1

CHAPTER 1

INTRODUCTION

1.1 Introduction

More than 100,000 new synthetic dyes have been produced after the first

synthetic dye, mauevin was found. Textile industries are the biggest consumers of

the total dyestuff market (Asad et al., 2007). These industries consume large amount

of water and are therefore a source of considerable colour pollution (McMullan et al.,

2001). Textile wastewater is a complex mixture of colorants (dyes and pigments)

and various organic compounds. It also contains high concentrations of heavy metals,

total dissolved solids and has higher chemical as well as biological oxygen demand.

Thus, textile wastewater is chemically very complex in nature (Sharma et al., 2007).

Colour in textile wastewater is a visible pollutant which may be resulted from

the presence of different colouring agents like dyes, inorganic pigments, tannins,

lignins and others. Among these, dyes are considered as xenobiotic compounds that

are very recalcitrant to biodegradation. The degradation products of textile dyes are

often carcinogenic. In addition, the absorption of light due to textile dyes creates

problems to photosynthetic aquatic plants and algae. The presence of the dyes in

aqueous ecosystems reduces the photosynthesis by impeding the light penetration

into deeper layers thereby deteriorating the water quality and lowering the gas

solubility (Anjaneyulu et al., 2005).

2

During textile processing, inefficiencies in dyeing cause a large amount of the

dyestuff being directly lost to the wastewater which ultimately release into the

environment. Therefore, the treatment of textile wastewater has been a major

concern. Many remediation technologies have been developed due to the

increasingly stringent environmental legislation. These include physicochemical

methods such as filtration, coagulation, carbon activated and chemical flocculation.

Despite the existence of a variety of chemical and physical treatment processes,

biological treatment of textile effluent is still seen as cost effective, environmentally

friendly and publicly acceptable treatment technology.

In recent years, new biological process including aerobic and anaerobic

bacteria and fungi for dye degradation and wastewater reutilization have been

developed (McMullan et al., 2001). Decolourization of azo dyes normally starts with

initial reduction or cleavage of azo bond anaerobically which turn to colourless

compounds. This is followed by complete degradation of aromatic amine under

aerobic condition. Therefore, anaerobic-aerobic processes are crucial for complete

mineralization of azo dye (Moosvi, 2007). The main aim of this study was to

investigate textile dye decolourizing and degradation potential of a selected mixed

culture, known as MicroClear.

3

1.2 Objectives of Research

The objectives of this study were:

1. To isolate and characterize the bacteria obtained from acclimatized mixed

culture of decolourizing bacteria.

2. To utilize selected mixed culture of decolourizing bacteria (MicroClear) in

the treatment of raw textile wastewater.

1.3 Scope of Study

Characterization of each bacteria isolated from the acclimatized mixed

bacterial culture was part of the research. Textile wastewater was treated using

selected mixed culture of decolourizing bacteria, MicroClear under sequential

facultative anaerobic and aerobic condition. The effectiveness of MicroClear in the

wastewater treatment was based on water quality before treatment and after treatment.

The significant water quality parameters included colour, pH, chemical oxygen

demand (COD), nitrate, phosphate, sulphate and total suspended solids (TSS).

4

CHAPTER 2

LITERATURE REVIEW

2.1 Modes of Bioremediation

Different modes of bioremediation of coloured effluents include

decolourization using mixed cultures, isolated organisms and isolated enzymes.

Bioremediation of colored effluents using mixed cultures, where a consortium of

different species is present, and dye decolorization may happen due to the synergistic

action of various microorganisms. An organism may cause biotransformation of a

dye, which consequently make it more accessible to another organism that otherwise

is not able to attack this dye but may stabilize the overall ecosystem. In this way, the

decolorization could mutually depend on the presence of several microorganisms and

on their synergistic action (Kandelbauer and Guebitz, 2005).

Similarly to isolated organisms, there are only a few expressed enzymes

directly involved in dye biotransformation. A single microorganism may be able to

decolorize the solution by breaking the structure of chromophore but complete

degradation is not achieved. The metabolic end products yielded during the

decolorization process may be toxic. If the unwanted metabolite can be used as a

nutrient source by other organisms, detoxification can be achieved (Kandelbauer and

Guebitz, 2005).

5

In enzyme remediation, specific enzymes are used to degrade pollutants.

They may be used after separated from the biomass. The actions of enzymes are

depending on the presence of substances such as cofactor, co-substrates or mediators.

Biochemical transformation of the dye may either occur extracellular if the enzymes

are excreted into the medium or intracellular, where the dye is readily transported

into the cell, demonstrating the impact of its bioavailability. A single enzyme or

group of enzymes may be involved in the decolourization process and the presence

of cofactors, co-substrates or mediators may improve the decolourization as well.

Figure 1.1 shows the important oxidative enzymes used for dye decolourization

(Kandelbauer and Guebitz, 2005). In general, any organism that secretes these

enzymes is a likely dye degradable microorganism.

Peroxidase

Dye + H2O2 Oxidized dye + H2O Laccase Dye + O2 Oxidized dye + H2O

Monooxygenase

Dye + O2 Hydroxylated dye + H2O

Dye + O2 Dioxygenase Bishyroxylated dye

Figure 1.1 Important oxidative enzymes used for dye decolourization

(Kandelbauer and Guebitz, 2005).

2.1.1 Anaerobic Bacterial Decolourization of Textile Dyes

Anaerobic bioremediation allows azo and other water-soluble dyes to be

decolourized. Many bacteria have been reported to readily decolourize dyes with

azo-based chromophores under anaerobic conditions. The reductive cleavage of azo-

linkage (–N=N–) by the bacteria results in dye decolourization and the production of

colourless aromatic amines. This process is catalyzed by a variety of soluble

cytoplasmic enzymes with low-substrate specificity which are known as

6

“azoreductases”. Under anoxic conditions, these enzymes facilitate the transfer of

electrons via soluble flavins to the azo dye, which is then reduced. Figure 1.2

illustrates the suggested mechanism for reduction of azo dyes by whole bacterial cells.

Whilst the anaerobic reduction of azo dyes is relatively easy to achieve, complete

mineralization of the molecule is difficult. Such decolourization may yield toxic

metabolic end products (McMullan et al., 2001). These toxic intermediate products

are generally degraded under aerobic condition. Therefore, anaerobic-aerobic

processes are crucial for complete mineralization of azo dyes.

Figure 1.2 Suggested mechanism for reduction of azo dyes by whole bacterial

cells (Pearce, 2003).

2.1.2 Anaerobic-aerobic Biodegradation of Dyes

Although anaerobic reduction of azo dyes is generally more satisfactory than

aerobic degradation, the intermediate products (carcinogenic aromatic amines) have

7

to be degraded through an aerobic process. In the first anaerobic stage, the azo dye is

readily reduced to the corresponding colourless aromatic amines. Then, aerobic

condition is required for complete degradation of the toxic intermediate compounds

into harmless products (McMullan et al., 2001). The aromatic compounds produced

by the initial reduction are then degraded via hydroxylation and ring opening in the

presence of oxygen (Doble and Kumar, 2005). Therefore, sequential

anaerobic/aerobic processes are important for complete mineralization of azo dyes

(Kodam et al., 2005).

2.2 Characteristics of Textile Effluent

Textile wastewater is extremely variable in composition due to the large

number of dyes and other chemicals used in the dyeing processes. In general, the

characteristics of a particular wastewater in addition to site-specific conditions, aid in

the selection and design of the most appropriate treatment facilities. Detailed

wastewater characterization is therefore an integral step in selecting wastewater

treatment methodologies (Reife et al., 1996). Wastewater is characterization can be

divided into physical and chemical. The most significant parameters in wastewater

from textile industry are COD (Chemical Oxygen Demand), BOD5 (Biological

Oxygen Demand), colour, pH, nitrogen, phosphorus, sulphate and suspended solids

(Tufekcu et al., 2007).

2.2.1 Physical Characterization

The physical characterization of wastewater involves solids content, turbidity

temperature, colour and odour (Oke et al., 2006).

8

2.2.1.1 Solids

Solids in the form of floating debris and grease and oil slicks show a highly

polluted waste stream and indicate untreated or ineffectively treated wastes

(Maheswari and Dubey, 2000). Solid in wastewater was formed according to the

relative size and condition of solid particles. The total solid material can be

classified into non-filterable and filterable solids factions. The non-filterable fraction

consists of settle able and non-settle able fraction and the filterable fraction consists

of total dissolved solids (TDS) and colloidal fraction. Each of these fractions

contains volatile (organic) and fixed (inert) fraction. Those are volatilized at high

temperature (600°C) are known as volatile solid whereas for those that are not are

known as fixed solids (Oke et al., 2006).

