CHAPTER 3
RESEARCH METHODOLOGY
3.1
General Research Methodology
General research methodology that had been used shows the research
procedure for the dissertation. The Figure 4.1 shows the stages of research
dissertation.
3.1.1
Preliminary Stage
The background and problem of study were determined as a major concern
for this stage. The treatment by using constructed wetland was implemented in term
of its environmental friendly by product. Besides of the performance efficiency, the
long term maintenances and operational cost were put into consideration. Last but
not least, the objective and scope of study were drawn up as guidance for the future
research in advanced wastewater treatment.
2
3.1.2
Literature Review Stage
The treatment concept and wastewater characteristic were determined at this
stage as a completing part for preliminary research. Hence, it shows some related
theories from references such as books, journals, magazines, etc.
3.1.3
Analysis Stage
At this stage, the treated effluent was determined towards the removal
efficiency. The analysis technique in regard to the standard methods was used.
3.1.4
Conclusion Stage
The conclusion of the study had been written up based on the result analysis,
which was taken out from the experimental works. Besides that, further
recommendations will be suggested for future research.
3.2
Constructed Wetland Design
The study was carried out by controlling and maintaining four laboratory
scales constructed wetlands (CW), (cells 1 to 4) as well as treatment system for the
domestic wastewater samples as shown in Figure 3.2. Two different vegetations were
covered the cells, which are: water hyacinth (Eichornia Crassipes) and Water
Lettuce (Pista Stratiotes L.).
Figure 4.1: Research Flowsheme
PROBLEM STATEMENT
OBJECTIVE AND SCOPE OF
STUDY
LITTERATURE REVIEW
WASTEWATER
CHARACTERISTIC
- Criteria
- Composition
WASTEWATER TREATMENT
- Physical
- Biological
- Chemical
- Treatment System
DESIGN PARAMETER
- Flow process
- Vegitation used
- Loading capacity
- Flowrate
- Process efficiency
CONSTRUCTED WETLAND
DESIGN
CONTROLLING SYSTEM OF CW
- Flow rate
- Retention time
- Water quality
EXPERIMENTAL DESIGN
ANALYSIS
NUTRIENT REMOVAL
BACTERIA REMOVAL
TREATMENT
EFFICIENCY
CONCLUSION
Figure
3.1: Research
flow
scheme
Source:
Dissertation
Research,
2003.
Sampling
point (S1)
Sampling
point (S2)
Feed Tank
Flow
rate
Sampling
point (S4)
control
Sampling
tank
point (S3)
Figure 3.2: The schematic flow for constructed wetlands
66
3.2.1
The Operations System for Constructed Wetlands
The system was done in 4 sets of different detention times and flow rate,
which were: set A (flow rate: 0.0015 l/s, detention time: 6 days); set B (flow rate:
0.0015 l/s, detention time: 4 days); set C (flow rate: 0.001 l/s, detention time: 9
days); and set D (flow rate: 0.001 l/s, detention time: 6 days). The operating of the
system is shown in Figure 3.3, 3.4, 3.5 and 3.5.
Water Hyacinth (E Crassipes)
Water Hyacinth (E Crassipes)
Water Lettuce (P Stratiotes L.)
Figure 3.3: The schematic flow for set A
Water Hyacinth (E Crassipes)
Water Hyacinth (E Crassipes)
Water Lettuce (P Stratiotes L.)
Figure 3.4: The schematic flow for set B
Water Hyacinth (E Crassipes)
Water Hyacinth (E Crassipes)
Water Lettuce (P Stratiotes L.)
Figure 3.5: The schematic flow for set C
Water Hyacinth (E Crassipes)
Water Lettuce (P Stratiotes L.)
Water Hyacinth (E Crassipes)
Figure 3.6: The schematic flow for set D
Figure 1
67
3.3
Biological Assays
The standard test for the coliform group may be carried out using the most
probability number technique. The complete most probability number technique
procedure for total coliform and fecal coliform has described below.
3.3.1
Sampling Procedures
To obtain an aseptic, representative sample for bacteriological examination:
(i)
The sample bottle was kept unopened after sterilization until the
sample is to be collected.
