Lab Manual

(For Physico-chemical & Microbiological Testing of

Water & Wastewater)

Environmental Engineering Laboratory

Department of Civil Engineering
Indian Institute of Technology Roorkee
 TABLE OF CONTENTS
Page

TABLE OF CONTENTS ................................................................................................................ II

Parameters ................................................................................................................................... 3
Temperature ................................................................................................................................ 5
Color & Odor .............................................................................................................................. 5
EC (Electrical Conductivity) ...................................................................................................... 5
pH (Potential of Hydrogen) ........................................................................................................ 6
DO (Dissolved Oxygen) (Aqueous Oxygen) .............................................................................. 8
Turbidity ..................................................................................................................................... 8
COD Test .................................................................................................................................. 10
BOD (Biochemical Oxygen Demand) ...................................................................................... 11
TN (Total Nitrogen) .................................................................................................................. 15
Ammonia-N (NH4-N)............................................................................................................... 16
Nitrate-N (NO3-N) ................................................................................................................... 17
TKN (Total Kjeldahl Nitrogen) ................................................................................................ 18
TP (Total Phosphorus) .............................................................................................................. 19
Ortho-P (Po4-P) ........................................................................................................................ 22
Alkalinity .................................................................................................................................. 23
Acidity ...................................................................................................................................... 24
TS (Total Solids) or Solids in Wastewater ............................................................................... 24
TSS Test .................................................................................................................................... 26
TDS (Total Dissolved Solids) ................................................................................................... 28
MLSS (Mixed Liquor Suspended Solids) & MLVSS (Mixed Liquor Volatile Suspended
Solids) ....................................................................................................................................... 28
TC (Total Coliform) & FC (Fecal Coliform)............................................................................ 30
SV30 & SVI .............................................................................................................................. 33
SOUR (Specific Oxygen Uptake Rate) of activated Sludge ..................................................... 33
FOOD TO MICROORGANISM (F/M) RATIO ...................................................................... 34
SRT (Sludge Retention Time) .................................................................................................. 35
Bacteriological Analysis ........................................................................................................... 35
FESEM characterization of PVA gel beads .......................................................................... 35
16S rRNA analysis of PVA gel beads .................................................................................. 36
Support material for biomass attachment ............................................................................. 37
References ................................................................................................................................. 53

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 Parameters
All the parameters were analyzed according to The “standard methods for the
examination of water and wastewater.” (22nd Edition. 2012).
1. Temperature
2. Color
3. Odor
4. EC (Electrical Conductivity)
5. Turbidity
6. TS (Total Solids)
a. TSS (Total Suspended Solids)
i. FSS (Fixed Suspended Solids)
ii. VSS (volatile Suspended Solids)
b. TDS (Total Dissolved Solids)
i. FDS (Fixed Dissolved Solids)
ii. VDS (Volatile Dissolved Solids)
TFS (Total Fixed Solids)
TVS (Total Volatile Solis)
7. MLSS (Mixed Liquor Suspended Solids)
8. MLVSS (Mixed Liquor Volatile Suspended Solids)
9. pH (Potential of Hydrogen)
10. DO (Dissolved Oxygen)
11. Alkalinity
12. Acidity
13. BOD (Biochemical Oxygen Demand)
a. BOD Total
b. BOD Soluble
14. COD (Chemical Oxygen Demand)
a. COD Total
b. COD Soluble
15. TP (Total Phosphorus)

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16. Ortho-P (Po4-P)
17. Ammonical-N (NH4-N)
18. Nitrate-N (NO3-N)
19. TKN (Total Kjeldahl Nitrogen)
20. TN = TKN + NO3-N
21. SV30 & SVI (Sludge Volume after 30 Minutes & Sludge Volume Index)
22. F/M ratio (Food to Microorganism)
23. TC (Total Coliforms)
24. FC (Fecal Coliforms)
25. E.coli
26. (ORP) Oxidation-Reduction Potential)
27. OUR
28. SOUR

