NorHabibahMohdRosliKPFS2006TTT

DEVELOPMENT OF BIOLOGICAL TREATMENT SYSTEM FOR

REDUCTION OF COD FROM TEXTILE WASTEWATER

MASTER DESSERTATION

Prepared by,

NOR HABIBAH BINTI MOHD ROSLI

Supervised by,

PM. DR. WAN AZLINA AHMAD

UNIVERSITI TEKNOLOGI MALAYSIA








A project report submitted in partial fulfillment of the requirements for the

award of the degree of Master of Science (Chemistry)

Faculty of Science

Universiti Teknologi Malaysia.

NOVEMBER, 2006

PSZ 19:17


BORANG PENGESAHAN STATUS TESIS JUDUL: DEVELOPMENT OF BIOLOGICAL TREATMENT SYSTEM FOR


SESI PENGAJIAN: 2005/2006

Saya NOR HABIBAH BINTI MOHD ROSLI

(HURUF BESAR)

mengaku membenarkan kertas projek ini disimpan di Perpustakaan Universiti Teknologi Malaysia dengan syarat-syarat kegunaan seperti berikut : 1. Hakmilik kertas projek adalah di bawah nama penulis melainkan penulisan sebagai projek

bersama dan dibiayai oleh UTM, hakmiliknya adalah kepunyaan UTM. 2. Naskah salinan di dalam bentuk kertas atau mikro hanya boleh dibuat dengan kebenaran bertulis

daripada penulis. 3. Perpustakaan Universiti Teknologi Malaysia dibenarkan membuat salinan untuk tujuan pengajian

mereka. 4. Kertas projek hanya boleh diterbitkan dengan kebenaran penulis. Bayaran royalti adalah

mengikut kadar yang dipersetujui kelak. 5. *Saya membenarkan/tidak membenarkan Perpustakaan membuat salinan kertas projek ini

sebagai bahan pertukaran di antara institusi pengajian tinggi. 6. ** Sila tandakan ( )

SULIT (Mengandungi maklumat yang berdarjah keselamatan atau kepentingan Malaysia seperti yang termaktub di dalam AKTA RAHSIA RASMI 1972)

TERHAD (Mengandungi maklumat TERHAD yang telah ditentukan oleh

organisasi/badan di mana penyelidikan dijalankan). TIDAK TERHAD

Disahkan oleh (TANDATANGAN PENULIS) (TANDATANGAN PENYELIA) Alamat Tetap: T.2 BLOK A, JLN TEMPUA, ASSOC. PROF. DR. WAN AZLINA AHMAD

FELDA SG. NEREK, 28030 Nama Penyelia

TEMERLOH, PAHANG.

Tarikh: Tarikh: CATATAN: * Potong yang tidak berkenaan.

** Jika Kertas Projek ini SULIT atau TERHAD, sila lampirkan surat daripada pihak berkuasa/organisasi berkenaan dengan menyatakan sekali tempoh tesis ini perlu dikelaskan sebagai SULIT atau TERHAD.

♦ Tesis ini dimaksudkan sebagai tesis bagi Ijazah Doktor Falsafah dan Sarjana secara penyelidikan, atau disertasi bagi pengajian secara kerja kursus dan penyelidikan, atau Laporan Projek Sarjana Muda (PSM)

ii

I declare that I have read this project report entitled “Development of Biological

Treatment System for Reduction of COD from Textile Wastewater” and in my opinion

this thesis is sufficient in terms of scope and quality for the award of the degree of

Master of Science (Chemistry).

Signature : …………………………………...

Name of Supervisor : PROF.MADYA DR. WAN AZLINA AHMAD

Date : …………………………………..

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I declare that this project report entitled “Development of Biological

Treatment System for Reduction of COD from Textile Wastewater” is the

result of my own research except as cited in the references. The project

report has not been accepted for any degree and is not concurrently

submitted in candidature of any other degree.

Signature : .............................................

Author : NOR HABIBAH BINTI MOHD ROSLI

Date : 7 NOVEMBER 2006

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Istiwewa untuk Mak Puteh binti Idris,

Abah Mohd Rosli bin Dahli,

dan adik-adik tersayang,

Nor Hazirah, Mohd Izzul Ihsan, Nur Fatihah, Nur Ain Izati.

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ACKNOLEDGEMENTS

I would like to thank Associate Professor Dr. Wan Azlina Ahmad for being my

supervisor for this postgraduate project. Thank you for giving me full support and gives

all the guidance that I need to complete the project.

I would also like to thank all Biotechnology lab members for giving the help and

co-operation needed to complete the experiment.

Besides that, I would like to thank my parents, Mohd Rosli Dahli and Puteh Haji

Idris, and my siblings for understanding my project and gives me a support. Also to all

my friends, Eina, Miha and Dura for giving help and supports.

Last but not least for Mohd Fuad Abdul Majid, thank you for your supports and

patient.

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ABSTRACT

Wastewater from industrial plants such as textile, electroplating and petroleum

refineries can contain various substances that tend to increase the chemical oxygen

demand (COD) of the wastewater. Various local agencies have placed limits on the

allowable levels of COD in industrial wastewater effluent. It is desired to develop a

process suitable for treating the wastewater to meet the regulatory limits. Various

methods are available for COD reduction in industrial wastewater such as precipitation,

incineration, chemical oxidation and biological oxidation. This project attempts to solve

the high concentration of COD in industrial wastewater using bacteria. The organic

matter that contributed to COD in the wastewater will be degraded by the bacteria under

aerobic conditions using batch process. The wastewater will be supplemented with an

agricultural liquid waste for bacterial growth and metabolism. This will ensure the

continual presence of bacteria to carry out degradation of organic matter in the

wastewater.

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ABSTRAK

Air sisa industri seperti industri tekstil, elektroplat, dan penapisan petroleum

mengandungi pelbagai bahan yang menyumbang kepada peningkatkan COD. Pelbagai

agensi tempatan telah meletakkan had ke atas tahap COD yang dibenarkan dalam

pengaliran keluar air sisa industri. Oleh itu, wujudnya keperluan untuk membangunkan

proses yang bersesuaian bagi merawat air sisa tersebut supaya mencapai tahap pelepasan

air sisa yang dibenarkan. Terdapat pelbagai kaedah yang boleh digunakan bagi

pengurangan COD dalam air sisa industri seperti pemendakan, pengoksidaan kimia, dan

pengoksidaan biologi. Projek ini dibangunkan untuk menyelesaikan masalah kandungan

COD yang tinggi dalam air sisa industri dengan menggunakan bakteria. Bahan organik

yang menyumbang kepada COD dalam air sisa akan dikurangkan oleh bakteria ini.

Sumber nutrien yang dibekalkan ialah air sisa nenas. Ini adalah untuk memastikan

perkembangan populasi bakteria yang berterusan bagi menjalankan proses pengurangan

bahan organik di dalam air sisa.

