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 + H2O2Laccase

Oxidized dye + H2O Oxidized dye + H2O Monooxygenase Hydroxylated dye + H2O Dioxygenase Bishyroxylated dye

Dye + O2 Dye + O2 Dye + O2

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 azolinkage (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 (600C) 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 (23%) and organic colourants (34%) 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).

12 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 BODremoval 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).

13 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.

16 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 121C, 101.3 kPa for 20 minutes. The medium was left to cool down until 55 C before being poured into petri dishes.

20 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 37C 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 37C 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 heatfixed. 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 121C, 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 (PO43-) 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. determination of phosphate was 3015 The program used for the of HACH DR 4000

program

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 m). 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 after filter (mg) before filter (mg) Volume of sample (L)

(Equation 3.1)

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 m) was used. The filter membrane was dried in an oven for 24 hours at 60C 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)

=

(BA) __ Volume of sample (L)

(Equation 3.2)

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 4C. 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 Colour (ADMI) pH COD (mg/L) TSS (mg/L) Nitrate (mg/L) Phosphate (mg/L) Sulphate (mg/L) 1st Sampling 1090 8.69 843 2100 28 256 327 2nd Second Sampling 1070 9.03 855 1400 57 264 333

Note: The lapse of time between 1st sampling and 2nd 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. sources of sulphur can be the use of sulphuric acid for pH control. Other

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) 0.05 0.01 0.15 0.20 0.25 0.30 Time(h) for Decolourization 48 24 24 12 12 24

1.4 1.2

0.05%(w/v)1

OD600nm

0.10%(w/v)0.8

0.15%(w/v)0.6

0.20%(w/v)0.4 0.2 0 0 1 2 3

0.25%(w/v) 0.30% (w/v)

Time (day)

Figure 4.1

Growth of bacteria with addition of different concentrations of yeast

extract into the textile wastewater medium.

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 A B C D E Shape Filiform Round Round Round Round Colour White Cream White Cream Yellow Orange Light yellow Orange Light yellow Orange Margin Thread-like Smooth Smooth Smooth Smooth Elevation Hilly Convex Convex Convex Convex

33 Table 4.4: Results of bacteria identification. Bacteria Label A B C D E Bacteria Staphylococcus sp. Staphylococcus sp., Bacillus sp. Escherichia sp. 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.

1.8 1.6 1.4

Facultative Anaerobic

60%

Colour Removal (%)

50% 40% 30%

OD 600nm

1.2 1 0.8 0.6 0.4 0.2 0 0 3 6 9 12 14 16 18 20 22 24 30 36 40

OD600 OD600nm Colour Removal

Aerobic20% 10% 0%

Time (h)

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

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.

1.8 1.6 1.4 1.2OD 600nm

40% 35% 30% 25% 20%COD Removal (%)

Facultative Anaerobic

Aerobic

1 0.8 0.6 0.4 0.2 0 0 3 6 9 12 14 16 18 20 22 24 30 36 40 15% 10% 5% 0%

OD600 OD600nm COD Removal

Time (h)

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 %

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.

1.8 1.6 1.4

10

Aerobic Facultative Anaerobic

9.5 OD600 OD600nm

OD 600nm

1.2 1 0.8 0.6 0.4 0.2 0 0

9

pH

pH

8.5

8

7.5 3 6 9 12 14 16 18 20 22 24 30 36 40

Time (h)

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.

38

1.8 1.6 1.4 1.2

60 50 40 30 20 10 0

Nitrate (mg/L)

Facultative Anaerobic Aerobic Facultative Anaerobic

90 80 70

OD 600nm

OD600nm OD600 (nm)Nitrate (mg/L)

1 0.8 0.6 0.4 0.2 0 0 6 12 18 24 30 36 40

Time (h)

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).

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).

1.8 1.6

300 250

1.2

200

1 0.8 0.6 0.4

Facultative Facultative Anaerobic Anaerobic Aerobic Aerobic

Phosphate (mg/L)

1.4

OD 600nm

OD600 OD600nm

150 100 50

Phospate

0.2 0 0 6 12 18 24 30 36 40 0

Time (h)

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

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.

41

1.8 1.6 1.4 1.2OD 600nm

800 700Sulphate (mg/L)

Facultative Anaerobic Aerobic

600 500 400 300 200 100 0

1 0.8 0.6 0.4 0.2 0 0 6 12 18 24 30 36 40

OD600nm OD600 Sulphate

Time (h)

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. process. Sulphate reduction was limited during the treatment

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

42 concentration of mix liquor volatile suspended solids (MLVSS) and mix liquor suspended solids (MLSS) during the treatment process (Figure 4.8).

1400 1200

2500

2000

MLVSS (mg/L)

1000 800 600 400 200 0 0 3 6 9 12 14 16 18 20 22 24 30 36 40 1500

MLSS (mg/L)

MLVSS (mg/L) MLSS (mg/L)

1000

Facultative Anaerobic

Aerobic500

0

Time (h)

Figure 4.8 treatment.

MLVSS and MLSS versus time of facultative anaerobic and aerobic

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 (cosubstrate) 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

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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50 Reife, A., Reife, A., and Freeman, H. S. (1996). Environmental Chemistry of Dyes and Pigments. USA: Wiley-Interscience. Rein, M. (2000). Industrial Wastewater Characteristics. Water Use and Management. Sweden: Ditt Tryckerii Uppsala AB.