Table 1. Methods to analyze Physico-chemical and microbiological parameters

Physico-chemical parameters
 pH, Turbidity, Alkalinity, Total solids APHA (21st Edition)
 Ammonia, Nitrate, Phosphorus, COD, APHA (21st Edition)
BOD, etc.
Microbiological parameters
 Total and Fecal Coliforms APHA (21st Edition)
 E.coli APHA (21st Edition)
 Microbiota Microbiota in Activated Sludge, Manual
by Eikelboom; Nishihara Corporation,
Japan.
Process operational parameters
 OUR, SOUR. Metcalf and Eddy, APHA (21st Edition).
 MLSS, MLVSS
 SVI, SV30

4
Note: - Those tests which require Spectrophotometer, they‟re called Colorimetric analysis.
Filtering Samples for Nutrients removal
Apparatus and material used:
1. Flasks
2. Funnels
3. Filter papers
4. Sample

Temperature

1
The observations of the temperature of sewage are useful in indicating solubility of
oxygen, which affects the transfer capacity of aeration equipment in aerobic systems, and the rate
of biological activity. Extremely low temperature affects adversely on the efficiency of
biological treatment systems and the efficiency of sedimentation. In general, under Indian
conditions, the temperature of the raw sewage is observed to be between 15 and 35°C at various
places in different seasons. Besides, oxygen is less soluble in warm water than in cold water.

Color & Odor

2
Historically the term “Condition” was used along with composition and concentration to
describe wastewater. Condition refers to the age of the wastewater, which is determined
qualitatively by its color and odor. Fresh wastewater is usually a light brownish-gray color.
However, as the travel time in the collection system increases and more anaerobic conditions
develop, the color of the wastewater changes sequentially from gray to dark gray, and ultimately
to black. When the color of the wastewater is black, the wastewater is often described as septic.
Some industrial wastewater may also add color to domestic sewage. In most cases, the gray, dark
gray, and black color of the wastewater is due to the formation of metallic sulfides, which form
as the sulfide produced under anaerobic conditions reacts with the metals in the wastewater.

EC (Electrical Conductivity)

3
The electrical conductivity (EC) of water is a measure of the ability of a solution to
conduct an electrical current. Because the ions transport the electrical current in solution, the
5
conductivity increases as the concentration of ions increases. In effect, the measured EC value is
used as a surrogate measure of total dissolved solids (TDS) concentration. At present, the EC of
water is one of the critical parameters used to determine the suitability of water for irrigation.
The salinity of treated wastewater to be used for irrigation is estimated by measuring its
electrical conductivity.
Note: - For measuring the Conductivity of Wastewater or water, we need a Conductivity meter,
and the unit is micro Siemens per Centimeter (µS/cm).

pH (Potential of Hydrogen)

4
In chemistry, pH is a scale used to specify how acidic or basic a water-based solution is.
Acidic solutions have a lower pH, while basic solutions have a higher pH. At room temperature
(25 °C), pure water is neither acidic nor basic and has a pH of 7.

The neutral value of the pH depends on the temperature, being lower than seven if the
temperature increases. The pH value can be less than 0 for very strong acids or greater than 14
for very strong bases. Measurements of pH are important in chemistry, agronomy, medicine,
water treatment, and many other applications.

pH is a measure of the hydrogen ion concentration of a solution. Solutions with a high

concentration of hydrogen ions have a low pH, and solutions with a low concentration of H+ ions
have a high pH. This may seem like a confusing way to express these relationships, and it is until
you understand what pH stands for. The equation that defines pH is given as follows:
pH = -log [H+] concentration,
This read as:
The pH is equal to minus the log of the H+ concentration.
For example, if the H+ concentration is deficient, let‟s say about 0.0000001M, then the pH is
pH= -log [0.0000001] which is the same as -log [1 X 10-7]
The term log [1 X 10-7] = -7
- (-7) = 7