The total solids in a wastewater consist of the insoluble or suspended solids

and the water soluble compounds. They may be organic matter and inorganic matter.

Total dissolved solid (TDS) are due to soluble materials whereas suspended solid (SS)

are discrete particles. The suspended solids content is found by drying and weighing

the residue removed by filtering of the sample. Suspended solids (SS) concentration

is the measure of the amount of floating matter in the wastewater. When this residue

is ignited the volatile solids are burned off. Volatile solids are presumed to be

organic matter, although some organic matter will not burn and some inorganic salts

break down at high temperatures. Organic matters mainly are proteins,

carbohydrates and fats. Around 40% to 65% of the solids in an average wastewater

are in suspension. Settable solids are those can be removed by sedimentation.

Usually about 60% of the suspended solids in a municipal wastewater are settle able

(Rein, 2000). Figure 2.4 shows the classification of total solids.

9

Figure 2.3 Classification of Total Solids (EEAA, 2002)

2.2.1.2 Turbidity

The dark colour of effluents is due to usage of dyes and chemicals, which

increases the turbidity of water body. Turbidity is a major factor in determining the

control of the process among the many wastewater facilities. It is a measurement of

the light-transmitting properties of water which is used to determine the quality of

waste discharges and natural waters with respect to colloidal and residual suspended

matter (Tchobanoglous et al., 2003). It is a measure of the extent to which light is

either absorbed or scattered by suspended matter in water, but it is not a direct

quantitative measurement of suspended solids. Turbidity measurement is an

important factor related to the quality of public water supply. It should be measured

in treated wastewater effluent if it is reused (Mamta, 1999). The measurement of

turbidity is based on comparison of the intensity of light scattered by a sample to the

intensity of light scattered by a standard solution under the same conditions.

Formazine solutions are used as standards for calibration (Tchobanoglous et al.,

2003).

10

2.2.1.3 Colour

Colour is a qualitative characteristic that can be used to assess the general

condition of wastewater. Colour is measured by comparison with standards (Rein,

2000). Colour in textile wastewater water may due to the presence phenolic

compounds such as tannins, lignins (2–3%) and organic colourants (3–4%) and with

a maximum contribution from dye and dye intermediates, which could be sulphur,

mordant reactive, cationic, dispersed, azo, acid, or vat dye (Anjaneyulu et al., 2005).

Colour in the wastewater can be classified into two categories (true and apparent

colours). Apparent colours are the total colour due to both turbidity and the colour of

the wastewater. True colour is the colour after filtration of the wastewater (Oke,

Okiofu and Otun, 2006). Wastewater that is light brown in colour is less than 6 hour

old, while a light-to-medium grey colour is characteristic of wastewaters that have

undergone some degree of decomposition or that have been in the collection system

for some time. Lastly, if the colour is dark grey or black, the wastewater is typically

septic and has undergone extensive bacterial decomposition under anaerobic

conditions. The blackening of wastewater is often due to the formation of various

sulphides, particularly, ferrous sulphide. This results when hydrogen sulphide

produced under anaerobic conditions combines with divalent metal, such as iron,

which may be present (Rein, 2000). The common unit of measurement of colour is

the platinum in potassium chloroplatinate (K2PtCl6). One milligram per liter Pt in

K2PtCl6 is one unit of colour (Sincero, 2003).

2.2.1.4 Odour

Odours may be generated in textile manufacturing especially during dyeing

and other finishing processes due to the use of oils, solvent vapors, formaldehyde,

sulfur compounds, and ammonia (World Bank Group, 2007). The odour of fresh

wastewater is usually not offensive, but a variety of odours compound are released

when wastewater is decomposed biologically under anaerobic conditions. The

principal odorous compound is hydrogen sulphide (the smell of rotten eggs). Odour

11

is measured by successive dilutions of the sample with odour-free water until the

odour is no longer detectable (Rein, 2000).

2.2.1.5 Temperature

Wastewater temperature is an important parameter because most wastewater

treatment schemes that include biological processes are temperature dependent. It

affects chemical and biological reactions and the solubility of gases such as oxygen.

The temperature of wastewater is different from season to season and also with

geographic location. In cold regions the temperature will vary from about 7 to 18 °C,

while in warmer regions the temperature vary from 13 to 24 °C. The temperature of

wastewater is usually higher than the water supply because warm municipal water

has been added. Generally, higher temperatures increase reaction rates and solubility

up to the point where temperature becomes high enough to inhibit the activity of

most microorganisms (around 35 °C) (Drinan and Whiting, 2001).

2.2.2 Chemical Characteristics

The main chemical characteristics of wastewater are divided into two classes,

inorganic and organic. The principal chemical tests for inorganic chemicals include

free ammonia, organic nitrogen, nitrites, nitrates, organic phosphorus and inorganic

phosphorus. Nitrogen and phosphorus are important because these two nutrients are

responsible for the growth of aquatic plants. Other tests such as chloride, sulphate

and pH are performed to determine the suitability of reusing treated wastewater and

in controlling the various treatment processes. Trace elements which include some

heavy metals are not determined routinely, but trace elements may be a factor in the

biological treatment of wastewater. Biochemical oxygen demand (BOD), chemical

oxygen demand (COD) and total organic carbon (TOC) are common laboratory

methods used today to measure gross amounts of organic matter (greater than 1 mg/l)

in wastewater (Rein, 2000).

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2.2.2.1 Biochemical Oxygen Demand (BOD)

Biochemical oxygen is an overall measurement of the biodegradable organic

matter in a wastewater indirectly via microbial oxygen consumption. This parameter

reflects both the rate at which organic matter is assimilated by microorganisms and

the quantity of organic carbon matter available to the microorganisms (Brooks et al.,

2003). It is an important analytical tool in determining the effects of effluents on

water treatment plants and surface water system and also in evaluating the BOD-

removal efficiency of such treatment systems. The test measures the molecular

oxygen utilized during a specified incubation period for the biochemical degradation

of organic material (carbonaceous demand) and the oxygen used to oxidize inorganic

material such as sulfides and ferrous iron. It also may measure the amount of oxygen

used to oxidize reduced forms of nitrogen (nitrogenous demand) unless their

oxidation is prevented by an inhibitor (APHA, 1999). BOD values usually refer to

the standard 5 days value, which is the carbonaceous stage (Brooks et al., 2003).

Higher BOD increases the natural level of microorganism activity, which

lowers dissolved oxygen concentration (Brooks et al., 2003). An effluent with a high

BOD can be harmful to a stream if the oxygen consumption is great enough to

eventually cause anaerobic conditions drops the level of dissolved oxygen. The rate

of oxygen used is not a measure of some specific pollutant. Rather, it is a measure of

the amount of oxygen required by aerobic bacteria and other microorganisms while

stabilizing decomposable organic matter. If the microorganisms are brought into

contact with a food supply such as human waste, oxygen is used by the

microorganisms during the decomposition. A low rate of use would indicate either

absence of contamination or that the available microorganisms are unable to

assimilate the available organic. A third possibility is that the microorganisms are

dead or dying (Vesilind and Rooke, 2003).

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2.2.2.2 Chemical Oxygen Demand (COD)

Chemical Oxygen Demand (COD) is a laboratory measurement of the

amount of oxygen used in chemical reactions that occur in water as a result of the

addition of wastes. It is commonly used to indirectly measure the amount of organic

compounds in water. Most applications of COD determine the amount of organic

pollutants found in surface water such as lakes and rivers, making COD a useful

measure of water quality. COD is expressed in milligrams per liter (mg/L) which

indicates the mass of oxygen consumed per liter of solution. A major objective of

conventional wastewater treatment is to reduce the chemical and biochemical oxygen

demand (Jennings and Sneed, 1996). The basis for the COD test is that nearly all

organic compounds can be fully oxidized to carbon dioxide, water and ammonium

with a strong oxidizing agent under acidic conditions. Due to its unique chemical

properties, the dichromate ion (Cr2072-

) is the specified oxidant in the majority of

cases. Dichomate ion (Cr2072-

) is reduced to the chromic ion (Cr3+

) in these tests

(Tchobanoglous et al., 2003).

The COD test can be performed in a few hours. However, the results of the

COD tests are usually higher that the corresponding BOD test for several reasons.

Biochemical oxygen demand measure only the quantity of organic material capable

of being oxidized while the chemical oxygen demand represents a more complete

oxidation (Tay, 2006). Many organic compounds which are dichromate oxidizable

are not biochemically oxidizable and certain inorganic substances such as sulfides,

sulfites, thiosulfates, nitrites and ferrous iron are oxidized by dichromate, creating an

inorganic COD which is misleading when estimating the organic content of the

wastewater (Michael, 1999).