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TABLE OF CONTENTS

CHAPTER TITLE PAGE

DECLARATION ii

DEDICATION iv

ACKNOWLEDGMENTS v

ABSTRACT vi

ABSTRAK vii

TABLE OF CONTENTS viii

LIST OF TABLES xii

LIST OF FIGURES xiii

LIST OF ABBREVIATIONS AND SYMBOLS xiv

I INTRODUCTION

1.1 Textile industry 1

1.2 Biodegradability of textile waste

1.2.1 Dyes 2

1.2.2 Surface active agents 3

1.2.3 Sizes 4

1.2.4 Finishing agents 4

1.2.5 Grease and oils 4

1.2.6 Complexing agents 5

1.3 Textile Wastewater Treatment

Current Technologies 5

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1.4 COD reduction

1.4.1 Definition of COD 6

1.4.2 COD and BOD 7

1.5 Methods of COD reduction

1.5.1 Physical method

1.5.1.1 Granular activated carbon 8

1.5.1.2 Membrane filtration 9

1.5.2 Chemical method

1.5.2.1 Ozonation 10

1.5.2.2 Electrochemical treatment 10

1.5.2.3 Fenton reagent 11

1.5.3 Biological method 11

1.5.4 Combined method 12

1.6 COD reduction by biological treatment

1.6.1 Biological treatment by fungi 13

1.6.2 Biological treatment by algae 14

1.6.3 Biological treatment by bacteria 15

1.7 Bacteria

1.7.1 Acinetobacter baumanni and

Acinetobacter calcoaceticus 16

1.7.2 Cellulosimicrobium cellulans 17

1.7.3 Mixed bacterial culture (consortium) 18

1.8 Objective and scope of studies 19

II EXPERIMENTAL

2.1 Materials

2.1.1 Glasswares and apparatus 20

2.1.2 Textile wastewater 20

2.1.3 Agricultural waste 21

x

2.1.4 Bacteria

2.1.4.1 Bacteria growth on plates 21

2.1.4.2 Bacteria liquid cultures 21

2.1.5 Growth media

2.1.5.1 Nutrient broth 22

2.1.5.2 Nutrient agar 22

2.1.5.3 Pineapple waste 22

2.2 Method

2.2.1 Characterization of microorganisms

2.2.1.1 Growth profile of single cultures 23

2.2.1.2 Growth profile of consortium 23

2.2.1.3 pH profile 24

2.2.2 Bacterial adaptation studies

2.2.2.1 Screening for bacterial tolerance

to textile wastewater 25

2.2.2.2 Bacterial survival in

textile wastewater 25

2.2.3 COD reduction using single culture

2.2.3.1 Liquid cultures 26

2.2.3.2 Preparation of freeze-dried culture 26

2.2.3.3 Freeze-dried culture experiment 27

2.2.4 COD reduction using mixed culture

2.2.4.1 Liquid culture 27

2.2.4.2 Effect of NB supplementation 28

2.2.4.3 Effect of pH 28

2.2.5 COD analysis

2.2.5.1 Materials 29

2.2.5.2 Reagents 29

2.2.5.2 Preparation of samples for analysis 30

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III RESULTS AND DISCUSSION

3.1 Characterizations of microorganisms

3.1.1 Growth profile 31

3.1.1.1 Growth profile of single culture 32

3.1.1.2 Growth profile of consortium 34

3.1.1.3 pH profiles 35

3.2 Bacterial adaptation studies

3.2.1 Screening for bacterial tolerance 37

To textile wastewater

3.2.2 Bacterial survival in textile wastewater 39

3.3 Characteristics of textile wastewater 43

3.4 COD reduction using single culture

3.4.1 Liquid culture 44

3.4.2 Freeze-dried culture 45

3.4.3 Cell dry weight determination 45

3.4.4 COD reduction using 45

freeze-dried culture

3.5 COD reduction using mixed culture

3.5.1 Liquid culture 48

3.5.2 Effect of NB supplementation 49

3.5.3 Effect of pH 51

3.6 Comparison with existing COD 52

reduction methods

IV CONCLUSION AND SUGGESTIONS 54

REFERENCES 57

xii

LIST OF TABLES

TABLE NO. TITLE PAGE

1.1 Textile wastewater characteristics 2

1.2 Textile wastewater treatment current technologies 6

3.1 Adaptation times for single culture and mixed culture 37

3.2 Total number of A.baumannii colonies developing on

nutrient agar plate from samples with different

concentrations of textile wastewater 40

3.3 Total number of A.calcoaceticus colonies developing on



3.4 Total number of C.cellulans colonies developing on



3.5 Total number of mixed culture colonies developing on



3.6 Characteristics of textile wastewater 43

3.7 COD reduction using liquid single culture 44

3.8 Types of system using mixed culture 48

3.9 COD reduction using mixed culture 49

3.9 Comparison with existing of the COD reduction

Conventional methods 52

xiii

LIST OF FIGURES

FIGURE NO. TITLE PAGE

3.1 Growth profiles of single culture 33

3.2 Growth profiles of bacterial consortium 35

3.3 pH profiles of single culture and consortium 36

3.4 Bacterial tolerance level for each culture upon adaptation

to increase concentrations of textile wastewater

based on OD measurement 39

3.5 Bacteria tolerance level for each culture upon adaptation

to increase concentrations of textile wastewater

based on viable count 43

3.6 COD reduction using freeze-dried culture 47

3.7 The effect of NB supplementation on COD reduction 50

3.8 The effect of various pH on COD reduction 51

xiv

LIST OF ABBREVIATIONS AND SYMBOLS

BOD Biochemical oxygen demand

COD Chemical oxygen demand

TSS Total suspended solid

NB Nutrient broth

OD Optical density

CFU Colony forming unit

L Liter

g Gram

mg milligrams

rpm Rotation per minute oC Degree Celsius

mL Milliliters

mg/L Milligrams per liter

kPa Kilo Pascal

ppm Parts per million

Cr (IV) Chromium (IV)

Cu (II) Copper (II)

DDW Distilled deionized water

TNTC Too numerous to count

IB Indigenous bacteria

xv

AB Acinetobacter baumannii

AC Acinetobacter calcoaceticus genospecies 3

CC Cellulosimicrobium cellulans

M Mixed culture

A.baumannii Acinetobacter baumannii

A.calcoaceticus Acinetobacter calcoaceticus genospecies 3

C.cellulans Cellulosimicrobium cellulans

1

CHAPTER I

INTRODUCTION

1.1 Textile industry

Textile industry can be classified into three categories, cotton, woolen, and

synthetic fibers depending upon the raw materials used (Babu et al., 2000). Textile

industries consume large volumes of water and chemicals for wet processing of textiles.

The chemical reagents used are very diverse in chemical composition, ranging from

inorganic compounds to polymers and organic products. In general, the waste water

from textile industry is characterized by high value of BOD, COD, pH, and colour

(Robinson et al., 2001).

Because of the high BOD, the untreated textile waste water can cause rapid

depletion of dissolved oxygen if it is directly discharged into the surface water sources.

Therefore the effluents with high COD level are toxic to biological life. The high

alkalinity and traces of chromium where it was employed in dyes adversely affect the

aquatic life and also interfere with the biological treatment process (Babu et al., 2000).

Table 1.1 shows common characteristics for effluent emanated from textile industries

(Zaharah et al,. 2004).

2

Table 1.1: Textile wastewater characteristics.

Parameter Unit Limit allowed for industrial

discharge (Standard B) _______________________________________________________________________

Temperature oC 40 pH - 5.5 -9.0 BOD mg/L 50 COD mg/L 100 TSS mg/L 100 Nitrate mg/L 7 Phosphate mg/L 0.2 Sulphate mg/L - Cr (III) mg/L 1.0 Cu (II) mg/L 1.0 * TSS-Total suspended solid Cr(III)-Cromium (III) Cu(II)-Copper(II)

1.2 Biodegradability of textile waste

The major sources of waste from textile operations can be contributed to the

following activities, raw wool scouring, yarn and fabric manufacture, wool finishing,

woven fabric finishing, and knitted fabric finishing. The chemicals normally used in

textile processing are as follows:

1.2.1 Dyes

The most studied step in textile processing regarding the treatment of the effluent

is the dyeing step. Dyeing causes an easy recognition of pollution via colour. A very

small amount of dye is visible in water, decreasing the transparency of the water. In the

environment this leads to inhibition of sunlight penetration and therefore of

photosynthesis. In addition, industrial textile waste water presents the additional

3

complexity of dealing with unknown quantities and varieties of many kinds of dyes, as

well as low BOD/COD ratios, which may affect the efficiency of the biological

decolorization (Babu et al., 2000).

1.2.2 Surface active agent

A series of research studies on biodegradation of surface active agents have been

reviewed. For the biodegradation of surface active agents (surfactants) their

hydrophobic groups are important. The hydrophobic ends are less fundamental for

biodegradation but fundamental for their water solubility (Bisschops et al., 2003).

Biodegradation of surfactants are important because they interfere with effective

oxygen transfer in biological treatment systems, they are characterized by relatively high

aquatic toxicity and contribute to final BOD or COD of treated effluents (Bisschops et

al., 2003).

Even though literature indicates that surfactants are more easily biodegradable

than ever, they are still very difficult to degrade. For example, the surfactant

alkylphenol ethoxylate is removed in activated sludge treatment and it was thought to be

biodegradable. This surfactant is resistant to attack by bacteria found in activated sludge

but it is effectively adsorbed on the sludge. The adsorption affected both the

biodegradability and elimination. Thus most of the surface active agents used today are

actually eliminated and not biodegraded by more than 80% 1 (Timothy et al., 1988).

4

1.2.3 Sizes

The hydrolysis of polyvinyl acetate (PVA) to yield water soluble alcohol

produces PVA. PVA resists biodegradation when it contains small amounts of acetate

groups in the polymer. The number and location of these residual groups may vary from

batch to batch, causing the biodegradability to vary considerably. A portion of PVA is

removed by sedimentation in the biological systems. Starch is readily biodegradable.

Polyacrylates and CMC are partially removed by sedimentation in activated sludge.

CMC degrades slowly with acclimatization. Polyacrylic and polyester sizes are

recalcitrant to biodegradation (Robinson et al., 2001).

1.2.4 Finishing agents

Many different chemicals are used in treating textile fabric to impart desirable

properties to final garment. The resin finishes have extremely low BOD values

compared to their COD values. This may be caused by the presence of toxic chemicals

or metal catalyst used in finish formulation. Most finishing agents are eliminated by

adsorption or sedimentation in the biological treatment plants and not by biodegradation

(Robinson et al., 2001).

1.2.5 Grease and oils

Oil and grease may present problems at the waste water treatment plant because

they adhere to particulate matter, causing it to float. Not all oils are equally

troublesome. Vegetable oils and natural grease tend to be fairly readily degradable.

5

The modern trend is towards lubricants based on petroleum and these materials

are very much less easily degraded. Generally pretreatment for removal of these

substances will not be required unless floating oil or grease is obviously present in the

effluent. The naphthenic and paraffinic mineral oils are biodegradable with

acclimatization. The silicon oil is not biodegradable. Waxes degrade slowly and the

fatty esters are biodegradable (Bisschops et al., 2003).

1.2.6 Complexing agents

EDTA (ethylene diamene tetraacetate), which is the most widely used

complexing agent, is very stable against oxidation and is not biodegradable. Due to its

low molecular weight it is not adsorbed on activated sludge and escapes with effluent of

the treatment plant (Timothy et al., 1988).

1.3 Textile Wastewater Treatment Current Technologies

There are many different unit operations employed for treatment of waste water

from textile industries. Pretreatment for removal of inorganic solids and floating

materials, flow equalization and neutralization, chemical coagulation, clarification, and

biological treatment are the most common unit operations employed in the textile waste

water treatment (Babu et al., 2000). For simplification, the operations treatment can be

classified into chemical, physical, and biological treatment. These approaches along

with their associated pros and cons are outlined in table 1.1 (Timothy et al., 1988).

6

__ Table 1.2. Textile Wastewater Treatment Current Technologies.

_____________________________________________________________________ Current technologies description advantages disadvantages

1. Biological Activated sludge, efficient BOD long residence times, large treatment Aerated lagoons, reduction aeration tanks, may

Land treatment, require nutrient. Extended aeration.

2. Chemical Percipitation-addition heavy metals, color removal varies with Precipitation of multivalent cations suspended solid, dye class, chemical with pH adjustment BOD, COD handling problems. 3. Activated carbon Passing water BOD, COD, expensive, long through a bed of color residence times, low carbon (as a adsorption capacity. pretreatment to further treatment) 4. Ultra filtration Permeation of water BOD, COD, membranes foul easily, under pressure color heavy metals not through specialized removed, need frequent polymeric membranes cleaning. 5. Ozone Ozone, generated BOD, COD, very expensive, heavy

by electrical color metals and solids discharged is used require separate to oxidize organics treatment.

