SOLID SUBSTRATE FERMENTATION (SSF) OF SAWDUST BY

ASPERGILLUS NIGER FOR CELLULASE PRODUCTON

Front cover

Mohd Amirul Bin Ghazali (24063)

A final project report submitted in partial fulfillment of the

Final Year Project II (STF 3015) Resource Biotechnology

Supervisor: Miss Nurashikin Suhaili

Co-supervisor: Dr. Micky Vincent

Resource Biotechnology Programme

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

2012

i

Acknowledgement

I would like to thank to my supervisor, Miss Nurashikin Suhaili who has given me an

opportunity to become one of her Final Year Project’s students. Endless thanks for her valuable

guidance, advices and encouragements towards the completion of this project. Special thanks to

my co-supervisor, Dr. Micky Vincent for his kindness to support and help me towards the

completion of this project.

I also would like to express my appreciation to the Department of Molecular Biology,

Universiti Malaysia Sarawak for giving me this opportunity to fulfill my Final Year Project. I

really appreciate all the materials, equipments, instruments and other facilities provided which

are necessary during the progression of my project. I would like to express my gratitude to the

master student in Biochemistry Laboratory and lab assistants for their help, support and

cooperation when this project was carried out.

Lastly, thank you to all the colleagues for their ideas and advice while we were working

together at the laboratory. Not forgetting, to my beloved family who has given me a lot of

spiritual and financial supports.

ii

List of Abbreviation

MEA

SSF

SmF

DNS

g

mL

µg

nm

pH

Malt Extract Agar

Solid state fermentation

Submerged fermentation

3,5-dinitrosalicylic acid

Gram

millilitre

microgram

nanometer

A measurement of the acidity or alkalinity of solution [p stands for “potenz” which

means the potential to be while H stands for Hydrogen]

iii

List of Tables and Figures

Table 1: Enzymatic activity of cellulase at different pH............................................................... 24

Table 2: Enzymatic activity of cellulase at different Moisture Content ....................................... 24

Table 3: Enzymatic activity of cellulase at different pH Incubation Time ................................... 24

Figure 1: Microscopic morphology of A. niger. ............................................................................. 7

Figure 2:7-day old A. niger on MEA .............................................................................................. 9

Figure 3:A. niger on sawdust after 9 days of incubation .............................................................. 10

Figure 4.1: Effect of incubation on the cellulase activity………………………………………………. 13

Figure 4.2: The effect of pH on the enzyme activity of cellulase………………………….…………….….15

Figure 4.3: Effect of initial moisture content on the cellulase…………………………………..….….….…17

Figure A.1: Glucose Standard Curve…………......................................................................………...23

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Table of Contents

Acknowledgement ........................................................................................................................... i

List of Abbreviation ........................................................................................................................ ii

List of Tables and Figures............................................................................................................... ii

Table of Contents ........................................................................................................................... iv

ABSTRACT .................................................................................................................................... 1

1.0 INTRODUCTION .................................................................................................................... 2

2.0 LITERATURE REVIEW ......................................................................................................... 4

2.1 Sawdust ................................................................................................................................. 4

2.2 Cellulase Production via Solid Substrate Fermentation (SSF) ............................................. 5

2.4 Aspergillus niger ................................................................................................................... 6

3.0 MATERIALS AND METHOD ................................................................................................ 8

3.1 Pretreatment of sawdust ......................................................................................................... 8

3.2 Microorganism ...................................................................................................................... 9

3.3 Solid Substrate Fermentation (SSF) .................................................................................... 10

3.3.1 Effect on moisture content ............................................................................................ 10

3.3.2 Effect on pH.................................................................................................................. 11

3.3.3 Effect of incubation time. ............................................................................................. 11

3.4 Extraction Enzyme .............................................................................................................. 11

3.5 Cellulase Assay ................................................................................................................... 12

3.6 Statistical analysis ............................................................................................................... 12

4.0 RESULTS AND DISCUSSION ............................................................................................. 13

4.1 Effect of initial moisture content on SSF of sawdust by A. niger ....................................... 13

4.2 Effect of pH on SSF of sawdust by A. niger ....................................................................... 15