2.2.2.3 Total Organic Carbon (TOC)

Total organic carbon (TOC) is defined as the amount if carbon covalently

bonded in organic compounds in a water sample. The TOC is a more suitable and

direct expression of total organics than either BOD or COD, but it does not provide

14

the same type of information. If a reproducible empirical relationship is established

between TOC values and either COD or BOD, the TOC can be used to estimate the

respective BOD or COD values. To determine the content of organically bonded

carbon, the organic molecules must be broken down to single carbon units and

converted into a simple molecular form that can be quantitatively measured. In order

to determine TOC, inorganic carbon (IC) must be either removed from the sample

(direct TOC method) or measured (indirect TOC method). With direct method, TOC

value can be obtained by removing IC and measuring the TOC value directly,

whereas with the indirect method IC and total carbon (TC) are measured and TOC is

obtained by subtracting IC from TC. Inorganic carbon can be eliminated by

acidifying the samples to a pH value of 2 or less in order to convert all the fractions

included in this category to carbon dioxide which is more easily removed from the

water sample. For IC determination, the sample can be injected into a separate

reaction chamber packed with phosphoric acid-coated quartz beads, where all the IC

is converted to carbon dioxide, which is then measured. Under there conditions,

organic carbon is not oxidized and only IC is measured (Nollet, 2007).

2.2.2.4 Nitrogen

Nitrogen in wastewater is most commonly present as bound organic

nitrogen. It is readily leached to groundwater by its solubility, mobility and stability

mean. It has an active role in the eutrophication process. Nitrogen in various forms

can deplete dissolved oxygen in receiving waters, stimulate aquatic plant growth,

exhibit toxicity toward aquatic life, present a public health hazard, and affect the

suitability of wastewater for reuse purposes. Besides, nitrogen in drinking water

poses a threat to human and animal health. Nitrate is a primary contaminant in

drinking water and can cause a human heath condition called Methemoglobinemia

(blue babies). This is due to the conversion of nitrate to nitrite by nitrate reducing

bacteria in the gastrointestinal tract. Oxidation by nitrite of iron in hemoglobin

forms methemoglobin. Since methemoglobin is incapable of binding molecular

oxygen, the result is a bluish tinge to the skin and suffocation or death may occur if

left untreated. The maximum contaminant level for nitrate in drinking water is 10.0

mg/L. (Patterson, 2003).

15

Biological treatment is required to convert the organic nitrogen. First

nitrogen is converted to ammonia, next to nitrite and follow by nitrate. Ammonia is

produced under anaerobic conditions while the nitrate is the product of aerobic

digestion. If nitrate is produced, the nitrogen reduction has come to a dead-end.

Wastewater treatment plant operators are interested in nitrogen compounds because

of the importance of nitrogen in the life processes of all plants and animals (Michael,

1999).

Total nitrogen is comprised of organic nitrogen, ammonia, nitrite and nitrate.

The organic fraction consists of a complex mixture of compounds such as amino

acids, amino sugars and proteins. Ammonia, organic nitrate, and nitrite are the most

important nitrogen forms in wastewater treatment. The nitrogen in these compounds

is readily converted to ammonium through the microbial action in the aquatic or soil

environment. Organic nitrogen is determined analytically using Kjeldahl method.

The aqueous sample is first boiled to drive off the ammonia and follow by digestion.

During digestion the organic nitrogen is converted to ammonium through the action

of heat and acid. Total Kjeldahl Nitrogen (TKN) is determined in the same way as

organic nitrogen except that the ammonia is not driven off before the digestion step.

Total Kjeldahl Nitrogen is therefore the total of the organic and ammonia nitrogen

(Tchobanoglous et al., 2003).

2.2.2.5 Phosphate

Phosphorus occurs in wastewater solely as various forms of phosphate. The

types of phosphate present typically are categorized according to physical

characteristic into dissolved and particulate factions and chemically into

orthophosphate, condensed phosphate and organic phosphate factions( usually on the

basis of acid hydrolysis and digestion) (Richard, 1991). Phosphorus is essential to

the growth of organisms. Phosphorus, in addition to nitrogen, is a nutrient which can

result in eutrophication of receiving streams and lakes. The discharge of wastewater

containing phosphorus may stimulate nuisance algal growths.

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Chemical-physical removal of phosphorous from wastewater is possible only

when the phosphorous is in the orthophosphate form. Although the organic and

condensed phosphates can be easily converted into orthophosphate by treating them

with strong, hot oxidizing acid conditions, this is not practical on a multi-million

gallon per day scale. Fortunately, most biological treatment processes perform the

conversion of the organic and condensed phosphates to orthophosphate. Most of the

orthophosphate salts are not water soluble and phosphorous reduction is achieved by

forming an insoluble salt. The most common methods are to form the insoluble

calcium, aluminum or iron phosphates and let the salt particles get caught in a floc

and settle to produce sludge (Michael, 1999).

2.2.2.6 Sulphate

Sulphur containing compounds have unpleasant smells and are often highly

toxic to animals and human. High sulphur concentration in wastewater effluent leads

to the formation of high concentration of sulphide that upset the anaerobic biological

organisms in wastewater (Ebenezer, 2007). The most important sources of sulphur

for commercial use are elemental sulphur, hydrogen sulphite and metal sulphides

(Tay, 2006). Oxidation of sulphur by microorganisms produces sulphuric acid which

can result in a dramatic reduction of pH. The generation of acidity, which results

from the microbial oxidation of sulphide minerals, is of great environmental

significance.

Many metals occur as sulphides and sulphides are the major mineralogical

form of many commercially important metals, such as copper, lead and zinc

(Johnson, 1995). Iron sulphides (most notably pyrite) are the most abundant

sulphide minerals. Iron sulphides are often associated with other metal sulphides in

ore deposits. The inadvertently process of these minerals during the mining

operation, ending up as waste materials in mineral tailings and in liquid effluent

(Burgess, 2002). The reduction of sulphate to hydrogen sulphide (H2S) under

anaerobic condition produces unpleasant odour and sewer-corrosion. This indirectly

causes the problems in handling and treatment of waste water (Tay, 2006).

Generally, water with a desirable fish fauna contains less than 90mg/l sulfate; waters

17

with less than 0.5 mg/l will not support algal growth. Drinking-water standards are

250 mg/l for sulfate (Brooks et al., 2003).

2.2.2.7 pH

The pH of water is affected by chemical reactions in aquatic systems. It also

represents thresholds for certain aquatic organisms. When the pH of water is exceed

7, it is indicative of alkaline water which normally occurs when carbonate or

bicarbonate ions are present. A pH below 7 represents acidic water. In natural

waters, carbon dioxide reactions are affecting pH level.

When carbon dioxide (CO2) either from the atmosphere or by respiration of

plants, carbonic acid is formed which dissociates into bicarbonate. Carbonate and H+

ions are then released and influencing pH .

The pH is an indication of the balance of chemical equilibrium in water and

affects the availability of certain chemicals or nutrients in water for uptake by plants.

The pH of water also directly affects fish and other marine life. Generally, toxic

limits are pH values less than 4.8 and exceed 9.2. Most freshwater fish seem to

tolerate pH values from 6.5 to 8.4. Most algae cannot survive at pH less more than

8.5 (Brooks et al., 2003).

18

CHAPTER 3

MATERIALS AND METHODS

3.1 Sampling of Textile Wastewater

Textile wastewater was collected from a textile company located at Batu

Pahat, Johor. The samples were transported in a container.

3.2 Storage of Textile Wastewater

After collection of wastewater from the factory, it was kept at 4 °C in order to

retard any activity of indigenous bacteria.

3.3 Aseptic Techniques

All work was done under aseptic condition to avoid contamination by other

microorganisms. Apparatus and media (where necessary) were autoclaved at 121 °C,

101.3 kPa for 20 minutes. Heat labile materials were filter sterilized. Transfers of

cultures were carried out in a laminar flow cabinet to avoid contamination. The

19

working area in the laminar flow cabinet was always sterilized by using alcohol

before used.

3.4 Microorganism

The mixed culture of decolourizing bacteria, known as MicroClear used in

the experiment was obtained from the broth culture of acclimatized bacteria in textile

wastewater. The mixed bacterial culture is mainly consisting of Bacillus sp.,

Paenibacillus sp., Achromobacter sp. and some indigenous bacteria which have not

been identified.

3.5 Media Preparation

3.5.1 Textile Wastewater Medium

The selected mixed culture of decolourizing bacteria was acclimatized in

textile wastewater. Textile wastewater medium was prepared by dissolving yeast

extract (0.2 % w/v) into the wastewater. The mix solution was then autoclaved at

121 °C, 101.3 kPa for 20 minutes.

3.5.2 Nutrient Agar (NA)

Nutrient agar (NA) was prepared by adding nutrient agar powder (20.0 g) in

the distilled water (1 L). The medium was then autoclaved at 121°C, 101.3 kPa for

20 minutes. The medium was left to cool down until 55 °C before being poured into

petri dishes.

