Cellulase production by two Streptomyces species

Scientific Journal for Damietta Faculty of Science 5 (1) 2015, 24-32 ISSN Print 2314-8594 ISSN Online 2314-8616 Cellulase production by two Streptomyces species Mohamed I. Abou-Dobara1*, Ahmed K.A. El-Sayed1, Amira A. El-Fallal1, and Mona A. Sauf2 1 Botany 2 Dept., Faculty of Sci., Damietta Univ., New Damietta, P.O. 34517, Egypt The higher institute for comprehensive professions, Mesallata, Libya Received: 24 May 2015 / Accepted: 20 June 2015 * Corresponding author: [email protected] Abstract Optimization of cellulase production by two actinobacterial species, identified as Streptomyces auranticus and Streptomyces minoensis, was studied on Hutchinson medium containing cellulose as carbon source. The optimal incubation period, temperature and initial pH recorded for maximum enzyme yield of Streptomyces minoensis were 6 days, 35oC and 8.0 respectively. Meanwhile, Streptomyces auranticus showed different conditions for maximum enzyme production (8 days, 35oC and pH 7.0). The best growth for the two isolates was recorded at 30 oC. The maximum cellulase production was observed in a medium containing carboxy-methylcellulose (CMC) and xylan as carbon sources for Streptomyces auranticus and Streptomyces minoensis respectively. The addition of tryptone as a nitrogen source exhibited a maximum cellulase activity for the two identified Streptomycetes species. Keywords: Cellulase, Streptomyces minoensis and Streptomyces auranticus, Optimization United States and Europe each consume 30% of the world output of enzymes. Approximately 75% of industrial enzymes are used for hydrolysis and depolymerization of complex natural substances (Kirk et al., 2002). Cellulase enzymes are produced from plant, animal and microbial sources. For commercial production, microbial enzymes have the enormous advantage of being scalable to high-capacity production by established fermentation techniques (Tahtamouni et al., 2006). Industrial applications of cellulases are in the textile polishing named "biopolishing" of fabrics such as production of the stonewashed look of denims, and in household laundry Introduction A wide variety of bacteria are known for their hydrolytic enzymes production with streptomycetes being the best known enzyme producers (Vinogradova and Kushnir, 2003). They are capable of secreting an array of different extracellular enzymes including cellulase, chitinases and xylanase. Cellulases are one of the most important industrial enzymes. They have attracted interest because of the diversity of their applications. In 2001, the world market for enzymes was over 1.5$ billion; this was doubled by the year 2008. The 24 Cellulase production by two Streptomyces species Scientific Journal for Damietta Faculty of Science 5 (1) 2015, 24-32 previous medium, and incubated at 30°C for up to 21 days. At the end of the incubation, the filter paper was examined if degraded and if any dark patches of growth are found in the filter paper. detergents to improve fabric softness and brightness (Hill et al., 2006). Moreover, they are used in animal feeds to improve nutritional quality and digestibility, in processing of fruit juices, and in baking; de-inking of paper is yet another emerging application (Ponnambalam et al., 2011). In addition, cellulase enzymes are involved in enzymatic hydrolysis of cellulose, one of the most abundant organic materials that can be converted to products with significant commercial interest. Bioconversion of cellulose to monomeric sugars has been intensively studied as researchers seek to produce bioethanol and biobased products, food and animal feeds, and many valuable chemicals (Barros et al., 2010). The present work aims to isolate and identify two cellulase producing actinobacteria and optimize the conditions required for maximum cellulase production. Growth condition and enzyme production Two