bioremediation of textile wastewater using microclear looi ngit chin 2009
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wastewaterTRANSCRIPT
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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).
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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.
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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).
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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).
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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
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“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
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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).
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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.
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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).
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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
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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
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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).
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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
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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).
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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
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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.
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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.
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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