The pH of raw water ≈ 6.4 + log

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 Look at the following Table
Hydrogen ion
pH Solutions with a pH of
concentration
.1M 1
.01M 2 coke and orange juice
.001 3
.0001 4
.00001 5 black coffee
.000001 6 Rain Water pH ~6
.0000001 7 pure water
.00000001 8 Natural water 6-8
.000000001 9 baking soda
.0000000001 10
.00000000001 11
.000000000001 12 household bleach
.0000000000001 13 oven cleaner

From this Table, you will notice a few relationships

 A difference of one pH unit (i.e., from pH 2 to pH 3) is a tenfold (10X) difference in H+

ion concentration.
 Pure water should have a pH of 7.0
 Solutions with a pH below 7.0 are termed acidic, and solutions with a pH above 7.0 are
termed basic.

5
The pH of the fresh sewage is slightly more than the water supplied to the community.
However, decomposition of organic matter may lower the pH, while the presence of industrial

7
wastewater may produce extreme fluctuations. Generally, the pH of raw sewage is in the range
of 5.5 to 8.0. The range of scale for aqueous systems is 0-14 at ambient temperature.
Note: - For measuring the pH of wastewater or water, we need a digital pH meter.

DO (Dissolved Oxygen) (Aqueous Oxygen)

The analysis for DO is a key test in water pollution and waste treatment process control.
DO depends on the temperature and partial pressure of oxygen in the air.
Note: - For measuring DO of Wastewater or water, we need a Digital DO meter.

Turbidity

6
Turbidity is an optical property that broadly describes the clarity or cloudiness of water.
It is related to color but has more to do with the loss of transparency due to the effect of
suspended particles and colloidal material. The presence of solids, for example, clay, silt finely
divided organic material, plankton, and other particulate material, cause turbidity.
Turbidity impacts on aquatic ecosystems by dispersing sunlight and reducing the oxygen
concentration. It also affects photosynthesis as well as the respiration and reproduction of fish.
Suspended particles also contribute to the adhesion of many heavy metals and other toxic
compounds. Turbidity is considered a measure of water quality: the more turbid the water, the
lower its quality.
Turbidity is measured in Nephelometric Turbidity Units (NTU) by a nephelometer or
turbidimeter, which uses a photocell placed at an angle of 90° to a light source to measure the
intensity of the light scattered when light passes through a water sample. Particle density is thus
a function of the light reflected by the particles to the photocell.
According to the World Health Organization (WHO), the turbidity of water for human
consumption should not exceed 5 NTU, and ideally, be below 1 NTU.

8
What causes turbidity?
Various parameters are influencing the cloudiness of the water. Some of these are:
- Phytoplankton
- Sediments from erosion
- Resuspended sediments from the bottom (frequently stir up by bottom feeders like carp)
- Waste discharge
- Algae growth
- Urban runoff
Treatment: -
7
Turbidity is commonly treated using either a settling or filtration process. Depending on
the application, chemical reagents will be dosed into the wastewater stream to increase the
effectiveness of the settling or filtration process. Potable water treatment and municipal
wastewater plants often remove turbidity with a combination of sand filtration, settling tanks,
and clarifiers.
In-situ water treatment or direct dosing for the treatment of turbidity is conventional when the
affected water bodies are dispersed (i.e., there are numerous water bodies spread out over a
geographical area, such as small drinking water reservoirs), when the problem is not consistent
(i.e., when there is turbidity in a water body only during and after the wet season) or when a low-

