. E-mail address: [email protected] (C.-W. Lin).
adaptable and can be easily cultivated. In this study, we tested their potential as sources of mono-sugars. During the degradation of lignin by microorganisms, some of the normally insoluble lignin becomes soluble in an aqueous medium. This effect has been detected using ligninolytic fungi (such as Roussoella, Anthostomella, Podosporium or Phanerochaete chrysosporium species) [11–14] and Streptomyces species [15,16]. Streptomycetes are Gram-positive mycelium-forming soil bacteria, which can produce and excrete antibiotics and wood-degrading enzymes [15–20]. This investigation involves the isolation and characterization of cellulolytic streptomyces strains from compost of bamboo shoots. The biodegradation capabilities of the obtained strains were also tested using various cellulose materials. Furthermore, the amount of reducing sugars produced by these strains was estimated. 2. Materials and methods 2.1. Source of microorganisms and culture conditions Original lignocellulose-degrading mixed cultures were isolated from a compost of bamboo shoot using the method that was described by Haruta et al. [21]. The strain ZQBC691 was then picked from a native community that was grown on a modified [22] and chemically defined solid medium that had the following composition; carboxymethylcellulose (CMC), 10 g/L with other
1876-1070/$ – see front matter ß 2011 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jtice.2011.08.004
F.-J. Chu et al. / Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 220–225
nutrients – KH2PO4, 2.0 g/L; (NH4)2SO4, 1.4 g/L; MgSO47H2O, 0.3 g/L; CaCl2, 0.34 g/L; FeSO47H2O, 0.005 g/L; ZnSO47H2O, 0.014 g/L; MnSO4H2O, 0.016 g/L; CoCl2H2O, 0.002 g/L; peptone, 1.0 g/L; urea, 0.3 g/L, and agar powder, 20 g/L. After a period of acclimation, the mixed culture was plated on a mineral medium, solidified with agar using 1% CMC or purified bamboo cellulose as a sole carbon source, and incubated at 30 8C. Colonies, which appeared after five days, were picked and transferred to new plates. A preliminary analysis of cellulolytic activity was performed using the Congo red dye method. The CMC or purified bamboo fiber agar plates were incubated at 30 8C for two to four days to allow cellulose to be secreted. At the end of the incubation, the agar medium was flooded with an aqueous solution of Congo red (1% w/v) for 30–60 min. The Congo red solution was then poured off, and the plates were flooded with 1.0 M NaCl for 60 min [23,24]. The formation of a clear zone of hydrolysis revealed cellulose degradation. The ratio of the diameter of the clear zone to that of the colony was measured to identify the producer of cellulase with the highest cellulase activity. The largest ratio was assumed to indicate the highest activity [25]. The initial screening of selected microorganisms preliminarily revealed that all strains can decompose CMC. Based on the ratio of the diameter of the enzyme ring to the diameter of the microbe’s colony ring of 2.5, the strain ZQBC691 was a representative microorganism that was better able to degrade amorphous cellulose [25]. One of the cellulose-degrading pure cultures was tentatively identified as Streptomyces griseoaurantiacus ZQBC691, based on the sequence of a 1400 bp fragment from its 16S rDNA gene and on a comparison with sequences that are extracted from the GenBank database using the BLAST facility of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/ BLAST). The accession number of the closest match to these sequences in the GenBank, as identified using the BLAST search tool, is DSM40430, with percentage similarity and E values which determine the matching score of 99% and 0.0, respectively. The pure strain was inoculated in 250 ml screw cap amber glass bottles that contained 100 ml of a mineral salts medium and a carbon source (such as CMC or other cellulose), sealed with Teflon Mininert valves. It was incubated at 30 8C with constant shaking at 150 rpm. Following the incubation, the pure S. griseoaurantiacus ZQBC691 was kept frozen at 70 8C in a mineral salts medium that contained 50% glycerol (v/v).
221
were considered in the treatment of the bamboo powder. A 2 g dry basis biomass sample was used in each pretreatment experiment. In this investigation, the amount of dry feedstock was pretreated at various temperatures (40, 60, 80, and 100 8C) for various residence times (30, 60, 90, and 120 min) at various sulfuric acid concentrations (0.1, 0.15, 0.2, and 0.25 M). Once the selected temperature and time were reached, the system was left to stand for a few minutes to allow the temperature to drop below room temperature, and the solid and liquid fractions were separated by filtration (0.22 mm filters). The pretreated solid residues were neutralized and detoxified using CaCO3 to remove the inhibitors from the hydrolysates and to limit their activities [31]. The neutralized filtrate and dried residues were then used as carbon sources in experiments to identify the biochemical products that could be obtained from pretreated bamboo. The liquid fraction from that was obtained by pretreatment (pre-hydrolysate) was analyzed to determine the monomeric sugar contents using high-performance liquid chromatography (HPLC, Precision Instruments, France). The determined optimal conditions for pretreating bamboo using H2SO4 as an acidic agent were 2 g of bamboo powder, 40 ml of H2SO4 (0.2 M) and heating at 100 8C for 60 min. 2.4. Batch growth experiments on various types of cellulose Maximum reducing sugar production rates were determined using batch incubation studies with the addition of various celluloses (avicel, bamboo powder, CMC, salicin, and pure bamboo cellulose). Experiments were conducted in 250-ml amber bottles containing 100 ml of a mineral salt medium. The pure strain ZQBC691 was then introduced to the same final biomass concentration in each bottle. Avicel, bamboo powder, CMC and salicin were used at 15 g/L final concentration (pure bamboo cellulose at 10 g/L) for all batch experiments. The cultures were incubated at a constant temperature of 30 8C and a shaking speed of 150 rpm. Samples were regularly taken to monitor the maximum reducing sugar production. 2.5. Analysis of reducing sugar
Natural bamboo shoots were obtained from the Industrial Technology Research Institute (Hsinchu City, Taiwan). Bamboo shoots were then cut into pieces of length 2–3 cm and mechanically ground into a micro-sized powder (smaller than 0.841 mm; 20 mesh). Purified bamboo shoot pulp (pure bamboo cellulose) was obtained from Chung Rhy Special Paper Co (Nantou County, Taiwan). Other carbon sources, including carboxymethylcellulose, salicin, and avicel, were purchased from Showa (Tokyo, Japan) and Fluka (Buchs, Switzerland). The lignocellulosic substrate was the carbon source. All other used chemicals and reagents were high-grade products.
The amounts of reducing sugars including mono-sugars (i.e., glucose, xylose and arabinose) were estimated using 3,5-dinitrosalicylic acid (DNS) in a modified procedure of Miller [32]. A 1.5 ml sample was placed into each of a set of 1.5 mL test tubes, which were then centrifuged at 35,060 g (14,000 rpm) for 10 min. One milliliter of supernatant that contained reducing sugar was mixed with 1 ml of prepared DNS solution; the mixture was then incubated at 100 8C in a dry bath incubator (Major Science, Taiwan) for 10 min [33,34]. The reaction was terminated using prepared ice, which sufficiently cooled the mixed solution after several minutes. Three milliliter of distilled water was added to the mixture and the optical density of the solution was measured at 550 nm (UV) using a blank of distilled water. The concentration of reducing sugar was estimated as being equivalent to the concentration of released glucose, determined from calibration curve of concentration versus optical density.