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3.6 Preparation of Bacterial Inoculum

Bacterial inoculum was prepared by inoculated the microorganism into

sterilized textile wastewater as the growth medium. The growth of bacteria was

enhanced by supplemented with different carbon and/or nitrogen source into the

wastewater medium. The carbon sources used included glucose, fructose, sucrose,

starch and sodium acetate whereas the nitrogen sources were yeast extract, nutrient

broth, ammonium chloride and ammonium sulphate. The range of concentrations

used was from 0.05 % to 0.3 % (w/v). The microorganisms that were inoculated in

the sterilized textile wastewater which was fully filled up the bottle was incubated

overnight at 37 °C without shaking in order to give a facultative anaerobic condition.

This was to allow maximum decolourization of textile wastewater before the culture

medium was transferred into a bigger flask for aerobic condition. The growth of

bacteria was monitored using spectrophotometer at 600nm. The bacteria culture was

ready to be used as the inoculum for the treatment of wastewater when the optical

density at 600nm reached 1.0 + 0.2.

3.7 Characterization of Bacteria

3.7.1 Isolation of Microorganisms

The acclimatized mixed culture of decolorizing bacteria was isolated by using

spread plate method. A serial dilution was done before inoculating the mixed culture

onto nutrient agar. Original inoculum was diluted in a series of dilution tubes. Five

dilution tubes were filled with 0.9mL of distilled water respectively. Then, 0.1mL of

sample was transferred into water blank followed by second transfer of 0.1mL of

sample from the first dilution into another dilution tube and so on until the dilution of

10-5

. A 0.01 mL of sample from each dilution was then spread evenly over the

surface of nutrient agar by using a sterilized glass spreader.

21

All plates were incubated at 37°C for 24 hours. The different colonies were

picked up with an sterile inoculating loop and transferred onto fresh nutrient agar.

All cultures were incubated at 37°C to obtain a pure culture.

3.7.2 Colony Morphology

Colony morphology was the initial step in identifying a bacterium. The

colony morphology of pure culture grown on the nutrient agar was examined for

their size, colour, shape, margin and elevation.

3.7.3 Cellular Morphology

a) Gram Staining

Smear of isolated pure culture on slides was prepared and subsequently heat-

fixed. Each smear was flooded with crystal violet for 1 minute. The crystal violet

was washed off with distilled water after 1 minute. This was followed by applying

iodine onto the smears. After 1 minute, the iodine was gently rinsed off with

distilled water. The smears were then decolourized by alcohol (95%) for about 15

seconds. The slides were counterstained with safranin for 20-30 seconds before

washing off. The slides were air dried at room temperature and ready for observation

under light microscope using oil-immersion technique. The colour and cell

morphology were observed. Gram negative cells will coloured pink-red while gram

positive cells appeared blue-purpled.

22

b) Spore Staining

Fresh bacterial culture was smeared onto slide and heat-fixed. The smears on

the slides were flooded with malachite green. A small piece of paper towel was

layered onto the slides and the slides were rest on top of a boiling water bath for 5

minute. The paper towel was removed when the slides were cooled. The stain was

later washed off with distilled water and the smears were counterstained with

safranin for 30-60 seconds before rinsed off with distilled water. The slides were

blotted dry with paper towel and observed under light microscope.

3.8 Biochemical Tests

Enzymatic activities of microorganism are widely used to differentiate and

characterize bacteria. Closely related bacteria can be separated into distinct species

by using biochemical tests. Specific enzyme secreted can reflect the taxonomy status

of the microorganism. The basis of differentiating one microorganism from the other

depends on the presence or absence of the enzyme. The standard methods for

biochemical tests are shown in Appendix 1 (Faddin, 1980).

3.9 Textile Wastewater Treatment

After the bacterial inoculum had been prepared, the textile wastewater was

ready to be treated by using sequential facultative anaerobic-aerobic batch system.

The textile wastewater was autoclaved at 121°C, 101.3 kPa for 20 minute in order to

kill the indigenous bacteria. Filter sterilized method was not used to sterilized the

wastewater because this may remove the dyes in the textile wastewater and affects

the results of the characterization of wastewater.

23

3.9.1 Sequential Facultative Anaerobic-aerobic Batch Treatment

The mixed bacterial culture (10 % v/v) was aseptically transferred into

sterilized raw textile wastewater medium supplemented with yeast extract (0.2 %

w/v). The culture medium was incubated at 37 °C ina facultative anaerobic

condition for optimum colour removal. After decolourization, it was transferred into

a conical flask in order to give an aerobic condition and incubated in an orbital

shaker at 37 °C. Samples were withdrawn at regular time intervals for analysis over

a 40 hours incubation period.

3.10 Laboratory Analysis

3.11 Determination of Chemical Oxygen Demand (COD)

The COD test was used to measure the organic matters in the wastewater. It

oxidized the reduced compounds in wastewater through a reaction with a mixture of

chromic and sulfuric acid. Thus, dichromate solution and silver sulphate solution

were prepared prior to COD determination. Dichromate solution was prepared by

dissolving 10.26 g of potassium dichromate (K2Cr2O7) in 500 mL of distilled water

and 167 mL of concentrated sulfuric acid (97 % H2SO4). A total of 33 g of mercury

sulfate (HgSO4) was then added into the mixture. The solution was cooled to 30 °C

and top up the solution with distilled water to a total volume of 1000 mL. Silver

sulphate solution was prepared by dissolving 5.05 g of silver sulphate (Ag2SO4) in

500 mL concentrated sulphuric acid (97 % H2SO4).

For COD determination, 2.5 mL of supernatant sample was transferred into

HACH test tube followed by 1.5 mL of dichromate solution and 3.5 mL of silver

sulphate solution. The tube was then shaken vigorously. Blank consisted distilled

water instead of wastewater sample was also prepared. The samples were then

heated at 150 °C for 2 hours by using a heater. The samples and blank were then let

24

it cooled down to room temperature before being analyzed by HACH DR 4000

Spectrophotometer using program number 2720 (HACH, 1997).

3.12 Determination of Colour Intensity (ADMI)

Wastewater supernatant sample (10 mL) was added into HACH test tube and

the colour intensity was measured by using the 1660 program of HACH DR 4000

spectrophotometer. The concentration of colour was compared to the blank (distilled

water).

3.13 Determination of Bacteria Growth

Growth of bacteria culture was determined in term of turbidity readings by

using spectrophotometer methods with optical density at 600 nm.

3.14 Determination of Nitrate (NO3-), Phosphate (PO4

3-) and Sulphate

(SO42-

) Content

Concentration of Nitrate in the sample was determined by using the 2530

program of HACH DR 4000 spectrophotometer. Wastewater (10 mL) was added

with Nitra Ver 5 Nitrate Reagent Powder Pillow and shake for 1 minute until the

mixture become homogenous. After that, the sample was allowed to stand for 5

minutes and the mixture was transferred into HACH sample cell. The concentration

of nitrate in the sample was compared to blank (distilled water) by using HACH DR

4000 Spectrophotometer. The amber colour resulted in the sample indicated nitrate

was present in wastewater (HACH, 1997). The same procedure was repeated for

25

phosphate and sulphate content determination. The program used for the

determination of phosphate was 3015 program of HACH DR 4000

Spectrophotometer and 3450 for sulphate. The reagents used were Phos Ver 3

Phosphate and Sulfa Ver 4 Reagent Powder Pillow respectively. The intense blue

colour indicated high concentration of phosphate in the wastewater while turbidity

resulted in the sample indicated the presence of sulphate in the wastewater (HACH,

1997).

3.15 Determination of Total Suspended Solid (TSS)

Total suspended solid was determined by filtered the well mixed wastewater

through weighing the nylon filter paper (0.45 µm). The residues retained on the filter

paper were dried to a constant weight at 103 °C to 105 °C for 24 hours. The increase

in weight of the filter paper represents the total suspended solids in the wastewater

sample (AHPA, 1989).

TSS = Weight of nylon paper - weight of filter paper (Equation 3.1)

after filter (mg) before filter (mg)

Volume of sample (L)

3.16 Determination of pH

Wastewater sample was added to a flask (50 mL) and the pH was measured

by using a pH meter. The pH electrode was rinsed with distilled water and calibrated

using standard solution before used (AHPA, 1989).

26

3.17 Determination of Biomass

Cellulose acetate membrane (0.2 µm) was used. The filter membrane was

dried in an oven for 24 hours at 60°C before used. Then, the pellet was aliquot with

3mL of distilled water and then filtered using filter housing. Next, the filter paper

containing biomass was dried in oven until constant weight was achieved. Biomass

was determined by using the equation below.