9
cost solution is required. In-situ treatment of turbidity involves the addition of a reagent;
generally, a flocculant evenly dispersed over the surface of the body of water. The flocs then
settle at the bottom of the water body where they remain or are removed when the water body is
drained. This method is commonly used at coal mines and coal loading facilities where
stormwater collection ponds have seasonal issues with turbidity. Some companies offer portable
treatment systems for in-situ water treatment or direct dosing of reagents.
Reagents: -
There are some chemical reagents that are available for treating turbidity. Reagents
available for treating turbidity include aluminum sulfate or alum (Al2 (SO4)3·nH2O), ferric
chloride (FeCl3), gypsum (CaSO4·2H2O), poly-aluminum chloride, long-chain acrylamide-
based polymers, and numerous proprietary reagents. The water chemistry must be carefully
considered when chemical dosing as some reagents, such as alum, will alter the pH of the water.
The dosing process must also be considered when using reagents as the flocs may be broken
apart by excessive mixing.
Procedure: -
1. Calibrate the turbidimeter (Hach Nephelometer) using standards in the working range.
2. Use Cell raiser for turbidity less than 10 NTU.
3. Shake well the sample.
4. Pour some amount of sample on Turbiditimeter‟s respected flask
5. Take the readings; the first reading is the exact one.

COD Test

COD is the amount of oxygen equivalent of dichromate, a specified oxidant that reacts
with the sample under controlled conditions.
Principle: -
A sample is digested (oxidized) with an excess of a known amount of potassium dichromate
(K2Cr2O7) in acid medium. The residual K2Cr2O7 or Cr+3 is determined
spectrophotometrically.
Apparatus and Reagents: -
1. Borosilicate culture tubes (16*100mm) with screw caps

10
 2. Block heater
3. Spectrophotometer for use at 600 nm and/or 420 nm
4. Digestion Solution or Oxidation agent
5. H2SO4 reagent (H2SO4+AgSO4)

Chemicals and Reagents required for COD Analysis: -

1. Oxidation agent - Potassium dichromate (K2Cr2O7) (0.25N)
a. Outlet: Low range (K2Cr2O7)
b. Inlet: High range (K2Cr2O7)
Making up of (K2Cr2O7 for COD) 10.216 gm K2Cr2O1 + 500ml Water 167 ml
H2SO4 + 33.3 gm HgSO4, Dissolve cool at room temperature and finally dilute it to
1Liter
2. Sulphuric Acid (98%) H2SO4 + COD
Making up of H2SO4+ COD  5.06 gm AgSO4 + 500ml H2SO4
Let stand 1-2 day to dissolve
AgSO4 = Silver Sulphate
Procedure: -
1. Take COD vials
2. Pour 2.5 ml Filtered sample for COD-Soluble or 2.5 ml Total Sample For COD-Total
3. Pour 1.5ml K2Cr2O7
4. Pour 3.5ml (H2SO4 + COD)
5. Digest it for 2hr - 150° C Temperature
6. Cool sample and standards to room temperature slowly.
7. Take Reading in Spectrophotometer (Calibrate the Device first)

BOD (Biochemical Oxygen Demand)

The BOD determination is an empirical test in which the relative oxygen

requirement of samples is determined over a period of time.
 Three days at 27 oC (BOD3(27°C))
 Five days at 20 oC (BOD5(20°C))

11
Principle:-
An airtight bottle of specified size is filled with the sample and incubated at 20 oC for five
days or 27 oC for three days. DO is measured initially and after incubation. BOD is computed
from the difference between initial and final DO.
Note: BOD is approximately 40-60% of COD.
The steps in the laboratory to measure BOD are: -
 Take a sample of waste; dilute with oxygen saturated water; add nutrients “and
microorganisms (seed) in case if the sample is Surface-water.”
 Measure dissolved oxygen (DO) levels over three days or five days.
 Temperature 20° C for five days or 27oC for 3 Days
 In the dark (prevents algae from growing)
 Final DO concentration must be > 2 mg/L.