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52

APPENDIX 1 Environmental Quality (Sewage and Industrial Effluents) Regulations, 1979 Maximum Effluent Parameter Limits Standard A and B

Parameters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Temperature pH BOD5 @ 20oC COD Suspended Solids Mercury Cadmium Chromium, Hexalent Arsenic Cyanide Lead Chromium, Trivalent Copper Manganese Nickel Tin Zinc Boron Iron (Fe) Phenol Free Chlorine Sulphide Oil and Grease

(Units)o

Standard A (1) 40 6.0 - 9.0 20 50 50 0.005 0.01 0.05 0.05 0.05 0.10 0.20 0.20 0.20 0.20 0.20 1.0 1.0 1.0 0.001 1.0 0.50 Not detectable B (2) 40 5.5 - 9.0 50 100 100 0.05 0.02 0.05 0.10 0.10 0.5 1.0 1.0 1.0 1.0 1.0 1.0 4.0 5.0 1.0 2.0 0.50 10.0

C -

mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l

53

1. Standard A for discharge upstream of drinking water take-off. 2. Standard B for inland waters.

APPENDIX 2

Treatment Time(h) 0 3 6 9 12 14 16 18 20 22 24 30 36 40 OD600 (nm) 0.202 0.315 0.364 0.366 0.437 0.746 0.960 1.130 1.429 1.525 1.561 1.332 1.282 1.204

Results of the Study

Optical Density at

600nm

54

Decolourization Time(h) 0 3 6 9 12 14 16 18 20 22 24 30 36 40 ADMI 1070 1000 550 560 520 560 490 490 490 480 630 530 560 580 % of Removal 0% 6.54% 48.60% 47.66% 51.40% 47.66% 54.21% 54.21% 54.21% 55.14% 41.12% 50.46% 47.66% 45.79%

pH Time(h) 0 3 6 9 12 14 16 18 20 22 24 30 36 40 pH 9.03 8.45 8.22 8.41 8.45 8.31 8.43 8.51 8.54 8.63 8.78 9.08 9.42 9.65

55

Chemical Oxygen Demand (COD) Removal Time(hr) 0 3 6 9 12 14 16 18 20 22 24 30 36 40 COD (mg/L) 855 840 803 841 832 822 819 815 798 644 615 576 563 559 COD Removal (%) 0% 1.75% 6.08% 1.63% 2.69% 3.86% 4.21% 4.68% 6.67% 24.68% 28.07% 32.63% 34.15% 34.61%

Nitrate, Phosphate and Sulphate Concentration Time(h) 0 6 12 18 24 30 36 40 Nitrate (mg/L)57 35 23 23 27 35 84 28

Phospate (mg/L)264 248 264.6 228.1 189.2 203.8 263.2 274.4

Sulphate(mg/L)333 269 329 682 528 715 465 691

56

Biomass Time(h) 0 3 6 9 12 14 16 18 20 22 24 30 36 40 W0 (mg) 404.5 430.0 452.4 466.2 434.6 461.4 444.0 447.4 443.8 479.3 425.4 431.9 443.3 465.1 W1 (mg) 406.8 431.9 454.4 468.8 437.2 464.5 447.0 450.3 446.9 483.1 429.1 435.5 447.1 468.7 Dry Cell Weight (mg/L)766.67

633.33 666.67 866.67 866.67 1033.33 1000.00 966.67 1033.30 1266.67 1233.33 1200.00 1266.67 1220.00

Total Suspended Solid (TSS) Time(h) 0 3 6 9 12 14 16 18 20 22 24 30 36 40 W0 (mg) 442.0 455.1 450.9 452.8 445.9 440.1 482.8 466.3 506.0 460.2 489.8 461 443.0 475.2 W1 (mg) 446.2 458.1 453.9 455.9 449.5 442.9 486.3 469.3 509.6 463.5 494.1 464.9 447.2 478.8 TSS (mg/L) 2100 1500 1500 1550 1800 1400 1750 1500 1800 1650 2150 1950 2100 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-phenyleneThe colony of

diaminehydrochloride) were added onto a piece of filter paper.

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 37C 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 37C 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 37C 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 Leisons 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 Leisons 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 37C 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 37C 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 37C for 24 hours. Reagent A (-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 Citrates slant agar and the tube was incubated at 37C 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 37C 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 37C 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% -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 37C 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 37C 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 Lactose, colour coloration sucrose and glucose

fermented, hydrogen sulfide has been produced.

Yellow slant, yellow butt and lifting Lactose,

sucrose

and

glucose

and/or cracking of media, no black fermented, hydrogen sulphide has not colouration been produced but gas has been produced. Yellow slant, yellow butt and no lifting Lactose, sucrose and glucose

and/or cracking of media, no black fermented, hydrogen sulphide has not colouration been produced nor gas production.

62

APPENDIX 4

Biochemical Test Results

Biochemical Tests Grams Staining Spore Staining Shape Oxidase Test Catalase Test Indole Test Nitrate Reduction Test Motility Test MacConkey Oxidation Fermentation Test Gelatin Liquefaction Test Citrate Test Voges-ProsKaur (VP) Test Methyl Red Test Urease Test + -

A + Cocci + + + + F

B + Cocci + + + + F

C + + Rod + + + + + + F

D Rod + + + + + F

E + + Cocci + + + F

+

+

+

+

-

-

-

+ -

-

-

+

+

+

+

+

: Positive result : Negative result

63 F : Fermentative