BIODEINKING OF THE MIXED OFFICE PAPER USING THE … of the Mixed Office Paper Using the Microbial... · significant difference for the deinking efficiency between chemical deinking

BIODEINKING OF THE MIXED OFFICE PAPER USING THE MICROBIAL

CONSORTIA DEVELOPED AND FORMULATED FROM THE UNIMAS

MICROBIAL COLLECTION

ELIANE CHOO YUAN SYN

This project is submitted in partial fulfillment of the requirements for the degree of

Bachelor of Science with Honours (Resource Biotechnology)

Resource Biotechnology (WS47)

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

2010

i

ACKNOWLEDGEMENT

First of all, I would like to express my sincere appreciation and gratitude to my cheerful and

helpful supervisor, Dr. Awang Ahmad Sallehin for his precious guidances, opinions and

comments throughout my final year project.

Next, I would like to convey utmost appreciation to all the postgraduates students and

research assistant in the Genetic Molecular Laboratory (GML): Mr George Deng (MSc), Mr

Frazer Midot MSc), Mr. Azlan (MSc), Mr Simon (MSc), Miss Lee Wai Ha (MSc), Miss

Noorhidayah Bt Nut (MSc), Miss Angeline (Research Assistant) as well as Miss Sheela Ak

Ungau (lab assistant of GML) for their helps, advises, explanations and suggestions.

Also, special thanks to Dr.Yuzine and Mr. Huzal Irwan b. Husin (lab assistant) for

allowing me to use the waterbath in Animal Research Lab 2. Furthermore, I also take this

opportunity to thanks all my friends and course-mates of Resource Biotechnology (2007-2010)

for giving me supports and assistance to overcome all kinds of problems during this project.

Last but not least, a thousand thanks to my beloved family members who always take

care of and support me throughout my undergraduate study in Universiti Malaysia Sarawak

(UNIMAS).

ii

Table of Contents

Page

Acknowledgement……………………………………………………………. i

Table of Contents ……………………………………………………………. ii

List of Table………..……………………………………………………….... iv

List of Figure…………………………………………………………………. vi

List of Abbreviations……………………………………………………….. vii

List of Appendixes………………………………………………………….. viii

Abstract / Abstrak…………………………………………………………… ix

1.0 Introduction 1

2.0 Literature Review 4

2.1 Bacillus spp ………………………………………...………………… 4

2.1.1 Bacillus licheniformis…………………………………………... 5

2.1.2 Bacillus amyloliquefaciens……………………………………. 5

2.2 Paper recycling 6

2.2.1 Deinking…………………………….......................................... 6

2.2.2 Chemical deinking……………………………………………… 8

2.2.3 Enzymatic deinking…………………………………………….. 10

2.3 Environmental issues in paper recycling……………………………… 11

3.0 Materials and Methods……………………………………………………….. 12

3.1 Bacteria source and culture……………………………………………. 12

3.1.1 Identification of the bacteria (Gram staining)…………………... 13

3.1.2 Preparing stock culture and working culture of the bacteria…… 13

3.2 Testing for thermostability and alkalotolerant of the bacteria ……….

3.2.1 Congo red screening…………………………………………….

3.2.2 DNS test…………………………………………………………

3.2.2.1 Preparation of glucose standard…………………………….

3.2.2.2 Enzyme activity…………………………………………….

3.2.3 Maintaining and culturing bacteria…………..…………………..

14

14

15

15

15

16

17

3.3 Trial deinking………………………………………………………….. 17

3.3.1 Paper source preparation………………………………………… 17

3.3.2 Biodeinking……………………………………………………... 18

iii

3.3.3 Controls……………………………………………………….. 18

3.4 Handsheets properties……………………………………………….. 18

3.5 Statistical analysis…………………………………………………… 19

4.0 Result and Discussion………………………………………………............

4.1 Identification of the bacteria (Gram staining)…………..……………

4.2 Testing for the thermostability and alkalotolerant of the bacteria…..

4.3 Congo Red test…………………………………………………….....

4.4 DNS test………………………………………………………………

4.5 Trial deinking…………………………………………………………

4.6 Statistical analysis……………………………...……………………..

4.6.1 Trial deinking using different number of bacteria…………......

4.6.2 Trial deinking using single bacteria ……………………………

4.6.3 Trial deinking using single different combination of bacteria ...

4.6.4 Comparison between the efficiency of media used: MSM and

distilled water…………………………………………………...

4.6.5 Comparison between the efficiency of chemical deinking and

biodeinking……………………………………………………..