2.3. Pretreatment of bamboo powder
2.6. Cellulolytic enzyme assays
The bamboo powder was pretreated to increase its contact surface area between microorganisms and lignocelluloses, increasing the efficiency of biodegradation of the lignocellulosic materials. The bamboo powder (<0.841 mm) was pretreated by a modified acid-pretreatment method [26,27]. Some pretreatment process parameters, including temperature, processing time, and acid concentration, affect biochemical products [28–30]. These factors
Carboxymethylcellulase (CMCase) activity was assayed using the method described by Miller et al. [35] with some modifications. Half of a milliliter of culture filtrate was added to the tube that contained 0.5 ml of 1% CMC, which was prepared in a 0.05 M sodium citrate buffer (pH 4.8) and incubated at 50 8C for 60 min in a dry bath. Appropriate controls were also run with the test. At the end of the incubation period, the tube was removed from the dry
2.2. Carbon sources and chemicals
F.-J. Chu et al. / Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 220–225
2.7. Analysis of sugars and organic acids Sugars (glucose, xylose) and organic acid (acetic acid, lactic acid) were identified using high-performance liquid chromatography with refractive index detection (HPLC-RID, Precision Instruments, France). Typically, the reacted solutions were poured into 1.5 ml plastic test tubes, and centrifuged at 35,060 g (14,000 rpm) for 10 min. The supernatant was sterilized and filtered through a 0.45 mm filter and then a 0.22 mm filter. 20– 40 mL of filtrate was extracted using a liquid-tight syringe and then injected into an HPLC-RID, which was equipped with a 300 7.8 mm MetaCarb H Plus column (Varian, USA). The column temperature was 50 8C; the mobile phase was 0.01N H2SO4; the flow rate was 0.4 ml/min, and the injected volume was 20 mL. The sugar and organic acid concentrations were estimated by extrapolating concentration versus area under the peak using standard curves which were obtained using HPLC. 3. Results and discussion 3.1. Pretreatment of bamboo powder using dilute sulfuric acid
12
2500
(a)
2000
9
1500 6 1000 3
500
0
Glucose, xylose and arabinose conc. (mg/L)
3.2. Endoglucanase activity Experiments were conducted on CMC substrates to test the amount of endoglucanase produced by S. griseoaurantiacus ZQBC691. The effects of temperature and pH in the liquid medium on the cellulase activity of S. griseoaurantiacus ZQBC691 were investigated. ZQBC691 gradually increased the rate of CMCase synthesis, and exhibited maximum activity at 96–120 h, after which time the activity of the enzyme slowly declined (data not shown). To elucidate the potential use of ZQBC691 as a cellulase producer, the effect of temperature on the production of crude cellulase was determined at various temperatures in the range 30– 100 8C at pH 5 (Fig. 2). ZQBC691 showed a best production for the amount of cellulase in term of CMCase activity between 40 and 60 8C with a maximum activity at 50 8C. The effect of pH on the activity of cellulose enzyme that was produced from ZQBC691 was examined at various values of pH from 3 to 10, as shown in Fig. 2. The enzyme exhibited high activity with an optimal pH of 5. The results indicate that ZQBC691 exhibited the highest CMCase activity (34.13–37.38 IU/ml) under optimal conditions of pH and 5 and 50 8C. Therefore, S. griseoaurantiacus ZQBC691 was preliminarily determined to be a mesospheric and slightly acid cellulosedecomposing microorganism. Table 1 compares the source of the
60
80
2500
(b) 12
2000
9
1500
6
1000
3
500
0
0 40
15
100
0 0.4
4000
(c) 9
3000
6
2000
3
1000
0
0 60
90
120
Pretreatment time (min)
Reducing sugar conc. (g/L)
12
30
2
4
Bamboo powder (g) Glucose, xylose and arabinose conc. (mg/L)
Reducing sugar conc. (g/L)
Temperature (ºC)
1
12
3000
(d) 9 2000 6 1000 3 0
Glucose, xylose and arabinose conc. (mg/L)
Reducing sugar conc. (g/L)
Bamboo powder was pretreated using dilute acid under various hydrolytic conditions to produce reducing sugar and monosaccharide. The amounts of reducing sugar and monosaccharide that were produced from the pre-hydrolyzed bamboo powder were recorded. As shown in Fig. 1, the amount of produced xylose generally increased with temperature, amount of bamboo powder, duration of pretreatment and concentration of sulfuric acid. However, the amounts of produced arabinose and glucose remained approximately constant as the magnitude of the aforementioned variables increased. As displayed in Fig. 1(a),
the highest concentration of reducing sugar was 8.67 g/L and the concentrations of produced xylose, arabinose and glucose followed the order, xylose (2,291 mg/L) > arabinose (273 mg/L) > glucose (27 mg/L) under conditions of 0.2 M H2SO4 solution, 2 g bamboo powder and 100 8C (hydrolysis temperature) for a reaction period of 1 h. Fig. 1(b) reveals that the amount of reducing sugar increased with the amount of bamboo powder. Moreover, the amounts of produced xylose, arabinose and glucose varied similarly, as shown in Fig. 1(a); the amount of xylose produced was the highest, while the amount of glucose produced was the lowest. The amount of reducing sugar produced varied little around 8.67–10.04 g/L when the pretreatment duration exceeded 60 min, as shown in Fig. 1(c). As presented in Fig. 1(d), the amount of reducing sugar produced increased with sulfuric acid concentration below 0.2 M, but decreased with increasing H2SO4 concentration above that value.
Glucose, xylose and arabinose conc. (mg/L)
bath, and the reaction was terminated after the assays by the DNS method [32]. One unit (IU) of CMCase activity corresponded to 1 mmol of glucose equivalent released per minute under the assay conditions [24]. Based on a calibration curve for glucose, one unit of enzyme activity was defined as the amount of enzyme that released 1 mmol of glucose per minute.
Reducing sugar conc. (g/L)
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0 0.1
0.15
0.2
0.25
H2SO4 conc. (M)
Fig. 1. Variations of amounts of produced reducing sugar and sugars under various conditions of hydrolysis of bamboo powder. (( ) reducing sugar; (&)glucose; (*) xylose; (~) arabinose).
F.-J. Chu et al. / Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 220–225
40
Table 2 Maximum amounts and rates of production of reducing sugar by Streptomyces griseoaurantiacus ZQBC691 in various conditions.
35
CMCase activity (IU/ml)
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Range
Items
30
CMC concentration (g/L)
25 20 15
Initial pH
5 Shaking speed (rpm)
2
4
6
8
10
12
80
100
120
pH Temperature (8C)
20
40
60
RSVmax (g/L/h)
0.325 0.530 0.787 1.100 1.902
0.0049 0.0095 0.0104 0.0257 0.0351
0.559 1.276 0.968 1.177 1.096 0.869
0.0036 0.0305 0.0141 0.0254 0.0237 0.0013
0 100 150
0.523 0.610 1.014
0.0038 0.0050 0.0086
20 30 45 60
1.148 1.173 0.962 0.962
0.0033 0.0192 0.0037 0.0004
4.35 5.32 6.07 6.96 7.51 9.59
10
0
RSmax (g/L)
2 4 8 10 15
Temperature (ºC) Fig. 2. Effects of temperature and pH on enzyme activity of cellulose that is produced by S. griseoaurantiacus ZQBC691. ((*) pH; (*) temperature).
RSmax: maximum reducing sugar; RSVmax: maximum reducing sugar production rate.
cellulolytic Streptomyces strain, the utilized carbon source by the strain, and endoglucanase (CMCase) activity that was calculated herein with the published data. The yield of CMCase that is produced by ZQBC691 on CMC was one quarter that of the CMCase that was produced by Streptomyces albolongus A5 (136.7 IU/ml). However, the CMCase yield of the strain ZQBC691 compares favorably with those of other strains.
culturing (Table 2). Therefore, ZQBC691 grows well on CMC in a neutral environment and less well in a low-acid or over-alkaline environment. Temperature is a well known factor that affects the growth of microorganisms. The initial pH of the medium was fixed at its optimal value, as determined in the preceding section, and the temperature was set to 20, 30, 45 and 60 8C. As shown in Table 2, ZQBC691 grew well on CMC in a mesophilic environment and less well in a more or less mesophilic environment. Reducing sugar concentrations clearly increased as the temperature increased. ZQBC691 was most effective at hydrolyzing CMC at a fixed temperature of 30 8C, with an RSmax and RSVmax of 1.173 g/L and 0.0192 g/L/h, respectively. The shaking speed of the orbital incubator could be controlled to enhance production of reducing sugar on CMC. The strain ZQBC691 grew well on CMC under both non-shaking and shaking conditions. Moreover, the amount of reducing sugar produced increased with shaking speed. As presented in Table 2, ZQBC691 was cultivated for a total of 160 h, and the RSmax and RSVmax increased from 0.109 g/L to 1.014 g/L and from 0.109 g/L/h to 0.0086 g/L/h, respectively, at a shaking speed of 150 rpm. The high viscosity of the CMC solution actually reduces the area of contact between the microorganism and the substrate. Shaking increases the number of opportunities for the microorganism to come into contact with its substrate; therefore, increasing the rates of hydrolysis of CMC and production of reducing sugar.