Biomass (mg/L) = ( B – A ) __ (Equation 3.2)

Volume of sample (L)

A = weight of filter membrane

B = weight of filter membrane with biomass

27

CHAPTER 4

RESULTS AND DISCUSSION

4.1 Textile Wastewater Characterization

Textile wastewater was collected from a textile company located at Batu

Pahat, Johor. Laboratory analysis on the sample was done within 24 hours upon

storage at 4°C. Water quality parameters measured included colour, COD, pH, and

TSS. In addition, the nitrate, phosphate and sulphate were also analyzed since they

are the indicator of treatability of wastewater by biological process. The element of

nitrogen and phosphorus are essential nutrients for the growth of microorganism, and

algae. The noxious algal blooms that occur on the surface waters is now much

concerned in controlling the amount of phosphorus compounds in wastewater before

discharged to the environment. Besides, insufficient nitrogen can also necessitate the

addition of nitrogen to make wastewater treatable. Furthermore, sulfate which is

reduced biologically under anaerobic condition to sulfide will combine with

hydrogen and form hydrogen sulfide (H2S). The accumulated H2S can be then

oxidized biologically to sulfuric acid which is corrosive to concrete sewer pipe.

Hence, the concentration of sulphate should be concerned in wastewater treatment.

The results of the characterization of wastewater were shown in the table below.

28

Table 4.1: Laboratory analysis of textile wastewater

Parameters 1st Sampling 2

nd Second Sampling

Colour (ADMI) 1090 1070

pH 8.69 9.03

COD (mg/L) 843 855

TSS (mg/L) 2100 1400

Nitrate (mg/L) 28 57

Phosphate (mg/L) 256 264

Sulphate (mg/L) 327 333

Note: The lapse of time between 1st sampling and 2

nd sampling was 4 months.

The colour of textile wastewater ranged from 1070 to 1090 ADMI. Colour is

due to the usage of certain dyes during the dyeing process in the textile industry.

Large amount of dyes textile sector are continuously released into wastewater stream

due to their poor absorbability to the fiber. The coloured industrial effluents cause

aesthetic and environmental problems by absorbing light and interfering with aquatic

biological activity. Colored pollutants also have been found toxic and carcinogenic

to human (Manu et al., 2002).

The hydrogen ion concentration is an important quality parameter of

wastewater. Biological activities and some chemical treatment process are usually

restricted by pH. Department of Environment (DOE) recommends pH value of range

5.5 to 9.0 for effluent to be discharge into stream. The pH values obtained from the

laboratory analysis showed that the textile wastewater was in the high alkaline range

and is not allowed to be discharged into stream based on DOE limit because it is

harmful to man and aquatic life if it is discharged untreated. Alkalinity in

wastewater may results from the presence of the hydroxides, carbonates, and

bicarbonates of elements such as calcium, magnesium, sodium, potassium or

ammonia. Besides, Borates, silicates and phosphate can also contribute to the

alkalinity (Brooks et al., 2003).

29

Oxygen demand is important because organic compounds are generally

unstable and maybe oxidized biologically and chemically to a stable relatively inert

end product. Chemical Oxygen Demand (COD) is a measure of pollutant loading in

terms of complete chemical oxidation using strong oxidizing agents, potassium

dichromate and concentrated sulphuric acid. The COD concentration of the

wastewater was in the range of 843 mg/L to 855 mg/L. High concentration of COD

observed in the wastewater might be due to the usage of organic or inorganic

chemicals which are oxygen demand in nature or variation in the process or method

of production (Oke et al., 2006).

The oxidized nitrogen compounds are usually present in low amount in

typical wastewater. The nitrate content in the wastewater was between 28 mg/L to

57 mg/L and phosphate was ranged from 256 mg/L to 264 mg/L. The nutrients in

the textile effluent were due to the dyebath additives containing nitrogen and

phosphorus such as urea, ammonium acetate, ammonium sulphate and phosphate

buffer. The concentration of sulphate was in between 327 mg/L and 333 mg/L. The

usage of sulphur or vat dye sodium sulphide and sodium hydrosulphide as reducing

agents in dyeing process resulted high sulphate level in textile effluent. Other

sources of sulphur can be the use of sulphuric acid for pH control. Excessive

nutrients (phosphorus and nitrogen) in wastewater causes problems like

eutrophication whereby algae grow excessively and lead to depletion of oxygen,

death of aquatic life and bad odours (Delee et al., 1998)

High TSS in textile wastewater is common. This is due to the removal of dirt,

waxes, vegetable matter and others. Soap, detergent, alkali, solvent and pesticides

may also be present. The result obtained from the laboratory analysis showed that

the TSS was very high in the effluent from textile industry. It was in between 1400

mg/L and 2100 mg/L. The value of TSS in the wastewater sample was exceeding the

standard limit allowed for industrial discharged (Appendix 1 DOE standard B).

Suspended solids are one of the important contaminants of concern in wastewater

treatment. It can lead to the development of sludge deposits and anaerobic condition

when untreated wastewater is discharged into the aquatic environment

(Cheremisinoff, 1995).

30

4.2 Effect of Carbon and Nitrogen Sources Addition on the Decolourization

of Textile Wastewater and Bacterial Growth

The performance of acclimatized mixed culture decolourizing bacteria,

MicroClear (10 % v/v) in decolorizing textile wastewater in the presence of an

additional carbon (glucose, fructose, sucrose, starch, sodium acetate) and nitrogen

sources (yeast extract, nutrient broth, ammonium chloride and ammonium sulphate)

(0.1 % w/v) were examined to obtain efficient and faster decolourization and bacteria

growth. Efficient decolourization and bacterial growth achieved within the shortest

period was when the yeast extract added to the culture medium. In contrast, less

decolourization and poor bacterial growth was obtained when other supplements of

carbon and nitrogen sources were added within 24 hours of incubation.

Addition of carbon sources seemed to be less effective in color removal. This

is probably due to the preferential assimilation of the added carbon sources over the

dye compound as the carbon source. On the other hand, organic nitrogen added as a

co-substrate can regenerate NADH which acts as an electron donor to reduce azo dye

by microorganism (Saratele et al., 2008).

4.3 Optimization of Bacterial Growth and Colour Removal with addition of

Yeast Extract

When using different carbon and nitrogen sources, addition of yeast extract

showed the best decolourization of textile wastewater and subsequently the growth of

the mixed culture of decolourizing bacteria. Different concentrations of yeast

subtract (0.05 % to 0.3 % w/v) was supplemented in the culture medium to obtain

optimum colour removal and bacterial growth. The results obtained showed that

yeast extract (0.2 % w/v) was efficient in enhancing growth with highest growth rate

and colour removal. It was also found that the decolourization efficiency increased

with increasing yeast extract concentration (from 0.05 % w/v to 0.25 % w/v) but only

31

slightly in the range of 0.25 % (w/v) to 0.30 % (w/v). Table 4.2 below illustrated the

period of time for colour removal and Figure 4.1 showed indirect bacterial growth

using spectrophotometer methods.

Table 4.2: Effects of different concentrations of yeast extract on colour removal.

Yeast Extract ( % w/v) Time(h) for Decolourization

0.05 48

0.01 24

0.15 24

0.20 12

0.25 12

0.30 24

Figure 4.1 Growth of bacteria with addition of different concentrations of yeast

extract into the textile wastewater medium.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 1 2 3

Time (day)

OD

60

0n

m

0.05%(w/v)

0.10%(w/v)

0.15%(w/v)

0.20%(w/v)

0.25%(w/v)

0.30% (w/v)

32

The growth of bacteria was good in the textile wastewater medium

supplemented with yeast extract (0.02 % w/v). This result may be implicated with

the ability of the bacteria to convert or transform partially degraded dye products

using specific enzymes into metabolic intermediates which can enter their central

metabolic pathway and can further be used to obtained energy for cellular activities

and growth of the bacteria (Idris et al., 2007).

4.4 Isolation and Characterization of Bacteria from Acclimatized Mixed

Culture in Textile Wastewater

In this study, 5 pure cultures of bacteria were successfully isolated from the

acclimatized mixed culture in textile wastewater by using streak plate method. The

bacteria were partially identified based on colony and cellular morphologies (Table

4.3) and also a series of biochemical tests (Appendix 4). The isolated strains were

partially identified as Streptococcus sp., Bacillus sp and Escherichia sp. (Table 4.4).

Table 4.3: Colony morphology of isolated bacteria.

Colony Shape Colour Margin Elevation

A Filiform White Cream Thread-like Hilly

B Round White Cream Smooth Convex

C Round Yellow Orange Smooth Convex

D Round Light yellow Orange Smooth Convex

E Round Light yellow Orange Smooth Convex

33

Table 4.4: Results of bacteria identification.

