MINISTRY OF EDUCATION and TRAINING CAN t

The optimal conditions for cellulase production by VK52 were determined as 300C, pH 8 and 4-day-incubation on the medium supplemented with 0.4 % yeast extract.. Results and discussion 7

MINISTRY OF EDUCATION & TRAINING

CAN THO UNIVERSITY

BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE

SUMMARY BACHELOR OF SCIENCE THESIS

THE ADVANCED PROGRAM IN BIOTECHNOLOGY

MSc DUONG THI HUONG GIANG TRAN NON NUOC

MSc VO VAN SONG TOAN Student code: 3064407

Session: 32 (2006-2010)

Can Tho, 2010

APPROVAL

MSc DUONG THI HUONG GIANG TRAN NON NUOC

MSc VO VAN SONG TOAN

Can Tho, November, 2010

PRESIDENT OF EXAMINATION COMMITTEE

ABSTRACT

Optimal selection of strain, fermentation conditions and substrate, which are very important for the successful production of cellulase by bacteria, were investigated in this study A total of 37 anaerobic bacterial isolates collected from soil, rumen cow, bagasse, rice husk was screened on modified Delafield medium, in which Whatman filter paper powder was used as substrate The result showed that the isolate VK52 adopted highest cellulase productivity among the others, illustrated by the largest haloes in diameter (20.5 mm) on Congo red-containing medium The optimal conditions for cellulase production by VK52 were determined as 300C, pH 8 and 4-day-incubation on the medium supplemented with 0.4 % yeast extract On laboratory scale the yield of endoglucanase and exoglucanase activities were 0.107 U/ml and 0.06 U/ml respectively, and 9.025% of substrate degradation There was a decrease of culture pH from 8 to 6.89 after cultivation Paper and cellulose powder were the best substrates for cellulase production by the isolate VK52 in comparision with rice straw, sugarcane bagasse and rice husk

Key words: anaerobic bacterial isolates, cellulase, filter paper powder, isolate VK52

CONTENTS

2 Materials and methods 3

3 Results and discussion 7 3.1 Screening of cellulase activity produced by anaerobic

3.5 Production of cellulases at laboratory scale by VK52

under optimal conditions

1 INTRODUCTION

Cellulose is the most abundant organic biomass on the earth (Tomme et al., 1995) It is the primary product of photosynthesis in terrestrial environments, and the most abundant renewable biological resource produced in the biosphere (100 billion dry tons/year) (Jarvis,

2003 and Zhang & Lynd, 2004) Cellulose is commonly degraded by

an enzyme called cellulase This enzyme is produced by several microorganisms, but commonly by bacteria and fungi (Bahkali, 1996; Shin et al., 2000 and Immanuel et al., 2006)

Complete enzymatic hydrolysis of cellulose requires synergistic action of 3 types of enzyme, namely cellobiohydrolase or exoglucanase, carboxymethycellulase (CMCase) or endoglucanase and ß-glucosidases (Bhat, 2000) Cellulases are used in textile industry for cotton softening and denim finishing; in laundry detergents for color care, cleaning, and anti-deposition; in the food industry for mashing; in the pulp and paper industries for de-inking, drainage improvement, and fiber modification They are also used for several pharmaceutical applications (Kirk et al., 2002) and (Cherry & Fidantsef, 2003)

Bacteria, which have significantly high growth rate in comparison to fungi, are well potential to be used for large scale cellulase production However, it has not been widely used Celluloytic property of some bacterial genera such as Cellulomonas, Cellovibrio, Pseudomonas, Sporosphytophaga spp (Nakamura and Kappamura, 1982); Bacillus and Micrococcus (Immanuel et al., 2006) were reported Enzyme production is firmly controlled in microorganisms; and these controls can be ameliorated for improving productivity Cellulase yields appear to depend on a complex

interaction of several factors like inoculum size, pH value, temperature, presence of inducers, medium additives, aeration, growth time and so on (Immanuel et al., 2006)

Therefore, this study was carried out to optimize the nutritional and environmental parameters for improving cellulase production by

the cellulolytic anaerobic bacteria

2 MATERIALS AND METHODS

2.1 Materials

- 37 anaerobic bacterial isolates used in this study had been

obtained from soil, cow rumen and sugarcane bagasse, and stored at

40C in Biotechnology Research&Development Institute, Cantho University

- Media:

M1 medium (modified Delafield medium) ( Ryckeoer et al, 2003) was used for the production of cellulose by the isolates It is composed (in g/l) of: Ground Whatman filter paper 10.0, (NH4)2SO4

1.0, K2HPO4 1.0, MgSO4.7H2O 0.5, NaCl 0.001, Agar 20.0

M2 medium was prepared as above without agar

- Chemicals: Ammonium sulfate (NH4)2SO4 (Merck), Bovin Serum Albumin (BSA) (Merck), cellulose powder (Himedia, India), yeast extract (India), K2HPO4 (Merck), MgSO4 (Merck), Congo Red (Merck), etc