4.3 Effect of incubations time on SSF of sawdust by A. niger ................................................. 17

5.0 CONCLUSION ....................................................................................................................... 19

REFERENCES ............................................................................................................................. 20

Appendix ....................................................................................................................................... 23

1

Solid Substrate Fermentation (SSF) of Sawdust by Aspergillus niger

for Cellulase Production

Mohd Amirul Bin Ghazali

Resource Biotechnology Programme

Faculty of Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

Sawdust waste is one of the highly potential agricultural wastes that can be used as substrate for the

production of cellulase via solid state fermentation. This research focuses on the relevant cultural

condition for optimum cellulase production by the locally isolated Aspergillus niger using sawdust as

substrate via solid state fermentation. The aim of this research is to study the optimum cellulase

production using sawdust as substrate. Different levels of pH, initial moisture content and incubation

time were set in order to assess their effects on cellulase production. The optimum pH for cellulase

production was seen at pH 5.5 with 0.0463 IU/mL cellulase activity. Cultures moistened at 75% (v/w)

gave 0.0669 IU/mL while the optimum incubation time was found between day 5 and day 7 where the

highest cellulase production of 0.0523 IU/mL was attained. These preliminary results proved the

feasibility of sawdust as potential substrate for cellulase production by the locally isolated Aspergillus

niger.

Key words: cellulase, solid substrate fermentation (SSF), Aspergillus niger, sawdust

ABSTRAK

Sisa habuk kayu merupakan salah satu sisa pertanian yang mempunyai potensi yang tinggi untuk

digunakan sebagai substrat bagi penghasilan enzim sellulas secara fermentasi keadaan pepejal.

Kajian ini berfokus kepada kondisi kultur yang yang relevan untuk penghasilan sellulas yang optimum

daripada pencilan Aspergillus niger tempatan. Tujuan kajian ini adalah untuk mengkaji penghasilan

sellulas secara optimum menggunakan habuk kayu sebagai substrat. Kadar kelembapan, pH dan masa

pengeraman yang berbeza telah telah digunakan untuk mengenal pasti kesan kondisi tersebut

terhadap penghasilan sellulas. pH optimum untuk penghasilan sellulas ialah pada pH 5.5 dangan

bacaan 0.0463I U/mL. kultur yang telah dilembabkan pada kadar 75% (v/w) memberikan bacaan

0.0669 IU/mL. Manakala masa pengeraman yang optimum telah direkodkan antara hari ke-5 dan ke-7

dengan pengahasilan paling tinggi pada bacaan 0.0523 IU/mL. Keputusan awalan ini membuktikan

habuk kayu berpotensi sebagai substrat untuk penghasilan sellulas daripada pencilan Aspergillus

niger tempatan.

Kata kunci: sellulas, fermentasi keadaan pepejal, Aspergillus niger, habuk kayu

2

1.0 INTRODUCTION

Agricultural wastes particularly those that are lignocellulosics, can be converted into

commercial products such as ethanol, enzyme and single cell protein. Lignocellulosics are

sources of carbohydrates, continually replenished by photosynthetic reduction of carbon

dioxide by sunlight energy. They are the most promising sources for production of energy,

food and various chemicals.

Malaysia spent a lot of money yearly on various types of enzymes for local industries

use and research purposes (Lee et al., 2011). This was because less attempts have been made

to produce the commercial enzymes using low cost resources. Solid state fermentation (SSF),

an alternative for submerged fermentation (SmF) for enzyme production, was found to be

more favorable, which can be performed under limited financial and labor requirements (Lee

et al., 2011). Agro industrial residuals have a great potential to be used as substrate in SSF not

only for enzymes production but also for other secondary metabolites. In this aspect, countries

like Malaysia with abundant agro-industrial residual especially logging industry will be at

great advantage.