_____________________________________________________________________________________

_

1.4 COD reduction

1.4.1 Definition of COD

Bacteria require oxygen to breathe and oxidize organic substances; the total

amount of which is called oxygen demand (Inoue, 2002). The level of COD is a

critically important factor in evaluating the extent of organic pollution in textile waste

water. Substantial sewer surcharges are often imposed when the local permitted limits

are exceeded. Depending on the types of fibers, dyes, and additives, various textile

operations will routinely generate water having high levels of COD (Timothy et al.,

1988).

7

In environmental chemistry, the chemical oxygen demand (COD) test is

commonly used to indirectly measure the amount of organic compounds in water,

making COD a useful measure of water quality. It is expressed in milligrams per liter

(mg/L), which indicates the mass of oxygen consumed per liter of solution. Older

references may express the units as parts per million (ppm) (Sawyer et al., 2003).

Therefore, COD measures the potential overall oxygen requirements of the waste water

sample including oxidizable components not determined in the BOD analysis (Timothy

et al., 1988).

1.4.2 COD and BOD

The biochemical oxygen demand (BOD) test measures oxygen uptake during the

oxidation of organic matter. It has wide applications in measuring waste loading of

treatment plants and in evaluating the efficiency of treatment processes. It is of limited

use in industrial waste water containing heavy metal ions, cyanide, and other substances

toxic to microorganisms.

Although COD is comparable to BOD, it actually measures chemically

oxidizable matter. Generally COD is preferred to BOD in process control applications

because results are more reproducible and are available in a few minutes or hours rather

than five days. In many industrial samples, COD testing may be the only feasible course

because of the presence of bacterial inhibitors or chemicals that will interfere with BOD

determination (Sawyer et al., 2003).

As dyeing process in textile is particularly difficult, acid dyes, fixing agents,

thickeners, finishing agents, are required for successful colorization and cause major

problems with the plant's effluents disposal in term of COD. Literature has shown that

the removal of non-colored agents in textile industry effluent is more problematic, in

terms of COD than visible dye agents (Walker et al., 2001).

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1.5 Methods of COD reduction

The main source of organic substances, which contributes to COD in the effluent

water, comes from the pretreatment of the raw materials. There are two ways to reduce

organic substances in the effluent; recovery of organic materials from the effluent

through ultrafiltration or electro-flocculation or by leasing using materials with low

content of soluble organic substances can be found (Prasad Modak, 1998). Many

approaches have been reported for COD removal of textile waste water ranging from

chemical, physical, biological method and combined method.

1.5.1 Physical method

The physical treatment generally can be achieved via adsorption. The common

adsorbents used in treating textile effluent are carbon, clay, and resin.

1.5.1.1 Granular activated carbon

In general granular activated carbon (GAC) processes have attracted interest for

municipal waste water treatment for the removal of small concentrations of low

molecular weight contaminants such as phenol from solution. But several researchers

have since studied textile waste water treatment using GAC. They concluded that GAC

adsorption was the best method for colour removal (Rozzi et al., 1999).

In many cases GAC adsorption is also coupled with ozonation or bio-ozonation

in order to improve the removal treatment for textile waste. However there are papers

which report, that GAC adsorption as sole treatment technique for COD removal from

textile effluent. The adsorbent used was the GAC Filtrasorb 400. The adsorbent was

9

washed in deionised water and sieved into 1000-4000 micrometer particle size range

before contacting with the adsorbate solutions (Walker et al., 2001).

1.5.1.2 Membrane filtration

This method has the ability to clarify, concentrate and to separate dye

continuously from textile effluent. But the concentrated residue left after separation

poses disposal problems, and high capital cost and the possibility of clogging, and

membrane displacements are disadvantages. This method of filtration is suitable for

water recycling within textile dye plant if the effluent contains low concentration of

dyes, but it is not effective to reduce dissolved solid content and COD content which

makes water re-use a difficult task (Tim et al., 2001).

1.5.2 Chemical method

The waste water discharged from a dyeing process in the textile industry exhibits

low BOD, high COD and high in colour. Hence, the need for chemical treatment is

necessary in order to produce a more readily biodegradable compound. Chemical colour

removal technique which are reported in the literature include adsorption by activated

carbon, electrochemical treatment, ozonation, chemical precipitation, membrane

filtration, hydrogen peroxide, Fenton’s reagent and reverse osmosis (Ghoreishi et al.,

2003).

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1.5.2.1 Ozonation

Ozone is a powerful oxidizing agent that may react with organic compounds

either directly or via radicals formed in a reaction chain as OH-radicals. Ozone can be

used in waste water field to reduce COD, colour, toxicity, and pathogens and to improve

waste water biodegradability and the coagulation-flocculation processes (Ciardelli et al.,

2001).

Regarding COD removal, ozone treatment with 40 g O3/m3 at contact times of 15

min and 30 min causes the COD reductions from 160 to 53 and 203 to 123 mg/L

respectively. In ozonation treatment, ozone generator will be fed with pure oxygen and

contact with reactor filled up with waste water. The most important parameter to

evaluate process feasibility is the ratio between the generated ozone and the eliminated

COD. According to literature this parameter ranged between 1 and infinity. Value

higher than 3 could make the oxidation process for COD elimination economically

unfeasible (Bes-Pia et al., 2004).

However, ozonation shows effective results in decolorizing textile waste water

compared to COD and BOD reduction. In addition, the cost of ozonation needs to be

further ascertained to ensure the competitiveness of this method (Bes-Pia et al., 2004).

1.5.2.2 Electrochemical treatment

In recent years, there has been increasing interest in the use of electrochemical

methods for the treatment of waste waters. Electrochemical methods have been

successfully applied in the purification of several industrial wastewaters as well as

textile effluent.

11

The electrolytic cell consisted of cathode and anode. In the past graphite was

frequently used as an anode as it is economical and gives satisfactory results. Recently,

titanium electrodes covered with very thin layers of electrodeposited noble metals have

been used. Besides titanium, ruthenium, rhodium, and iridium mixture can also be

applied as electrocatalysts for electro-oxidation of pollutants present in waste waters,

making it possible to destroy organic pollutants which are difficult to eliminate

biologically, such as phenols and surfactants (Bes-Pia et al., 2004).

1.5.2.3 Fenton reagent

Fenton’s reagent is a suitable chemical means of treating waste waters which are

resistant to biological treatment or is poisonous to live biomass. Chemical separation

uses the action of sorption or bonding to remove dissolved dyes from textile effluent and

has been shown to be effective in colour removal and also COD removal. But one major

disadvantage of this method is generation of sludge through the flocculation of the

reagent and the dye molecules used in dyeing stage (Tim et al., 2001).

The high cost and disposal problems have opened for development of new

techniques. Application of conventional biological processes in the treatment of textile

waste water has been extensively reported.

1.5.3 Biological method

Industrial biological waste water treatment systems are designed to remove the

dissolved organic load from the waters using microorganisms. The microorganisms

used are responsible for the degradation of the organic matter. Biological treatment

encompasses basically aerobic and anaerobic treatment.

12

In most waste waters, the need for additional nutrient seldom appears, but in an

aerobic operation, oxygen is essential for success operation of the systems. The most

common aerobic processes are activated sludge and lagoon, active or trickling filters,

and rotating contactors.

In contrast, anaerobic treatment proceeds with breakdown of the organic load to

gaseous products that constitute most of the reaction products and biomass. Anaerobic

treatment is the result of several reactions; the organic load present in the waste water is

first converted to soluble organic material which in turn is consumed by acid producing

bacteria to give volatile fatty acids and carbon dioxide and hydrogen. Further more

anaerobic processes are also sensitive to temperature (Sawyer et al., 2003).

In general, when comparing a conventional aerobic treatment with anaerobic

treatment systems the advantages of anaerobic systems are (Lema et al., 2001):

i. Lower treatment costs and production energy

ii. High flexibility, since it can be applied to very different types of effluents

iii. High organic loading rate operation

iv. Smaller volumes of excess sludge

v. Anaerobic organisms can be preserved for a long time, which make it

possible to treat waste water that are generated with longer pauses in

between

1.5.4 Combined method

Individual approach in reducing COD level show high tolerance but the

combination technique would give better performances. Commonly applied treatment

methods for COD removal from textile wastewater consisted of integrated processes

involving various combinations of biological, physical, and chemical methods (.Azbar et

al., 2004).

13

A rapid combined method of physical-chemical method has been developed for

the determination of COD. The method involved removal by flocculation and

precipitation of colloidal matter that normally passes through membrane filters.

For example study by Lin et al., 1996, indicated that the combined chemical

treatment methods are very effective and are capable of elevating the water quality of

the treated waste effluent to the reuse standard of the textile industry. While study by

Ghoreishi et al., 2003 indicated that combined chemical-biological method showed

higher efficiency and lower cost for the new technique of COD reduction. But

generally, these integrated treatment methods are efficient but not cost effective (Azbar

et al., 2004).

1.6 COD reduction by biological treatment

Treatment of the textile waste water via biological methods can be achieved by

three approaches, involving fungi, algae, and bacteria. Although the biological

treatment of waste water is important, it cannot completely remove soluble components,

such as endocrine disrupters and pesticides (Ilyin et al., 2004).

1.6.1 Biological treatment by fungi

Waste water from textile industries constitutes a threat to the environment in

large parts of the world. The degradation products of textile dye are often carcinogenic

(Azbar et al., 2004). A key feature in the degradation processes involves generation of

activated oxygen forms that can carry out the initial attack on the stable structure. Wood

rotting fungi have interesting properties in the sense they are capable to degrade lignin

which is polymeric structure with a lot of aromatic rings (Nilsson et al., 2006).