SAMPLING PRECAUTIONS BOD: -

 If the analysis begins within 2 hours of sample collection, the sample does not need to be
cooled. Otherwise, the sample must be cooled to <4°C immediately after it has been
taken.
 The time to analyze must not exceed 6 hours. If this is not possible, the duration and
temperature of storage must be noted. Exclude all air.
 The duration of bulk sampling is restricted to 24 hours.
 A sample is taken using a clean, dry vessel. The sampling volume is at least one liter.
 If possible, the sample should not be frozen. Deep-frozen samples result in lower
measured values (up to 10% lower: Damage to microorganisms).
Reagents: -
 Phosphate buffer Solution: 8.5 gm KH2PO4 + 21.75 gm K2HPO4 + 33.4 gm
Na2HPO4.7H2O +1.7 gm NH4Cl
Dissolve in 500 ml Distilled water and dilute to 1L. The pH should be 7.2 without
further adjustment
 Magnesium Sulphate solution (MgSO4): Dissolve 22.5 gm MgSO4.7H2O in
Distilled water and dilute to 1L.

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  Calcium chloride solution (CaCl2): Dissolve 27.5 CaCl2 in distilled water and
dilute to 1L.
 Ferric Chloride Solution (FeCl3): Dissolve 0.25 gm FeCl3.6H2O in Distilled
water and dilute to 1L.
Procedure: -
1. Place the desired volume of water (tap or distilled water) in a suitable bottle and add 1ml
each of Nutrients for one liter of water.
2. Before using, bring dilution water saturated with DO by shaking in a partially filled bottle
or by aerating with organic free filtered air for 1 hour.
Note: Do not store prepared Dilution water for more than 24 h.
3. Fill the sample of suitable dilution on BOD bottle
BOD measurement (measurable with various dilutions):
By using percent mixtures By direct pipetting into 300-ml bottles
Range of BOD, Range of BOD,
% mixture mL
mg/L mg/L
0.01 20,000-70,000 0.02 30,000-105,000
0.02 10,000-35,000 0.05 12,000-42,000
0.05 4,000-14,000 0.10 6,000-21,000
0.1 2,000-7,000 0.20 3,000-10,500
0.2 1,000-3,500 0.50 1,200-4,200
0.5 400-1,400 1.0 600-2,100
1.0 200-700 2.0 300-1,050
2.0 100-350 5.0 120-420
5.0 40-140 10.0 60-210
10.0 20-70 20.0 30-105
50.0 10-35 50.0 12-42
100.0 4-14 100.0 6-21
0-7 300.0 0-7

4. Fill the remained parts of BOD bottle with Seed Water

13
 5. Measure DO of Seed water
6. Put the BOD bottles on BOD Incubator on 27oC for three days or 20oC for five days.
( DOi  DO f )  ( B1  B2 ) f
BOD 5 d , 20 deg 
(mg / L) 
P
B1 and B2 = Initial and final DO of the control run with seed only
volume of seeded dil. water in the seeded sample
f 
volume of seeded dil. water in seeded blank
volume of wastewater in sample
P
total combined volume
Using seed in BOD analysis and the way to use it in the BOD dilution method:8 -
9
The term “seed” refers to microorganisms that consume biodegradable organic matter in
samples for the measurement of Biochemical Oxygen Demand (BOD).
Domestic wastewater (influent) or effluent from biological water treatment plants (before
disinfection) provides the best source of seed and give the most reproducible results. Other
sources, such as industrial wastewater, may not have enough microorganisms or may contain
toxins that prevent the organisms from growing. If sewage is not available, prepare a seed
solution from a freeze-dried capsule such as PolySeed®. Whatever source of seed is used, it will
exert some demand. Therefore a seed control must be measured to correct for this demand. Older
versions of the Standard Methods for the Examination of Water and Wastewater indicated that
Dissolved Oxygen (DO) uptake of seeded dilution water should be between 0.6-1.0 mg/L; newer
versions indicate a minimum of 2.0 mg/L. Refer to the current Standard Methods or your local
regulatory official to verify this requirement.
When determining BOD, it is necessary to have a population of microorganisms that can oxidize
(or consume) the biodegradable organic matter present in the sample. If there is too little seed
present in the sample, complete consumption of biodegradable matter may not occur, resulting in
inaccurate results. In samples such as influent samples and flowing waters before disinfection,
this is not a problem as the sample will contain sufficient bacteria to do the job. However, in
certain sample types, such as some industrial wastes, high-temperature wastes, and treated
effluent, there is not enough bacterial activity to consume the material that is present. In these
cases, a seed must be added. A seed is simply a solution that contains a sufficient population of