starch-nitrate agar discs of each Streptomyces 5-7 old days culture grown at 30oC were inoculated in flasks containing 50ml of Hutchinson medium supplemented with 0.5 % (w/v) cellulose. The cultures were incubated under 150rpm shaking at 30oC for 14 days. The enzyme activity of the medium filtrate was assayed. Enzyme assay Cellulase activity was quantified according to Miller (1959) with some modifications. A reaction mixture composed of 0.2mL crude enzyme solution and 1mL of 1.0% cellulose dissolved in distilled water and an aliquot of diluted enzyme preparation. The mixture was incubated at 50°C in water bath for 15min. The determination of reducing sugar released during the incubation mixture was detected by the dinitrosalicylic acid method of Miller (1959). One ml of dinitrosalicylic acid reagent was added to 1ml of the clarified reaction mixture and standards. After mixing the mixtures were boiled in a water bath for 5min. After cooling to room temperature, the optical density of the coloured product at 550nm was recorded. Calibration curve constructed using D-glucose standard in the range of 0-5μmol ml-1 were used. One unit of cellulase activity was defined as the amount of enzyme that released 1μmol of glucose per minute under the above assay conditions. Enzyme and substrate controls were included routinely. Material and Methods Isolation technique Soil samples collected from different localities in Egypt and Libya were subjected for actinobacteria-cellulase producers. Standard dilution plate technique was applied. Isolates were purified by streak-plating on starch nitrate agar plates following the method of Waksman, 1959. Colonies of actinobacteria were selected, isolated, purified and maintained as spore suspensions in 20% (v/v) glycerol at -20°C for subsequent investigation (Hopkins et al., 1985). The medium used for isolation, cultivation and stock maintenance of isolated strains was starch nitrate agar medium (Waksman, 1959). It contained (g/L): soluble starch, 20; KNO3, 2; K2HPO4, 1; NaCl, 0.5; MgSO4.7H2O, 0.5; FeSO4.7H2O, 0.01; CaCO3, 3; agar, 20; and distilled water up to 1L. Identification of the selected Streptomyces isolates Screening for cellulase producing actinomycetes strains The streptomycetes isolates used in this investigation was identified according to International Streptomyces Project (Shirling and Gottlieb, 1968a; 1968b; 1969; 1972; Pridham and Tresner, 1974a; 1974b; Bergey’s Manual of Systematic Bacteriology, 1989). A preliminary analysis of cellulolytic activity was conducted using grown strains on Hutchinsonmedium has the composition (g/L): Ca (NO3)2, 2.5; K2HPO4, 1; MgSO4.7H2O, 0.3; NaCl, 0.1; FeSO4.7H2O, 0.01; CaCl2, 0.1 and distilled water up to 1L, containing filter paper strip (1.0 X 10.0cm) as carbon source. The pH was adjusted to 7.0 with 1.0 N NaOH. A loopfull of streptomycetes cultures from agar plates was inoculated into glass tubes containing 5ml of the Electron microscopy studies Electron microscopy was performed using the cover slip technique. The cover slip was cut with a glass file and a suitable fragment with growth on 25 Cellulase production by two Streptomyces species Scientific Journal for Damietta Faculty of Science 5 (1) 2015, 24-32 surfaces of starch nitrate agar cultures was chosen. It was mounted on a specimen-tube, coated with gold-palladium under vacuum and examined with a scanning electron microscope (Joel ISM-5300) operating at 10KV. Statistical analysis Analysis of variance (one-way ANOVA) was used to identify statistically significant differences in cellulase activity and culture growth with incubation period, temperature, pH, carbon source and nitrogen source. All statistical analyses were performed using SPSS 18.0 software (SPSS, 2006). Optimization of culture conditions An attempt was also made to determine the optimal culture conditions such as pH, temperature, incubation period and