14

An advantage with these organisms is that they produce and excrete ligninolytic

enzymes that are non-specific with regards to aromatic structure. This means that they

are capable of degrading mixtures of aromatic compounds (Axelsson et al., 2006). The

application of white-rot fungi in large scale waste treatment, however, has been impeded

owing to the lack of appropriate reactor systems capable of coping with rather slow

fungal degradation, loss of extra cellular enzymes and mediators with discharged water,

and excessive growth of fungi (Faisal et al., 2006). Also, the fungi need to be supplied

with an external carbon source in order to be able to degrade polycyclic aromatic

hydrocarbons (Nilsson et al., 2006).

Besides white-rot fungi, obligate and facultative marine fungi occurring in

coastal marine environments can be an important source for use in bioremediation of

saline soil and waste water. The decolorization of colored effluents by lignin-degrading

fungus isolated from sea grass detritus have been reported (D’Sauze et al., 2006).

1.6.2 Biological treatment by algae

The most rapid and extensive degradation of lignin caused by certain fungi,

particularly white-rot fungi occurs in highly aerobic environments. However the high

glucose requirement of the microorganisms appears to be the major drawback of fungal

treatment. Therefore algae have been suggested to replace fungi to remove colour and

AOX from textile waste (Tarlan et al., 2002).

Algae are microscopic, photosynthetic organisms which typically inhabit aquatic

environments, soil, and other exposed locations (Mohan et al., 2002). Algal action was

reported to represent a natural pathway for the conversion of lignin to other materials

within the carbon cycle (Tarlan et al., 2002).

15

The potential of commonly available green algae belonging to species Spirogyra

was investigated as viable biomaterials for biological treatment of simulated synthetic

azo dye (reactive Yellow 22) effluents. The ability of the species in removing colour

was dependent both on the dye concentration and algal biomass (Mohan et al., 2002).

Green algae belonging to Chlorella and diatom species have also been reported being

applied in textile effluent treatment (Tarlan et al., 2002).

1.6.3 Biological treatment by bacteria

The waste water management strategy for the future should meet the benefits of

humanity safety, respect principles of ecology, and compatibility with other habitability

systems (Ilyin et al., 2004). For these purpose the waste water management

technologies, relevant to application of the biodegradation properties of bacteria are of

great interest. The selection of bacterial species for the biological treatment depends

upon the chemical composition of the dye and the alkalis and salts used in the dyeing

methods (Babu et al., 2000).

Escherichia coli is a common bacteria found in the environment has been used

for treating textile effluent. These bacteria grow very fast and double after every 20

minutes under favorable conditions of pH in the range of 6 to 8 and optimal temperature

at 35oC. COD reduction by these bacteria has achieved more than 60% removal (Babu

et al., 2000).

16

1.7 Bacteria

1.7.1 Acinetobacter baumanni and Acinetobacter calcoaceticus

The genus Acinetobacter has undergone numerous taxonomic changes over the

years and currently comprises at least 23 genospecies of clinical interest, of which only

nine (Acinetobacter calcoaceticus, Acinetobacter baumannii, A. haemolyticus, A. junii,

A. lwoffii, A. radioresistens, A. schlinderi, and A. ursingii) have been given a name

(Krawczyk et al., 2002). The bacteria used in this study are Acinetobacter calcoaceticus

genospecies 3 and Acinetobacter calcoaceticus have been isolated from textile waste.

Acinetobacter calcoaceticus genospecies 3 can be found in soil, water, sewage

and in hospital environments such as respiratory and urinary drainage equipment,

disinfectants and body fluids. It was formerly known as Achromobacter anitratus (Pal

et al., 1981). A.cakcoaceticus is a non-motile, coccobacillary, strictly aerobic and Gram

negative bacteria. It uses a variety of carbon sources for growth and can be cultured

relatively on simple media including tripticase soya agar and nutrient agar. The

optimum temperature for growth is at 30 oC (Bergogni-Berezin, 1995).

A. calcoaceticus is an opportunistic pathogen that has been reported to have

caused serious and sometimes fatal infections. It can be recovered occasionally from

human specimens such as urine. However, this pathogen appears to have little invasive

power and depend upon a pre-existing break in the normal body defenses like a surgical

wound to cause disease (Ana Isabel et al., 2004).

Clinical isolates associated with nosocomial outbreaks, particularly from

respiratory infections, blood cultures, and wounds, are mostly identified as

Acinetobacter baumannii. This genomic species alone is responsible for more than 80%

of all Acinetobacter infections in the hospital environment. While the Acinetobacter

calcoaceticus is considered mostly as an environmental species (Lagatolla et al., 1998).

17

A. baumannii is the second most common form of peritonitis caused by Gram

negative bacteria. Along with other members of this genus, A. baumannii is known to

harbor a variety of plasmid of different sizes, although most of them appear to be rather

small about less than 23 kb (Dorcey et al., 1998).

1.7.2 Cellulosimicrobium cellulans

The third bacterium used in this study is Cellulosimicrobium cellulans, also

known as Cellulomonas cellulans, Oerkoviaxanthineolytica, and Arthrobacter luteus

(Pau Ferrer, 2005).

The species has undergone several taxonomic changes since its first description

in 1923. It was first known as Brevibacterium fermentans, and two case reports have

been published describing meningitis and sepsis due to this bacterium in infants and

children. Some years later, B. fermentans was renamed Oerskovia xanthineolytica, and

a few case reports were published on infections with Oerskovia spp., such as

endocarditis, pyonephrosis, endophthalmitis, pneumonia, meningitis, parenteral

nutrition-related septicemia, catheter-related septicemia, and peritonitis. The genera

Oerskovia and Cellulomonas were united into the unique genus Cellulomonas in 1982,

and O. xanthineolytica became Cellulomonas cellulans. Finally, in 2001, Cellulomonas

cellulans was reclassified as Cellulosimicrobium cellulans (Beate Heym et al., 2005)

C.cellulans is a Gram positive rod shape bacteria and known as facultative

anaerobic. It is size ranges from 0.9-5.0 mm. The optimal temperature for growth is at

30 oC. It is a Gram positive bacterium with the rod shape. After exhaustion of the

medium, the rods transform into shorter rods or even spherical cells. C.cellulans is a

non-motile and chemo-organotrophic bacterium (Schumann et al., 2001).

18

1.7.3 Mixed bacterial culture (consortium)

Although pure culture is reported to be effective in treating textile waste water, it

is probable that a mixed culture or consortium would be more effective in degrading

toxic compounds in textile waste water. A mixed culture can adapt better to changing

conditions during growth. As an example, Cellulosimicrobium cellulans will dominate

over Acinetobacter calcoaceticus genospecies 3 and Acinetobacter baumannii due to its

facultative anaerobic properties. Since A. baumannii and A. calcoaceticus genospecies 3

are anaerobic bacteria, they require oxygen for growth. C. cellulans is a facultative

anaerobic and it can survive both with and without oxygen supply.

The different conditions of textile waste water after certain times may affect the

growth of the consortia. Thus, Cellulosimicrobium cellulans will predominate over the

other two bacteria in a mixed culture when three organisms are used in treating textile

waste water. Also under anaerobic consortia, reducing equivalents are formed by

fermentative bacteria (dos Santos et al., 2006). Therefore, a consortium may be more

effective in treating textile waste water.

19

1.8 Objective and scope of studies

The main objective of this research is to reduce the COD level of textile waste

water using a biological method employing bacteria which was isolated from a local

textile industry. The system used is a mixed culture or consortium consisted of three

different bacteria, Acinetobacter baumanni, Acinetobacter calcoaceticus genospecies 3,

and Cellulosimicrobium cellulans.

Studies on the ability of the individual and consortia adapting to the textile waste

will be carried out. This is to obtain the tolerance level of the bacteria to the textile

waste. Subsequently, studies on the ability of the consortium in reducing the COD level

will be monitored using liquid cultures and freeze dried cultures. Finally, developed

method will be compared with conventional COD reduction methods.

20

CHAPTER II

EXPERIMENTAL

2.1 Materials

2.1.1 Glassware and apparatus

All the glassware used in this study was washed with nitric acid (10%) before

use. Sterilization of the glassware and other apparatus were carried out by autoclaving

at 121oC, 103 kPa for 15 minutes (Fedegari, Italy).

2.1.2 Textile wastewater

The textile wastewater used in this study was obtained from Kim Fashion &

Knitwear Textile Sdn. Bhd. Senawang, Negeri Sembilan.

21

2.1.3 Agricultural waste

The agricultural waste used in this study was pineapple waste, which was

obtained from Lee Pineapple Manufacturing Industry, Skudai, Johor.

2.1.4 Bacteria

The bacteria used in this study, Acinetobacter baumannii, Acinetobacter

calcoaceticus genospecies 3 and Cellulosimicrobium cellulans was isolated from textile

wastewater as described by Zainul Akmar Zakaria and Nur Humaira’ Lau Abdullah

(2006).

2.1.4.1 Bacterial growth on plates

A loopful of the pure bacterial culture was inoculated aseptically by streaking

onto sterile nutrient agar plate and incubated in Memmert incubator at 30oC for 24

hours. Preparation of Nutrient Agar plates was as described in 2.1.4.2.

2.1.4.2 Bacterial liquid cultures

A loopful of bacterial culture from Nutrient Agar (NA) plate was inoculated

aseptically into Erlenmeyer flask (250 mL) containing fresh sterile Nutrient Broth (25

mL) and incubated at 30oC, 200 rpm (Certomat, B.Braun) for 24 hours. Upon growth,

the bacterial culture was used in the subsequent experiments.

22

2.1.5 Growth media

2.1.5.1 Nutrient Broth

Nutrient Broth was prepared by dissolving 8 g of NB (Merck®) in distilled

deionized water (DDW) (1 L) and sterilized by autoclaving at 121oC, 103 kPa for 15

minutes. The sterilized culture medium was then kept at 5oC.

2.1.5.2 Nutrient Agar

Nutrient agar (NA) plate was prepared by dissolving 20 g of NA powder

(Merck®) in distilled deionized water (1 L) and sterilized by autoclaving at 121oC, 103

kPa for 15 minutes. The agar was then cooled down to about 50oC before being poured

in sterile Petri dish and incubated for 24 hours in a Memmert incubator.