(ii)
The bottle stopper and hood or cap was removed as one unit. Do not
touch or contaminate the cap or neck of the bottle.
(iii)
The sample bottle was submerged in the water to be sampled.
(iv)
The sample bottle to be filled approximately ¾ full, but not less than
100 mL. The ample air space was left to allow the sample to be
mixed by shaking prior to testing.
(v)
Aseptically the stopper or cap was replaced on the bottle.
(vi)
On a sample tag or field data sheet, the date, time, and location of
sampling, was recorded as well as the sampler's name and any other
descriptive information pertaining to the sample.
3.3.2
Sample Preservation and Storage
Examination of bacteriological water samples should be performed
immediately after collection. If testing cannot be started within one hour of sampling,
the sample should be iced or refrigerated at 4°C or less. If samples are iced during
transport or storage, use only enough ice to maintain the required preservation
temperature. Excess ice can submerge the sample bottles after melting and
potentially contaminate the sample.
68
The maximum recommended holding time for fecal coliform samples from
wastewater is 6 hours. If the shipping time of the samples is consistently greater than
the recommended holding time, consider doing on-site testing for fecal coliforms.
The storage temperature and holding time should be recorded as a part of the test
data.
3.3.3
Multiple Tube Fermentation Technique
The Multiple Tube Fermentation (MPN) technique for fecal coliform testing
is useful in determining the fecal coliform density in most water, solid or semisolid
samples. It is recognized as the method of choice for any samples which may be
controversial (enforcement related). The technique is based on the most probable
number of bacteria present in a sample which produces gas in a series of
fermentation tubes with various volumes of diluted sample. The MPN is obtained
from charts based on statistical studies of known concentrations of bacteria.
The technique utilizes a two-step incubation procedure. The sample dilutions
are first incubated in lauryl (sulfonate) tryptose broth for 24-48 hours. Positive
samples are then transferred to EC broth and incubated for an additional 24 hours.
Positive samples from this second incubation are used to statistically determine the
MPN from the appropriate reference chart.
3.3.4
Equipment and Reagents Equipment
The following equipment and glassware will be needed to perform the MPN
procedure:
(i)
Autoclave
(ii)
Dry heat sterilizer
(iii)
Incubator
(iv)
Water bath or heat sink incubator, 44.5°C Triple beam balance, 0.1 g
accuracy
(v)
Fermentation tubes and shell vials
69
3.3.5
(vi)
Dilution bottles
(vii)
Serological pipettes, graduated at 1.0 and 1.1 mL
(x)
Serological pipettes, graduated at 10.0 and 11.0 mL
(xi)
Transfer loops
(xii)
Corrosion resistant test tube racks
(xii)
Bunsen burner or alcohol lamp
Reagents
The following broths and chemicals will be needed to perform the MPN procedure:
(i)
Reagent grade water
(Dehydrated lauryl sulfonate tryptose, LST broth)
(ii)
Dehydrated EC broth
(Potassium dihydrogen phosphate, KH2PO4)
3.3.6
Preparation of Sterile Media Broths
(i)
Lauryl Sulfonate Tryptose (LST) Broth
For most wastewater effluent samples, the lauryl sulfonate tryptose
broth can be prepared by dissolving 35.6 grams of dehydrated media
in 1 liter of distilled water. If the volume of sample being tested is
greater than 1 mL per fermentation tube, the strength of the broth
must be increased to maintain the correct proportions.
(ii)
EC Broth
The EC broth can be prepared by dissolving 37.0 g of dehydrated EC
media in 1 liter of distilled water.
70
3.3.7
Preparation of Fermentation Tubes
After the broths are prepared, the fermentation tubes should be prepared by
dispensing 10 mL of broth into each fermentation tube. This volume should be
sufficient to partially cover the inverted, inner test tube after sterilization.
Sterilization procedures for culture media are discussed above. After sterilization,
refrigerate the prepared fermentation tubes at 10°C or less until they are needed
incubate fermentation tubes prepared and stored in this manner at 35 +/-0.5°C for 24
hours prior to use. Discard any tubes in which the inverted, inner test tube is not
completely filled.