4.6.6 Comparison between trial deinking using single bacteria and in

combinations………………………………………………….

4.7 Overall discussion…………………………………………………….

20

20

21

21

23

27

28

30

35

39

44

48

52

54

5.0 Conclusion and Recommendation…………………...……………………….. 57

6.0 References …………………………………………………………………… 59

7.0 Appendices…………………………………………………………………… 62

iv

List of Table

List Table

Page

Table 1 Chemical compounds and their functions. 9

Table 2 Reaction mixture for glucose standard curve. 16

Table 3 Designation of bacteria A. 30

Table 4 Designation of bacteria B. 35

Table 5 Designation of bacteria C. 39

Table 6 Designation of bacteria for trial deinking using single

bacterial and in combinations.

44

Table 7 Absorbance reading (OD575nm) of glucose standard at

different concentration (mg/ml).

63

v

List of Figures

List Figure Page

Figure 1 Diagram for deinking system. Adapted from Thompson (1992) 7

Figure 2 Isolate M7 (1000X) 20

Figure 3 Isolate M5 (1000X) 20

Figure 4 Bacillus pumilus (1000X) 20

Figure 5 Bacillus licheniformis P7 (1000X) 20

Figure 6 Bacillus amyloliquefaciens UMAS 1002 (125X) 20

Figure 7 Growth was observed on the plate inoculated with the bacteria

culture after 24 hour incubation in 50°C

21

Figure 8 The agar plate was spotted with bacteria cultured in MSM with

CMC (above) and MSM with filter paper (below)

22

Figure 9 &10 Agar plate (MSM + CMC) spotted with bacteria culture (24 hour)

incubated in 37 °C (left) and 50°C (right)

22

Figure 11 &12 Congo Red result for agar plate (MSM + CMC) spotted with

bacteria culture (24 hour) incubated in 37 °C (left) and 50°C (right)

22

Figure 13 &14 Congo Red result for agar plate (MSM + CMC) spotted with

bacteria culture (48 hour) incubated in 37 °C (left) and 50°C (right)

22

Figure 15&16 Samples for DNS tests 23

Figure 17&18 Ink were floating in the media during the deinking process 27

Figure 19&20 Handsheets prepared after three days of incubation 27

Figure 21 Graph of glucose conversion rate for enzyme CMCase versus time

of incubation with media distilled water

31


of incubation with media MSM

31

Figure 23 Graph of glucose conversion rate for enzyme FPase versus time of

incubation with media distilled water

32


incubation with media MSM

32


of incubation with media distilled water 36



36



37



37


of incubation with media distilled water

40

Figure30 Graph of glucose conversion rate for enzyme CMCase versus time


40



41

vi



41

Figure 33&34 Comparison for the glucose conversion rate (CMCase activity) for

the same bacteria cultured at different media, using single bacteria

type, for day one (left) and day three (right)

45

Figure 35&36 Comparison for the glucose conversion rate (CMCase activity) for

the same bacteria cultured at different media, using various

combination of bacteria, for day one (left) and day three (right)

45

Figure 37&38 Comparison for the glucose conversion rate (FPase activity) for the

same bacteria cultured at different media, using single bacteria type,

for day one (left) and day three (right)

46

Figure 39&41 Comparison for the glucose conversion rate (FPase activity) for the

same bacteria cultured at different media, using various combination

of bacteria, for day one (left) and day three (right)

46

Figure 41 Handsheet from trial deinking using B.amyloliquefaciens UMAS

1002

49

Figure 42 Positive control (chemical deinking) from single bacteria trial

deinking

49

Figure 43 Handsheet from trial deinking using Bacillus spp. isolate M5. 49

Figure 44 Negative control (blank media, without bacteria inoculum) 49

Figure 45 Handsheet from trial deinking using bacteria combination C9. 50

Figure 46 Positive control (chemical deinking) from trial deinking using

bacteria in combinations

50

Figure 47 Handsheet from trial deinking using bacteria combination C5 50

Figure 48 Negative control (blank media, without bacteria inoculum) 50

Figure 49 Comparison between deinking using the bacteria individually and in

various combination, for media MSM on day one of incubation

52


various combination, for media MSM on day three of incubation

52


various combinations, for media distilled water on day one of

incubation

53


various combinations, for media distilled water on day three of

incubation

53

vii

List of Abbreviations

CMC Carboxy Methyl Cellulose

oC Degree celcius

DNS Dinitrosalicyclic Acid

OD Optical Density

g Gram

LB Luria Bertani

M Molar or molarity (moles of solute per liter of solution)