3.3. Effects of CMC, pH, temperature and shaking speed on production of reducing sugar Table 2 presents the maximum amounts and rates of production of reducing sugar by ZQBC691 in various conditions for each amount of CMC used. The maximum concentration of reducing sugar (RSmax) was 1.902 g/L, obtained with 15 g/L CMC, corresponding to a reducing sugar production rate (RSVmax) of 0.0351 g/L/h. Evidently, the concentration of reducing sugar that is produced by CMC hydrolysis slowly increased with the concentration of CMC in the liquid medium. According to the data in Table 2, the biodegradation rate of CMC was low, and was lowest for RSmax (0.559 and 0.869 g/L) and RSVmax (0.0036 and 0.0013 g/L/h) when S. griseoaurantiacus ZQBC691 was cultured in a low-acid (pH 4.35) or over-alkaline (pH 9.59) environment. When ZQBC691 grew on CMC at pH values from 5.32 to 7.51, the RSmax and RSVmax were in the range of 1.276–1.096 g/L and 0.0305–0.0237 g/L/h, respectively, over 75 h
Table 1 Comparison in source of Streptomyces strain and used carbon source between this study and the literatures. Strain Streptomyces ZQBC691 Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces
griseoaurantiacus ruber viridobrunneus SCPE-09 sp. sp. J2 albolongus A5
Streptomyces griseus B1
Source
Carbon
CMCase activity (IU/ml)
Temperature (8C)
pH
References
Bamboo shoot compost and bamboo cellulose (Taiwan) Marine sediments (Egypt) Soil sample (Brazil) Soil sample (Canada) Soil sample (Jordan) Saw dust (Bangladesh)
CMC, pure bamboo cellulose, bamboo powder, salicin, avicel CMC, rice straw Sugar cane bagasse, wheat bran CMC, filter paper, avicel CMC CMC, cellobiose octaacetate, filter paper, salicin Filter paper, hardwood powder (mango), softwood powder (deodar)
34.13–37.38
50
5
This study
25.6 2.0 11.8 0.432 136.7
40 50 50 60 35
6 4.9 6.5 7 6.5
[17] [16] [15] [20] [18]
0.44–3.72
37–42
5
[19]
Soil sample (India)
F.-J. Chu et al. / Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 220–225
2.0
3.4. Biodegradation of purified bamboo and its byproduct by S. griseoaurantiacus ZQBC691 The results of an investigation of purified bamboo (pure bamboo cellulose) demonstrate that S. griseoaurantiacus ZQBC691 grew well on pure bamboo cellulose and also formed such byproducts as lactic acid and acetic acid. Fig. 3 reveals that the aforementioned byproducts accumulated over time, reducing the pH value of the reacted solution. The amounts of those byproducts gradually increased with the original bamboo concentration. These products were produced in large amounts at a pure bamboo cellulose concentration of 2 g/L and in the largest amounts when 10 g/L pure bamboo cellulose was used (data not shown). The highest concentrations of produced lactic acid and acetic acid were 238 mg/L and 94 mg/L, formed after 144 and 100 h, respectively. The pH of the reacted solution substantially fell from 5.63 to 5.01 during 144 h of fermentation. Reducing sugar and mono-sugar such as glucose also accumulated as products of hydrolysis and the fermentation process. When 10 g/L pure bamboo cellulose was used, the amounts of reducing sugar and glucose reached their highest values of 0.563 and 0.260 g/L at 121 h of hydrolysis. Thereafter, their amounts gradually decreased, revealing that ZQBC691 used own products as a source of carbon. Furthermore, the amounts of byproducts gradually increased from 121 h of fermentation to 144 h. The results indicate that ZQBC691 can use mono-sugars in the reducing sugar to generate byproducts. As the data also show, hydrolysis of the pure bamboo cellulose yielded glucose. ZQBC691 also produced b-glucosidase. 3.5. Growth of ZQBC691 on various types of cellulose To evaluate the ability of cellulose by S. griseoaurantiacus ZQBC691 to degrade various types of cellulose, CMC, pure bamboo cellulose, bamboo powder, salicin and avicel were individually used as the sole carbon source at concentrations from 10 to 15 g/L. Fig. 4 indicates that soluble CMC at 15 g/L was associated with the maximum reducing sugar rate (1.691 g/L/h), while crystalline cellulose (pure bamboo cellulose, salicin, bamboo powder and avicel) yielded lower rates. These results indicate that ZQBC691 exhibited a weak ability to grow on crystalline cellulose, including avicel, salicin, pure bamboo cellulose and bamboo powder. Fig. 4 shows that RSVmax reached approximately 0.317 g/L/h at a salicin concentration of 15 g/L, revealing that S. griseoaurantiacus ZQBC691 can produce b-D-glucosidase, which hydrolyzes salicin and releases glucose as a reducing sugar.
8
600
6 400
pH
Concentration (mg/L)
500
300 4 200 100
2
0 0
20
40
60
80
100
120
140
160
Time (h) Fig. 3. Biodegradation of pure bamboo cellulose and its byproducts. ((*) reducing sugar; (*) glucose; (!) lactic acid; (~) acetic acid; (&) pH; pure bamboo cellulose concentration was 10 g/L).
Maximum reducing sugar rate (g/L/h)
224
1.5
1.0
0.5
0.0 CMC
Pure bamboo cellulose
Salicin
Bamboo powder
Avicel
Type of cellulose Fig. 4. Hydrolysis of various types of cellulose by S. griseoaurantiacus ZQBC691. CMC, salicin, bamboo powder and avicel concentrations were 15 g/L each; Pure bamboo cellulose concentration was 10 g/L.
ZQBC691 can use avicel to produce exoglucanases, thereby converting crystalline cellulose into reducing sugar. However, avicel was degraded only to a limited extent, as indicated by the low RSVmax of around 0.051 g/L/h (Fig. 4). ZQBC691 converts natural crystalline celluloses, such as pure bamboo cellulose and bamboo powder, into a reducing sugar and glucose, which result is attributable to the release of endoglucanases to the amorphous regions of cellulose and the subsequent conversion of b-Dglucosidases to glucose. Therefore, the strain produced enzymes (exoglucanases, endoglucanases and b-D-glucosidases) that hydrolyzed the natural lignocelluloses, including pure bamboo cellulose and bamboo powder, to produce reducing sugars and glucose. Moreover, the maximum reducing sugar production rate was low (0.136 g/L/h) using fresh bamboo powder, but was significantly increased to 0.468 g/L/h using pretreated bamboo (pure bamboo cellulose). The elevated reducing sugar rate was believed to be due to a reduced amount of lignin after the pretreatment. 4. Conclusions The novel strain S. griseoaurantiacus ZQBC691 was isolated and screened from a compost of bamboo waste at 30 8C. S. griseoaurantiacus ZQBC691 was a mesophilic, aerobic, and slightly acid cellulose-degrading microorganism. The optimal conditions under which the strain produced the most reducing sugar were an initial pH of 5.3, a temperature of 30 8C, a shaking-speed of 150 rpm and the use of 15 g/L CMC. ZQBC691 acted on CMC to generate endoglucanase; the optimal conditions for this process were pH 5 (37.38 IU/ml) at 50 8C (34.13 IU/ ml). ZQBC691 grew well on amorphous cellulose (CMC) but less well on crystalline cellulose (avicel, natural bamboo). However, ZQBC691 hydrolyzed pure bamboo cellulose; produced a monosugar, such as glucose, and fermented reducing sugar to form a biochemical product such as lactic acid or acetic acid. The results suggest that extensive research should be performed into the generation of ethanol by this strain. The use of a co-culture system is one means by which ZQBC691 can combine with other microbes (Zomomonas mobilis) that ferment reducing sugar (glucose) in a single step to convert purified-bamboo materials into ethanol.