- Facilities: Pbi-international autoclave (Germany), Orion

420A pH meter (USA), Incucell incubator (Germany), Laminar Flow (France), etc

- Method: Preliminary qualification of enzymatic activity from

37 isolates was done following the method described by Laurent et

al (2000) 20µl inoculums of each isolate were spotted on ground filter paper solid (GFPS) medium The plates were incubated at 30°C

for 5 days Cellulase activity was detected by staining the plates with Congo red dye (0.1 g/l) for 15 minutes The plates were then washed with a 1M NaCl solution to visualize cellulase activity, which was indicated by a bright orange halo surrounding the colony against a red medium background The diameters of the haloes, which were

proportional to cellulose activity, were measured The isolates

generating highest cellulase activity would be used for further

experiments

2.2.2 Effect of temperature and pH on cellulase production by

the selected isolates

- Objective: To determine the temperature and pH optimum

for cellulase production of the selected bacterial isolate from experiment 2.2.1

- Method: 20µl of primary culture was inoculated on a series

of GFPS plates whose pH varying from 4 to 11 The plates were incubated in different temperature: 300C, 350C, 400C, 450C, 500C The cellulolytic enzyme production of the selected isolates was determined according to Laurent et al (2000) after 5 days Obtained data were subjected to two-way ANOVA for significant different

2.2.3 Effect of yeast extract content on cellulase production of

selected isolates

- Objective: To determine the appropriate content of yeast

extract added to cultivation medium for highest cellulase production

- Method:

300 µl of bacteria primary culture were inoculated into falcons containing 15 ml M2 medium supplemented with different yeast extract content: 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6% The pH of the media was adjusted to pH optimum selected in experiment 2.2.2

After incubation at optimum temperature chosen in experiment 2.2.2, cell-free culture supernatants were obtained by centrifugation at 7,000 rpm for 20 minutes at 40C as crude enzyme mix

Enzyme activity and protein content were measured by the methods of Nelson – Somogyi (1944) and Bradford (1976),

respectively

2.2.4 Effect of fermentation time on cellulase production of

selected bacteria

- Objective: To determine the optimal cultivation time for

highest cellulase production

- Method: 15ml M2 medium (supplemented optimal yeast

extract concentration in experiment 2.2.3) inoculated with isolates as previous experiment were incubated at selected optimal temperature Crude enzyme mix was sampled after 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 days; and then was determined for cellulose activity and protein content as described above

2.2.5 Cellulase production by selected bacteria at laboratory

scale under determined optimal conditions

- Objective: To conduct an effective cellulase production

protocol at laboratory scale

- Method: Small scale experiments were carried out in conical

flasks (1000 ml) Each flask contained 800 ml cellulase production medium prepared as previous experiment After inoculation with 24

ml of selected anaerobic bacterial isolates (at the density of circa approximately (c.a.) 3.5 x 106 CFU/ml), the flasks were incubated at optimal temperature and cultivation time that were selected from previous experiments Crude enzyme mix was obtained and

examined for cellulose activity and protein content as previous experiments

2.2.6 Effect of cellulose-containing substrates on cellulase

production by seleted bacteria under optimal conditions

- Objective: To study the effect of different carbon sources on

cellulase production

- Method: Various cellulose-abundant substrates including

cellulose powder, rice straw, sugarcane bagasse, and rice husk were used as carbon sources in cellulase production medium Falcons supplemented optimal yeast extract concentration were inoculated with 300 µl of the inoculums These falcons were incubated under optimal conditions derived from previous experiments Then, cellulolytic activity of crude enzyme mix and protein content was determined as above

2.3 Experimental Design and Statistical Analysis

Raw data were analyzed by Analysis of Variance (ANOVA) in completely randomized design (CRD) mode using Statgraphics 3.0 software All experiments were performed in triplicates Least Significant Difference (LSD) test was used for comparison between the means

3 RESULTS AND DISCUSSION

3.1 Screening of cellulase activity produced by anaerobic

bacterial isolates

Cellulase productivity of 37 anaerobic bacterial isolates was

shown in Figure 3’ After 5 days of incubation at 300

C on M1 medium, 34 out of 37 (92%) isolates were confirmed as cellulose positive, 11 of which namely VK31, VK2, VK44, VK43, VK85, VK52, VK49, VK39, VK13, VK76, VK72, VK84 shown very strong cellulolytic activity (indicated by higher than 10 mm diameter halo zones) Isolate VK52 gave highest activity, as seen by a halo with 20.5 mm in diameter, thus it was used for later experiments