Large quantities of lignocellulosic wastes are generated through forestry, agricultural

practices and industrial processes, particularly from agro-allied industries such as breweries,

paper pulp, textile and timber industries. These wastes are generally accumulated in the

environment thereby causing pollution problems (Abu et al., 2000). Among the ways to

overcome the pollution problem caused by the accumulation of such wastes is by utilizing

them as substrates for bioconversion. One of the potential bioprocesses that can be initiated

from lignocellulosic agro waste is production of cellulase. Cellulase is generally known used

for commercial food processing in coffee. It also performs hydrolysis of cellulose during the

3

drying of vanilla beans (Waliszewski et al., 2007). Furthermore, cellulase is widely used in

textile industry and as components for laundry detergents (Sukumaran et al., 2005). It is also

applied in the pulp and paper industry for various purposes, and they are even used for

pharmaceutical applications. Currently, industrial demand for cellulase is being met by the

means of submerged fermentation (SmF) processes. Development of economical process for

cellulase production systems is hindered by the high cost of pure cellulose plus low yields of

cellulase for SmF processes.

Therefore, these scenarios necessitate the employment low cost yet reliable alternative

substrate such as for economical production of cellulase. The emphasis of this research project

was to study the production of cellulase production from sawdust via SSF using A. niger. The

parameters analyzed were incubation time, temperature, pH and inoculum size. Therefore the

specific objectives were:

i. to study the feasibility of sawdust as substrate for cellulase production by A. niger via

solid substrate fermentation.

ii. to identify the optimum conditions of cellulase production from sawdust by A. niger

via solid substrate fermentation.

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2.0 LITERATURE REVIEW

2.1 Sawdust

Wood industry plays important role in the socio-economic development of the country.

The increasing production of wood indicates the rising amount of its waste as well. Sawdust is

a byproduct produced from the wood processing industry. Generally sawdust composed of

fine particles of wood. Sawdust has a variety of practical uses, such as in agriculture mulch

and also in the manufacturing of the particle board.

According to Gan and Ho (1995), in wood based industry in Malaysia, removal of

wood waste is an important activity and at certain extent, it affects the productivity and

profitability of the mill operation. However, removal of waste can cause pollution in long term

period of time. The abundance of sawdust waste is hence seen here as a good supporting factor

in considering its usage as potential substrate for bioprocesses.

Plant biomass, especially sawdust is being researched as one of the desirable

alternative raw materials because of their low cost and easy availability (Musatto and Teixera,

2010). There are many advantages of the employment of agro-industrial waste as substrate for

bioconversion process. According to Singh et al., (2009) the use of agro-industrial as

substrates contribute to the cost reduction of enzyme production at the same time will

increase the awareness on energy recycling and conservation.

There are several works reported in the literature concerning the use of sawdust as

substrate for bioprocess. In addition Devi and Kumar (2012) reported on the use of sawdust

for cellulase production. Research done by Victor et al., (2003) employed sawdust as substrate

5

to yield cellulase in optimum condition. Sawdust also being cultivated for production xylanase

by Arthrobacter sp. (Murugan et al., 2011)

2.2 Cellulase Production via Solid Substrate Fermentation (SSF)

SSF is conveniently applied as the mean of cultivation for bioprocess that utilize agro-waste as

medium. SSF is defined as a cultivation that occurs on a non-soluble material that acts both as

support and a source of nutrient, with less amount of water and with the action of fermenting

agent (Manpreet et al., 2005). The fermentation takes place in the absence or near absence of

free water flowing. This condition resembles the natural habitat of the microorganism which

facilitates a better and more productive cultivation

SSF have several advantages over submerged fermentation (SmF) such as high

fermentation productivity, high end-concentration of products and stability, low catabolic

repression, cultivation of microorganism specifically for insoluble substrate and also low

demand on sterility due to low water activity used (Holker et al., 2004). Thus, SSF is seen as a

potential mean to yield high amount of bio product.

Cellulase is a group of hydrolytic enzymes which hydrolyze the β-glycosidc bonds of

native cellulose and related cellooligosaccharides (Jayant, 2011). It is the key enzyme for

hydrolysis of cellulosic materials. In the production of ethanol from cellulosic biomass

cellulase production was regarded as the most expensive step, accounting for about 40% of the

total cost (Spano et al., 1978). Cellulase production from various waste cellulosic materials

using different cellulolytic microfungi is being vigorously studied for cost reduction strategies

(Abu et al., 2000). Although a large number of microorganisms like fungi, bacteria and

6

actinomycetes are capable of degrading cellulose, only a few of them can produce significant

quantities of cell-free enzyme fractions capable of complete hydrolysis of cellulose in vitro

(Jayant, 2011).