carbon and nitrogen source requirements for their maximum growth and activities. The biomass yield and cellulase production of the selected isolate was recorded. Microbial growth under different growth factors was assayed. Cell pellets were dried in hot air oven at 80°C to a constant weight. The dry cell weight per 50ml of culture broth was used to determine microbial growth. Effect of incubation period: To determine the optimum incubation period of the isolates for maximum enzyme production, the supernatants were collected after 2, 4, 6, 8, 10 and 12 days of incubation and assayed as before. The growth was also recorded. Effect of initial pH: To determine the optimum medium pH, for maximum enzyme production, selected medium of different pH (4, 5, 6, 6.5, 7, 7.5, 8, 8.5, 9, and 9.5) was inoculated with the isolate. The growth and cellulase production were assayed as before. Effect of temperature: To determine the optimum temperature for enzyme production the culture medium was incubated at 25°C, 30°C, 35°C and 40°C, at optimum pH and incubation period. The effect of temperature on growth and cellulase production was recorded as before. Effect of carbon source: Different carbon sources; cellulose, CMC, xylan, glucose, maltose, sucrose, starch and xylose (5g/L) was added separately as a sole carbon source. The effects of these carbon sources in the production of cellulase, biomass yield, were recorded. Effect of nitrogen source: For this purpose, a range of different nitrogen sources includes, (NH4)2SO4, Ca(NO3)2, NaNO3 and KNO3, were added in equimolecular nitrogen weights equivalent to the nitrogen content of 2.5 gL-1 Ca(NO3)2 of Hutchinson medium.. Other nitrogen source like peptone (14% N), tryptone (13.5% N), yeast extract (09% N) and beef extract (12.5% N) were added separately. The effects of these nitrogen sources in the production of cellulase, biomass yield, were recorded. Results Screening of cellulase producers Screening of actinomycetes was conducted using the filter paper if degraded and if any dark patches of growth are found on the filter paper as a preliminary study for choosing the best cellulase producers. After 21 days of incubation, two isolates out of five actinomycetes were selected for the highest rupture and dark patches on filter paper. The cellulolytic activity of the selected strains was confirmed under submerged fermentation indicating the highest cellulose degradation. Isolates identification The selected two actinobacterial isolates were identified as Streptomyces auranticus and Streptomyces minoensis according to their morphology, under light and scanning microscope (Fig. 1), and biochemical characteristics (Table 1). Factors affecting the growth and cellulase activity Different effects, such as the incubation period, pH, temperature, and different sources of carbon and nitrogen, were studied on growth and production of cellulase activities of S. minoensis and S. auranticus in Hutchinson medium containing cellulose (0.5%). Effect of the incubation period The time course for the production of cellulase activity is shown in Figure 2 for both S. auranticus and S. minoensis, respectively. The cellulase activity was highly significantly increased (P< 0.0001) during the growth of the organism, with the maximum production of enzyme detected at 8 days and 6 days (0.3286 U/ml and 0.2189 U/ml) and the maximum growth (0.329 g/50ml and 26 Cellulase production by two Streptomyces species Scientific Journal for Damietta Faculty of Science 5 (1) 2015, 24-32 0.349 g/50ml) for S. auranticus and S. minoensis, respectively. After 8 days, cellulase activity was significantly declined by S. minoensis (0.0796 U/ml), while it reached the minimal level (0.15 U/ml) after 10 days by S. auranticus. Table 1. Cultural, morphological and physiological characteristics of S. auranticus and S. minoensis Characters S. auranticus S. minoensis Aerial