Figure 3’ Diameters of haloes (mm) in Congo Red-staining

assay generated by 37 anaerobic bacterial isolates

9.7 11.3 6.5

10.3

15.7 9.5

7.7 9.7 9.3 12.3

17.7 6.5

10.3 3.8

1.3

7.8

20.5 12.2

12.5 13.8 11.2 12.3 2.2

8.3 8.3 6.0 0.0

5.5 3.2

5.3 0.0

5.3 6.7 4.8 0.0

3.2 Effect of temperature and pH on cellulase production by VK52

The cellulase produce by VK52 seemed to be completely inhibited at strong acidic pH (below 5) or alkaline pH (above 10) The activity was also decreased at temperatures higher than 300C, and completely lost at 550C (Figure 5’) High was achieved at

neutral of slightly alkaline pH at 300C Ariffin et al (2006) reported that pH had significant influence on the enzyme biosynthesis of the bacteria, and the stability of the enzyme molecules themselves, which resulted in the change of its activity

Figure 5’ Effect of pH and temperature on cellulose

These results are similar to those from Abou–Taleb et al

(2009) who reported that the cellulase produced by Bacillus

alcalophilus S39 had highest activity at 300C and pH 7 Likewise,

Fred (1972) reported that pH 8 was optimal for cellulase production

by Thermomonospora curvata

3.3 Effect of yeast extract content on cellulase production of

selected anaerobic bacterial isolates

The influence of yeast extract supplement was clarified in

Figure 6, in which enzyme activity was increased with addition of ≤

0.4% of yeast extract Maximum activity of 0.067 U/ml was obtained

when 0.4% (w/v) yeast extract was added into cellulase production

medium (3-fold increase in cellulose activity than the control)

Figure 6 Effect of yeast extract on cellulase

The decrease of cellulase activity (Figure 6) at supplemetary yeast extract of more than 0.4% might be due to the overproduction

of reducing sugars (glucose) and cellobiose through product inhibition in enzyme kinetics (Howell and Mangat, 1978) This is supported by the results in Figure 7, in which there was a significant increase of protein content when yeast extract was added to cultivation medium The maximum protein content of 99.35 µg/ml was gained when 0.4% (w/v) yeast extract was supplied into medium

Figure 7 Variation of protein content by yeast extract

44.4456.39

99.35

69.2649.91

Yeast extract was reported to act as a stimulator for the producing of cellulase (Amtul, 1989), (Azzaz, 2009), (Abou-Taleb et al., 2009) The appropriate yeast extract content added into medium depends on different cultures Ammut (1989) determined optimum yeast extract for cellulase production by Cellulomonas flavigena was 0.2%, while Abou–Taleb (2009) indicated that 0.7% yeast extract

was the best for B alcalophilus S39 and B amyloliquefaciens C23 For most efficient cellulase production by Clostridium sp M7, 0.5% yeast extract was chosen by Lee and Blackburn (1975)

3.4 Effect of fermentation time on cellulase production by

VK52

The change of cellulase activity over cultivation time was recorded in Figure 8, in which the activity was rapidly increased during the first 4 days and gradually decreased afterwards The maximum cellulase activity of 0.064 U/ml was obtained after 4 days

of cultivation The activity loss might be due to denaturation of the enzyme when it was exposed to the fermentation conditions different than those of physical cellular metabolism (Krishna, 1999), (Liu and Yang, 2007) In addition, the depletion of nutrients and substrates in medium could also cause the reduction in cellulase production

(Laurent et al., 2000) Furthermore, the accumulation of cellobiose products might also be as an inhibitor to both endoglucanase and β-

glucosidase as reported in other studies (Howell and Mangat, 1978)

Figure 8 Effect of cultivation time on cellulase

215.37

156.11 136.11 127.96 115.74 109.63

On the other hand, the production of protein over time also got similar scheme, in which it increased rapidly in the first 4 days and yielded maximum value of 215.37 µg/ml, and gradually decreased later on (Figure 9) Nutritional depletion could be the cause of this phenomenon Ray et al (2007) claimed the cellulase productivity by Bacillus subtilis CY5 and Bacillus circulan TP3 reached maximum after 96h, with corresponding activities of 30.5 U/ml and 25 U/ml However, optimumal cultivation time is different among species For example, maximum CMCase and Avicelase activities of Cellulomonas flavigena (10 U/ml and 1.2 U/ml, respectively) were achieved after 72h of fermentation (Amtul, 1989); while Abhaykumar (1992) reported that highest CMCase activity of Vibrio agar-liquefaciens was determined at 0.09 IU/ml after 9 days

3.5 Cellulase production VK52 at laboratory scale in selected

optimal conditions

A laboratory-scale production (c.a 800 ml culture in 1 litter conincal flasks) of cellulose by VK52 was set up under the optimal conditions mentioned above Various parameters of this process were determined and shown in Table 4’ The activity (in U/ml) and protein

content (in µg/ml) were higher than those obtained from 15 ml culture described in previous experiments

The decrease of pH from 8 to 6.89 after 4 days could be explained by the appearance of acidic secondary products during fermentation, which in turn caused the activity loss of the enzymes later on Similar results were reported in the study of Krishna (1999)