Basic and applied studies on cellulase enzymes have demonstrated their

biotechnological potentials such as in food, poultry, brewing and wine making, agriculture,

biomass refining, pulp and paper, textile, and laundry.

2.4 Aspergillus niger

Filamentous fungi are the class of microorganisms preferred for enzyme production in both

SSF and SmF. In SSF, they filamentous fungi are the most important group of microorganism

used in SSF process owing to their physiological, enzymological and biochemical properties.

The hyphal mode of fungi of fungal growth and their good tolerance high osmotic pressure

condition make fungi efficient and competitive in natural micro flora for bioconversion of

solid substrate. Thus, these factors support the choice of A. niger as a candidate for cultivation

in this research in term of its availability and efficiency for enzyme production. Moreover,

filamentous fungi have better growth at low water activities compared to yeast and bacteria

(Molitoris, 2000)

A. niger is a filamentous fungus that commonly occurs in the environment and it is

generally regarded as nonpathogenic. Figure 1 shows the microscopic observation of A. niger.

It grows aerobically on organic matter and tolerates a wide range of temperature and pH. A.

7

niger has a long history of use as a source of citric acid and enzymes used in food processing.

The growth of this fungus depends on the influence of temperature, water activity and pH.

A. niger has been used in several research in SSF. Chinedu et al., (2008) reported that

A. niger has been use as inocula in fermentation sawdust and sugar cane pulp for production of

cellulase and xylanase. Moreover, Mrudula and Murugammal (2011) cultivated coir waste for

production of cellulase by A. niger. Besides, Acharya et al., (2008) reported about the

cellulase production using sawdust as substrate by A. niger.

Figure 1: Microscopic morphology of A. niger. Image retrieved from http://bjpsbiotech.edublogs.org/labs-

activities/beano-lab/

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3.0 MATERIALS AND METHOD

3.1 Pretreatment of sawdust

The sawdust waste was collected from sawmills in Kota Samarahan area. The substrate was

sundried for two days in order to reduce the moisture content and make them more susceptible

to grind. The sample was soaked in 1% (w/v) sodium hydroxide solution at a ratio of 1:10

(substrate: solution) for 3 hours at room temperature. After that, the sawdust was washed to

release the chemicals and autoclaved at 121 ºC for 1 hour which very crucial for sterilization

process. The treated substrate was filtered and washed successively with distilled water until

the wash water is achieved neutral condition. Then, the substrate was dried in the oven until a

constant weight is achieved. Finally, the substrate was ground and sieved with 0.1mm mesh

sieve before to be used in SSF.

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3.2 Microorganism

The A. niger strain that was isolated from sago hampas was obtained from Department of

Molecular Biology, Faculty of Resource Science and Technology, UNIMAS. The fungus was

subcultured on Malt Extract Agar (MEA). The fungus was grown at 37ºC for 5-7 days for the

development of spores as showed in Figure 2. The spore grown on MEA was harvested using

0.001% (w/v) Tween-80 and the spore concentration was determined by using

haemacytometer. Inoculum was prepared in the form of spore suspension with standardized

concentration of 1 x 106

spores/mL medium.

Figure 3Figure 3.1: 7-days growth old A. niger on Malt Extract Agar

(MEA).

Figure 2: 7-day old A. niger on MEA

Figure 2:7-day old A. niger on MEA.

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3.3 Solid Substrate Fermentation (SSF)

In this work, SSF culture was developed by using 5 g of sawdust supplemented with 1 mL of

mineral salt solution. The cultures were incubated at various incubation times, pH and initial

moisture content (as mentioned in Section 3.3.1, 3.3.2 and 3.3.3 respectively). Figure 3 shows

A. niger cultivated on sawdust.

Figure 3: A. niger on sawdust after 9 days of incubation.

3.3.1 Effect on moisture content

Optimization of initial moisture content was carried out by incubating the A. niger cultures at

60%, 65%, 70%, 75% and 80% (v/w). The moisture content of the substrate was examined by

adding using distilled water at different ratio between the substrate. After regular intervals,

enzyme assay was performed as mentioned in Section 3.5.