MSM Minimal Salt Media

min Minute(s)

mL Mililiter

mg Miligram

mM MiliMolar

µl Microliter

% Percentages

rpm Revolution per minute

TAPPI Technical Association of the Pulp and Paper Industry

V/W Volume / Weight (concentration)

W/V Weight / Volume (concentration)

viii

List of Appendixes

List Appendix

Page

Appendix 1 Flow chart of methodology 62

Appendix 2 Glucose standard curve 63

Appendix 3 Gram Staining 63

Appendix 4 Formula of media 64

Appendix 5 Comparing the significant different of the number of

bacteria used

66

Appendix 6 Comparing the significant of trial deinking using single

bacteria

69

Appendix 7 Comparing the significant of the bacteria combination used 74

Appendix 8 Comparison between the efficiency of media used : MSM

and distilled water (single bacteria deinking)

84

Appendix 9 Data for Trial deinking using various number of bacteria

culture, for investigating enzyme CMCase (upper) and

enzyme FPase (lower), using two media, distilled water and

MSM, respectively.

85

Appendix 10 Data for Trial deinking using single bacteria culture, for

investigating enzyme CMCase (upper) and enzyme FPase

(lower), using two media, MSM and distilled water,

respectively.

87

Appendix 11 Data for Trial deinking using various combination of

bacteria culture, for investigating enzyme CMCase and

enzyme FPase , using two media, distilled water (upper) and

MSM (lower), respectively.

89

ix

Biodeinking of mixed office paper using the microbial consortia developed and

formulated from the UNIMAS Microbial Collection

Eliane Choo Yuan Syn Resource Biotechnology Programme Faculty of Resource Science and Technology Universiti Malaysia Sarawak

ABSTRACT One of the main obstacles in paper recycling is the removal of ink particles- the deinking process. Conventional

deinking using chemicals may cause environment problems and pose potential health hazard. Thus, biodeinking

emerged as an alternative to chemical deinking. Biodeinking involves the usage of enzymes for the deinking

process. Studies have shown that microorganisms such as Vibrio sp. and fungi can be used in biodeinking as they

produce enzymes (pectinases, hemicellulases and cellulases) that facilitate the ink removal. Hence, this study was

conducted to investigate the ability of Bacillus spp. in biodeinking of mixed office papers, using a consortium of

Bacillus spp. The Bacillus spp studied were Bacillus licheniformis P7, Bacillus amyloliquefaciens UMAS 1002,

Bacillus pumillus, Bacillus spp. isolate M5 and isolate M7. The consortium was formulated based on their ability

to withstand high temperature and alkali condition and the deinking efficiency. Deinking efficiency was

evaluated based on the enzyme activity, which was measured by the DNS method. The enzymes investigated in

this study were CMCase and FPase. From the trial deinking, B. amyloliquefaciens UMAS 1002, Bacillus spp.

isolate M5 and isolate M7 were among the high enzyme producers and the combination C9, which consists of

this three species was the most ideal bacteria consortia for the deinking. From the study, it was deduced that

biodeinking using Bacillus spp. in combination was better than using them individually. As there was no obvious

significant difference for the deinking efficiency between chemical deinking and biodeinking, enzymatic

deinking should be given priority as it can reduce chemicals usage and more environmental friendly.

Keywords: Paper recycling, biodeinking, Bacillus spp., mixed office paper, enzyme activity, deinking efficiency

ABSTRAK

Salah satu halangan utama dalam pengitaran-semula kertas ialah proses penanggalan dakwat atau

penyahdakwatan. Penyahdakwatan tradisonal menggunakan bahan kimia yang berpotensi mencemarkan alam

dan membahayakan kesihatan. Justeru itu, penyahdakwatan secara biologikal muncul sebagai alternatif. Proses

ini melibatkan penggunaan enzim dalam penyahdakwatan. Pelbagai kajian telah menunjukkan yang

mikroorganisma seperti Vibrio sp. dan fungi dapat digunakan dalam penyahdakwatan secara biologikal kerana

menghasilkan enzim (pektinase, hemiselulase and selulase) yang dapat membantu menanggalkan dakwat kertas.

