Studying the optimum incubated conditions of anaerobic bacteria in rice husk

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MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOG Y R & D INSTITUTE SUMMARY OF BACHELOR THESIS THE ADVANCE BIOTECHNOLOG Y PROGRAM STUDYING THE OPTIMUM INCUBATED CONDITIONS OF ANAEROBIC BACTERIA IN RICE HUSK SUPERVISOR VO VAN SONG TOAN DUONG THI HUONG GIANG STUDENT VAN HUU LOC Student code: 1065586 Session: 32 (2006-2010) Can Tho, 2010 APPROVAL SUPERVISOR STUDENT VO VAN SONG TOAN VAN HUU LOC DUONG THI HUONG GIANG Can Tho, December 3, 2010 PRESIDENT OF EXAMINATION COMMITTEE Ass.Prof. Cao Ngoc Diep ABSTRACT Twelve anaerobic isolates were screened for cellulolytic capacity on rice husk. The soil isolate, annotated as 84, gave the highest cellulase activity. The growth rate peaked at 96h of incubation, with the cell density of 35.11 x10 7 CFU/ml. Species identification by Microlog system 4.20.04 (Biolog, 2004), with the kit GN2 revealed that the isolate is belong to the Acinetobacter genera. This bacteria adopted high hydrolytic activity on rice husk at 30 0 C, pH 7; which was enhanced by induction with 1% lactose. The cellulase production of the isolate 84 reached peak at the fifth day. Final protein content, CMCase and avicelase activities were determined as 0.19 mg (in 15ml of cultural solution), 0.197 U/ml and 0.181 U/ml, respectively. The cellulose hydrolytic yield was 5,46% at 5 days of incubation. Key word: Acinetobacter, cellulase, lactose, rice husk i CONTENTS Content 1. Introduction 2. Materials and methods 3. Results and discussion 3.1. Determination of cellulose content of rice husk 3.2. Screening of anaerobic bacteria capable to degrade rice husk 3.3. Selection of the isolates exposing highest cellulolytic activities 3.4. Growth curve of the isolate 84 3.5. Identification of the scientific name of the isolate 84 3.6. pH-temperature optimum for the growth of isolate 84 3.7. Lactose induction of cellulase production of the isolate 84 3.8. Study of the optimum cultivation time for the isolate 84 3.9. The cellulolytic capacity of isolate 84 on different substrates 4. Conclusions 5. Suggestions References ii ii 1 4 8 8 8 9 10 11 12 13 14 16 19 19 1. INTRODUCTION Most agricultural wastes of crop plants, particularly cereals, are rich in lignocellulosic materials, of which cellulose is the main constituent. Association of cellulose with lignin, another complex polymeric molecule composed of phenylpropanoid units, form lignocelluloses. Also hemicellulose is another major celulosic component. Bacterial enzymatic degradation of these materials is extremely slow because of the stable structure of the lignocelluloses complex. Besides, there are only a few species of bacteria and fungi which are able to breakdown such materials. Enzymatic degradation of cellulose requires the synergystic action of a family of cellulolytic enzymes that have been classified into three major groups: endoglucanases (CMCase, EC 3.2.1.4) whcih cleaves internal β-1,4-glycosidic bonds, exoglucanase (Avicelase, EC 3.2.1.91) which releases cellobiose from the nonreducing end of cellulose, and cellobiase (β-1,4-glycosidase, EC 3.2.1.2) which hydrolyses cellobiose to glucose (Wood and Bhat, 1998). Viet Nam is the second largest rice exporter in the world. The rice industrial process generates a large amount of fibrous byproducts and wastes, especially rice husk. This material is mainly composed of 25-30% lignin, 10-15% hemicellulose and 35-40% cellulose (Elijah T. Iyagba et.al, 2009). In the Mekong Delta, it has been approximately estimated to 3,600,000 tons per year. Treatment of these wastes is partially done by using as fuel, while the major part is discarded to the environment. To contribute to enviroment solving problem, this research aimed at selection of the most potential bacteria which are able to 1 degrade rice husk and optimization of culture conditions for highest cellulolytic activities of the selected bacteria. 2 Objectives: - To isolate, select and identify anaerobic bacterial isolates having high cellulolytic activity. - To study the optimum conditions for the cellulase production. - To study the degradation ability of the selected isolates on 3 different substrates: rice husk, sugarcane bagasse, and rice straw. 