Bacteria Label Bacteria

A Staphylococcus sp.

B Staphylococcus sp.,

C Bacillus sp.

D Escherichia sp.

E Staphylococcus sp.

4.5 Water Quality Analysis

The textile wastewater samples were collected and analyzed at the interval of

3 hours for 40 hours of incubation period. The consecutive sampling was designed

to evaluate the variation in COD and colour values of the textile wastewater by the

treatment of consortium. The ability of the consortium to reduce the other main

wastewater parameters such as total suspended solids, pH and biomass were also

being investigated.

4.5.1 Analysis of Decolourization of Textile Wastewater in Sequential

Facultative Anaerobic and Aerobic Condition

The effectiveness of microbial decolourization was affected by the

adaptability and activity of selected microorganisms. Time course of effluent

decolourization was studied along with the growth of consortium. Figure 4.2 showed

the decolourization of textile wastewater during facultative anaerobic and aerobic

stage along with the growth of bacteria. Significant of colour removal up to 51.40 %

from the 1070 ADMI value was occured after 12 hours of incubation time at 37 °C in

facultative anaerobic condition even the bacteria showed little growth. However, no

34

significance changes were detected in the following aerobic stage where the colour

removal was only increased to 3.74 %. In general, the selected mixed culture of

decolourizing bacteria had significantly decolourized textile wastewater in

facultative anaerobic condition.

Figure 4.2 Decolourization in sequential facultative anaerobic and aerobic

condition along with bacterial growth.

The presence of co-substrate such as yeast extract may act as electron donors

that facilitate reduction of azo dye. Under anaerobic condition, for example, the

selected consortium can reduce azo compounds to form the corresponding amines

using azoreductase which ultimately cause decolourization. The presence of oxygen

normally inhibits the azo bond reduction activity since aerobic respiration may

dominate the use of NADH (electron donor) and thus hinder electron transfer from

NADH to the azo bonds. Reported of no further degradation under anaerobic

condition, however the aromatic amines can further degraded under the aerobic

condition (Pazdzior et al., 2008). This may be implied the decolourization was

mainly occurred during facultative anaerobic condition.

However, there was an increase in colour during aerobic stage after 24 hours

of incubation. This was probably due to the aeration of a reduced dye solution

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 3 6 9 12 14 16 18 20 22 24 30 36 40

Time (h)

OD

600n

m

0%

10%

20%

30%

40%

50%

60%

Co

lou

r R

em

ov

al (%

)

OD600

Colour

Removal

OD600nm

Facultative

Anaerobic

Aerobic

35

causing the colour of solutions to darken. This is probably due to aromatic amines

produced from the reduction of azo dyes which are unstable in the presence of

oxygen. This may cause the oxidation of the hydroxyl groups and of the amino

groups to quinines and quinine imines. These compounds can undergo dimerisation

or polymerization leads to the formation of new, darkly coloured chromophores

which are the unwanted byproducts. Besides, textile wastewater which complex in

nature containing dye and various auxiliaries, salt and sulfates might have an

inhibitory effect on the anaerobic decolourization (Pearce et al., 2003).

4.5.2 Analysis of COD Removal

Figure 4.3 showed the removal of COD during facultative anaerobic and

aerobic condition during growth of bacteria.

Figure 4.3 Removal of COD under facultative anaerobic and aerobic condition.

The COD removal showed similar trend as the growth profile. COD

concentration was decreased from initial value of 855 mg/L to 803 mg/L (or 6.08 %

Facultative

Anaerobic

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 3 6 9 12 14 16 18 20 22 24 30 36 40

Time (h)

OD

600n

m

0%

5%

10%

15%

20%

25%

30%

35%

40%

CO

D R

em

ov

al (%

)

OD600

COD Removal

OD600nm

Facultative

Anaerobic

Aerobic

36

COD removal) after 6 hours incubation time under facultative anaerobic condition,

which was a phase of low COD degradation (lag phase) followed by a exponential

stage of COD degradation after 20 hours incubation in aerobic condition. The COD

concentration was further reduced to 559 mg/L or 34.61 % COD reduction during

aerobic stage within 40 hours of incubation time. The results showed that COD

concentration was significantly being removed during late exponential and early

stationary phase of bacterial growth in 20 hours.

Observed COD reduction of 34.61 % indicated a partial mineralization of

dyes mixture in the textile wastewater. Aerobic conditions are required for the

complete mineralization of the reactive azo dye molecule as the simple aromatic

compounds produced by the initial reduction are degraded via hydroxylation and

ring-opening in the presence of oxygen. The bacterial population in mixed culture

showed degrading ability for the pollutants in textile effluent by utilizing them as

their nutrient. Each strain in mixed culture played an important role in

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

2006).

However, there was an increase in COD effluent was observed for 3 hours

during facultative anaerobic condition. This was due to the soluble microbial

product which in turn contributed to the COD value in the effluent. Nevertheless, the

presence of inorganic compounds may also cause the variation of COD measurement

(Ghasimi et al., 2008).

4.5.3 Analysis of pH

The pH of wastewater was found to increase through out the treatment

process. The pH of the medium was shifted to the alkaline range from 9.03 to 9.96

which were above the level allowed by legistration (Appendix 1). The mostly likely

explanation for the increase in pH may due to the formation of ammonia from

37

aromatic amine during biodegradation under aerobic condition (Sandhya et al., 2005).

The formations of hydrogen carbonate (HCO3-

) due to the reaction of hydroxide

(OH-) with CO2 produced during anaerobic degradation also cause the alkalinity of

the effluent (Movahedya et al., 2007). Figure 4.4 showed the changes in pH values

throughout the treatment process.

Figure 4.4 pH of textile wastewater throughout the treatment process.

4.5.4 Analysis of Nitrate

Removal of nitrate up to 59.65 % was achieved after 12 hours of incubation

time under facultative anaerobic condition from the initial value of 57 mg/L.

However, the concentration of nitrate was increased when continued to aerobic stage.

Figure 4.5 illustrated the changes of nitrate concentration during the treatment

process under facultative anaerobic and aerobic condition.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 3 6 9 12 14 16 18 20 22 24 30 36 40

Time (h)

OD

60

0n

m

7.5

8

8.5

9

9.5

10

pH

OD600

pH

OD600nm

Facultative

Anaerobic

Aerobic

38

Figure 4.5 Concentration of nitrate during the treatment process.

Nitrate removal is commonly performed by denitrification. Nitrate is usually

converted to nitrogen (and nitrogen dioxide as a byproduct) via anaerobic respiration

in which nitrate serves as an alternate electron acceptor for the oxidation of organic

compounds. The results showed nitrate removal decreased during the aerobic phase.

This was due to the presence of oxygen which competes with nitrate as an electron

acceptor in the energy metabolism of cells. It is generally accepted that an anaerobic

condition is required for microbial denitrification to take place. Therefore, this had

explained that nitrate removal only happened successfully during facultative

anaerobic stage (Sabina, 2002).

Another probable reason for the increased concentration of nitrate might also

due to cellular lysis of microorganisms under nutrient depleting condition which

resulted in the release of large amount of protein in the effluent. The loss of biomass

has triggered the reduction in nitrification performance (Yogalakshmi et al., 2006).

Facultative

Anaerobic

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 6 12 18 24 30 36 40

Time (h)

OD

60

0n

m

0

10

20

30

40

50

60

70

80

90

Nit

rate

(m

g/L

)

OD600 (nm)

Nitrate (mg/L)

OD600nm

Facultative

Anaerobic

Aerobic Aerobic

39

4.5.5 Analysis of Phosphate

Results showed maximum reduction of phosphate was only 28.41 % after 24

hours incubation from initial concentration at 264 mg/L to 189.2 mg/L under aerobic

condition (Figure 4.6).

Figure 4.6 Concentration of phosphate during the treatment process.

Phosphorus is normally found in wastewater as phosphate (orthophosphate,

condensed phosphate, organic phosphate fractions), and it can be eliminated either by

precipitation and/or adsorption or by luxury uptake that is phosphate accumulation

by bacteria in excess of immediate need. Luxury uptake typically occurs during the

limitation of nutrient other than phosphate and of a source of carbon and energy.

However, luxury uptake is a highly unlikely event. Only a small amount of

phosphorus is used for cell metabolism and growth which is 1 to 2% of the total

suspended solids mass in the mixed liquor.