2.1.5.3 Pineapple waste

The liquid pineapple waste used in this study was collected from Lee Pineapple

Skudai, Johor. Before use, the liquid waste was filtered using a sieve to separate the

remaining solid waste from the pineapple waste solution. The liquid obtained was then

centrifuged at 9000 rpm and 4oC for 5 minutes (B.Braun, SIGMA 4K-15) to remove any

remaining solids. The clear liquid waste was then kept in the refrigerator for further use.

23

2.2 Method

2.2.1 Characterization of microorganisms

2.2.1.1 Growth profile of single cultures

Growth of single culture, Acinetobacter baumannii, Acinetobacter calcoaceticus

genospecies 3 and Cellulosimicrobium cellulans was monitored using pineapple waste

as growth medium. The single active cultures (10% inoculum) were grown in

Erlenmeyer flasks (500 mL) containing 80% neutralized pineapple waste and 10% of

textile wastewater. All flasks were incubated at 30oC, 200 rpm for 24 hours. The

samples were complimented with cell-free control set. At various intervals, cultures (3

mL) were aseptically pipetted into 5 mL test tubes for optical density measurement at

600 nm using spectrophotometer (Genesys 20). Samples were monitored at various

interval of the incubation period. A graph of OD600 against time was plotted to obtain

the growth curve of the bacteria.

2.2.1.2 Growth profile of mixed bacterial culture

The growth profile of mixed bacterial culture (10% inoculum) consisting of

Acinetobacter baumannii, Acinetobacter calcoaceticus genospecies 3 and

Cellulosimicrobium cellulans was monitored. The individual bacterial cultures were

first grown as described in section 2.1.3.2. Then 33% of each culture was inoculated

into Erlenmeyer flasks (500 mL) containing 80% neutralized pineapple waste

supplemented with 10% textile wastewater. All flasks were incubated at 30oC, 200 rpm

for 24 hours. The samples were complimented with cell-free control set. At various

intervals, cultures (3 mL) were aseptically pipetted into 5 mL test tubes for optical

density measurement at 600 nm. Samples were monitored at various interval of the

24

incubation period. A graph of OD600 against time was plotted to obtain the growth curve

of the mixed culture.

2.2.1.3 pH profile

The same procedure as described in section 2.2.1.1 and 2.2.1.2 for single culture

and mixed culture respectively was carried out. At various time intervals (3 mL)

cultures were aseptically pipetted into (5 mL) test tubes for pH measurement using a pH

meter (Mettler Toledo 320). A pH profile of the single cultures and mixed bacterial

culture was then plotted.

2.2.2 Bacterial adaptation studies

Bacterial adaptation studies were carried out using bacteria grown in pineapple

waste in increasing concentration of textile wastewater (1%, 2.5%, 5%, 7.5%, and 10%).

The transfer of culture from one concentration to the next was made when the respective

bacteria was in the exponential phase of growth.

25

2.2.2.1 Screening for bacterial tolerance in textile wastewater

The overnight active cultures (10% inoculum) were incubated in Erlenmeyer

flasks (250 mL) containing 89% neutralized pineapple waste and 1% textile wastewater

at 30oC, 200 rpm. At various time intervals, the turbid sample (3 mL) was pipetted

aseptically for OD600 measurements. When the culture has reached exponential phase,

the culture (10% inoculum) was transfered into Erlenmeyer flasks (250 mL) containing

neutralized pineapple waste and textile wastewater in increased concentration as

described in section 2.2.2 at 30oC, 200 rpm. A graph of bacterial tolerance in textile

effluent was obtained by plotting OD600 against respective concentrations of textile

wastewater.

2.2.2.2 Bacterial survival in textile wastewater

Serial dilutions of the bacterial cultures obtained from section 2.2.2.1 were made

and 0.1 mL each of the respective cultures was plated onto NA plates using the spread

plate technique. The plates were then incubated at 30oC for 24 hours before

enumeration of the colonies were made.

2.2.3 COD reduction using single cultures

Chemical oxygen demand (COD) reduction studies were made using bacterial

cultures adapted to 2.5% textile wastewater as described in 2.2.2.1. The single culture

used in this studies are Acinetobacter baumannii, Acinetobacter calcoaceticus

genospecies 3 and Cellulosimicrobium cellulans. These experiments were conducted in

non sterile condition and samples were complemented with un-treated textile effluent as

a control.

26

2.2.3.1 Liquid cultures

Adapted bacteria (10% inoculum) was incubated in Erlenmeyer flasks (250 mL)

containing 90% textile wastewater (pH unmodified) at room temperature, 200 rpm for 5

days (Kim et al., 2003). At various time intervals, (10 mL) samples were pipetted

aseptically for COD analysis as described in section 2.2.5.

2.2.3.2 Preparation of freeze-dried cultures

The freeze-dried cultures were prepared from liquid bacterial cultures adapted to

2.5% textile wastewater. Overnight active culture (10% inoculum) was incubated in

Erlenmeyer flasks (1L) containing 2.5% textile wastewater and 87.5% neutralized

pineapple waste. All flasks were incubated at 30oC, 200 rpm until exponential phase

was reached. This was determined by OD600 measurement.

Then the culture was centrifuged at 9000 rpm and 4oC for 5 minutes (B.Braun,

SIGMA 4K-15) to separate the bacterial pellet from the mixed solution of textile

wastewater and pineapple waste. The pellet was then transferred into 10 mL centrifuged

tube. The culture was then kept in the freezer at -20 oC. Frozen bacteria were then

freeze-dried at -47oC using freeze-drier (Cryst Alpha 1-2) and the dry weight was

recorded.

27

2.2.3.3 Freeze-dried culture experiment

This study was conducted using freeze-dried bacteria as described in section

2.2.3.2. Bacteria at various concentrations such as 5 mg/L, 10 mg/L, 15 mg/L, 20 mg/L,

and 30 mg/L were used. The freeze-dried bacteria (10% inoculum) at various

concentrations were incubated in Erlenmeyer flasks (250 mL) containing 100% of textile

wastewater. All flasks were incubated at room temperature, 200 rpm for 5 days.

Samples (10 mL) were pipetted at various time intervals for COD analysis as described

in section 2.2.5.

2.2.4 COD reduction using mixed cultures

In this experiment, two types of bacterial mixed culture consisting of

Acinetobacter baumannii, Acinetobacter calcoaceticus genospecies 3, and

Cellulosimicrobium cellulans at the following ratios were used. i.e. 1:1:1 and 1:1:3

respectively. Adapted mixed cultures at respective ratios (10% inoculum) were

incubated in Erlenmeyer flasks (500 mL) containing 90% of textile wastewater at room

temperature, 200 rpm for 30 days (Chaturvedia et al., 2006). At various intervals of

time, samples (25 mL) were pipetted and centrifuged to separate the cultures from the

textile wastewater. Then COD analysis as described in section 2.2.5 was carried out.

Sample was complimented with untreated textile effluent as a control.

28

2.2.4.1 Effect of Nutrient Broth supplementation

In this study, cultures were supplemented with increasing concentration (5%,

10%, 15%, 20%, and 30%) of NB. Adapted mixed culture at ratio 1:1:1 (10% inoculum)

was incubated in Erlenmeyer flasks (500 mL) containing 85% textile wastewater (pH

unmodified) and 5% NB. All flasks were incubated at room temperature, 200 rpm for 5

days. At various intervals of time, (25 mL) samples were pipetted for COD analysis as

described in section 2.2.5. Sample was complimented with untreated textile effluent as a

control.

2.2.4.2 Effect of pH

The pH of textile wastewater was varied from pH 3 to pH 11. Adapted mixed

cultures at ratio 1:1:1 (10% inoculum) was incubated in Erlenmeyer flasks (500 mL)

containing textile wastewater (90%) at respective pH. All flasks were incubated at room

temperature, 200 rpm for 5 days. At various time intervals, (25 mL) samples were

pipetted for COD analysis as described in 2.2.5. Sample was complimented with

untreated textile effluent as a control.

2.2.5 Chemical Oxygen Demand analysis

COD is an indication of overall oxygen load that a wastewater will impose on

streams. COD is equal to the amount of dissolved oxygen that a sample will absorb

from a hot acidic solution containing potassium dichromate and mercuric ions.

29

2.2.5.1 Materials

The materials used were COD test tube (10 mL), pipette (10 mL) and volumetric

flask (25 mL).

2.2.5.2 Reagents

The reagents used in COD analysis are standard potassium dichromate, sulphuric

acid (QRëC), mercury sulphate (Scharlau), silver sulphate (Scharlau) and acid silver

sulphate.

2.2.5.2.1 Preparation of standard potassium dichromate

Standard potassium dichromate was prepared by dissolving 12.259 of potassium

dichromate (GPR™), previously dried at 105oC for 2 hours, in DDW and make up to 1

liter.

2.2.5.2.2 Preparation of acid silver sulphate

Acid silver sulphate was prepared by dissolving 10.5 g of silver sulphate in

concentrated sulphuric acid (2.5 L). The mixed solution was then stirred for 1 to 2 days.

30

2.2.5.3 Preparation of samples for analysis

The apparatus used was COD reactor (Bioscience, Inc.) and spectrophotometer

(Hach DR/4000 U) in which diluted samples (2.5 mL) was contacted, in a vial, with the

mercury sulphate, (3.5 mL) prepared acid silver sulphate and (1.5 mL) standard

potassium dichromate which was then held at 150oC for two hours. After cooling the

sample was then analyzed in the spectrophotometer at 620 nm (Walker et al,. 2001 and

Georgiou et al,. 2005).