3 2. MATERIALS AND METHODS 2.1. Materials - Devices: + Spectrophotometer (Pharmacia LKB – Ultrospec, USA) + Eppendoft centrifuge (Rotor – Germany). + Eppendoft thermal shaker (Eppendorf – Germany). + Refrigerator and some laboratory equipments + Microlog system 4.20.04 (Biolog, 2004) - Samples: rice husk from rice mill factory in Can Tho city. - Materials for culture medium: CMC, K 2 HPO4 , MgSO4 .7H2 O, Lactose, NaCl, agar, Ammonium sulphate, grinded rice husk… - Chemicals: Congo red, HCl, Javen solution, Bovin Serum Albumin (BSA), Tris-HCl (Sigma), Sodium hidroxide (NaOH), Bromophenol blue, Acetic acid, Nelson reagent, Braford reagent … 2.2. Methods a) Determination of cellulose content in rice husk + Objective: To define the cellulose content in rice husk. + Method: - Collecting rice husk from rice processing factory in Can Tho city - Grinding rice husk into powder. - Define the cellulose content by using NaOH 0.5%, HCl 10% and javel solution. - Determining the cellulose content. b) Screening of anaerobic bacteria capable to degrade rice husk + Objective: To obtain the pure isolates of anaerobic bacteria + Method: 4 - Culturing 12 isolates on rice husk agar medium, 4-5 times repeated until the pure culture is obtained. - Morphologically description of bacterial colonies c) Selection of the isolates exposing highest cellulolytic activities + Objective: To select some isolates that expose highest hydrolytic activities + Method: - 5 µl of bacteria solution was dropped into the wells made on the Petri disks of rice husk agar medium and incubate for 5 days. - Congo Red was used for screening of the hydrolytic zones surrounding bacteria colonies. - Measuring the diameters of those zones to estimate the cellulolytic activities. d) Studying the growth rate of selected isolates + Objective: To determine the density of bacteria by the time + Method: - Bacteria was inoculated into the broth of rice husk medium. - Every day, 100µl of culture solution was taken and diluted into 10, 102 , 103 …108 times, then grow on petri dishes and the number of bacteria was counted based on the number of colony formed. e) Identification of the scientific name of selected isolates + Objective: To define the scientific name of selected isolates + Method: - Isolate a pure culture of selected isolates. Due to Gram staining the testing MicroPlate GN2 was selected. 5 - Put the colony of those isolates into 1 tube to compare the turbidity with the standard tube. - Inoculate 150µl above solution to MicroPlate. - Incubate 300 C. After 24h, read the result by MicroStation Reader. f) Studying the effects of pH and temperature on the production of selected bacteria + Objective: To define the optimum pH and temperature for the cellulase production of the selected bacteria + Method: - 5 µl of bacteria suspension was dropped into the readymade wells on the rice husk agar medium. The pH of the medium was in a range of 4, 5, 6, 7, 8, 9, 10, 11. Incubate 5 days at the different temperature of 25, 30, 35, 40, 45, 500 C. - Using congo red to screen the hydrolytic sphere zones surrounding bacterial colonies on rice husk agar medium. - Measuring the diameters of hydrolytic spheres to estimate the cellulolytic activity. g) Studying the effect of lactose concentration on the cellulase production of the selected bacteria + Objective: To define optimum lactose concentration for the cellulase production of those bacteria + Method: - 5 µl of bacteria suspension was dropped into the readymade wells on the rice husk agar medium with lactose concentration of 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5%. Incubate for 5 days. 6 - Cellulolytic activities were determined as described in the above experiment. h) Studying the optimum time for cellulase production. + Objective: To determine the optimum incubation time when the enzyme productivity is high. + Method: - Inoculate the bacteria into 27 tube of rice husk liquid medium. - The cellulolytic activities were tested every day for protein content by Bradford method and enzyme activity by Nelson Somogyi method. - Determination of hydrolysis yield by the remained rice husk in the culture media. i) Studying the cellulolytic activities of the selected isolates on different substrates + Objective: To define the hydrolytic ability of bacteria in 3 different substrates such as rice husk, sugarcane bagasse, and rice straw. + Method: - Inoculate the bacteria in liquid medium of rice husk, sugarcane bagasse and rice straw. - Testing the protein content by Bradford method and enzyme activities by Nelson Somogyi method. 