Most phosphates are removed during the aerobic period when the

accumulated nitrate is completely denitrified under the anoxic condition. Under

anaerobic condition, phosphate accumulating bacteria requires an electron acceptor

for metabolic activity. Therefore, electron acceptor is obtained by hydrolysis of

polyphosphate in the cells and subsequently released the phosphate from cells to the

Facultative

Anaerobic

Aerobic

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 6 12 18 24 30 36 40

Time (h)

OD

60

0n

m

0

50

100

150

200

250

300

Ph

os

ph

ate

(m

g/L

)

OD600

Phospate

OD600nmFacultative

Anaerobic Aerobic

40

medium (Choi and Yoo, 2000). In the biological phosphate removal, phosphate

release is prerequisite for the phosphate uptake which is store as polyphosphate

granules in the microbial cells (Fuhs et al., 1975).

Phosphate removal was not achieved to higher level. This was probably

inhibited by nitrate. In the anaerobic stage, nitrate reduces phosphate release and in

the aerobic stage it diminishes its uptake. Denitrification has more capability than

phosphorus release with respect to the competition of substrate. This is because

nitrate will be utilized as a final electron acceptor in the growth of on-polyphosphate

heterotrophs. Therefore, the amount of substrate available for polyphosphate

organisms is reduced and hence the removal of phosphorus is lowered (Radjenovic et

al., 2007). Besides, the autolysis of microorganism during death phase also

contributed to the increased concentration of phosphate (Yogalakshmi et al., 2006).

4.5.6 Analysis of Sulphate

The concentrations of sulphate fluctuated throughout the incubation period

and no significant sulphate removal being achieved. The removal of sulphate was

only 19.22 % after 6 hours of incubation under facultative anaerobic condition.

Figure 4.7 below showed the concentrations of sulphate during the treatment process.

Facultative

Anaerbic

41

Figure 4.7 Concentration of sulphate throughout the treatment process

Microbial removal of sulphate primarily involves reduction of sulphate to

sulphides. The sulphide produced is then biologically oxidized to elemental sulphur.

Microorganisms are utilizing hydrogen and organic substances as electron donors

and sulfates as acceptors. Sulphate reduction was limited during the treatment

process. This was due to denitrification yields more energy in the process of

anaerobic respiration, denitrifiers have competitive advantage and thus sulphate

reduction should be limited until nitrate has been depleted (Whitmire and Hamilton,

2005).

.

4.5.7 Analysis of MLVSS and MLSS

When the concentration of microorganisms is relatively high, the mixture of

suspended microbes, wastewater treated and other substances, both dissolved and

suspended is referred to as mix liquor suspended solids (MLSS). The term “mix

liquor volatile suspended solids” (MLVSS) is used to design that portion of the

MLSS that is active microbes (Woodard, 2001). This study revealed the

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 6 12 18 24 30 36 40

Time (h)

OD

600n

m

0

100

200

300

400

500

600

700

800

Su

lph

ate

(m

g/L

)

OD600

Sulphate

OD600nm

Facultative

Anaerobic

Aerobic

42

concentration of mix liquor volatile suspended solids (MLVSS) and mix liquor

suspended solids (MLSS) during the treatment process (Figure 4.8).

0

200

400

600

800

1000

1200

1400

0 3 6 9 12 14 16 18 20 22 24 30 36 40

Time (h)

ML

VS

S (

mg

/L)

0

500

1000

1500

2000

2500

ML

SS

(m

g/L

)

MLVSS (mg/L)

MLSS (mg/L)

Figure 4.8 MLVSS and MLSS versus time of facultative anaerobic and aerobic

treatment.

Figure 4.8 showed what happened in a batch system in which at the initial

stage, substrate and nutrients were present in excess and only a small amount

biomass was present in the bioreactor. As substrate was being taken, four distinct

growth phases should be established. Results obtained showed that MLSS was

drastically being removed under aerobic condition after 12 hours treatment using

mixed culture. The percentage of removal was 33.33 % from the initial high

concentration of TSS at 2100 mg/L. The final concentration of TSS was reduced to

1400 mg/L.

During facultative anaerobic stage, the major part of the organic load (co-

substrate) was consumed anaerobically to reduce the azo dye in the textile

wastewater. Complete biodegradation of organic compounds was not achieved

during the anaerobic stage due to a lack mineralization of the aromatic amines and

hence bacteria were not growing well under anaerobic condition (Tan, 2001). The

Facultative

Anaerobic

Aerobic

43

concentration of MLVSS and MLSS were decreased 18.18 % and 28.57 % from their

initial value of 770 mg/L and 2100 mg/L respectively.

During aerobic phase, bacteria cells were multiplying as resulted aromatic

amines during anaerobic stage were consequently served as main substrate for the

microorganisms to grow. In this stage, both MLVSS and MLSS started to rise up

exponentially and reached to their maximum level of 1270 mg/L and 2150 mg/L

respectively.

After 30 hours of incubation, stationary phase was achieved where the

biomass concentration remains relatively constant with time. The growth of bacteria

remained stable or retarded was due to the death of cells. The MLVSS was

decreased to 1230 mg/L. In the death phase, the substrate had been depleted and

therefore no growth was being observed. The concentration of MLVSS and MLSS

had further decreased to 1800 mg/L and 1220 mg/L respectively after 40 hours. Both

biomass and concentration of TSS will continued to reduce if the experiment is

prolonged.

Reduction of MLSS was due to the decomposition of organic constituents by

the bacteria. Besides, the increase of MLSS observed may due to slow growth and

death of bacteria and also the non-biodegradable part of substrate (Ghasimi et al.,

2008).

44

CHAPTER 5

CONCLUSION

5.1 Conclusion

In conclusion, the acclimatized mixed culture of decolourizing bacteria had

successfully been isolated and characterized. The bacteria were partially identified

as Staphylococcus sp, Bacillus sp. and Escherichia sp. The ability of these strains to

decolourize the textile wastewater indicated that these bacteria were able to utilize

the dyes in the textile wastewater as their carbon and energy source.

The results obtained had showed the selected mixed culture of decolourizing

bacteria had the ability to treat the textile wastewater. Essential co-substrate (yeast

extract 0.2% w/v) was needed to obtain good colour removal and bacterial growth.

The efficiency of the mixed culture in wastewater treatment can be determined from

the reduction of measured water quality parameters such as colour, COD, pH, and

TSS, nitrate, phosphate and sulphate. However, most of the water quality parameters

did not fulfill the discharge limit allowed by the Department of Environment (DOE)

standard B. Therefore, improvement of the sequential facultative anaerobic-aerobic

batch system is required to further improve the water quality.

45

5.2 Future Work

Further studies on sequential aerobic-anaerobic continuous systems instead of

batch system can be carried out to improve the wastewater treatment. The factors

affecting the colour and COD removal can be investigated in order to increase the

efficiency of mixed culture to treat the wastewater. Besides, strict anaerobic

condition is suggested for the treatment system instead of facultative anaerobic.

Ecotoxicity test can also be done on the textile effluent and finally the products of

degradation can be analyzed by using High Performance Liquid Chromatography

(HPLC).

46

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52

APPENDIX 1

Environmental Quality (Sewage and Industrial Effluents) Regulations, 1979

Maximum Effluent Parameter Limits Standard A and B

Parameters (Units) Standard

A (1) B (2)

1 Temperature oC 40 40

2 pH - 6.0 - 9.0 5.5 - 9.0

3 BOD5 @ 20oC mg/l 20 50

4 COD mg/l 50 100

5 Suspended Solids mg/l 50 100

6 Mercury mg/l 0.005 0.05

7 Cadmium mg/l 0.01 0.02

8 Chromium, Hexalent mg/l 0.05 0.05

9 Arsenic mg/l 0.05 0.10

10 Cyanide mg/l 0.05 0.10

11 Lead mg/l 0.10 0.5

12 Chromium, Trivalent mg/l 0.20 1.0

13 Copper mg/l 0.20 1.0

14 Manganese mg/l 0.20 1.0

15 Nickel mg/l 0.20 1.0

16 Tin mg/l 0.20 1.0

17 Zinc mg/l 1.0 1.0

18 Boron mg/l 1.0 4.0

19 Iron (Fe) mg/l 1.0 5.0

20 Phenol mg/l 0.001 1.0

21 Free Chlorine mg/l 1.0 2.0

22 Sulphide mg/l 0.50 0.50

23 Oil and Grease mg/l Not detectable 10.0

53

1. Standard A for discharge upstream of drinking water take-off.

2. Standard B for inland waters.

APPENDIX 2

Treatment Results of the Study

Optical Density at 600nm

Time(h) OD600 (nm)

0 0.202

3 0.315

6 0.364

9 0.366

12 0.437

14 0.746

16 0.960

18 1.130

20 1.429

22 1.525

24 1.561

30 1.332

36 1.282

40 1.204

54

Decolourization

Time(h) ADMI % of Removal

0 1070 0%

3 1000 6.54%

6 550 48.60%

9 560 47.66%

12 520 51.40%

14 560 47.66%

16 490 54.21%

18 490 54.21%

20 490 54.21%

22 480 55.14%

24 630 41.12%

30 530 50.46%

36 560 47.66%

40 580 45.79%

pH

Time(h) pH

0 9.03

3 8.45

6 8.22

9 8.41

12 8.45

14 8.31

16 8.43

18 8.51

20 8.54

22 8.63

24 8.78

30 9.08

36 9.42

40 9.65

55

Chemical Oxygen Demand (COD) Removal

Time(hr) COD (mg/L) COD Removal (%)