3.3.8
Presumptive Test
The first step of the MPN procedure for fecal coliform testing is called the
presumptive test. In this test, samples or serial sample dilutions are inoculated into a
series of fermentation tubes. The fermentation tubes are then incubated at 35 +/0.5°C. The tubes are observed at the end of 24 and 48 hours for gas production. Any
tube showing gas production during this test indicates the possible presence of
coliform group bacteria and is recorded as a positive presumptive tube. All positive
presumptive tubes are transferred to EC broth fermentation tubes to confirm the
presence of fecal coliform bacteria.
All positive presumptive tubes should be carried into the fecal coliform
confirming test procedure. Transfer should be made as soon as the gas production is
noted in a fermentation tube.
3.3.9
Fecal Coliform Confirming Test
In the confirming test procedure for fecal coliform bacteria, the positive
presumptive cultures are transferred to EC broth, which is specific for fecal coliform
bacteria. Any presumptive tube transfer which shows gas production after 24 (+/-
71
20C) confirm the presence of fecal coliform bacteria in that tube and is recorded as a
positive confirmed tube.
3.3.10 Calculation of Most Probable Number (MPN)
The calculation of the MPN test results requires the selection of a valid series
of three consecutive dilutions. The number of positive tubes in each of the three
selected dilution inoculations is used to determine the MPN/100 mL. In selecting the
dilutions to be used in the calculation, each dilution is expressed as a ratio of positive
tubes per tubes inoculated in the dilution, i.e. 3 positive/5 inoculated (3/5). There are
several rules to follow in determining the most valid series of dilutions. In the
following examples, four dilutions were used for the test.
(i)
The selected highest dilution showing all positive results (no lower
dilution showing less than all positive) and the next two higher
dilutions.
(ii)
If a series shows all negative values with the exception of one
dilution, the series that places the only positive dilution was
selected in the middle of the selected series.
(iii)
If a series shows a positive result in a dilution higher than the selected
series, it should be incorporated into the highest dilution of the
selected series.
After selecting the valid series, the MPN/100 mL was determined by
matching the selected series with the same series on the MPN reference chart (See
Table 3-1). If the selected series does not match the sample dilution series at the top
of the MPN reference chart, the results must be calculated using the following
formula:
MPN/100 mL = MPN from chart  (mL sample for first column of chart/mL
sample in first dilution of the selected
series)
72
Table 3.1: MPN reference table
Table 3.1: MPN Referance Table (MPN/100mL)
Sample Volume
Sample Volume
MPN
10 mL
1 mL
0.1 mL
10 mL
1 mL
0.1 mL
0
0
0
0
2
0
0
0
0
1
3
2
0
1
0
0
2
6
2
0
2
0
0
3
9
2
0
3
0
1
0
3
2
1
0
0
1
1
6.1
2
1
1
0
1
2
9.2
2
1
2
0
1
3
12
2
1
3
0
2
0
6.2
2
2
0
0
2
1
9.2
2
2
1
0
2
2
12
2
2
2
0
2
3
16
2
2
3
0
3
0
9.4
2
3
0
0
3
1
13
2
3
1
0
3
2
16
2
3
2
0
3
3
19
2
3
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
1
1
1
1
2
2
2
2
3
3
3
3
0
1
2
3
0
1
2
3
0
1
2
3
0
1
2
3
3.6
7.2
11
15
7.3
11
15
19
11
15
20
24
16
20
24
29
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
0
0
0
0
1
1
1
1
2
2
2
2
3
3
3
3
0
1
2
3
0
1
2
3
0
1
2
3
0
1
2
3
MPN
9.1
14
20
26
15
20
27
34
21
28
35
42
29
36
44
53
23
39
64
95
43
75
120
160
93
150
210
290
240
460
1100+
1100++
Source: APHA (2002)
3.4
Analytical Procedures
The analysis used to characterize wastewater vary from quantitative chemical
determinations. Spectroscopy as instrumental methods of the analysis was used to
73
carry out the experiments. The details concerning for the analysis has referred to
Standard Methods for the Examination of Water and Wastewater, APHA, 21st edition
(2002).