7 3. RESULTS AND DISCUSSION 3.1. Cellulose content of rice husk The cellulose content of the rice husk explored in the experiment was 41,06%. This amount is equivalent to the result of Elijah et.al (34.34 - 43.80%). Lignin contaminant was also present in the analyzed sample that made the measurement not 100% accurate. According to Sun et.al (2004), materials harboring 44.7% of cellulose would contain ca. 5.7% hemicelluloses and 1.6% lignin. Besides, rice husk also consists of crude protein, carbohydrates, lipid and inorganic compounds. Cellulose content is also different among rice species. Hwang and Chandra (1996) showed that Japanese possessed higher holocellulose (hemicelluloses and cellulose) than Indian rice. 3.2 Screening of anaerobic bacteria capable to degrade rice husk Morphological observation has been revealed that, among 12 isolates tested, there were seven of smooth colony form (63, 64, 66, 67, 76, 80, 84), two of curled form (65, 83), two of wavy form (72, 85) and one of lobated form (62). Regarding the elevation, there were five isolates with umbonated elevation, five isolates with flat and two isolates with raised elevation. All 12 isolates are Gram negative. They can be monococci, diplococci, and streptococci groups, of which 50% were motile. 8 Gram negative coccus Gram negative dip lococcic Gram negative streptococci Figure 1. Gram staining and cell forms of the tested isolates 3.3 Selection of the isolates exposing highest cellulolytic activities Table 1. Cellulolytic capacity of the investigated isolates Isolates Diameter of hydrolytic zone (mm) 62 6.00g 63 10.67de 64 6.00g 65 7.00 f 66 11.33cd 67 10.33e 72 12.00bc 76 11.67bc 80 6.00g 83 6.00g 84 13.33a 85 12.33b Notes: Values are mean of triplicates. Means with different subscripts within a column are statistically significant different at 5%. 9 According to the data on table 1, it is obvious that the isolate 84 adopted highest cellulolytic activities with 13.33 mm diameter of the hydrolysis zone. While the isolate 85 was lower in hydrolysis activities (12.33mm). Four isolates named 62, 64, 80, and 83 had no hydrolytic zone, indicating that there is no cellulase production. Thus, the isolate 84 could be grown on rice husk medium for cellulase production. According to Trần Thị Ánh Tuyết and Trương Quốc Huy (2010), the hydrolytic zone diameter of Bacillus subtilis was 22 mm on CMC-agar medium. The difference might be due to the fact that CMC has simpler molecule structure than the lignocelluloses complex so that cellulose breakdown would be easier. Conclusively, the isolate 84 was chosen for the later experiments. 3.4. Growth curve of the isolate 84 The lowest density obtained at 0h. The figure (Figure 2) did not show the lag phase, it might conclude that the chosen period (24h) was too large to obtain the lag phase. Stable phase was reached after 96 hours and stayed remained until 144 hours .After 24h, the log of numbers of bacteria increased and reached the peak at 96h, followed by the death phase. In the lag phase, the bacteria needed time to adapt with new medium. In log phase, the bacteria density increase was geometric progression while the growing time was arithmetic progression. The actual rate of this growth (i.e. the slope of the line in the figure) depended upon the growth conditions, which affected the frequency of cell division events and the survival of daughter cells. 10 Figure 2. Growth curve of isolate 84 Exponential growth could not continue indefinitely because the medium was then nutrients-depleted and enriched with wastes. Consequently, the bacteria entered the stationary phase, in which growth rate was equal to death rate. The growth rate slows resulted from nutrient depletion and accumulation of toxic products. Soon later, the nutrient exhaustion made the bacteria enter the death phase, in which bacteria density started decreasing. There was no general rule for this phase. The study in Pythium aphanidermatum (Onuh and Ohazurike, 2000) also showed that the time to get the maximum density of this fungus was 96h. Then at the day of 7, the density reduces 50%. The statistic result indicated the difference between 96h and 120h was not significant. So, 96h was the best time to get the highest density of bacteria. 3.5. Identification of the scientific name of the isolate 84 11 Initially, the data obtained from MicroStation Reader showed that isolate 84 was Acinetobacter sp. The bacteria belong to the phylum Proteobacteria, class Gammaproteobacteria, order Pseudomonadales, family Moraxellaceae, and genus Acinetobacter. Non-motile Acinetobacter species are oxidase-negative and occur in pairs under microscopy. They are important soil organisms because of the contribution to mineralization of aromatic compounds. Reisolation and identification gave the same results. Ekperigin (2007) reported that Acinetobacter anitratus gave the highest cellulase activity of 0.48 U/ml. Hrenović et.al (2005) showed that the Acinetobacter calcoaceticus had ability to accumulate phosphor, which helped improve the waste water treatment process. Acinetobacter isolates are capable to degrade a wide range of aromatic compounds. 