Oleh itu, kajian ini dijalankan untuk mengkaji keupayaan Bacillus spp. dalam penyahdakwatan secara biologikal

terhadap campuran kertas pejabat dengan menggunakan konsortia Bacillus spp. Antara Bacillus spp. yang dikaji

termasuklah Bacillus licheniformis P7, Bacillus amyloliquefaciens UMAS 1002, Bacillus pumillus, Bacillus spp.

isolate M5 dan isolate M7. Pembentukan konsortia ini bergantung kepada keupayaan backeria tersebut menahan

suhu tinggi dan keadaan alkali serta keberkesanan dalam penyahdakwatan. Keberkesanan penyahdakwatan

adalah berdasarkan aktiviti enzim yang disukat dengan kaedah DNS. Dua enzim yang dikaji ialah CMCase dan

FPase. Dalam percubaan penyahdakwatan, didapati Bacillus amyloliquefaciens UMAS 1002, Bacillus spp.

isolasi M5 dan isolasi M7 adalah antara penghasil enzim yang terbanyak dan kombinasi bakteria yang paling

ideal ialah C9 yang terdiri daripada tiga jenis bakteria ini. Daripada kajian ini, dapat disimpulkan bahawa

penyahdakwatan menggunakan bakteria secara kombinasi adalah lebih baik daripada secara individu.

Memandangkan tiada perbezaan nyata antara penyahdakwatan menggunakan kaedah kimia dan biologi,

penyahdakwatan secara biologikal harus diberi keutamaan kerana ia dapat mengurangkan penggunaan bahan

kimia dan lebih mesra alam.

Kata kunci: Pengitaran-semula kertas, penyahdakwatan secara biological, Bacillus spp., campuran kertas pejabat,

aktiviti enzim, keberkesanan penyahdakwatan

1

1.0 INTRODUCTION

Paper is the cornerstone of the development process, crucial to literacy and important to

increased cultural and business exchange (Murtedza, 1999). Over the year, the sustained

economic growth, increased population, higher living standard, rising literacy levels and

demands for packaging materials have boost up the development of pulp and paper

industry. As early as in 1960, it was realized that the consumption of pulp and paper

products in Malaysia, which were mainly imported, will increased tremendously. Thus,

government have established the pulp and paper industry locally. The plantation of various

high quality timber species, such as Pinus caribaea var. hondurensis, Acacia mangium and

Gmelina arborea were initiated to ensure the supply of raw materials locally. However, in

the long run, the land availability, high capital investment and long gestation gap has lead

to the need to search for other source of raw materials for this industry.

Subsequently, this has triggered the efforts of looking into utilizing recycled

papers or secondary fibers as a new source of raw materials for the industry (Lee et al,

2006). It is hoped that this may help to overcome the high demand of fibers for

papermaking industry and serve as solution for the increased paper waste produced yearly.

In paper recycling, the most important step involved is the removal of the ink

particles or deinking process. Previously, the deinking process of waste-papers, mostly

newspaper, books, and magazines were rather simple because they used vegetables-oil

based ink, which can be removed via the conventional method. However, the introduction

of photocopier machines and laser printers has contributed to the high content of laser-

printed papers among the mixed office paper-wastes (MOP), which makes the deinking

step more difficult (Vyas & Lachke, 2003). This is because the ink or toner used

polymerised with the paper fibers during the high temperature while printing and is more

2

difficult to be removed and detached during recycling process compared to conventional

vegetables oil-based ink (Pala et al, 2003; Fricker et al, 2007).

Conventionally, the ink-removing or deinking process in paper recycling uses

various chemicals such as sodium hydroxide, sodium silicate, hydrogen peroxide,

chelating agents and surfactants to remove the ink (Venugopal, 1997). However, over the

year, the usage of these chemicals has begun to spark off debates among environmentalists

as they feared that these chemicals used may lead to costly waste water treatment (Lee et

al, 2006), environment pollution and impose long-term health hazards (Rix et al, 1997).

Thus, many researchers began to look at the possibility of using a biological-based

method to de-ink. In 1992, Prasad et al reported that enzymes can serve as alternative to

remove the ink and more environmental friendly. In addition, studies on the capabilities of

various microorganisms such as Vibrio sp. and fungi to produce enzymes (pectinases,

hemicellulases and cellulases) that can facilitate the ink removal also adds on to the

possible usage of enzymes in the deinking process (Mohandass & Raghukumar, 2005;

Soni et al, 2008). Thus, this led to emergence of enzymatic deinking or biodeinking .

