DEVELOPMENT OF BIOLOGICAL TREATMENT SYSTEM FOR
REDUCTION OF COD FROM TEXTILE WASTEWATER
NOR HABIBAH BINTI MOHD ROSLI
UNIVERSITI TEKNOLOGI MALAYSIA
iv
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.
v
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.
vi
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.
vii
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.
viii
TABLE OF CONTENTS
CHAPTER
I
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
INTRODUCTION
1.1
Textile industry
1.2
Biodegradability of textile waste
1.3
1
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
Textile Wastewater Treatment
Current Technologies
5
ix
1.4
COD reduction
1.4.1
Definition of COD
1.4.2 COD and BOD
1.5
1.5.2
1.7
II
Physical method
1.5.1.1 Granular activated carbon
8
1.5.1.2 Membrane filtration
9
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
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
Bacteria
1.7.1
1.8
7
Methods of COD reduction
1.5.1
1.6
6
Acinetobacter baumanni and
Acinetobacter calcoaceticus
16
1.7.2
Cellulosimicrobium cellulans
17
1.7.3
Mixed bacterial culture (consortium)
18
Objective and scope of studies
19
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
2.2
Growth media
2.1.5.1 Nutrient broth
22
2.1.5.2 Nutrient agar
22
2.1.5.3 Pineapple waste
22
Method
2.2.1
2.2.2
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
Bacterial adaptation studies
2.2.2.1 Screening for bacterial tolerance
to textile wastewater
25
2.2.2.2 Bacterial survival in
textile wastewater
2.2.3
2.2.4
2.2.5
25
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
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
COD analysis
2.2.5.1 Materials
29
2.2.5.2 Reagents
29
2.2.5.2 Preparation of samples for analysis 30
xi
III
RESULTS AND DISCUSSION
3.1
Characterizations of microorganisms
3.1.1
3.2
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
Bacterial adaptation studies
3.2.1
Screening for bacterial tolerance
37
To textile wastewater
3.2.2
Bacterial survival in textile wastewater
3.3
Characteristics of textile wastewater
3.4
COD reduction using single culture
39
43
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
3.6
COD reduction using mixed culture
3.5.1 Liquid culture
48
3.5.2
49
Effect of NB supplementation
3.5.3 Effect of pH
51
Comparison with existing COD
52
reduction methods
IV
CONCLUSION AND SUGGESTIONS
REFERENCES
54
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
3.2
Total number of A.baumannii colonies developing on
37
nutrient agar plate from samples with different
concentrations of textile wastewater
3.3
40
Total number of A.calcoaceticus colonies developing on
nutrient agar plate from samples with different
concentrations of textile wastewater
3.4
40
Total number of C.cellulans colonies developing on
nutrient agar plate from samples with different
concentrations of textile wastewater
3.5
41
Total number of mixed culture colonies developing on
nutrient agar plate from samples with different
concentrations of textile wastewater
41
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
3.5
39
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
o
Degree Celsius
C
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
pH
BOD
COD
TSS
Nitrate
Phosphate
Sulphate
Cr (III)
Cu (II)
o
C
mg/L
mg/L
mg/L
mg/L
mg/L
mg/L
mg/L
mg/L
40
5.5 -9.0
50
100
100
7
0.2
1.0
1.0
Cr(III)-Cromium (III)
Cu(II)-Copper(II)
* TSS-Total suspended solid
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
large
treatment
Activated sludge,
efficient BOD
long
Aerated lagoons,
Land treatment,
Extended aeration.
reduction
aeration tanks, may
require nutrient.
2. Chemical
Precipitation
Percipitation-addition
of multivalent cations
with pH adjustment
heavy metals,
suspended solid,
BOD, COD
color removal varies with
dye class, chemical
handling problems.
3. Activated carbon
Passing water
through a bed of
carbon (as a
pretreatment to
further treatment)
BOD, COD,
color
expensive, long
residence times, low
adsorption capacity.
4. Ultra filtration
Permeation of water
under pressure
through specialized
polymeric membranes
BOD, COD,
color
membranes foul easily,
heavy metals not
removed, need frequent
cleaning.
residence
times,
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).
8
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).
10
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
2
Growth (OD600)
1.75
1.5
1.25
1
0.75
0.5
0.25
0
0
2
4
6
8
10 12 14 16 18 20 22 24 26
Time (hour)
control
A.baumannii
A.calcoaceticus
C.cellulans
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
2
Growth (OD600)
1.75
1.5
1.25
1
0.75
0.5
0.25
0
0
2
4
6
8
10 12 14 16 18 20 22 24 26
Time (hour)
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).
8
7
6
pH
5
4
3
2
1
0
0
2
4
6
8
10 12 14 16 18 20 22 24 26
Time (hour)
control
C.cellulans
A.baumannii
A.calcoaceticus
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
Acinetobacter baumannii
Acinetobacter calcoaceticus
Cellulosimicrobium cellulans
Consortium
3
3
3
3.5
3
3
3
3.5
3.5
3.5
3.5
4
3.5
3.5
3.5
4
*consortium – mixed culture consisting of of A.baumannii, A.calcoaceticus and C.celulans.