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Acknowledgements The authors would like to thank the Industrial Technology Research Institute of the Republic of China, Taiwan, for financially supporting this research. Ted Knoy is appreciated for his editorial assistance. References [1] Sims R. Biomass and resources bioenergy options for a cleaner environment in developed and developing countries. London, UK: Elsevier Science; 2003. [2] Lynd LR, Laser MS, Bransby D, Dale BE, Davison B, Hamilton R, et al. How biotech can transform biofuels. Nat Biotechnol 2008;26:169–72. [3] Gamage J, Lam H, Zhang Z. Bioethanol production from lignocellulosic biomass: a review. J Biobased Mater Bioenergy 2010;4:3–11. [4] Hendriks ATWM, Zeeman G. Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresour Technol 2009;100:10–8. [5] Wyman CE. Ethanol from lignocellulosic biomass: technology, economics, and opportunities. Bioresour Technol 1994;50:3–16. [6] Li X, Pan X. Hydrogels based on hemicellulose and lignin from lignocellulose biorefinery: a mini-review. J Biobased Mater Bioenergy 2010;4:289–97. [7] Fu J. Moso bamboo in China. ABS Mag 2000;21:12–7. [8] Scurlock JMO, Dayton DC, Hames B. Bamboo: an overlooked biomass resource. Biomass Bioenergy 2000;19:229–44. [9] Bystriakova N, Kapos V, Stapleton C, Lysenko I. Bamboo biodiversity. UNEP World Conservation Monitoring Centre and International Network for Bamboo and Rattan; 2003. [10] Kigomo B. Guidelines for growing bamboo. Kenya Forestry Research Institute; 2007. pp. 1–41. [11] Sin MKW, Hyde KD, Pointing SB. Comparative enzyme production by fungi from diverse lignocellulosic substrates. J Microbiol 2002;40:241–4. [12] Zhou D, Hyde KD. Fungal succession on bamboo in Hong Kong. Fungal Diversity 2002;10:213–27. [13] Tanaka K, Hirayama K, Yonezawa H, Hatakeyama S, Harada Y, Sano T, et al. Molecular taxonomy of bambusicolous fungi: Tetraplosphaeriaceae, a new pleosporalean family with Tetraploa-like anamorphs. Stud Mycol 2009;64: 175–209. [14] Singh D, Zeng J, Laskar DD, Deobald L, Hiscox WC, Chen S. Investigation of wheat straw biodegradation by Phanerochaete chrysosporium. Biomass Bioenergy 2011;35:1030–40. [15] Antai SP, Crawford DL. Degradation of softwood, hardwood, and grass lignocelluloses by two Streptomyces strainst. Appl Environ Microbiol 1981;42:378– 80. [16] Vinha F, Gravina-Oliveira M, Franco M, Macrae A, Silva Bon E, Nascimento R, et al. Cellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substrate. Appl Biochem Biotechnol 2010;1–12.
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[17] El-Sersy NA, Abd-Elnaby H, Abou-Elela GM, Ibrahim HAH, El-Toukhy NMK. Optimization, economization and characterization of cellulase produced by marine Streptomyces ruber. Afr J Biotechnol 2010;9:6355–64. [18] Das SK, Alam MZ, Manchur MA, Anwar MN. Effect of nutrients and cellobiose octaacetate on cellulolytic enzyme productions by Streptomyces albolongus. Bangladesh J Microbiol 2007;24:70–2. [19] Arora A, Nain L, Gupta JK. Solid-state fermentation of wood residues by Streptomyces griseus B1, a soil isolate, and solubilization of lignins. World J Microbiol Biotechnol 2005;21:303–8. [20] Jaradat Z, Dawagreh A, Ababneh Q, Saadoun I. Influence of culture conditions on cellulase production by Streptomyces spstrain J2). Jordan J Biol Sci 2008;1:141–6. [21] Haruta S, Cui Z, Huang Z, Li M, Ishii M, Igarashi Y. Construction of a stable microbial community with high cellulose-degradation ability. Appl Microbiol Biotechnol 2002;59:529–34. [22] Mandels M, Reese ET. Induction of cellulase in Trichoderma viride as influenced by carbon sources and metals. J Bacteriol 1957;73:269–78. [23] Teather RM, Wood PJ. Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microbiol 1982;43:777–80. [24] Ghose TK. Measurement of cellulase activities 1987;59:257–68. [25] Kim Y-S, Jung H-C, Pan J-G. Bacterial cell surface display of an enzyme library for selective screening of improved cellulase variants. Appl Environ Microbiol 2000;66:788–93. [26] Leenakul W, Tippayawong N. Dilute acid pretreatment of bamboo for fermentable sugar production. J Sustain Energy Environ 2010;1:117–20. [27] Zhuang XS, Qi W, Yuan ZH, Wang Q, Tan XS. An investigation of cellulose hydrolysis under extremely low acids. J Biobased Mater Bioenergy 2010;4: 35–9. [28] Kumar P, Barrett DM, Delwiche MJ, Stroeve P. Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind Eng Chem Res 2009;48:3713–29. [29] Yang B, Wyman CE. Pretreatment: the key to unlocking low-cost cellulosic ethanol. Biofuels Bioprod Biorefining 2008;2:26–40. [30] Lin C-W, Wu C-H, Tran D-T, Shih M-C, Li W-H, Wu C-F. Mixed culture fermentation from lignocellulosic materials using thermophilic lignocellulose-degrading anaerobes. Process Biochem 2011;46:489–93. [31] Torii N, Okai A, Shibuki K, Aida TM, Watanabe M, Ishihara M, et al. Production of D-glucose from pseudo paper sludge with hydrothermal treatment. Biomass Bioenergy 2010;34:844–50. [32] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959;31:426–8. [33] Breuil C, Saddler JN. Comparison of the 3,5-dinitrosalicylic acid and NelsonSomogyi methods of assaying for reducing sugars and determining cellulase activity. Enzyme Microb Technol 1985;7:327–32. [34] Lindsay H. A colorimetric estimation of reducing sugars in potatoes with 3,5dinitrosalicylic acid. Potato Res 1973;16:176–9. [35] Miller GL, Blum R, Glennon WE, Burton AL. Measurement of carboxymethylcellulase activity. Anal Biochem 1960;1:127–32.
Contents lists available at SciVerse ScienceDirect
Journal of the Taiwan Institute of Chemical Engineers journal homepage: www.elsevier.com/locate/jtice
Hydrolysis of bamboo cellulose and cellulase characteristics by Streptomyces griseoaurantiacus ZQBC691 Feng-Jen Chu a, Chi-Wen Lin a,*, Yet-Po I a, Chih-Hung Wu b, Ding-Hsuan Chen c a
Department of Safety, Health and Environmental Engineering, National Yunlin University of Science and Technology, Yunlin 64002, Taiwan Graduate School of Engineering Science and Technology, National Yunlin University of Science and Technology, Yunlin 64002, Taiwan c Capital Engineering Corporation, Kaohsiung 802, Taiwan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 April 2011 Received in revised form 7 August 2011 Accepted 16 August 2011 Available online 19 September 2011
This investigation studies the cellulolytic characteristics of a microbial isolate native to Taiwan. The bacterium was isolated from a compost of bamboo shoots in Taiwan and identified as Streptomyces griseoaurantiacus ZQBC691 using morphological criteria and 16S rRNA sequence. The strain ZQBC691 grew vigorously on amorphous cellulose (carboxymethylcellulose – CMC) and produced endoglucanase. Less endoglucanase was produced on crystalline cellulose (avicel). Under the optimal growth condition with a of 5.3, a shaking speed of 150 rpm, a culture temperature of 30 8C, and a CMC concentration of 15 g/L, the final concentration of reducing sugar reached 1.60 g/L. Additionally, the strain ZQBC691 can hydrolyze purified bamboo fiber to generate glucose, and ferment monosugars to produce products including lactic acid and acetic acid. Moreover, data obtained in an enzyme activity test indicated that at the optimal temperature of 50 8C strain ZQBC691 generated 34.13 IU/ml of endoglucanase on CMC, and 37.38 IU/ml at the optimal pH of 5. ß 2011 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Keywords: Bioenergy Bamboo cellulolytic microbes Cellulose Isolation Reducing sugar
1. Introduction Lignocellulosic biomass, a potential alternative bioenergy resource, is a renewable and globally abundant organic raw material [1–3]. The exact composition of lignocelluloses depends on their sources, but comprises mostly cellulose, hemicelluloses and lignin. Lignocelluloses are produced from a variety of hardwood, softwood, forestry residues, agricultural residues and municipal solid waste [4–6]. Therefore, they are the most promising feedstock for producing energy, food and chemicals, and they may be used in self-sustainable processes and products. Bamboos are useful woody plants that belong to the subfamily Bambusoideae of the family Gramineae. They are known to be the fastest growing woody plant with a growth rate of 30–100 cm per day [7]. Ninety percent of bamboo in Asia is found in Southeast Asia [8]. The largest number of species is found in China (626 species), followed by India (102 species) and Japan (84 species) [9]. Taiwan’s location is ideal for growing bamboo, which distributes in very large quantities throughout the island [10] and is regarded an important forestry product. Some species in Taiwan are fast growers with high yields and high cellulose contents. They are very
* Corresponding author. Tel.: +886 5 534 2601x4425; fax: +886 5 531 2069. E-mail address: [email protected] (C.-W. Lin).