In this study, instead of focusing on biodeinking using an individual

microorganism alone, this study aimed at investigating the biodeinking potential of the

Bacilllus spp. using a consortium of various Bacillus species which includes Bacillus

licheniformis P7 and Bacillus amyloliquefaciens UMAS 1002 to evaluate the deinking

efficiency of the bacteria used individually and in different combinations. Besides that, the

ability of the bacteria to withstand high temperature and alkaline condition would be an

added advantage as deinking is usually performed under these conditions. Subsequently,

this may be applied in large scale biodeinking process to increase the efficiency of

deinking process for recycling waste-papers, which in turn minimizes the cost for paper

recycling.

3

The objectives of this study are:

1. To test for Bacillus spp. that can withstand high temperature and alkali condition.

2. To investigate the ability of Bacillus spp. in removing ink particles of the MOP.

3. To perform trial biodeinking using the thermophilic and alkaliphilic Bacillus spp.

4. To evaluate the effectiveness of biodeinking using the Bacillus spp. individually

and in combinations.

5. To compare the deinking efficiency between chemical deinking and enzymatic

biodeinking.

4

2.0 LITERATURE REVIEW

2.1 Bacillus spp.

Bacillus spp. is the rod-shaped, aerobic or facultative aerobic, endospore - forming

gram positive bacteria (Claus and Berkeley, 1986). Majority of the Bacillus spp. are

mesophiles, which grows well at 30°C. Some of them are thermophiles, which grow at

55 °C - 60 °C. They can either be acidophiles or alkaliphiles.

According to Arbrige et al (1993), Bacillus spp. is a class of bacteria that has the

ability to produce extracellular enzymes such as protease, amylase, cellulose and other

degradative enzymes. Priest (1977; 1992) defined extracellular enzymes as enzymes that

are secreted by microorganisms, outside the cell into the surrounding medium of the cells.

These enzymes can be easily extracted from the microorganisms. Thus, Bacillus spp. has

been extensively use in industrial applications, especially in laundry and food industry. For

example, the alkaline cellulases with optimal activities at pH 8.5-9.5 produced by Bacillus

strain KSM-19, KSM-64 and KSM-520 for laundry detergent (Horikoshi, 1999).

Schallmey et al (2004) also reported that Bacillus spp. can produce alkaline enzymes such

as proteases, amylases, xylanases and cellulases that can tolerate high temperature. These

enzymes had been largely applied in detergent industry and de-hairing of leather. Besides

that, the use of some Bacillus spp. as industrial producers of vitamin riboflavin and

supplement poly-γ-glutamic acid has demonstrated their ability to secrete large amount of

extracellular enzymes and a higher growth rate.

Hence, these examples and characteristics have illustrated the potential of Bacillus

spp. for being utilizes in other industries, such as the paper recycling industry. In this study,

among the Bacillus spp. used are Bacillus licheniformis P7, Bacillus amyloliquefaciens

UMAS 1002 (isolated from the sago pith waste), Bacillus pumilus, and two Bacillus spp.

isolated from Paku Hot Spring, Lundu.

5

2.1.1 Bacillus licheniformis

Bacillus licheniformis is a rod-shaped, gram positive bacteria. William et al (1990)

reported that this group of bacteria was thermophiles and alkaliphiles that can withstand

high temperature and alkali condition. In his study, he showed that Bacillus licheniformis

can grow well in temperature ranging from 45 °C - 50 °C and pH about 8.0. Hence, it is

very suitable to be used in deinking process which is generally performed in high

temperature and alkali condition. Bacillus licheniformis had been widely studied in the

production of penicillinases (Priest, 1977). It produces the enzyme protease, which is used

extensively as de-hairing agent in the leather industry, as well as in detergent industry

(Schallmey et al , 2004). The Bacillus licheniformis used in this study is Bacillus

licheniformis P7 isolated by Dayang Iqliema (2008).

2.1.2 Bacillus amyloliquefaciens

According to Priest (1977), Bacillus amyloliquefaciens is a strain of Bacillus spp. that has

the ability to secrete high level of liquefying alpha amylase and protease. It is found that

this bacteria is closely similar to Bacillus subtillis (Claus &Berkeley, 1986). According to

Apun et al (2000), Bacillus amyloliquefaciens has cellulolytic and amylolytic properties,

suggesting that it produced the enzyme amylase and cellulase. A study by Jong (1997) and

Apun et al (2000) shown that it was able to hydrolyse sago pith waste into reducing sugars

in a minimal fermentation medium. Furthermore, this bacteria strain is able to produce

high level of amylase and cellulase enzymes by utilizing the sago pith waste as its carbon

source. This rendered it to be a suitable candidate for biodeinking process. The Bacillus

amyloliquefaciens used in this study was Bacillus amyloliquefaciens UMAS 1002 isolated

by Apun et al (2000).