3.4.1
Reagent and Equipment Requirement
The following tables show that the chemical reagent, equipment and supplies
which had been used to determine the nutrient from water sample.
Table 3.2: The reagent to determine the nitrite component in water sample
Using Powder Pillows
Quantity Required
Required Reagents and Standards
Per Test
NitriVer 3 Nitrite reagent powder pillow
1 pillow
Using AccuVac Ampuls
Quantity Required
Required Reagents and Standards
Per Test
NitriVer 3 Nitrite reagent accuVac ampul
1 ampul
Source: APHA (2002)
Unit
100/kg
Unit
25/pkg
Table 3.3: The equipment and supplies to determine the nitrite
component in water sample
Using Powder Pillows
Quantity Required
Required Equipment and Supplies
Per Test
DR/4000 1-Inch cell adapter
1
Sample cells, matched pair, 1-inch, glass with stoppers
2
Using AccuVac Ampuls
Quantity Required
Required Equipment and Supplies
Per Test
Beaker, 50-mL
1
DR/4000 Accuvac ampul adapter
1
sample cell, with cap (zeroing vial)
1
Source: APHA (2002)
Unit
each
pair
Unit
each
each
each
74
Table 3.4: The reagent to determine the nitrate component in water sample
Using Powder Pillows
Quantity Required
Required Reagents and Standards
Per Test
NitraVer 5 Nitrate reagent powder pillow (for 10 mL sample) 1 pillow
Using AccuVac Ampuls
Quantity Required
Required Reagents and Standards
Per Test
NitriVer 5 Nitrate reagent AccuVac ampul
1 ampul
Source: APHA (2002)
Unit
100/pkg
Unit
25/pkg
Table 3.5: The equipment and supplies to determine the nitrate
components in water sample
Using Powder Pillows
Quantity Required
Required Equipment and Supplies
Per Test
Beaker, 50- mL
1
DR/4000 AccuVac ampul cell adapter
1
Sample cells, 10-mL with cap (zeroing vial)
1
Stopper, for 18-mm tube
1
Using AccuVac Ampuls
Quantity Required
Required Equipment and Supplies
Per Test
DR/4000 1-inch cell adapter
1
Sample cells, matched pair, 1-inch, glass with stopper
2
Source: APHA (2002)
Unit
each
each
each
6/pkg
Unit
each
pair
Table 3.6: The reagent to determine the nitrogen ammonia
component in water sample
Required Reagents and Standards
Nessler reagent
Mineral stabilizer
Polyvinyl alcohol dispersing agent
Water, deionized
Quantity Required
Unit
Per Test
2 mL
500 mL
6 drops
50 mL *SCDB
6 drops
50 mL SCDB
25 mL
4 liters
Source: APHA (2002)
75
Table 3.7: The equipment and supplies to determine the nitrogen ammonia
component in water sample
Quantity Required
Per Test
Cylinder, graduated, mixing, 25 mL
2
DR 4000 1-inch cell adapter
1
Pipet, scrological, 1 mL
2
Pipet filter, safety bulb
1
Source: APHA (2002)
Required Equipment and Supplies
Unit
each
each
each
each
Table 3.8: The reagent to determine the phosphorus component in water sample
Quantity Required
Per Test
PhosVer 3 Phosphate reagent powder pillows, 10 mL
1 pillow
Water, deionized
varies
Source: APHA (2002)
Required Reagents and Standards
Unit
100/pkg
4 liters
Table 3.9: The equipment and supplies to determine the phosphorus
component in water sample
Quantity Required
Per Test
DR 4000 test tube adapter
1
Funnel, micro
1
Pipet, volumetric, Class A, 4.00 mL
1
Pipet, volumetric, Class A, 5.00 mL
1
Pipet filter, safety bulb
1
Test tube rack
1-3
Test 'N tube vials
1
Source: APHA (2002)
Required Equipment and Supplies
3.4.2
Unit
each
each
each
each
each
each
25/pkg
Methodology
The nutrient components such as nitrite, nitrate, ammonia nitrogen and
phosphorus were determined by using DR 4000 spectrophotometer model 48000.
The experiments were conducted according to the instrument manual prepared by
Hach Company, Iowa, United State.