31

CHAPTER III

RESULTS AND DISCUSSION

3.1 Characterization of microorganisms

3.1.1 Growth profile

The growth of microorganism is defined as an increase in the number of

microbial population, which can also be measured as an increase in microbial mass. The

growth cycle of the batch culture can be divided into several distinct phases called the

lag phase, exponential (log) phase, stationary phase and death phase (Harley and

Prescott, 1990).

Microorganisms require energy to maintain cellular functions. Pineapple waste

as carbon source contain high concentrations of glucose and fructose that can be utilized

to generate energy required to serve this function (Nester et al., 2001). Pineapple waste

is used as an alternative growth medium due to its availability and abundances in Johor.

It has been reported that the sugar content in pineapple waste stored at room

temperature and refrigerator lasted for only 3 days. As the storage time increases, the

concentrations of both sugars decrease. This is due to the consumption of the sugars by

indigenous bacteria present in the pineapple waste. The best method for preserving the

32

pineapple waste is by treating with 5% of ethanol to kill the indigenous bacteria

(Nordiana Nordin, 2006).

3.1.1.1 Growth profile of single cultures

The growth profiles of three different bacteria, Acinetobacter baumanni,

Acinetobacter calcoaceticus genospecies 3 and Cellulosimicrobium cellulans were

monitored based on optical density measured at 600 nm at various intervals for 24 hours

using pineapple waste as growth medium. The experiment was conducted at neutral pH

because this is the optimum condition for the growth of most common bacteria referred

to as neutrophiles (Nester et al., 2001).

The growth of Acinetobacter baumanni, Acinetobacter calcoaceticus

genospecies 3 and Cellulosimicrobium cellulans (Figure 3.1) showed a typical profile

consisting of the lag, exponential and stationary phase. The lag phase for all strains

were not pronounced possibly due to the inoculation of an exponentially growing culture

into the same medium under the same conditions of growth, thus exponential phase

commences immediately (Harley and Prescott, 1990).

33

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Time (hour)

Gro

wth

(OD

600)

A.baumannii A.calcoaceticus C.cellulanscontrol

Figure 3.1: Growth profile of single culture.

The time required for all three bacteria to reach the stationary phase was between

12 to 14 hours respectively. The growth patterns especially the rate of the exponential

growth could be influenced by the environmental conditions such as temperature and

components of the culture medium as well as the genetic characteristics of the bacteria

(Harley and Prescott, 1990).

34

3.1.1.2 Growth profile of mixed culture

The growth profile of the mixed culture of bacteria consisting of Acinetobacter

baumannii, Acinetobacter calcoaceticus genospecies 3 and Cellulosimicrobium

cellulans is shown in Figure 3.2. The growth was monitored based on OD600 at various

intervals for 24 hours.

Same growth conditions as single culture were observed for mixed culture. The

lag phase did not pronounced and therefore the exponential phase pronounced

immediately due to the inoculation of an exponentially growing culture into the same

medium under the same conditions of growth.

The survival and growth of a microbial species in a mixed culture depends on the

nature and extent of interactions with the other species present. Amongst the types of

interactions which can occur between different species in a mixed culture are neutralism,

competition, commensalisms, mutualism, symbiosis, amensalism and predation (Sterritt

and Lester, 1988).

From the results obtained, the highest OD for mixed culture was 1.931. OD unit

are proportional to cell number. The more cell present, the more turbid the suspension

(Madigan et al,. 2000). Compared with single cultures (Figure 3.1), mixed culture

showed higher OD600 and this indicated that, mixed culture exhibited more cells.

From the results obtained, the best time to transfer culture for adaptation studies

was at the exponential phase which was at fourth hour. At this phase, cells are usually at

their active phase and thus are desirable for studies of enzymes or other cell components

(Madigan et al,. 2000). The time required for mixed culture to reach the stationary

phase was between 12 to 14 hours.

35

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Time (hour)

Gro

wth

(OD

600)

control mixed culture

Figure 3.2: Growth profile of bacterial consortium of Acinetobacter baumannii,

Acinetobacter calcoaceticus genospecies 3 and Cellulosimicrobium cellulans.

3.1.1.3 pH profiles

The pH profiles for single culture and mixed culture is shown in Figure 3.3. In a

batch culture, pH can change during growth as a result of metabolic reactions that

consume or produce acidic or basic substances as wastes (Madigan et al., 2000). At the

beginning of the growth, all three single cultures and mixed culture showed a decrease in

pH value from neutral to acidic environment. This might be due to the production of

acetic acid, lactic acid and ethanol from the pineapple waste (Madigan et al., 2000).

After 8 hours the pH value of both single and mixed cultures remained stationary with

an average in the range of 4.19 to 5.15.

36

The pineapple waste was acidic with pH 4.23. It was reported that optimum pH

range for bacteria is at pH between 5 and 7.5 (Inoue, 2002). Therefore the pineapple

waste was neutralized upon using. Upon neutralization, the pineapple waste showed a

change in colour from pale yellow to dark brown. This phenomenon occurs due to the

Maillard browning reaction. The Maillard is a non-enzymatic reaction between carbonyl

moieties of the sugar molecule with amino acid or lysine which gives the final product

of brown nitrogenous polymers called melanoidins (Hodge, 1953).

0

1

2

3

4

5

6

7

8

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Time (hour)

pH

A.baumannii A.calcoaceticuscontrol C.cellulans mixed culture

Figure 3.3: pH profile of single cultures and mixed culture.

37

3.2 Bacterial adaptation studies

3.2.1 Screening for bacterial tolerance to textile wastewater

Adaptation studies were carried out by growing the bacteria in pineapple waste

supplemented with 1%, 2.5%, 5%, 7.5%, and 10% of textile wastewater. The objective

to the adaptation of a microbial community to pollution, is to compare the degradation

potential and the presence of degradative genes within bacterial communities in the field

before and after a specific pollutant spill has occurred (Eva et al,. 2003).

As shown in Table 3.1, the adaptation time for all single culture reached at 3 to 4

hour whereby, for consortium, the adaptation times are longer where they reached within

3.5 to 4 hour. This might be due to the competitions for nutrients for each member of

the consortia, whereas the activities of microorganisms are greatly affected by the

chemical and physical conditions of their environment. The environment factors that

could be considered such as temperature, pH, water availability, nutrients and oxygen

(Madigan et al,. 2000). Within these time ranges, cultures were transferred to the

increased concentration of wastewater (Eva et al,. 2003). The indication is shown by the

change of colour of the cultures where the brown colour turns to cloudy solution. In the

mean times, OD measurements for those cultures were carried out and the results

obtained were compared with respective growth profiles. This to ensure the cultures

were in the mid of exponential phase whereby, at this phase cultures were at their

healthier state.

Table 3.1: Adaptation times for single cultures and mixed culture.

Bacteria Adapted time (hour) Textile wastewater (%) 1 2.5 5 7.5 10

Acinetobacter baumannii 3 3 3.5 3.5 4 Acinetobacter calcoaceticus 3 3 3.5 3.5 4 Cellulosimicrobium cellulans 3 3 3.5 3.5 4 Consortium 3.5 3.5 4 4 4 *consortium – mixed culture consisting of of A.baumannii, A.calcoaceticus and C.celulans.

38

The purpose of adaptation is to allow bacterial community to rapidly adapt to

their new environment (Eva et al,. 2003). There are several mechanisms, or

combinations by which microbial communities can adapt to their environment. Firstly,

there can be an increase in population size of those organisms that tolerate or even

degrade the compound by induction of appropriate genes. Secondly, the cells can adapt

through mutations of various kinds, such as single nucleotide changes or DNA

rearrangements that result in resistance to or degradation of the compound. Thirdly,

they may acquire genetic information from either related or phylogenetically distinct

populations in the community. Eventually, the individual cells best suited to resist or

degrade the compounds will be selected and sweep through the population until they

constitute a larger fraction of the total microbial community than before the presence of

the compounds (Eva et al,. 2003).

Figure 3.4 showed the optical density at 600 nm for each culture upon adaptation

to increase concentration of textile wastewater. From 1% to 2.5% textile wastewater the

OD600 are decreased. At this rate, all single cultures and mix culture are adapting to

their new environment. Upon increasing the textile wastewater concentration up to

7.5%, there are an increased in OD600 and the maximal value obtained are less than 1.

Compared with growth medium without textile wastewater the value obtained is more

than 1 (Nordiana Nordin, 2006). High OD value indicated the cells are increased in

populations (Madigan et al,. 2000). At 10% concentration, the OD600 started to decline.

Therefore, based on OD measurement, the best tolerance levels for all the bacteria are at

7.5% of textile wastewater whereby, Acinetobacter baumannii showed the greatest

OD600.

39

-0.2

0

0.2

0.4

0.6

0.8

1

0 2.5 5 7.5 10 12.5

textile wastewater (%)

OD

600

A.baumannii A.calcoaceticus C.cellulans consortium

Figure 3.4 Bacteria tolerance level for each culture upon adaptation to increase

concentration of textile wastewater based on OD measurement.

3.2.2 Bacteria survival in textile wastewater

These experiments were conducted prior to concern whether Acinetobacter

baumannii, Acinetobacter calcoaceticus genospecies 3, Cellulosimicrobium cellulans

and consortium of those three bacteria can survive in textile wastewater.

Upon adaptation, plate count was performed on the nutrient agar plates that had

been spread with the samples at various textile wastewater concentrations. The results

are shown in Table 3.2 to 3.5.

40

Table 3.2: Total number of A.baumannii colonies developing on nutrient agar plate from samples with different concentration of textile wastewater.