mass color pink grey Melanoid pigment on: tyrosine, peptone yeast Colour and pigmentation and synthetic media Reverse side pigment Pale yellow Grey Soluble pigment Spore chain flexuous Straight Spore morphology Spore surface Smooth Smooth Arabinose + + Xylose + + Inositol + + Mannitol + + Carbon source utilization Fructose + + Rhamnose + + Sucrose + + Raffinose ± + Potassium nitrate + + L-valine + + L-threonine + + L-serine + + L-Methionine + + Nitrogen source utilization L-histidine + + Hydroxy proline + + L-proline + + L-cysteine + ± L-phenylalanine + + Milk coagulation + + Milk peptonization + + Starch hydrolysis + + Urea utilization + + Physiological properties Gelatin liquification + Melanin/L-tyrosine Cellulose degradation + + Esculin degradation + + (+) good, (±) Little, (-) nil. Fig 1. Scanning electron micrograph showing the two isolated streptomycetes growth after 7 days at 30°C. (A) S. auranticus and (B) S. minoensis isolates. 27 Cellulase production by two Streptomyces species Scientific Journal for Damietta Faculty of Science 5 (1) 2015, 24-32 minoensis (0.266 U/ml). The arrangement of different carbon sources in descending order according to its effect on cellulase production for S. auranticus was CMC, cellulose, maltose, glucose, xylan, starch, xylose and then sucrose, with activity ranging from 0.207 U/ml to 0.0278U/ml. In contrast, the carbon source arrangement for S. minoensis was xylan, cellulose, maltose, CMC, glucose, xylose, starch and sucrose, with activity varying from 0.266 U/ml to 0.0259 U/ml. Effect of different nitrogen sources The nitrogen sources effect on the production of cellulase by S. auranticus and S. minoensis isolates was investigated (Figure 6). The highest cellulase activity and growth (0.292 U/ml-0.0852 g/50ml for S. auranticus and 0.412 U/ml - 0.119 g/50ml for S. minoensis) were recorded with highly significantly (P< 0.0001) when tryptone was used as the nitrogen source. The arrangement of different nitrogen sources in descending order according to its effect on cellulase production by S. auranticus was tryptone, yeast extract, beef extract, peptone, potassium nitrate, sodium nitrate, calcium nitrate and finally ammonium sulphate. The descending order of the different nitrogen sources effect on cellulase production by S. minoensis was tryptone, beef extract, peptone, yeast extract, sodium nitrate, calcium nitrate, ammonium sulphate and then potassium nitrate. Fig 2. Effect of different incubation periods on cellulase production and growth of S. auranticus (A) and S. minoensis (B). Effect of different initial pH The optimum pH for cellulase production (Figure 3) was 7 and 8 with the maximum level of enzyme activity 0.2049 U/ml and 0.297 U/ml for S. auranticus and S. minoensis, respectively. On the other hand, the maximum growth (0.3139 g/50ml and 0.356 g/50ml) occurred highly significantly (P< 0.0001) at pH 7 and pH 8 for S. auranticus and S. minoensis, respectively. Cellulase activity was completely inhibited at pH 9.5 for S. auranticus. Effect of temperature The production of cellulase by S. auranticus and S. minoensis was highly affected by raising temperature of 25oC up to 40oC (Figure 4). The optimum temperature for the enzyme production was 35oC (0.1568 U/ml and 0.233U/ml), while the maximum growth was at 30oC (0.4866 g/50ml and 0.4502 g/50ml) by S. minoensis and S. auranticus, respectively. Cellulase production was gradually increased significantly (P< 0.0001) with increasing temperature for both S. auranticus and S. minoensis. Effect of different carbon sources The effect of a range of carbon sources on the growth and production of cellulase by S. auranticus and S. minoensis was varied (Figure 5). The best carbon sources for enzyme activity were found to be carboxymethylcellulose (CMC) for S. auranticus (0.207 U/ml) and xylan for S. Fig 3. Effect of different pH on the cellulase production and growth of S. auranticus (A) and S. minoensis (B). 