adaptable and can be easily cultivated. In this study, we tested their potential as sources of mono-sugars. During the degradation of lignin by microorganisms, some of the normally insoluble lignin becomes soluble in an aqueous medium. This effect has been detected using ligninolytic fungi (such as Roussoella, Anthostomella, Podosporium or Phanerochaete chrysosporium species) [11–14] and Streptomyces species [15,16]. Streptomycetes are Gram-positive mycelium-forming soil bacteria, which can produce and excrete antibiotics and wood-degrading enzymes [15–20]. This investigation involves the isolation and characterization of cellulolytic streptomyces strains from compost of bamboo shoots. The biodegradation capabilities of the obtained strains were also tested using various cellulose materials. Furthermore, the amount of reducing sugars produced by these strains was estimated. 2. Materials and methods 2.1. Source of microorganisms and culture conditions Original lignocellulose-degrading mixed cultures were isolated from a compost of bamboo shoot using the method that was described by Haruta et al. [21]. The strain ZQBC691 was then picked from a native community that was grown on a modified [22] and chemically defined solid medium that had the following composition; carboxymethylcellulose (CMC), 10 g/L with other
1876-1070/$ – see front matter ß 2011 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jtice.2011.08.004
F.-J. Chu et al. / Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 220–225
nutrients – KH2PO4, 2.0 g/L; (NH4)2SO4, 1.4 g/L; MgSO47H2O, 0.3 g/L; CaCl2, 0.34 g/L; FeSO47H2O, 0.005 g/L; ZnSO47H2O, 0.014 g/L; MnSO4H2O, 0.016 g/L; CoCl2H2O, 0.002 g/L; peptone, 1.0 g/L; urea, 0.3 g/L, and agar powder, 20 g/L. After a period of acclimation, the mixed culture was plated on a mineral medium, solidified with agar using 1% CMC or purified bamboo cellulose as a sole carbon source, and incubated at 30 8C. Colonies, which appeared after five days, were picked and transferred to new plates. A preliminary analysis of cellulolytic activity was performed using the Congo red dye method. The CMC or purified bamboo fiber agar plates were incubated at 30 8C for two to four days to allow cellulose to be secreted. At the end of the incubation, the agar medium was flooded with an aqueous solution of Congo red (1% w/v) for 30–60 min. The Congo red solution was then poured off, and the plates were flooded with 1.0 M NaCl for 60 min [23,24]. The formation of a clear zone of hydrolysis revealed cellulose degradation. The ratio of the diameter of the clear zone to that of the colony was measured to identify the producer of cellulase with the highest cellulase activity. The largest ratio was assumed to indicate the highest activity [25]. The initial screening of selected microorganisms preliminarily revealed that all strains can decompose CMC. Based on the ratio of the diameter of the enzyme ring to the diameter of the microbe’s colony ring of 2.5, the strain ZQBC691 was a representative microorganism that was better able to degrade amorphous cellulose [25]. One of the cellulose-degrading pure cultures was tentatively identified as Streptomyces griseoaurantiacus ZQBC691, based on the sequence of a 1400 bp fragment from its 16S rDNA gene and on a comparison with sequences that are extracted from the GenBank database using the BLAST facility of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/ BLAST). The accession number of the closest match to these sequences in the GenBank, as identified using the BLAST search tool, is DSM40430, with percentage similarity and E values which determine the matching score of 99% and 0.0, respectively. The pure strain was inoculated in 250 ml screw cap amber glass bottles that contained 100 ml of a mineral salts medium and a carbon source (such as CMC or other cellulose), sealed with Teflon Mininert valves. It was incubated at 30 8C with constant shaking at 150 rpm. Following the incubation, the pure S. griseoaurantiacus ZQBC691 was kept frozen at 70 8C in a mineral salts medium that contained 50% glycerol (v/v).
221
were considered in the treatment of the bamboo powder. A 2 g dry basis biomass sample was used in each pretreatment experiment. In this investigation, the amount of dry feedstock was pretreated at various temperatures (40, 60, 80, and 100 8C) for various residence times (30, 60, 90, and 120 min) at various sulfuric acid concentrations (0.1, 0.15, 0.2, and 0.25 M). Once the selected temperature and time were reached, the system was left to stand for a few minutes to allow the temperature to drop below room temperature, and the solid and liquid fractions were separated by filtration (0.22 mm filters). The pretreated solid residues were neutralized and detoxified using CaCO3 to remove the inhibitors from the hydrolysates and to limit their activities [31]. The neutralized filtrate and dried residues were then used as carbon sources in experiments to identify the biochemical products that could be obtained from pretreated bamboo. The liquid fraction from that was obtained by pretreatment (pre-hydrolysate) was analyzed to determine the monomeric sugar contents using high-performance liquid chromatography (HPLC, Precision Instruments, France). The determined optimal conditions for pretreating bamboo using H2SO4 as an acidic agent were 2 g of bamboo powder, 40 ml of H2SO4 (0.2 M) and heating at 100 8C for 60 min. 2.4. Batch growth experiments on various types of cellulose Maximum reducing sugar production rates were determined using batch incubation studies with the addition of various celluloses (avicel, bamboo powder, CMC, salicin, and pure bamboo cellulose). Experiments were conducted in 250-ml amber bottles containing 100 ml of a mineral salt medium. The pure strain ZQBC691 was then introduced to the same final biomass concentration in each bottle. Avicel, bamboo powder, CMC and salicin were used at 15 g/L final concentration (pure bamboo cellulose at 10 g/L) for all batch experiments. The cultures were incubated at a constant temperature of 30 8C and a shaking speed of 150 rpm. Samples were regularly taken to monitor the maximum reducing sugar production. 2.5. Analysis of reducing sugar
Natural bamboo shoots were obtained from the Industrial Technology Research Institute (Hsinchu City, Taiwan). Bamboo shoots were then cut into pieces of length 2–3 cm and mechanically ground into a micro-sized powder (smaller than 0.841 mm; 20 mesh). Purified bamboo shoot pulp (pure bamboo cellulose) was obtained from Chung Rhy Special Paper Co (Nantou County, Taiwan). Other carbon sources, including carboxymethylcellulose, salicin, and avicel, were purchased from Showa (Tokyo, Japan) and Fluka (Buchs, Switzerland). The lignocellulosic substrate was the carbon source. All other used chemicals and reagents were high-grade products.