6

2.2 Paper recycling

Compact Oxford English Dictionary (2008) defined paper as material manufactured in thin

sheets from the pulp of wood or other fibrous substances, used for writing or printing on or

as wrapping material. Meanwhile, recycle can be interpreted as a process of converting

waste into a usable form or to reclaim a material from waste. Thus, paper recycling can be

inferred as re-use or converting waste-papers into usable form.

Paper recycling has been introduced as a strategy in solid waste management. It

serves as alternative to landfill (reducing waste materials produced) and incineration for

domestic and industrial waste management. In the same time, paper recycling assists in

reduction of deforestation and wood consumption, this indirectly contributes to the

conservation of the resources (trees). Furthermore, recycling waste paper can minimize

energy usage.

According to Robert (1996), recycled paper has become one of the major sources

for paper and pulp industry. Lee et al (2006) has also suggested that recycled paper can be

used as the raw material for paper and pulp industries. It provides a cheap and readily

available source, especially in the countries that encounter wood deficiency problem.

However, one of the main obstacles for paper recycling is the deinking process that will

affect the quality of the paper produced (Vyas & Lachke, 2003).

2.2.1 Deinking

Deinking is a process of removing printed ink particles from a paper source (Thompson,

1992). It is the most important chemical process involved in waste-paper preparation for

high quality grades. Overall, there are four main stages in deinking, namely repulping with

the associated ink removal from the fibers, cleaning to remove the ink from the stock,

7

separation of residual ink contaminants from the fibers stock and bleaching (Biermann,

1993).

Deinking is performed by combining mechanical, chemical and thermal forces. It

begins with the pulping stage, in which the waste-papers are blended in water. The high

temperature, chemicals and water assists in the swelling of the fibers. It usually begins

with the adjustment of fibers suspension to a high pH using alkali, which causes the

swelling of the fibers. The alkali help in saponifying the vehicle of the printing ink and

allowing the release of the pigment contained in the ink. At some point, sodium hydroxide

is added to facilitate the dispersion of the pigment and prevents the agglomeration of the

released ink particles. Alkali also assists in breaking down the sizing chemical in the paper,

which are often bonded via alkali sensitive ester links. As a result, the attached ink and

other compound of the fibers start cracking off and detached. Further blending and

churning accelerate the separation of ink from fibers. After pulping, the ink is removed via

the washing, flotation and dispersion process. A diagram of deinking system is shown

below.

Pulping

Water and waste-papers are blended in large pulper. Swollen fibers rub against

each other, starting the process of ink removal.

Flotation

Stock is aerated and foaming agents (such as 3-5% saponified fatty acid) are

added to attract ink particles to air bubbles. The air bubbles with the ink particles

will rise to the surface and removed by the action of a slow rotating paddle.

8

Basically, there are two methods for deinking, namely chemical deinking and enzymatic

deinking. Conventionally, chemicals are being used to remove the ink particles.

2.2.2 Chemical deinking

Chemical deinking is the conventional method for removing ink particles. It involves

usage of chemical compounds such as sodium hydroxide, sodium silicate, hydrogen

peroxide, chelating agents and surfactants for deinking. Following are the examples of

these chemicals and their functions.

Washing

Removes smaller ink particles, clays and other paper filler by washing them out

with water.

Bleaching

Stock undergoes bleaching to improve brightness. Usually involved usage of

hydrogen peroxide, sodium hypochlorite and other chlorination.

Dispersion

Stock is thickened, and any remaining ink is dispersed into very small particles.

Figure 1: Diagram for deinking system. Adapted from Thompson (1992).

9

Chemical

compound

Function

sodium

hydroxide

(NaOH)

Increases pH of the solution to alkali condition, help in swelling of

the paper fibers and releasing of the ink particles from the fibers

hydrogen

peroxide (H202)

Decolorised chromophores produced at the alkaline pH in the pulp

and enhance the brightness of the pulp (‘System’, 1997). Hydrogen

peroxide reacts with the sodium hydroxide to produce the perhydroxy

anion (HOO-), which is an active bleaching chemical (Stevens &

Hsieh, 1994).

H202 + NaOH HOO- + Na

+ + H 20

sodium silicate

(NaiOJ) or water

glass

Acts as buffer to minimize the fluctuation of pH in which hydrogen

peroxide is active and helps prevent the ink particles from re-attach

back on the fibers.

Chelating agent

(EDTA)

Forms soluble complexes with heavy metal ions, reducing the

decomposition of hydrogen peroxide by the heavy metals and

subsequently increases paper brightness (Stevens & Hsieh, 1994).