28 Cellulase production by two Streptomyces species Scientific Journal for Damietta Faculty of Science 5 (1) 2015, 24-32 Fig 6. Effect of different nitrogen sources on the cellulase production and growth of S. auranticus (A) and S. minoensis (B). Fig 4. Effect of different temperatures on the cellulase production and growth of S. auranticus (A) and S. minoensis (B). Discussion Streptomyces species have always been a source of thousands of bioactive compounds. Enzymes are one of the important products of this unusual group of bacteria. Streptomyces sp. with potential cellulolytic activity is subjected to produce cellulase in liquid culture (Chellapandi and Jani, 2008). S. auranticus and S. minoensis strains exhibited the highest ratio of dark patches or rupture to filter paper strip compared with the isolates, indicating a higher level of cellulose activity. Cellulase production by Streptomyces auranticus and S. minoensis increased during the growth of the cultures in Hutchinson medium with the maximum production that detected after 8 and 6 days incubation, respectively. After this period, the activities of the enzymes decreased. The observed peaking and troughing of the production of extracellular enzymes might be attributed to the differences in the timing of induction of separate components of the cellulase system, the products of action of one component inducing the synthesis of another, differential inhibition by products of substrate hydrolysis, differential inactivation by proteases, or variation in the pH during cultivation conditions (Tuohy and Coughlan, 1992; Wang et al., 1993). Prolonged incubation periods (7, 8 days) required to obtain maximum enzymatic production by the studied Streptomyces isolates were in agreement with Arunachalam et al. (2010). Also, the maximum yield of endoglucanase activity was Fig 5. Effect of different carbon sources on the cellulase production and growth of S. auranticus (A) and S. minoensis (B). 29 Cellulase production by two Streptomyces species Scientific Journal for Damietta Faculty of Science 5 (1) 2015, 24-32 found to be CMC and xylan for S. auranticus and S. minoensis, respectively. The highest CMC-ase activity (233.56 U mL-I) was recorded with the crude enzyme when CMC used as a carbon source and the lowest CMC-ase activity (11.11 U mL-I) when sawdust and rice bran used as a carbon source reported by (Alam et al., 2004). The cellulase productivity by S. auranticus and S. minoensis was on maximum level when tryptone was used as the nitrogen source. This was in correlation with the findings of many other workers who found that maximum cellulase productivity was obtained by Bacillus pumilus BpCRI 6, Pseudomonas flourescens, Monascus purpureus and Streptomyces sp. BRC2 when tryptone was added as an organic nitrogen source to the production medium (Bakare et al., 2005, Chellapandi and Jani, 2008; Daniel et al., 2008). Also Bacillus subtilis KO strain gave maximum cellulase productivity when tryptone was added to the production medium (Shabeb et al., 2010). The cellulases identified by both isolates would be fully characterized in a future work in order to investigate their usefulness in the industrial purposes. obtained after 7 days (Azzedine et al., 2013). On the other hand, the production of the CMCase by E. coli JM109/DL-3 in submerged fermentations took only 3 days, which resulted in an increase in productivity of CMCase and decrease in its production cost (You-Jung et al., 2012). However, our results for S. minoensis are in agreement with that obtained by Streptomyces griseorubens which exhibited high cellulase production after 6 days (Prasad et al., 2013). Temperature and pH values were found to be important parameters that influenced enzyme activities and production (Odeniyi et al., 2009). Cellulase enzyme from S. auranticus and S. minoensis was