DILUTION 10-5 10-6 10-7

TEXTILE WASTE CONCENTRATION, (%)

1 2 1 2 1 2

Pineapple waste + 1% textile waste + AB

AB:TNTC IB : 174

AB:TNTC IB : 174

AB: 2 IB : 40

AB: 2 IB : 53

AB: 0 IB : 2

AB: 0 IB : 3

Pineapple waste + 2.5% textile waste + AB

AB: 168 IB : 33

AB: 168 IB : 33

AB: 86 IB : 8

AB: 11 IB : 4

AB: 22

AB: 2


AB: 5 IB : 1

AB: 6

AB: 2

AB: 2

AB: 1

AB: 1

Pineapple waste + 7.5% textile waste + AB

AB: 186

AB: 124

AB: 18

AB: 15

AB: 3

AB: 5


AB: 100

AB: 85

AB: 5

AB: 9

AB: 1

AB: 1

AB – A.baumannii IB – Indigenous bacteria TNTC – too numerous to count; > 300 colonies Table 3.3: Total number of A.calcoaceticus colonies developing on nutrient agar plate from samples with different concentration of textile wastewater.

DILUTION 10-5 10-6 10-7


1 2 1 2 1 2

Pineapple waste + 1% textile waste + AC

AC: TNTC IB : 174

AC: TNTC IB : 174

AC: TNTC IB : 40

AC: TNTC IB : 53

AC: 153 IB : 2

AC: 107 IB : 3

Pineapple waste + 2.5% textile waste + AC

AC: TNTC IB : 33

AB: TNTC IB : 33

AC: 195 IB : 8

AC: 199 IB : 4

AC: 22

AC: 23


AC: 16 IB : 1

AC: 23

AC: 5

AC: 6

AC: 0

AC: 0

Pineapple waste + 7.5% textile waste + AC

AC: 21

AC: 28

AC: 4

AC: 4

AC: 0

AC: 0


AC: 72

AC: 38

AC: 2

AC: 4

AC: 1

AC: 0

AC – A.calcoaceticus IB – Indigenous bacter

41

Table 3.4: Total number of C.cellulans colonies developing on nutrient agar plate from samples with different concentration of textile wastewater.

DILUTION 10-5 10-6 10-7


1 2 1 2 1 2

Pineapple waste + 1% textile waste + CC

CC: TNTC IB : 174

CC: TNTC IB : 174

CC: 137 IB : 40

CC: 109 IB : 53

CC: 26 IB : 2

CC: 17 IB : 3

Pineapple waste + 2.5% textile waste + CC

CC: TNTC IB : 33

CC: TNTC IB : 33

CC: 84 IB : 8

CC: 88 IB : 4

CC: 22

CC: 7


CC: 198 IB : 1

CC: 160

CC: 28

CC: 30

CC: 2

CC: 1

Pineapple waste + 7.5% textile waste + CC

CC: 100

CC: 63

CC: 10

CC: 9

CC: 4

CC: 1


CC: 61

CC: 68

CC: 10

CC: 7

CC: 0

CC: 0

CC – C.cellulans IB – Indigenous bacteria Table 3.5: Total number of mixed culture colonies developing on nutrient agar plate from samples with different concentration of textile wastewater.

DILUTION 10-5 10-6 10-7


1 2 1 2 1 2

Pineapple waste + 1% textile waste + M

M:TNTC IB : 174

M:TNTC IB : 174

M: 75 IB : 40

M: 86 IB : 53

M: 8 IB : 2

M: 8 IB : 3

Pineapple waste + 2.5% textile waste + M

M:TNTC IB : 33

M:TNTC IB : 33

M: 179 IB : 8

M: 178 IB : 4

M: 8

M: 10


M: 191 IB : 1

M: 180

M: 8

M: 7

M: 4

M: 0

Pineapple waste + 7.5% textile waste + M

M: 112

M: 152

M: 14

M: 15

M: 4

M: 0


M: 29

M: 14

M: 2

M: 3

M: 2

M: 0

M – A.baumannii + A.calcoaceticus + C.cellulans IB – Indigenous bacteria

42

From the results obtained, indigenous bacteria from the pineapple waste were

observed at 1% and 2.5% textile. At higher concentration the presence of indigenous

bacteria was not observed. The highest colony forming unit (CFU) for A.baumannii was

at 2.5% with 1.0 x 109 per mL. Acinetobacter calcoaceticus genospecies 3,

Cellulosimicrobium cellulans and consortium also showed the highest CFU at 2.5% with

2.1 x 109 per mL, 1.5 x 109 per mL and 1.7 x 109 per mL respectively. These indicated

that the best condition for adaptation was at 2.5% textile wastewater.

The dominant strain in the mixed culture could probably be distinguished by

referring to CFU measurement of each of the single strain involved. Acinetobacter

calcoaceticus genospecies 3 showed the highest CFU therefore, the dominacy strain was

Acinetobacter calcoaceticus genospecies 3.

Based on the results in the tables of colony forming unit, a graph for screening

the best tolerance level based on CFU were plotted (Figure 3.5). Results obtained

supported that the best condition for adaptation was at 2.5% textile wastewater. These

results were used in subsequent COD reduction studies.

43

-5000

0

5000

10000

15000

20000

25000

0 2.5 5 7.5 10 12.5

textile wastewater

CFU

(10

6 )

A.baumannii A.calcoaceticus C.cellulans consortium

Figure 3.5: Bacterial tolerance level based on viable cell count.

3.3 Textile waste water characteristics

Real textile wastewater used in this study was taken from Senawang, Negeri

Sembilan. Wastewater was sampled and analysed for COD, pH and colour. As shown

in Table 3.6, the COD levels are above the permitted limit (100 mg/L). In this study

COD level will be reduced using developed techniques.

Table 3.6: Characteristics of textile wastewater.

Parameters Unit Textile wastewater pH - 9.38 COD mg/L 600 Colour Pt-Co 1050

44

3.4 COD reduction using single culture

3.4.1 Liquid cultures

Results obtained from section 3.2.2 were used for COD reduction studies. Single

culture adapted to 2.5% textile wastewater (10% inoculum) was introduced to the textile

effluent without pH adjustment. The experiment was conducted under non sterile

condition.

Table 3.7: COD reduction using liquid single culture.

COD reduction (%) 1 2 3 4 5 (day) A.baumannii 20 27 40 48 60 A.calcoaceticus 18 25 39 50 67 C.cellulans 18 26 33 46 58

Table 3.7 shows COD reduction using single culture. A.baumannii,

A.calcoaceticus and C.cellulans which were locally isolated microorganisms showed

excellent pollutant removal capabilities. COD reduction was observed up to 60%, 67%

and 58% for A.baumannii, A.calcoaceticus and C.cellulans respectively after 5 day

treatment for each case. The results suggested that A.calcoaceticus showed the best

performance in reduction of COD for liquid single culture.

45

3.4.2 Freeze dried culture

It is important to preserve microorganisms, partly due to the danger of reduction

biodiversity in natural environments and partly due to their expanding use in teaching,

research, and biotechnology. Therefore freeze-drying is a convenient method for the

preservation and long term storage of microorganisms (Malik, 1995). Freeze-dried

bacteria have been reported to survive for as long as 35 years. T he survival rates differ

according to genus, but it is unknown whether they differ among species (Miyamoto et

al., 2000). In this study, the reduction of COD using single freeze-dried culture from

textile effluent was reported.

3.4.3 Cells dry weight determination

The dry weight of all three single cultures, Acinetobacter baumannii,

Acinetobacter calcoaceticus genospecies 3 and Cellulosimicrobium cellulans was

determined to be 1.15 mg, 2.35 mg and 2.20 mg respectively.

3.4.4 COD reduction using freeze-dried culture

The different biomass concentration (5 mg, 10 mg, 15 mg, 20 mg and 30 mg) in

reducing COD level of textile effluent will be observed. This experiment was conducted

using batch process under non sterile condition for 5 days. This was similar condition

and time interval as reported by Kim et al,. (2003).

46

From the results obtained (Figure 3.6), at day 1, A.baumannii showed 46% of

COD reduction whereby, C.cellulans and A.calcoaceticus showed 80% and 83% of

COD reduction respectively. By increasing the biomass concentrations the COD

removal efficiency was decreased. This might be due to the increase in nutrient needed

for the bacteria activity whereby, by freezing the growth medium were removed from

the cells. Therefore, the bacteria were trying to survive and generate energy by utilizing

the nutrients available in textile effluent and at certain rate the bacteria cannot survive

and the COD tend to increase.

From this study, COD reduction will stop predominantly between 3 and 4 day

after the start of the treatment and 24 hours of interaction was determined as the best

treatment time for treating textile effluent using freeze-dried culture. The textile effluent

vary greatly in their chemical contents, and therefore their interactions with

microorganisms depend on the chemistry of the pollutant compounds and the microbial

biomass (Robinson et al,. 2001).

These results suggest that the advantages of using freeze-dried with lower

concentration of biomass are higher levels of COD reduction and shorter treatment time.

The use of biomass has its advantages, especially if the textile effluent containing very

toxic compounds.

Furthermore, compared with liquid culture, freeze-dried culture exhibited better

COD reduction. This might be due to the presence of Triple Super Phosphate and Urea

as reported by Babu et al,. (2000), and hence environment for bacterial growth in the

textile effluent using freeze-dried culture are favorable. This was supported by

Robinson et al,. (2001), the biomass is also effective when conditions are not favorable

for growth and maintenance of the microbial population.

47

05

101520253035404550

0 1 2 3 4 5

day

)

n (%

uctio

D re

d

CO

(a)

01020

30405060

708090

0 1 2 3 4 5

day

CO

D re

duct

ion

(%)

(b)

0102030405060708090

0 1 2 3 4 5

day

CO

D re

duct

ion

(%)

5mg 10mg 15mg 20mg 30mg

(c)

Figure 3.6: COD reduction of freeze-dried culture a) A.baumannii, b) A.calcoaceticus

and c) C.cellulans.

48

3.5 COD reduction using mixed culture

3.4.1 Liquid culture

Results obtained from section 3.2.2 were used for COD reduction studies. Mixed

culture adapted to 2.5% textile wastewater (10 % inoculum) was introduced to the textile

effluent without pH adjustment. The experiment was conducted under non sterile

condition. The experiment was conducted using liquid culture at different ratios as

described in Table 3.8 and the COD reduction performance was monitored for 30 days

as described by Chaturvedia et al., (2006).