0 855 0%

3 840 1.75%

6 803 6.08%

9 841 1.63%

12 832 2.69%

14 822 3.86%

16 819 4.21%

18 815 4.68%

20 798 6.67%

22 644 24.68%

24 615 28.07%

30 576 32.63%

36 563 34.15%

40 559 34.61%

Nitrate, Phosphate and Sulphate Concentration

Time(h) Nitrate (mg/L) Phospate (mg/L) Sulphate(mg/L)

0 57 264 333

6 35 248 269

12 23 264.6 329

18 23 228.1 682

24 27 189.2 528

30 35 203.8 715

36 84 263.2 465

40 28 274.4 691

56

Biomass

Time(h) W0 (mg) W1 (mg) Dry Cell Weight (mg/L)

0 404.5 406.8 766.67

3 430.0 431.9 633.33

6 452.4 454.4 666.67

9 466.2 468.8 866.67

12 434.6 437.2 866.67

14 461.4 464.5 1033.33

16 444.0 447.0 1000.00

18 447.4 450.3 966.67

20 443.8 446.9 1033.30

22 479.3 483.1 1266.67

24 425.4 429.1 1233.33

30 431.9 435.5 1200.00

36 443.3 447.1 1266.67

40 465.1 468.7 1220.00

Total Suspended Solid (TSS)

Time(h) W0 (mg) W1 (mg) TSS (mg/L)

0 442.0 446.2 2100

3 455.1 458.1 1500

6 450.9 453.9 1500

9 452.8 455.9 1550

12 445.9 449.5 1800

14 440.1 442.9 1400

16 482.8 486.3 1750

18 466.3 469.3 1500

20 506.0 509.6 1800

22 460.2 463.5 1650

24 489.8 494.1 2150

30 461 464.9 1950

36 443.0 447.2 2100

40 475.2 478.8 1800

57

APPENDIX 3

Standard Methods for Biochemical Tests

a) Catalase Test

The colony of bacteria was picked up aseptically using inoculating loop and

placed on a slide. A drop of hydrogen peroxide was added onto the colony

adherering the slide. The formation of gaseous bubbles was observed. Bubbling

indicated presence of catalase.

b) Oxidase Test

A few drops of oxidation solution (1% tetramethyl-p-phenylene-

diaminehydrochloride) were added onto a piece of filter paper. The colony of

bacteria was picked aseptically and gently rubbed onto the filter paper wetted with

oxidation solution. The formation of dark purple colour shows positive result

whereas a negative result does not display any colour changes.

c) Motility Test

A pure culture was stabbed into a motility test medium with a sterile

inoculating loop to a dept of halp inch. The medium was then incubated at 37°C for

24 to 48 hours. Motile organisms will migrate from the stabbed line diffuse into the

58

medium causing turbidity. Non motile organism will grow along the stab line only

while the surrounding medium remains clear.

d) Urease Test

Fresh culture was inoculated onto the surface of urea slant agar and incubated

at 37°C for 24 hours. The colour changes of medium to pinkish colour indicating

positive result. No colour change for negative result.

e) Gelatin Liquefaction Test

Fresh and heavy culture was stabbed into the Nutrient Gelatin Stab Medium

to a dept of half to one inch. The universal bottle with the medium was then

incubated at 37°C for 24 hours up to 14 days. After 14 days, the bottles were kept in

refrigerator for 1 hour to determine whether liquefaction of gelatin has occurred.

The medium was let cool to room temperature. Liquefaction of the medium

indicated a positive result while solidify of medium indicated negative result.

f) Oxidation-Fermentation Test

Hugh and Leison‟s OF basal medium was prepared and glucose was used as

the carbohydrate source. The glucose medium (10% w/v) was filtered sterilized and

added into Hugh and Leison‟s OF basal medium. The fresh culture was transferred

into the medium by stabbing using inoculating loop until approximately 1cm from

the bottom of the universal bottle. This medium used for each culture was duplicated

whereby one of two inoculated media would be overlaid with 1mL of sterile paraffin

oil to exclude oxygen. The inoculated media were then incubated at 37°C for 24

hours. Oxidative bacteria will change the green colour of open universal bottle to

yellow while the colour of sealed tube remains green. Fermentative bacteria would

both open and sealed tube into yellow colour.

59

g) Methyl Red (MR) Test

Fresh culture was inoculated into MR broth and incubated at 37°C for 24

hours. A few drops of MR reagent was added into the culture the result was

observed immediately. Positive result displayed red colour while negative result

gives a yellow colour. Orange colour indicated variable result.

h) Voges-ProsKaur (VP) Test

Fresh culture was inoculated into VP broth and incubated at 37°C for 24

hours. Reagent A (α-naphtol in ethanol), 0.6mL and Reagent B (potassium

hydroxide), 0.2mL were added into overnight culture. The culture was shaken gently

and examines the colour. Positive result showed eosin-pink colour.

i) Citrate Test

Fresh culture was streaked onto Simmon Citrate‟s slant agar and the tube was

incubated at 37°C for 24 to 48 hours. Growth with an intense blue colour on the

slant indicated a positive result while negative result is shown by no change of colour

on the green colour slant.

j) MacConkey

Fresh culture was streaked onto the surface of MacConkey afar and incubated

at 37°C for 24 to 48 hours. The growth and colour changes of colony on MacConkey

agar were observed. The appearance of bacterial colonies on the medium indicated

60

positive result. Pinkish colonies showed that the bacteria were able to utilize lactose

while whitish colonies indicated that the bacteria are non-lactose fermenter.

k) Nitrate Reduction

Heavy inoculum of fresh culture (1mL) was added into nitrate broth and

incubated at 37°C for 24 to 48 hours. After that, 5 drop of reagent A (0.8 %

sulphanilic acid in acetic acid) and 5 drops of reagent B (0.5% α-Napthylamine in

acetic acid) was added into the medium and shaken gently. Red colour developed

within 1 to 2 minutes indicated positive result. If no colour changes occur,

approximatedly 20mg of zinc powder was added into the solution and the tube was

shake vigorously. The tube was allowed to stand at room temperature for 10 to 15

minutes. No colour changes indicated positive result while red colour occurred

within 1 to minutes give negative result.

l) Indole Test

Fresh culture was inoculated into casein medium and incubated at 37°C for

24 to 48 hours. Then, 5 drops of Kovac Reagent was added to the inoculated casein

medium and shaken gently. Positive result displayed a red ring at the surface of

medium in the alcoholic layer while there is no colour development at the alcoholic

layer for negative result.

61

m) Triple Sugar Iron (TSI) Test

Fresh culture was streak onto Triple Sugar Iron slant agar. The tubes were

then incubated at 37°C for 24 to 48 hours. The expected results are shown in the

table below.

Expected results for Triple Sugar Iron (TSI) Test.

Red sland and red butt, no black colour

No fermentation of glucose, sucrose or

lactose, no hydrogen sulfide produced.

Red slant and black butt No lactose or sucrose fermentation,

hydrogen sulfide has been produced

Red slant with yellow butt No lactose or sucrose fermentation,

lactose is fermented; no hydrogen

sulfide has been produced.

Yellow slant, yellow butt and black

colour coloration

Lactose, sucrose and glucose

fermented, hydrogen sulfide has been

produced.

Yellow slant, yellow butt and lifting

and/or cracking of media, no black

colouration

Lactose, sucrose and glucose

fermented, hydrogen sulphide has not

been produced but gas has been

produced.

Yellow slant, yellow butt and no lifting

and/or cracking of media, no black

colouration

Lactose, sucrose and glucose

fermented, hydrogen sulphide has not

been produced nor gas production.

62

APPENDIX 4

Biochemical Test Results

Biochemical Tests A B C D E

Gram‟s Staining + + + - +

Spore Staining - - + - +

Shape Cocci Cocci Rod Rod Cocci

Oxidase Test - - + - -

Catalase Test + + + + +

Indole Test - - + + -

Nitrate Reduction Test + + + + +

Motility Test + + + + -

MacConkey + + + + +

Oxidation Fermentation

Test

F F F F F

Gelatin Liquefaction

Test

+ + + + -

Citrate Test - - + - -

Voges-ProsKaur (VP)

Test

- - - - -

Methyl Red Test - - - - -

Urease Test + + + + +

“+” : Positive result

“-” : Negative result

63

“F” : Fermentative