found to be active over a pH range of 7-9 with maximum activity at pH7 and pH8, respectively. This result is considerably similar to previous results reported by Azzedine et al. (2013) who found that cellulase enzyme from Streptomyces sp. (B-PNG23) was active over a pH7, also enzymatic activity was observed at alkaline pH (8-9). Similar results were reported by Rahna and Ambili (2011); Immanuel et al. (2006); GoKhan-Coral et al. (2002); Akiba et al. (1995); Prasetson and Doelle (1987); and Garcia-Martinez et al. (1980). However Solingen et al. (2001) studied the alkaline novel Streptomyces species isolated from east African soda lakes which showed an optimal pH of 8, while the Cellulomonas sp isolated by Irfan et al. (2012) recorded optimum activity at pH 7.5. Trichoderma viridae which produces cellulase presented an optimum activity at pH 8.0 as reported by Iqbal et al. (2011). Cellulase activity between pH 6.0 and 10.0 is useful in the textile industry (Kochavi et al., 1990) and in detergents (Suominen et al., 1993). The temperature has a great effect on the enzyme activity. S. auranticus and S. minoensis showed a maximum cellulase activity at 35°C within an optimum range 30°C-40°C. Alam et al. (2004) recorded a heavy growth and high cellulase activity by S. omiyaensis at 35°C-40°C. The maximum growth of mesophilic organisms at 35oC was reported by Shibli (2002). The enzyme CMCase showed a good production between 20°C to 40°C with maximum activity at 35°C for Streptomyces sp. strain NEAE-D by El-Naggar and Abdelwahed (2012). The optimum temperature recorded for maximum cellulase productivity at 35°C for Bacillus subtilis CBTK 106; Bacillus spp. B21 (Amritkar et al., 2004; Krishna, 1999) and Pseudomonas fluorescense (Bakare et al., 2005). The best carbon sources of the enzyme activity and the growth of the organism were Conclusion It can be concluded that the two isolates S. auranticus and S. minoensis can be used for the production of useful substances by degrading cellulosic agro-wastes. Consequently, two goals would be achieved; getting rid of the continuously added agricultural wastes and recovery of bioenergy from degraded cellulose. Acknowledgment Mona AS was supported by a Libyan Government Scholarship at Botany Department, Faculty of Science, Damietta University. The SPSS statistical analysis was manipulated by the aid of Prof. A.A. Khedr at Damietta University. References Akiba S, Kimura Y, Yamamoto K, Kumagap H (1995) Purification and characterization of a proteaseresistant cellulase from Aspergillus niger. J. Fermen. Bioengin. 79: 125-132. 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Suominen P, Mantyla A, Karhunen T, Hakola S, Nevalainen H (1993) High frequency one-step gene ‫الملخص العربي‬ ‫ إنتاج إنزيم السليوليز من نوعين من اإلستربتوميسيس‬:‫عنوان البحث‬ 2‫سوف‬ ‫ مني علي مصطفي‬،1‫ أميرة علي الفالل‬،1‫ أحمد قاسم عبدالصمد السيد‬،1‫محمد إسماعيل أبو دبارة‬ ‫ مصر‬-‫ دمياط الجديدة‬- ‫ جامعة دمياط‬- ‫ كلية العلوم‬- ‫ قسم النبات‬1 ‫ المعهد العالى للمهن الشاملة – مسالتة – ليبيا‬2 ‫يهدف البحث الي عزل اكتينوبكتريا لها القدرة علي انتاج انزيم تحلل السليوليوز و دراسة تﺄثير ظروف‬ ‫ وتمت الدراسة بﺄستخدام العوامل المدروسه من‬.‫النمو وتركيب الوسط الغذائي على إنتاج هذا اإلنزيم‬ ‫درجات حراره و حموضة وغيرها علي بيئة هتشينسون الغذائية التى تحتوى على مادة السليلوز كمصدر‬ ‫ فترة‬:‫ أظهرت النتائج أن الظروف المثلى إلنتاج إنزيم السليوليز للساللتين كانت كاألتى‬.‫وحيد للكربون‬ ‫ لكل من إستربتوميسيس أورانتيكس‬8 ،7 ‫ أيام واألرقام الهيدروجينية كانت‬8‫ و‬6 ‫التحضين المثلى هى‬ .‫ هى المثلى إلنتاج اإلنزيم للكائنين‬o‫م‬35 ‫ و كانت درجة الحرارة‬.‫وإستربتوميسيس مينوينسيس على التوالى‬ ‫أفضل المصادر الكربونية إلنتاج إنزيم السليوليز كان مادة الكربوكسي ميثيل سليلوز للساللة‬ ‫استربتوميسيس اورانتيكس ومادة الزيالن للساللة واستربتوميسيس مينوينسيس كمواد كيميائية نقية‬ .‫ أما مادة التربتون كانت أحسن مصدر نيتروجيني إلنتاج إنزيم السليوليز لكال الكائنين‬.3.5% ‫بتركيزات‬ 32