14
bacteria. Hach offers PolySeed®, a seed capsule that can be added to samples.
The seed requires proper pH, temperature control, and nutrients such as phosphorus, calcium,
and magnesium for adequate growth. Hach nutrient buffer pillows provide the necessary
nutrients and pH.
Example: - If 3 ml of wastewater pours at 300 ml BOD bottles, what are F and P?
F= = 0.99 P= = 0.01

For unseeded sample, BOD is calculated as: -

BOD (mg/L) =

Where: -
D1 = Initial DO (mg/L)
D2 = Final DO (mg/L)

TN (Total Nitrogen)

Nitrogen Types: -
1. Ammonia Nitrogen
2. Nitrate Nitrogen (NO3)
3. Nitrite Nitrogen (NO2)
4. Organic Nitrogen

Organic Nitrogen: - Urea, nucleic acids, proteins; concentration varies from a few hundred
µg/L in lakes to >20 mg/L in raw sewage.
Total-Nitrogen = inorganic-Nitrogen + organic-Nitrogen
Inorganic Nitrogen = Ammonium + Nitrate + Nitrite
Or:
TN = TKN + No3 –N
TN = NH4-N+No3-N +No2-N + Org-N
TKN = NH4-N + Org- N

15
 Ammoniacal-N (NH4-N)

10
Ammoniacal nitrogen (NH3-N) is a measure for the amount of ammonia, a toxic
pollutant often found in landfill leachate and waste products, such as sewage, liquid manure, and
other liquid organic waste products. It can also be used as a measure of the health of the water in
natural bodies such as rivers or lakes, or in human-made water reservoirs. The term is used
widely in waste treatment and water purification systems.
Two methods: -
Method #01: -
1. 50ml (5ml Filtrated sample + 45ml D.W)
2. Add one drop EDTA Solution
3. Add 1ml Nessler Reagent)
(Yellow Color Appears)
4. Put 10 ml sample on 1” Cuvette glass
5. Measure Reading on Spectrophotometer (Calibrate the Device first)

Method #02: -
1. 25ml (5ml Filtrated Sample +20ml D.W)
2. Add 3 drops PVA
3. Add three drops Mineral stabilizer
4. Add 1ml Nessler Reagent
(Yellow Color Appears)
5. Put 10 ml sample on 1” Cuvette glass
6. Measure Readings on Spectrophotometer (Calibrate the Device first)

D.W (Distilled water)

EDTA (Ethylene Diamine Tetraacetic acid)
PVA (Polyvinyl Alcohol)

16
 Nitrate-N (NO3-N)

11
Nitrates are a form of nitrogen, which is found in several different forms in terrestrial
and aquatic ecosystems. These forms of nitrogen include ammonia (NH3), nitrates (NO3), and
nitrites (NO2). Nitrates are essential to plant nutrients, but in excess amounts, they can cause
significant water quality problems. Together with phosphorus, nitrates in excess amounts can
accelerate eutrophication, causing dramatic increases in aquatic plant growth and changes in the
types of plants and animals that live in the stream. This, in turn, affects dissolved oxygen,
temperature, and other indicators. Excess nitrates can cause hypoxia (low levels of dissolved
oxygen) and can become toxic to warm-blooded animals at higher concentrations (10 mg/L) or
higher) under certain conditions. The natural level of ammonia or nitrate in the surface water is
typically low (less than 1 mg/L); in the effluent of wastewater treatment plants, it can range up to
30 mg/L.
Sources of nitrates include wastewater treatment plants, runoff from fertilized lawns and
cropland, failing on-site septic systems, runoff from animal manure storage areas, and industrial
discharges that contain corrosion inhibitors.
A nitrate test is a chemical test used to determine the presence of nitrate ion in solution.
Levels of nitrate can be expressed in either of two ways: “nitrate as nitrogen” (symbol: NO3-N)
or simply as nitrate (NO3). To convert NO3-N to NO3 in parts per million (ppm, or mg/L),
multiply NO3-N by 4.427.
Nitrate = Nitrate Nitrogen x 4.43
Nitrate Nitrogen = Nitrate x 0.226
Procedure: -
1. 50ml (5ml Filtrated Sample + 45ml D.W)
2. Add 1ml 1N HCL
3. Pour sample on Quartz Cuvette glass
4. Measure Reading on Spectrophotometer (Calibrate the Device first)