The amounts of reducing sugars including mono-sugars (i.e., glucose, xylose and arabinose) were estimated using 3,5-dinitrosalicylic acid (DNS) in a modified procedure of Miller [32]. A 1.5 ml sample was placed into each of a set of 1.5 mL test tubes, which were then centrifuged at 35,060 g (14,000 rpm) for 10 min. One milliliter of supernatant that contained reducing sugar was mixed with 1 ml of prepared DNS solution; the mixture was then incubated at 100 8C in a dry bath incubator (Major Science, Taiwan) for 10 min [33,34]. The reaction was terminated using prepared ice, which sufficiently cooled the mixed solution after several minutes. Three milliliter of distilled water was added to the mixture and the optical density of the solution was measured at 550 nm (UV) using a blank of distilled water. The concentration of reducing sugar was estimated as being equivalent to the concentration of released glucose, determined from calibration curve of concentration versus optical density.
2.3. Pretreatment of bamboo powder
2.6. Cellulolytic enzyme assays
The bamboo powder was pretreated to increase its contact surface area between microorganisms and lignocelluloses, increasing the efficiency of biodegradation of the lignocellulosic materials. The bamboo powder (<0.841 mm) was pretreated by a modified acid-pretreatment method [26,27]. Some pretreatment process parameters, including temperature, processing time, and acid concentration, affect biochemical products [28–30]. These factors
Carboxymethylcellulase (CMCase) activity was assayed using the method described by Miller et al. [35] with some modifications. Half of a milliliter of culture filtrate was added to the tube that contained 0.5 ml of 1% CMC, which was prepared in a 0.05 M sodium citrate buffer (pH 4.8) and incubated at 50 8C for 60 min in a dry bath. Appropriate controls were also run with the test. At the end of the incubation period, the tube was removed from the dry
2.2. Carbon sources and chemicals
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2.7. Analysis of sugars and organic acids Sugars (glucose, xylose) and organic acid (acetic acid, lactic acid) were identified using high-performance liquid chromatography with refractive index detection (HPLC-RID, Precision Instruments, France). Typically, the reacted solutions were poured into 1.5 ml plastic test tubes, and centrifuged at 35,060 g (14,000 rpm) for 10 min. The supernatant was sterilized and filtered through a 0.45 mm filter and then a 0.22 mm filter. 20– 40 mL of filtrate was extracted using a liquid-tight syringe and then injected into an HPLC-RID, which was equipped with a 300 7.8 mm MetaCarb H Plus column (Varian, USA). The column temperature was 50 8C; the mobile phase was 0.01N H2SO4; the flow rate was 0.4 ml/min, and the injected volume was 20 mL. The sugar and organic acid concentrations were estimated by extrapolating concentration versus area under the peak using standard curves which were obtained using HPLC. 3. Results and discussion 3.1. Pretreatment of bamboo powder using dilute sulfuric acid
12
2500
(a)
2000
9
1500 6 1000 3
500
0
Glucose, xylose and arabinose conc. (mg/L)
3.2. Endoglucanase activity Experiments were conducted on CMC substrates to test the amount of endoglucanase produced by S. griseoaurantiacus ZQBC691. The effects of temperature and pH in the liquid medium on the cellulase activity of S. griseoaurantiacus ZQBC691 were investigated. ZQBC691 gradually increased the rate of CMCase synthesis, and exhibited maximum activity at 96–120 h, after which time the activity of the enzyme slowly declined (data not shown). To elucidate the potential use of ZQBC691 as a cellulase producer, the effect of temperature on the production of crude cellulase was determined at various temperatures in the range 30– 100 8C at pH 5 (Fig. 2). ZQBC691 showed a best production for the amount of cellulase in term of CMCase activity between 40 and 60 8C with a maximum activity at 50 8C. The effect of pH on the activity of cellulose enzyme that was produced from ZQBC691 was examined at various values of pH from 3 to 10, as shown in Fig. 2. The enzyme exhibited high activity with an optimal pH of 5. The results indicate that ZQBC691 exhibited the highest CMCase activity (34.13–37.38 IU/ml) under optimal conditions of pH and 5 and 50 8C. Therefore, S. griseoaurantiacus ZQBC691 was preliminarily determined to be a mesospheric and slightly acid cellulosedecomposing microorganism. Table 1 compares the source of the
60
80
2500
(b) 12
2000
9
1500
6
1000
3
500
0
0 40
15
100
0 0.4
4000
(c) 9
3000
6
2000
3
1000
0
0 60
90
120
Pretreatment time (min)
Reducing sugar conc. (g/L)
12
30
2
4
Bamboo powder (g) Glucose, xylose and arabinose conc. (mg/L)
Reducing sugar conc. (g/L)
Temperature (ºC)
1
12
3000
(d) 9 2000 6 1000 3 0
Glucose, xylose and arabinose conc. (mg/L)
Reducing sugar conc. (g/L)
Bamboo powder was pretreated using dilute acid under various hydrolytic conditions to produce reducing sugar and monosaccharide. The amounts of reducing sugar and monosaccharide that were produced from the pre-hydrolyzed bamboo powder were recorded. As shown in Fig. 1, the amount of produced xylose generally increased with temperature, amount of bamboo powder, duration of pretreatment and concentration of sulfuric acid. However, the amounts of produced arabinose and glucose remained approximately constant as the magnitude of the aforementioned variables increased. As displayed in Fig. 1(a),
the highest concentration of reducing sugar was 8.67 g/L and the concentrations of produced xylose, arabinose and glucose followed the order, xylose (2,291 mg/L) > arabinose (273 mg/L) > glucose (27 mg/L) under conditions of 0.2 M H2SO4 solution, 2 g bamboo powder and 100 8C (hydrolysis temperature) for a reaction period of 1 h. Fig. 1(b) reveals that the amount of reducing sugar increased with the amount of bamboo powder. Moreover, the amounts of produced xylose, arabinose and glucose varied similarly, as shown in Fig. 1(a); the amount of xylose produced was the highest, while the amount of glucose produced was the lowest. The amount of reducing sugar produced varied little around 8.67–10.04 g/L when the pretreatment duration exceeded 60 min, as shown in Fig. 1(c). As presented in Fig. 1(d), the amount of reducing sugar produced increased with sulfuric acid concentration below 0.2 M, but decreased with increasing H2SO4 concentration above that value.
Glucose, xylose and arabinose conc. (mg/L)
bath, and the reaction was terminated after the assays by the DNS method [32]. One unit (IU) of CMCase activity corresponded to 1 mmol of glucose equivalent released per minute under the assay conditions [24]. Based on a calibration curve for glucose, one unit of enzyme activity was defined as the amount of enzyme that released 1 mmol of glucose per minute.
Reducing sugar conc. (g/L)
222
0 0.1
0.15
0.2
0.25
H2SO4 conc. (M)
Fig. 1. Variations of amounts of produced reducing sugar and sugars under various conditions of hydrolysis of bamboo powder. (( ) reducing sugar; (&)glucose; (*) xylose; (~) arabinose).
F.-J. Chu et al. / Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 220–225
40
Table 2 Maximum amounts and rates of production of reducing sugar by Streptomyces griseoaurantiacus ZQBC691 in various conditions.
35
CMCase activity (IU/ml)
223
Range
Items
30
CMC concentration (g/L)
25 20 15
Initial pH
5 Shaking speed (rpm)
2
4
6
8
10
12
80
100
120
pH Temperature (8C)
20
40
60
RSVmax (g/L/h)
0.325 0.530 0.787 1.100 1.902
0.0049 0.0095 0.0104 0.0257 0.0351
0.559 1.276 0.968 1.177 1.096 0.869
0.0036 0.0305 0.0141 0.0254 0.0237 0.0013
0 100 150
0.523 0.610 1.014
0.0038 0.0050 0.0086
20 30 45 60
1.148 1.173 0.962 0.962
0.0033 0.0192 0.0037 0.0004
4.35 5.32 6.07 6.96 7.51 9.59
10
0
RSmax (g/L)
2 4 8 10 15
Temperature (ºC) Fig. 2. Effects of temperature and pH on enzyme activity of cellulose that is produced by S. griseoaurantiacus ZQBC691. ((*) pH; (*) temperature).