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3.3.2 Effect on pH

Optimization of pH was carried out by adjusting the initial of the culture pH of the culture at

4, 4.5, 5, 5.5 and 6. Upon sampling at certain intervals, enzyme assay were performed as

mentioned in Section 3.5.

3.3.3 Effect of Incubation Time

The effect of incubation time on cellulase activity was examined at different time intervals

namely 24, 48, 72, 96, 120, 144, 168, 192, and 216 hours, respectively, at temperature of 30

ºC. Upon sampling, cellulase assay was performed as mentioned in Section 3.5.

3.4 Extraction Enzyme

Extraction was done by mixing 50 mL of 0.01M, phosphate buffer (pH7) to the sampled

cultures. The slurry suspension was mixed thoroughly. Then the slurry suspension was

centrifuged at 6000 rpm at 4ºC for 23 minutes (Conti et al., 2001). After that, the slurry

suspension was filtered through 0.45 µm filter and kept at -20ºC for 24 hour prior cellulase

assays. Assays were performed in triplicates and the results were expressed as mean of

triplicate values.

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3.5 Cellulase Assay

The cellulase assay includes 0.5 mL of diluted enzyme solution, 1mL 0.1M citrate buffer at

pH 4.8 and a strip of Whatman No. 1 filter paper that has been curled round a glass. The

reaction mixture was incubated for 1 hour at 50°C. After incubation 3 mL DNS reagent was

added. The mixture was then boiled for 15 minutes. After the mixture has cooled, 1 mL

Rochelle salt was added. The absorbance was measured at 550nm using spectrophotometer

(UVmini1240v series, Shibasu Cooperation, Japan). The blank used was 0.5 mL of denatured

enzyme from the sample that was added instead of enzyme filtrate.

3.6 Statistical analysis

Statistical analysis was done by using Analyse-it software (version 2.26 Analyse-it, Inc.,

Leeds, UK). The mean values were compared by applying Tukey method via One-Way

Analysis of Variance (ANOVA).

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4.0 RESULTS AND DISCUSSION

4.1 Effect of initial moisture content on SSF of sawdust by A. niger

In this study, the initial moisture content of the culture was adjusted at 60% (v/w),

65%(v/w), 70%(v/w), 75%(v/w) and 80%(v/w). The fermentation was incubated for 6 days.

As different moisture content was tested, the highest cellulase production was found at 75%

(v/w) initial moisture content with the concentration of 0.0669 IU/mL. The production of

cellulase was significantly reduced in when the moisture level was less than 75% (v/w).The

second most optimal initial moisture level was 80% (v/w) moisture with the cellulase activity

of 0.0516 IU/mL. Moreover, there is no significant different observed in the maximum

production of cellulase between 75% (v/w) and 80% (v/w). Therefore, from this study, it can

be deduced that the optimal range of initial moisture content for cellulase production moisture

content for cellulase production from sawdust by A. niger was between 75% (v/w) and 80%

(v/w) (Figure 4.1).

SSF technique offers significant advantages in reducing the risk of contamination as

the ratio of water to substrate is comparatively low. And most bacterial species are unable to

grow at low water activity. Sawdust is mostly insoluble in water. Therefore water will have to

be absorbed into the substrate particles, which can be used by the fungi like A. niger to growth

and to undergo metabolic activity (Pandey, 1992). Different substrate may have different rate

of water absorbance

The rate of water absorbance by the different substrates varies from one to another.

Water plays an important role to swell the substrate and facilitates good utilization of

substrates for the A. niger to grow. Too high moisture level is believed to have reduced the

porosity of substrate, thus limiting the oxygen transfer into the substrate (Raimbault and

14

Alazard, 1980). In contrast, a low moisture content level might cause reduced solubility of the

nutrients of the solid substrate and lower degree of swelling and a higher water tension

(Lonsane et al., 1985). As indicated in Figure 4.1, the cellulase production was optimum

when the moisture content was set at higher levels which were 75% (v/w) to 80% (v/w).

Hence, it can be assumed that the degree of hydration of the sawdust plays an

important role on the growth of the fungi and subsequently for the enzyme production.

Figure 4.1: Effect of initial moisture content on cellulase activity.