3.6 pH-temperature optimum for the growth of isolate 84 Figure 3 showed that the pH-temperature optimum for the growth of isolate 84 was 300 C, pH 7. The corresponding hydrolytic zone diameter was 15mm. Isolates with diameters of 6 mm, which was the diameter of the wells, had no cellulolytic activity. This isolate 84 did not produced cellulases at strong acidic (≤ 4) and alkaline pH (≥ 11), thus the isolates have cellulase production capability at a wide pH range. High temperatures resulted in activity loss of cellulase, but a minor hydrolysis activity at 500 C had been at slightly acidic pH (5 and 6). Thus, the isolate 84 might belong to temperate thermopilic bacteria. Chakraborty et.al (2000) claimed that the pH optimum of Cellulomonas sp was pH 7 in the temperature range of 30-370 C. Similar results were obtained from the study of Trần Thị Ánh Tuyết and Trương Quốc Huy (2010), in which the 12 optimum conditions for Bacillus subtilis growth were pH 7 and 300 C. Figure 3. Hydrolytic diameters, presenting relative cellulase activity of isolate 84 at different pH and temperatures 3.7 Lactose induction of cellulase production of the isolate 84 The cellulolytic activity could be significantly improved when 1% or 1.25% (v/w) lactose was added to the medium, as seen in the corresponding hydrolytic diameter of 20.4 mm in comparison to the control (15.67 mm) and previous experiment (15 mm). Hence, the relative cellulase activity could be increased 1.3 times. Higher lactose concentration (1.75% and 2.5% (w/v)), in contrast, resulted in activity loss when the halo diameters were decreased to 14,7mm and 12,53 mm, respectively. It means that lactose was probably an inducer of the cellulase biosynthesis of the bacteria at appropriate concentration. Morikawa et.al (1995) indicated that lactose made the cellulase activity increased 2-3 times. Study of Alazzeh et.al (2009) 13 also proved that β-galactosidase activity of Lactobacillus reuteri could increase by lactose induction. According to Mandels and Reese (1956) lactose was a much better inducer for cellulase activity than the others. On lactosecontaining medium, cellulose hydrolysis yield increased as sugar concentration rises to 0.75 per cent and then leveled off. Hydrolytic diameter (mm) Lactose concentration (%) Figure 4. The induction effect of lactose on cellulase production of the isolate 84 3.8 Study of the optimum cultivation time for the isolate 84 Highest total protein content of 0.19 mg was achieved after 5 days (Figure 5), which corresponded to the stationary phase of the bacteria (Figure 2). This density was high, but in 5th day, this density was not change. Later reduction in protein content resulted from denaturation, proteolysis and cell death. Protein content, especially cellulase content, is remarkably high in cellulase-producing fungi like Trichoderma. According to Kazuhisa (1997) the cellulase 14 content of T. reesei KDG-3 mutant in semi-batch culture was 36.7 mg/ml. Total protein content (mg) Time (day) Figure 5. Total protein content over time The endo- and exocellulase enzymatic activities of the isolate 84 was simultaneously determined for CMCase and avicelase using CMC and Avicel, respectively, as substrates. Maximal activity was fit with the protein content, which peaked at day 5 of fermentation process (Figure 6). It was observed that endoglucanse (CMCase) activity was always slightly higher than exoglucanase activity (avicelase). The molecular structure of CMC was simple and specific for endoglucanase, so the endoglucanase activity may more dominant than exoglucanase. Highest activities of these two were determined as 0.197 U/ml and 0.181 U/ml, respectively after 5 days. Ram et.al (1991) reported that Clostridium thermocellum isolate SS8 secreted both CMCase and avicelase when cultured in cellulose-containing medium, and about 85% cellulase was extracellular enzyme. 15 Avicelase activity was increased in the presence of DTT and Ca2+. Conclusively, the final hydrolytic productivity of the process was 5.46% after 8 days. Activity(U/ml) Ti me (day) Figure 6. CMCase and avicelase activities of isolate 84 over time 3.9 The cellulolytic capacity of isolate 84 on different substrates In this study, isolate 84 efficiently hydrolyzed the rice husk. The bacteria were also viable on the media containing other substrates like sugarcane bagasse and rice straw, but did not generate the hydrolytic zone. The bacteria was isolated from soil and continuously cultured on rice husk-containing medium, by which they possibly gained the specificity to this substrate. 16
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