RSmax: maximum reducing sugar; RSVmax: maximum reducing sugar production rate.
cellulolytic Streptomyces strain, the utilized carbon source by the strain, and endoglucanase (CMCase) activity that was calculated herein with the published data. The yield of CMCase that is produced by ZQBC691 on CMC was one quarter that of the CMCase that was produced by Streptomyces albolongus A5 (136.7 IU/ml). However, the CMCase yield of the strain ZQBC691 compares favorably with those of other strains.
culturing (Table 2). Therefore, ZQBC691 grows well on CMC in a neutral environment and less well in a low-acid or over-alkaline environment. Temperature is a well known factor that affects the growth of microorganisms. The initial pH of the medium was fixed at its optimal value, as determined in the preceding section, and the temperature was set to 20, 30, 45 and 60 8C. As shown in Table 2, ZQBC691 grew well on CMC in a mesophilic environment and less well in a more or less mesophilic environment. Reducing sugar concentrations clearly increased as the temperature increased. ZQBC691 was most effective at hydrolyzing CMC at a fixed temperature of 30 8C, with an RSmax and RSVmax of 1.173 g/L and 0.0192 g/L/h, respectively. The shaking speed of the orbital incubator could be controlled to enhance production of reducing sugar on CMC. The strain ZQBC691 grew well on CMC under both non-shaking and shaking conditions. Moreover, the amount of reducing sugar produced increased with shaking speed. As presented in Table 2, ZQBC691 was cultivated for a total of 160 h, and the RSmax and RSVmax increased from 0.109 g/L to 1.014 g/L and from 0.109 g/L/h to 0.0086 g/L/h, respectively, at a shaking speed of 150 rpm. The high viscosity of the CMC solution actually reduces the area of contact between the microorganism and the substrate. Shaking increases the number of opportunities for the microorganism to come into contact with its substrate; therefore, increasing the rates of hydrolysis of CMC and production of reducing sugar.
3.3. Effects of CMC, pH, temperature and shaking speed on production of reducing sugar Table 2 presents the maximum amounts and rates of production of reducing sugar by ZQBC691 in various conditions for each amount of CMC used. The maximum concentration of reducing sugar (RSmax) was 1.902 g/L, obtained with 15 g/L CMC, corresponding to a reducing sugar production rate (RSVmax) of 0.0351 g/L/h. Evidently, the concentration of reducing sugar that is produced by CMC hydrolysis slowly increased with the concentration of CMC in the liquid medium. According to the data in Table 2, the biodegradation rate of CMC was low, and was lowest for RSmax (0.559 and 0.869 g/L) and RSVmax (0.0036 and 0.0013 g/L/h) when S. griseoaurantiacus ZQBC691 was cultured in a low-acid (pH 4.35) or over-alkaline (pH 9.59) environment. When ZQBC691 grew on CMC at pH values from 5.32 to 7.51, the RSmax and RSVmax were in the range of 1.276–1.096 g/L and 0.0305–0.0237 g/L/h, respectively, over 75 h
Table 1 Comparison in source of Streptomyces strain and used carbon source between this study and the literatures. Strain Streptomyces ZQBC691 Streptomyces Streptomyces Streptomyces Streptomyces Streptomyces
griseoaurantiacus ruber viridobrunneus SCPE-09 sp. sp. J2 albolongus A5
Streptomyces griseus B1
Source
Carbon
CMCase activity (IU/ml)
Temperature (8C)
pH
References
Bamboo shoot compost and bamboo cellulose (Taiwan) Marine sediments (Egypt) Soil sample (Brazil) Soil sample (Canada) Soil sample (Jordan) Saw dust (Bangladesh)
CMC, pure bamboo cellulose, bamboo powder, salicin, avicel CMC, rice straw Sugar cane bagasse, wheat bran CMC, filter paper, avicel CMC CMC, cellobiose octaacetate, filter paper, salicin Filter paper, hardwood powder (mango), softwood powder (deodar)
34.13–37.38
50
5
This study
25.6 2.0 11.8 0.432 136.7
40 50 50 60 35
6 4.9 6.5 7 6.5
[17] [16] [15] [20] [18]
0.44–3.72
37–42
5
[19]
Soil sample (India)
F.-J. Chu et al. / Journal of the Taiwan Institute of Chemical Engineers 43 (2012) 220–225
2.0
3.4. Biodegradation of purified bamboo and its byproduct by S. griseoaurantiacus ZQBC691 The results of an investigation of purified bamboo (pure bamboo cellulose) demonstrate that S. griseoaurantiacus ZQBC691 grew well on pure bamboo cellulose and also formed such byproducts as lactic acid and acetic acid. Fig. 3 reveals that the aforementioned byproducts accumulated over time, reducing the pH value of the reacted solution. The amounts of those byproducts gradually increased with the original bamboo concentration. These products were produced in large amounts at a pure bamboo cellulose concentration of 2 g/L and in the largest amounts when 10 g/L pure bamboo cellulose was used (data not shown). The highest concentrations of produced lactic acid and acetic acid were 238 mg/L and 94 mg/L, formed after 144 and 100 h, respectively. The pH of the reacted solution substantially fell from 5.63 to 5.01 during 144 h of fermentation. Reducing sugar and mono-sugar such as glucose also accumulated as products of hydrolysis and the fermentation process. When 10 g/L pure bamboo cellulose was used, the amounts of reducing sugar and glucose reached their highest values of 0.563 and 0.260 g/L at 121 h of hydrolysis. Thereafter, their amounts gradually decreased, revealing that ZQBC691 used own products as a source of carbon. Furthermore, the amounts of byproducts gradually increased from 121 h of fermentation to 144 h. The results indicate that ZQBC691 can use mono-sugars in the reducing sugar to generate byproducts. As the data also show, hydrolysis of the pure bamboo cellulose yielded glucose. ZQBC691 also produced b-glucosidase. 3.5. Growth of ZQBC691 on various types of cellulose To evaluate the ability of cellulose by S. griseoaurantiacus ZQBC691 to degrade various types of cellulose, CMC, pure bamboo cellulose, bamboo powder, salicin and avicel were individually used as the sole carbon source at concentrations from 10 to 15 g/L. Fig. 4 indicates that soluble CMC at 15 g/L was associated with the maximum reducing sugar rate (1.691 g/L/h), while crystalline cellulose (pure bamboo cellulose, salicin, bamboo powder and avicel) yielded lower rates. These results indicate that ZQBC691 exhibited a weak ability to grow on crystalline cellulose, including avicel, salicin, pure bamboo cellulose and bamboo powder. Fig. 4 shows that RSVmax reached approximately 0.317 g/L/h at a salicin concentration of 15 g/L, revealing that S. griseoaurantiacus ZQBC691 can produce b-D-glucosidase, which hydrolyzes salicin and releases glucose as a reducing sugar.
8
600
6 400
pH
Concentration (mg/L)
500
300 4 200 100
2
0 0
20
40
60
80
100
120
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Time (h) Fig. 3. Biodegradation of pure bamboo cellulose and its byproducts. ((*) reducing sugar; (*) glucose; (!) lactic acid; (~) acetic acid; (&) pH; pure bamboo cellulose concentration was 10 g/L).
Maximum reducing sugar rate (g/L/h)
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1.5
1.0
0.5
0.0 CMC
Pure bamboo cellulose
Salicin
Bamboo powder
Avicel
Type of cellulose Fig. 4. Hydrolysis of various types of cellulose by S. griseoaurantiacus ZQBC691. CMC, salicin, bamboo powder and avicel concentrations were 15 g/L each; Pure bamboo cellulose concentration was 10 g/L.