Table 3.8: Types of system using mixed culture.

Types of system Cultures ratio System 1 ab:ac:cc System 2 ab:ac:3cc * ab-A.baumannii ac- A.calcoaceticus cc-C.cellulans

System 1 consist of each single culture at same ratio whereas system 2, the ratio

for C.cellulans was increased by three times. Since A. baumannii and A. calcoaceticus

genospecies 3 are aerobic bacteria, they require oxygen for growth whereby, C. cellulans

is a facultative anaerobe and it can survive both with and without oxygen supply. The

different conditions of textile wastewater after certain times may affect the growth of the

consortia. Thus, Cellulosimicrobium cellulans will predominate over the other two

bacteria when the three microorganisms were used as a mixed culture in treating textile

wastewater. Under anaerobic consortia, reducing equivalents are formed by dominated

bacteria. Therefore, System 2 may be more effective in treating textile wastewater (dos

Santos et al., 2006).

49

As shown in Table 3.9 at day 7 to day 21, system 2 exhibited higher COD

reduction compared to system 1. At day 30 system 1 and system 2 managed to reduce

the COD up to 94% and 95 % respectively. Although both systems showed high

efficiency in COD reduction of the textile effluent, system 1 was selected for subsequent

studies. The results suggests that selected microorganism are important factors in the

textile wastewater treatment process (Kim et al,. 2003). Instead of that, the use of

system 1 could minimized the quantity of culture required and thus will be cost

effective.

As reported by Chaturvedia et al., (2006), the bacterial population in mixed

culture showed degrading potential for these pollutants by utilizing the pollutants as

their nutrients. In this case the responsible pollutants are textile effluents. Chaturvedia et

al., (2006) also described that each strain in mixed culture played a major role in

bioremediation of effluent. Therefore, the values of COD were reduced.

Compared to single culture, using mixed culture exhibited better performance

where the rate of COD reduction is increased by about 30%. The mixed culture was

able to reduce the COD level of textile wastewater having diverse and complex

structures at faster rate than individual cultures (Kehra et al,. 2005).

Table 3.9: COD reduction using mixed culture.

Types of system COD Reduction (%) day 7 day 14 day 21 day 30 System 1 69 85 90 94 System 2 78 88 92 95

50

3.4.2 Effect of NB supplementation

In this study, Nutrient Broth which is a complex culture media was selected as

supplement. Nutrient Broth as culture medium provide carbon and nitrogen source

needed for microorganism metabolism. The nutrients present in the culture medium

provide the microbial cell with the ingredients required for the cell to produce more cells

like itself (Madigan et al., 2000).

Different initial concentrations of NB were used to determine their effect on

COD removal using mixed culture at ratio of 1:1:1, Acinetobacter baumannii,

Acinetobacter calcoaceticus genospecies 3 and Cellulosimicrobium cellulans

respectively. The experiment was conducted for 5 day and this was similar as described

by Azbar et al., (2004). The results as presented in Figure 3.7 indicated that NB

supplementation was significant for COD removal. NB supplementation at 5% gave the

highest reduction with 66% of COD removal. However, the increasing of NB

supplementation decreased the COD reduction efficiency of the mixed culture.

Growth factors are organic compounds that are required in very small amounts

and only by some cell. Growth factors include vitamins, amino acids, purines and

pyrimidines. Although most of microorganisms are able to synthesize all of these

compounds, some microorganisms require one or more of them to preform from the

environment (Madigan et al., 2000).

51

0

10

20

30

40

50

60

70

0 5 10 15 20 25 30 35

Nutrien Broth (%)

CO

D re

duct

ion

(%)

Figure 3.7: The effect of NB supplementation on COD reduction

3.4.2 Effect of pH

Each organism has a pH range within which growth is possible and usually has a

well defined pH optimum (Inoue, 2002). Therefore, effect of pH of textile wastewater in

COD reduction was performed. The experiment was carried out at pH between 3 and

11. Figure 3.8 shows the effect of various pH on COD removal using mixed culture at

ratio as mention in section 3.3.1.1. The initial pH of textile wastewater was 9.23.

COD reduction efficiency varied from 52% up to 61% during batch treatment.

Acidic condition showed better reduction efficiency and highest COD reduction was

observed at pH 5. It was reported that optimum pH range for bacteria is at pH between 5

and 7.5 (Inoue, 2002). There was no significant increase in COD removal in alkaline

conditions, which resulted in 56% and 55% of COD reduction at pH 9 and pH 11

respectively. At neutral pH condition 54% of COD removal was observed.

52

515253545556575859606162

3 4 5 6 7 8 9 10 11 12 13

pH

CO

D re

duct

ion

(%)

Figure 3.8: The effect of various pH on the COD reduction.

3.6 Comparison with existing COD reduction methods

Many attempts have been made to treat textile wastewater using conventional

wastewater treatment methods. Several methods have been developed to treat textile

wastewater, but most of them do not effectively treat the wastewater. Therefore, this

method which based on biological technique has been developed whereby locally

isolated bacterium has been exploited. The efficiency of this biological technique

compared with other conventional biological treatment methods is shown in Table 3.10.

53

Table 3.10: Comparison of the COD reduction efficiency with other

biological conventional methods.

Treatment COD reduction Treatment Reference technique (%) time (day) Bacteria mixed culture 95 30 This study Bacteria single culture 83 1 This study (freeze-dried) Bacteria single culture 67 5 This study Bacteria mixed culture 86 30 Chaturvedi etal 2006 Bacteria mixed culture 42.4 2 Kim et al., 2003 Bacteria mixed culture 63.6 2 Kim et al., 2003 with support media Bacteria spp. 62 - Babu et al,. 2000

Biological technique proposed by Babu et al., (2000) achieved 62% of COD

removal. 42.4% and 63.6% COD reduction was reported by Kim et al., 2003 for bacteria

without support media and bacteria with support media respectively. This suggest that

support media is advantageous for achieving efficient treatment of textile wastewater.

Kim et al., 2003 also reported that upon using support media and selected

microorganisms, prevent the generation of large amount of mixed liquor suspended

solids.

An improvement in COD reduction was achieved using the technique developed

in this study. This was demonstrated by both mixed culture systems that showed high

COD removal efficiency, but long treatment time is required. Textile wastewater

treatment using single culture was also presented efficient COD reduction whereby,

faster treatment time was achieved. The other advantages of this developed technique

are the exploitation of locally isolated bacterium and the used of growth medium which

is the pineapple waste, a cheap carbon source.

54

CHAPTER IV

CONCLUSION AND SUGGESTION

4.1 Conclusion

The bacteria used in this study Acinetobacter baumanni, Acinetobacter

calcoaceticus genospecies 3 and Cellulosimicrobium cellulans were locally isolated

bacteria. In COD reduction study, two systems were used which are single culture and

mixed culture supplemented with pineapple waste as growth media.

All the single cultures and mixed culture used in this study were adapted to

textile wastewater prior to use to give faster retention time. The maximal tolerance level

was at 2.5% of textile wastewater. Based on the CFU, Acinetobacter calcoaceticus

genospecies 3 showed highest survival rate with 2.1 x 109 CFU per mL.

Study using single strain was conducted using liquid culture and freeze-dried

culture. An efficient COD reduction was observed using both types of culture whereby,

in both studies, Acinetobacter calcoaceticus genospecies 3 exhibited higher COD

removal compared with other strain. Upon using freeze-dried culture, lower biomass

concentration resulted in higher COD reduction. On the other hand, faster treatment

time was also achieved. 2 day and 5 day treatment time were achieved for freeze-dried

culture and liquid culture respectively.

55

The ability of mixed culture in reducing COD of textile wastewater was also

conducted. The mixed culture consisted of Acinetobacter baumanni, Acinetobacter

calcoaceticus genospecies 3 and Cellulosimicrobium cellulans. This experiment was

conducted using mixed culture at different ratios. An improvement of COD reduction

by about 20% was achieved using mixed culture for both ratios compared to single

culture. The excellent results were achieved but long retention time is required whereby,

this is not applicable in industrial practices.

In stead of that, System 1 showed higher COD reduction compared to system 2.

Therefore system 1 is selected for subsequent COD reduction experiments. The use of

system 1 could minimized the quantity of culture required and thus will be cost

effective.

The effect of nutrients supplementation and pH conditions of the mixed culture

were observed. Upon NB supplementation, it was significant result in COD level. The

reduction of COD can be increased by supplying the mixed culture with 5% of NB.

Thus, acidic conditions were more favorable in reducing the COD level and the best

condition was at pH 5.

56

4.2 Suggestions

From the study of this research, several potential developments for the COD

reduction of textile wastewater were identified. The Acinetobacter calcoaceticus

genospecies 3 and mixed culture that was used in this study exhibited high tolerance in

reducing the COD level. Therefore, these cultures should be further used of continuous

treatment system for textile effluents.

The potential of the property of Acinetobacter calcoaceticus genospecies 3 and

mixed culture to reduce COD level in textile effluent should be evaluated, with a special

emphasis on complete metabolism pathway. This microbe should be exploited for the

reduction of COD level for other contaminated industrial effluents.

Instead of reducing COD, used of Acinetobacter calcoaceticus genospecies 3

and mixed should be exploited in treating other parameter of textile wastewater (BOD,

colour, total suspended solid and pH).

A more complete study should be performed on the operative parameters for the

COD reduction i.e. temperature and support media. It is reported, by the use of support

media efficient textile wastewater treatment will be achieved even prevent the

generation of suspended solids.

Besides, this study was conducted based on the batch process. Although it gave

good performances in reducing the COD level of, other process should be implemented,

for an example column processes that could possibly give better COD reduction.

This study was conducted using pineapple waste as growth medium. On the

other hand, a study using other available agricultural waste should be performed for

COD removal such as molasses, sugar cane and monosodium glutamate.

57

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