However, this method had causes several environmental impacts such as waste water

treatment, water pollution and long-term health hazards (risk of cancer). According to Lee

et al (2006), the chemicals released from deinking may increase cost for waste water

treatment. For example, NaOH used will increase the Chemical Oxygen Demand (COD)

and Biological Oxygen Demand (BOD) of water, which need to be treated before

discharging into the river (‘System’, 1997). Meanwhile, some of the chemicals used

Table 1: Chemical compounds and their functions.

10

contains mutagenic agent. For example, the 3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-

TCDD) and polychlorinated dibenzofurans (PCDFs) found in the effluent water from

paper mills (Rix et al, 1997). A study by Rix et al in 1997 shown that the workers from

the paper making industry has an increase risk for cancer, especially pharyngeal cancer

and Hogkin’s disease. Moreover, it was found that the ferns near the contaminated river

have a high incident of chromosomal mutation. Hence, enzymatic biodeinking is

introduced as an alternative.

2.2.3 Enzymatic Biodeinking

An alternative for chemical deinking is the usage of enzymes to remove the inks, known as

enzymatic biodeinking. It involves the usage of enzymes in the deinking process and

reduces usage of chemicals. This method is more environmental friendly because the

enzymes are readily biodegraded and unlikely to pose any serious environmental impacts.

Enzymatic biodeinking has been highlighted in a study by Welt and Dinus (1994)

as they reported some methods for detachment of the ink particles from paper pulp using

hemicellulases, lipase, protease, amylase and xylanase. It is suggested that the enzymes

facilitates the ink removal through the peeling effect caused by the endoglucanase enzyme

(Vyas & Lachke, 2003). This enzyme hydrolyzes the fiber-ink bonding regions, cut the

cellulose polysaccharide chain at several amorphous sites and generating non-reducing

ends of the chain. Then, during deinking, it loosens the fibers and helps in releasing the ink

trapped between the fibers. Besides that, it also acts at the frazzled surface of fibers and

releases short fibers from it. As a result, the cellulose fibers and the ink particles are being

split apart and hence released the ink particles. These properties facilitate the ink removal

of the waste-papers.

11

In 2002, Pe’lach et al (2001) showed that cellulase can improve the deinking

efficiency as compare to the chemical deinking. Moreover, studies using fungi Fusarium

sp. (Vyas & Lachke, 2003) and bacteria Vibrio alginolyticus (Mohandass & Raghukumar,

2005) demonstrated that these microorganisms can effectively remove the ink from the

waste-papers. Since Bacillus spp. is known for its ability to produce these extracellular

enzymes (Priest, 1992), this bacteria may be utilized in the biodeinking process. As the

paper recycling process is usually conducted in alkali condition and high temperature, the

thermophilic and alkalophilic Bacillus spp. are preferred.

2.3 Environment issues in paper recycling

According to Roberts (1996), the main problems of paper recycling are the production of

ink-rich slurry that must be disposed and the contents of the effluent or waste water

released. Paper recycling involves many steps that require extensive usage of hazardous

chemicals, such as the chlorine-based chemical during bleaching process that contains high

amount of hazardous organochlorine (Roberts, 1996). This may lead to the toxic pollution

to the water system. Furthermore, the high level of chlorine released can cause the

formation of toxic polychlorinated compound that will bio-accumulated in living organism.

Beside that, there is also the possibility that the effluent discharge contains a

certain amount of biodegradable nutrients, i.e. carbohydrates. Upon discharge into the

water system, the micro-aquatic flora and fauna metabolize these organic molecules for

growth. The amount of dissolved oxygen will decreased, causing a high biochemical

oxygen demand (BOD) level that will eventually impose significant effect to the

ecological balance in the water system.

12

3.0 MATERIALS AND METHODS

The laboratory work was done at Genetic Molecular Laboratory in FSTS, University

Malaysia Sarawak from October 2009 to April 2010. Before starting the project, all the

equipments were sterilized by autoclaving at 121°C for 15 minutes in the autoclaving

room at FRST, 2nd

floor. All the work was done inside the Laminar Flow Hood to ensure

sterility. The overall flow chart of the methodology involved in this study was shown in

Appendix 1.

3.1 Bacteria Source and Culture

Five types of Bacillus spp. were used in this study, namely Bacillus licheniformis P7,

Bacillus amyloliquefaciens UMAS 1002, Bacillus pumilus, and two Bacillus spp. isolated

from Paku Hot Spring, Lundu, designated as isolate M5 and M7.