ZQBC691 can use avicel to produce exoglucanases, thereby converting crystalline cellulose into reducing sugar. However, avicel was degraded only to a limited extent, as indicated by the low RSVmax of around 0.051 g/L/h (Fig. 4). ZQBC691 converts natural crystalline celluloses, such as pure bamboo cellulose and bamboo powder, into a reducing sugar and glucose, which result is attributable to the release of endoglucanases to the amorphous regions of cellulose and the subsequent conversion of b-Dglucosidases to glucose. Therefore, the strain produced enzymes (exoglucanases, endoglucanases and b-D-glucosidases) that hydrolyzed the natural lignocelluloses, including pure bamboo cellulose and bamboo powder, to produce reducing sugars and glucose. Moreover, the maximum reducing sugar production rate was low (0.136 g/L/h) using fresh bamboo powder, but was significantly increased to 0.468 g/L/h using pretreated bamboo (pure bamboo cellulose). The elevated reducing sugar rate was believed to be due to a reduced amount of lignin after the pretreatment. 4. Conclusions The novel strain S. griseoaurantiacus ZQBC691 was isolated and screened from a compost of bamboo waste at 30 8C. S. griseoaurantiacus ZQBC691 was a mesophilic, aerobic, and slightly acid cellulose-degrading microorganism. The optimal conditions under which the strain produced the most reducing sugar were an initial pH of 5.3, a temperature of 30 8C, a shaking-speed of 150 rpm and the use of 15 g/L CMC. ZQBC691 acted on CMC to generate endoglucanase; the optimal conditions for this process were pH 5 (37.38 IU/ml) at 50 8C (34.13 IU/ ml). ZQBC691 grew well on amorphous cellulose (CMC) but less well on crystalline cellulose (avicel, natural bamboo). However, ZQBC691 hydrolyzed pure bamboo cellulose; produced a monosugar, such as glucose, and fermented reducing sugar to form a biochemical product such as lactic acid or acetic acid. The results suggest that extensive research should be performed into the generation of ethanol by this strain. The use of a co-culture system is one means by which ZQBC691 can combine with other microbes (Zomomonas mobilis) that ferment reducing sugar (glucose) in a single step to convert purified-bamboo materials into ethanol.
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Acknowledgements The authors would like to thank the Industrial Technology Research Institute of the Republic of China, Taiwan, for financially supporting this research. Ted Knoy is appreciated for his editorial assistance. References [1] Sims R. Biomass and resources bioenergy options for a cleaner environment in developed and developing countries. London, UK: Elsevier Science; 2003. [2] Lynd LR, Laser MS, Bransby D, Dale BE, Davison B, Hamilton R, et al. How biotech can transform biofuels. Nat Biotechnol 2008;26:169–72. [3] Gamage J, Lam H, Zhang Z. Bioethanol production from lignocellulosic biomass: a review. J Biobased Mater Bioenergy 2010;4:3–11. [4] Hendriks ATWM, Zeeman G. Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresour Technol 2009;100:10–8. [5] Wyman CE. Ethanol from lignocellulosic biomass: technology, economics, and opportunities. Bioresour Technol 1994;50:3–16. [6] Li X, Pan X. Hydrogels based on hemicellulose and lignin from lignocellulose biorefinery: a mini-review. J Biobased Mater Bioenergy 2010;4:289–97. [7] Fu J. Moso bamboo in China. ABS Mag 2000;21:12–7. [8] Scurlock JMO, Dayton DC, Hames B. Bamboo: an overlooked biomass resource. Biomass Bioenergy 2000;19:229–44. [9] Bystriakova N, Kapos V, Stapleton C, Lysenko I. Bamboo biodiversity. UNEP World Conservation Monitoring Centre and International Network for Bamboo and Rattan; 2003. [10] Kigomo B. Guidelines for growing bamboo. Kenya Forestry Research Institute; 2007. pp. 1–41. [11] Sin MKW, Hyde KD, Pointing SB. Comparative enzyme production by fungi from diverse lignocellulosic substrates. J Microbiol 2002;40:241–4. [12] Zhou D, Hyde KD. Fungal succession on bamboo in Hong Kong. Fungal Diversity 2002;10:213–27. [13] Tanaka K, Hirayama K, Yonezawa H, Hatakeyama S, Harada Y, Sano T, et al. Molecular taxonomy of bambusicolous fungi: Tetraplosphaeriaceae, a new pleosporalean family with Tetraploa-like anamorphs. Stud Mycol 2009;64: 175–209. [14] Singh D, Zeng J, Laskar DD, Deobald L, Hiscox WC, Chen S. Investigation of wheat straw biodegradation by Phanerochaete chrysosporium. Biomass Bioenergy 2011;35:1030–40. [15] Antai SP, Crawford DL. Degradation of softwood, hardwood, and grass lignocelluloses by two Streptomyces strainst. Appl Environ Microbiol 1981;42:378– 80. [16] Vinha F, Gravina-Oliveira M, Franco M, Macrae A, Silva Bon E, Nascimento R, et al. Cellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic biomass as inducer substrate. Appl Biochem Biotechnol 2010;1–12.
225
[17] El-Sersy NA, Abd-Elnaby H, Abou-Elela GM, Ibrahim HAH, El-Toukhy NMK. Optimization, economization and characterization of cellulase produced by marine Streptomyces ruber. Afr J Biotechnol 2010;9:6355–64. [18] Das SK, Alam MZ, Manchur MA, Anwar MN. Effect of nutrients and cellobiose octaacetate on cellulolytic enzyme productions by Streptomyces albolongus. Bangladesh J Microbiol 2007;24:70–2. [19] Arora A, Nain L, Gupta JK. Solid-state fermentation of wood residues by Streptomyces griseus B1, a soil isolate, and solubilization of lignins. World J Microbiol Biotechnol 2005;21:303–8. [20] Jaradat Z, Dawagreh A, Ababneh Q, Saadoun I. Influence of culture conditions on cellulase production by Streptomyces spstrain J2). Jordan J Biol Sci 2008;1:141–6. [21] Haruta S, Cui Z, Huang Z, Li M, Ishii M, Igarashi Y. Construction of a stable microbial community with high cellulose-degradation ability. Appl Microbiol Biotechnol 2002;59:529–34. [22] Mandels M, Reese ET. Induction of cellulase in Trichoderma viride as influenced by carbon sources and metals. J Bacteriol 1957;73:269–78. [23] Teather RM, Wood PJ. Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microbiol 1982;43:777–80. [24] Ghose TK. Measurement of cellulase activities 1987;59:257–68. [25] Kim Y-S, Jung H-C, Pan J-G. Bacterial cell surface display of an enzyme library for selective screening of improved cellulase variants. Appl Environ Microbiol 2000;66:788–93. [26] Leenakul W, Tippayawong N. Dilute acid pretreatment of bamboo for fermentable sugar production. J Sustain Energy Environ 2010;1:117–20. [27] Zhuang XS, Qi W, Yuan ZH, Wang Q, Tan XS. An investigation of cellulose hydrolysis under extremely low acids. J Biobased Mater Bioenergy 2010;4: 35–9. [28] Kumar P, Barrett DM, Delwiche MJ, Stroeve P. Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind Eng Chem Res 2009;48:3713–29. [29] Yang B, Wyman CE. Pretreatment: the key to unlocking low-cost cellulosic ethanol. Biofuels Bioprod Biorefining 2008;2:26–40. [30] Lin C-W, Wu C-H, Tran D-T, Shih M-C, Li W-H, Wu C-F. Mixed culture fermentation from lignocellulosic materials using thermophilic lignocellulose-degrading anaerobes. Process Biochem 2011;46:489–93. [31] Torii N, Okai A, Shibuki K, Aida TM, Watanabe M, Ishihara M, et al. Production of D-glucose from pseudo paper sludge with hydrothermal treatment. Biomass Bioenergy 2010;34:844–50. [32] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959;31:426–8. [33] Breuil C, Saddler JN. Comparison of the 3,5-dinitrosalicylic acid and NelsonSomogyi methods of assaying for reducing sugars and determining cellulase activity. Enzyme Microb Technol 1985;7:327–32. [34] Lindsay H. A colorimetric estimation of reducing sugars in potatoes with 3,5dinitrosalicylic acid. Potato Res 1973;16:176–9. [35] Miller GL, Blum R, Glennon WE, Burton AL. Measurement of carboxymethylcellulase activity. Anal Biochem 1960;1:127–32.