10
4
4
4
4
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
1
0.8
OD600
0.6
0.4
0.2
0
0
2.5
5
7.5
10
12.5
-0.2
textile wastewater (% )
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.
10-6
1
AB:TNTC
IB : 174
AB:TNTC
IB : 174
AB: 168
IB : 33
2
2
AB: 2
IB : 40
AB: 2
IB : 53
AB: 0
IB : 2
AB: 0
IB : 3
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
+
+
AB: 186
AB: 124
AB: 18
AB: 15
AB: 3
AB: 5
+
+
AB: 100
AB: 85
AB: 5
AB: 9
AB: 1
AB: 1
Pineapple waste + 1%
textile waste + AB
Pineapple waste +
2.5% textile waste +
AB
Pineapple waste + 5%
textile waste + AB
Pineapple waste
7.5% textile waste
AB
Pineapple waste
10% textile waste
AB
DILUTION
10-5
1
2
10-7
1
TEXTILE WASTE
CONCENTRATION,
(%)
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
1
2
10-6
1
2
AC:
TNTC
IB : 174
AC:
TNTC
IB : 174
AC:
TNTC
IB : 40
AC:
TNTC
IB : 33
AB:
TNTC
IB : 33
AC: 16
IB : 1
Pineapple waste + 7.5%
textile waste + AC
Pineapple waste + 10%
textile waste + AC
TEXTILE
WASTE
CONCENTRATION,
(%)
Pineapple waste + 1%
textile waste + AC
Pineapple waste + 2.5%
textile waste + AC
Pineapple waste + 5%
textile waste + AC
AC – A.calcoaceticus
10-7
1
2
AC:
TNTC
IB : 53
AC: 153
IB : 2
AC: 107
IB : 3
AC: 195
IB : 8
AC: 199
IB : 4
AC: 22
AC: 23
AC: 23
AC: 5
AC: 6
AC: 0
AC: 0
AC: 21
AC: 28
AC: 4
AC: 4
AC: 0
AC: 0
AC: 72
AC: 38
AC: 2
AC: 4
AC: 1
AC: 0
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.
TEXTILE
WASTE
CONCENTRATION,
(%)
Pineapple waste + 1%
textile waste + CC
DILUTION
10-5
1
2
10-6
1
2
10-7
1
2
CC:
TNTC
IB : 174
CC:
TNTC
IB : 174
CC: 137
IB : 40
CC: 109
IB : 53
CC: 26
IB : 2
CC: 17
IB : 3
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
Pineapple waste + 10%
textile waste + CC
CC: 61
CC: 68
CC: 10
CC: 7
CC: 0
CC: 0
Pineapple waste + 2.5%
textile waste + CC
Pineapple waste + 5%
textile waste + CC
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
1
2
10-6
1
M:TNTC
IB : 174
M:TNTC
IB : 174
M:TNTC
IB : 33
2
10-7
1
2
M: 75
IB : 40
M: 86
IB : 53
M: 8
IB : 2
M: 8
IB : 3
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
Pineapple waste + 10%
textile waste + M
M: 29
M: 14
M: 2
M: 3
M: 2
M: 0
TEXTILE
WASTE
CONCENTRATION,
(%)
Pineapple waste + 1%
textile waste + M
Pineapple waste + 2.5%
textile waste + M
Pineapple waste + 5%
textile waste + M
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
25000
20000
CFU (10 6)
15000
10000
5000
0
0
2.5
5
7.5
10
12.5
-5000
textile wastewater
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
COD
Colour
mg/L
Pt-Co
9.38
600
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.
A.baumannii
A.calcoaceticus
C.cellulans
1
COD reduction (%)
2
3
4
5
20
18
18
27
25
26
40
39
33
48
50
46
(day)
60
67
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
50
CO D reductio n (%)
45
40
35
30
25
20
15
10
5
0
0
1
2
(a)
3
4
5
3
4
5
3
4
5
day
90
COD reduction (%)
80
70
60
50
40
30
20
10
0
0
1
2
day
COD reduction (%)
(b)
90
80
70
60
50
40
30
20
10
0
0
1
2
day
(c)
5mg
10mg
15mg
20mg
30mg
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
System 2
* ab-A.baumannii
ab:ac:cc
ab:ac:3cc
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
System 1
System 2
day 7
COD Reduction (%)
day 14
day 21
day 30
69
78
85
88
94
95
90
92
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
70
COD reduction (%)
60
50
40
30
20
10
0
0
5
10
15
20
25
30
35
Nutrien Broth (%)
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.
COD reduction (%)
52
62
61
60
59
58
57
56
55
54
53
52
51
3
4
5
6
7
8
9
10
11
12
13
pH
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
technique
Bacteria mixed culture
Bacteria single culture
(freeze-dried)
Bacteria single culture
Bacteria mixed culture
Bacteria mixed culture
Bacteria mixed culture
with support media
Bacteria spp.
COD reduction
(%)
Treatment
time (day)
Reference
95
83
30
1
This study
This study
67
86
42.4
63.6
5
30
2
2
This study
Chaturvedi etal 2006
Kim et al., 2003
Kim et al., 2003
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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