The Bacillus amyloliquefaciens UMAS 1002 and Bacillus licheniformis P7 were

obtained from stock culture from Mr. Mohd Suhaib and Mr. Nikson Chong from

Proteomic Laboratory, respectively. The Bacillus amyloliquefaciens UMAS used was

isolated by Apun et al (2000), Bacillus licheniformis P7 was isolated by Ms Dayang

Iqliema (2008), Bacillus pumilus was obtained from GML stock culture and two Bacillus

spp. isolate M5 and M7 were isolated by Ms Dayang Shareney (2010) from Paku Hot

Spring, Lundu.

13

3.1.1 Identification of the bacteria (Gram staining)

Upon obtaining the bacteria stock, gram staining test was performed on the bacteria for

identification. The bacteria were grown in Luria Broth (LB) for overnight and the

overnight fresh culture were inoculated (streak plate) in minimal agar plate consisting of

0.2 % yeast extract, 0.1 % potassium dihydrogen phosphate (KH2PO4), 0.5 % magnesium

sulphate (MgSO4), 2.0 % agar and 0.5 % carboxymethyl cellulose (CMC). After overnight

incubation, a single colony was picked and transferred onto a glass slide, and gently mix

with a drop of distilled water. Then, the glass slide was passed over the Bunsen burner

several times for fixation (drying) and allowed to cool down before staining. The staining

procedure was shown in Appendix 3.

3.1.2 Preparing stock culture and working culture of the bacteria

Glycerol stock culture and working culture were prepared from the stock culture obtained

from Proteomic Lab. First, the bacteria were inoculated into Luria Broth (LB) in universal

bottles and incubated in 37 °C incubator for 24 hour. The temperature of 37 °C was used

as most bacteria grow in this temperature and shaking allows the nutrient to be distributed

evenly to the bacteria and facilitates growth. After an overnight incubation, it was

observed that the LB had turned turbid, indicating bacteria growth.

For the glycerol stock, the glycerol used was 20 % (v/v) of the sample. 4.0 ml of

the overnight culture of the bacteria eg. Bacillus licheniformis P7 was pipetted into the

universal bottle and added with 1.0 ml of 40 % glycerol. The mixture was inverted several

times. Then, 1.0 ml of the mixture was pipetted into a 2.0 ml microcentrifuge tube. This

procedure was repeated for Bacillus amyloliquefaciens UMAS 1002, Bacillus pumilus,

isolate M5 and M7. Duplicated was done for each bacteria. Then, the microcentrifuge

tubes were freezed in liquid nitrogen and kept in -20 °C freezer.

14

For the working cultures, two methods were used. The first method was culturing

the overnight bacteria culture in minimal agar plate with 0.5 % CMC. The second method

was the direct storage of the fresh culture in LB. Both the plates and bottles were incubated

in 37 °C incubator. After overnight incubation, they were stored in -4 °C freezer.

3.2 Testing for thermostability and alkalotolerant of the bacteria

From the working culture, the bacteria were inoculated in LB and grown in 37 °C for 24

hours. Duplicate were done for each sample. The turbidity of LB indicated the growth of

the bacteria.

For the testing of thermostability and alkalotolerant of the bacteria, two types of

media were used, namely minimal salt media (MSM) with 0.5 % filter paper and minimal

salt media (MSM) with 0.5 % CMC. After adjusted to pH 8.0 using 1 M sodium hydroxide

(NaOH), the media were autoclaved using the autoclave machine. Then, the overnight

culture of the bacteria were inoculated into the media, and incubated overnight at 50 °C,

under 100 rpm for 24 hours. After incubation, only the bacteria that can withstand high

temperature and alkali condition survived. The viability of the bacteria culture was

determined by observing the growth of bacteria on minimal agar plate with 0.5 % CMC

and from the Congo Red Test.

3.2.1 Congo red screening

The Congo red screening or also known as CMCases test was performed to verify the

presence of cellulase-producing bacteria. Firstly, minimal agar plate with 0.5 % CMC was

prepared. Then, the bacteria culture from section 3.2 was spotted onto the agar plate (10

spots for each media), using sterile needle stick and grown overnight at 37 °C and 50 °C,

respectively. After overnight incubation, the agar plates were observed for bacteria growth

and potential colonies were marked.

Documents

BIODEINKING OF THE MIXED OFFICE PAPER USING THE … of the Mixed Office Paper Using the Microbial... · significant difference for the deinking efficiency between chemical deinking