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

60% 65% 70% 75% 80%

En

zym

ati

c a

ctiv

ity

(IU

/mL

)

Moisture content (v/w)

15

4.2 Effect of pH on SSF of sawdust by A. niger

In this study, the medium used in SSF was initially adjusted to pH values of 4 , 4.5, 5,

5.5 and 6 and the fermentation was conducted for 6 days. Throughout the range tested in this

work, the highest cellulase production was found at pH 5.5 with the enzymatic activity of

0.0463 IU/mL. The maximum production of cellulase was significantly reduced when the pH

was lowered from 5.5 to 4. The second highest point of cellulase production was at pH 5 with

the enzymatic activity of 0.0403 IU/mL. Moreover, there is no significant different was seen

in the maximum production of cellulase achieved in the pH region between pH 5 and 5.5.

Therefore, in Figure 4.2 it can be suggested the optimal pH for production of cellulase lies

from pH 5 to 5.5.

From the result obtained, it can be concluded that the optimum condition for A. niger

to grow and produce high amount of cellulase is in acidic condition. Clarke & Stone (1965)

published a result with a rather broad range which was between pH 4 and pH 6 for the

production of cellulase by A. niger.

On the other hand, Ikeda et al., (1973) conclude that cellulase activity was optimum at

pH 2.5. Hurst et al., (1976) reported that the H+ concentration which indicates the acidity of

the fermentation had a role in the stability of the cellulase produced by the A. niger.

Moreover, the H+ concentration may also influence the optimum production of cellulase.

16

Figure 4.2: Effect of pH on the cellulase activity.

0

0.01

0.02

0.03

0.04

0.05

0.06

4 4.5 5 5.5 6

En

zym

ati

c a

ctiv

ity

(IU

/mL

)

pH

17

4.3 Effect of incubations time on SSF of sawdust by A. niger

The cellulase production was analyzed for 9 days (216h) and was assayed at 1 day

(24h) interval. As illustrated on Figure 4.3, in general there was a rapid rise in the cellulase

activity after 48 hours of incubation until the peak point was reached at 144th

hour. These

results are in agreement with the reports made by Devi and Kumar (2012). Following that, the

cellulase activity decreased gradually towards the end of the duration tested. This is because

further increase in the incubation time might reduce the enzyme production. This is due to the

depletion of macro and micronutrients in the fermentation medium with the lapse in time,

which stressed the fungal physiology resulting in the inactivation of secreting machinery of

the enzymes (Nochure et al., 1993). The highest production of cellulase was determined on

day 6 (144th

h) where 0.0523 IU/mL was achieved. There was also no significant difference

between the production of cellulase on day 6 and 8. It is expected that the highest production

of cellulase might be within that range. Hence, it can be suggested that further investigation s

should be done in finding the more exact time that favours optimal production of cellulase.

Therefore, short incubation time necessary for optimal production was observed at 72 h in

SSF. Short incubation period for enzyme production offers the potential for inexpensive

production of enzyme

18

Figure 4.3: Effect of incubation on the cellulase activity.

0

0.01

0.02

0.03

0.04

0.05

0.06

24 48 72 96 120 144 168 192 216

En

zym

ati

c a

ctiv

ity

(IU

/mL

)

Incubation Time (hour)

19

5.0 CONCLUSION

Cellulase is an important industrial enzyme which has wide applications. In this work,

the feasibility of employing sawdust as substrate for cellulase production by A. niger via SSF

was demonstrated. The results showed the ability of A. niger to degrade the sawdust and

produce cellulase. The optimum pH for cellulase production was observed at pH 5.5 with

0.0463 IU/mL cellulase activity. Cultures moistened at 75% (v/w) gave 0.0669 IU/mL while

the optimum incubation time was found between day 5 and day 7 where the highest cellulase

production of 0.0523 IU/mL was obtained.

The study of optimum conditions for SSF using sawdust as substrate is important in

order to find an economical way to produce cellulase particularly in large scale. In addition, it

also provide beneficial time management, reduce capital cost and energy saving. In future,

more parameters should be incorporated into the optimization studies such as inoculum size,

amount of nitrogen source and temperature. The cultivation can also be carried out under SmF

method.