17
 TKN (Total Kjeldahl Nitrogen)

12
Understanding Total Nitrogen: Three forms of nitrogen are commonly measured in
water bodies: ammonia, nitrates, and nitrites. Total nitrogen is the sum of total Kjeldahl nitrogen
(ammonia, organic and reduced nitrogen) and nitrate-nitrite. It can be derived by monitoring for
organic nitrogen compounds, free-ammonia, and nitrate-nitrite individually and adding the
components together. An acceptable range of total nitrogen is 2 mg/L to 6 mg/L, though it is
recommended to check tribal, state, or federal standards for an adequate comparison of your
data.
Generally, nitrogen content in the untreated sewage is observed to be in the range of 20 to
50 mg/L measured as TKN.
Procedure: -
1. Take 40ml sample (B)
2. Add 5ml Acid (2.5ml HNO3 + 2.5ml H2SO4)
3. Acid hydrolyze the sample (Digest it) on Hotplate (10 – 20 ml) (1 hour)
4. Make it to 70ml by D.W
5. Take 2ml Sample (C)
6. Add one drop TKN Indicator
7. Add 6-10 drop 8N KOH for blue color appearing
8. Add I drop (1N NaOH/KOH) (no role)

18
 9. Make it to 20ml by D.W
10. Add three drops PVA + 3 drops Mineral Stabilizer
11. Makeup 25ml by D.W
12. Add 1ml Nessler Reagent (Yellow Color appears)
13. Put 10 ml sample on 1” Cuvette glass
14. Take Reading in Spectrophotometer (Calibrate the Device first)

A = Readings from Spectrophotometer

B = 40ml Sample
C = 2ml Sample
13
Note: - The following correlation is used to estimate influent TKN on the basis of the COD
data (Eq. 1). Also, Soluble Kjeldahl nitrogen (SKN) is being estimated in a similar way, using a
correlation with ammonium data (Eq. 2).
TKN = 0.0189COD + SNH (1)
SKN = SNH/0.82 (2)
Where SNH = Ammonium nitrogen

TP (Total Phosphorus)

Phosphorus is removed from wastewater because it provides a nutrient or food source for algae.

Dead algae can cause serious oxygen depletion problems in receiving streams, which in turn can

kill fish and other aquatic life. Also, algae can cause taste and odor problems in drinking water

supplies. Phosphorus removal techniques can be divided into three main categories: physical,

chemical, and biological. Physical methods have proved to be either too expensive, as in the

cases of electrodialysis and reverse osmosis, or inefficient, removing only 10% of the total

phosphorus (TP) (Yeoman et al., 198814). Chemical removal techniques have the problem of

chemical costs and high sludge production (Helness and Ødegaard, 199915). EBPR (Enhanced

Biological Phosphorus Removal), The biological nutrient removal process is a so-called A2O
19
process (Johannesburg process). Biological methods can remove up to 98% of the TP.

Phosphorus Sources in Wastewater or Surface water

1. Human Waste
2. Detergent
3. Fertilizers

Method #01: Procedure: -16

1. Start the DRB200 Reactor (Digester). Preheat to 150 °C
2. Add 5 ml of sample to the Total Phosphorus Test vial.
3. Add the contents of one Potassium persulfate powder pillow for Phosphonate to the vial.
4. Put the cap on the vial, shake to dissolve the powder.
5. Insert the vial into the reactor (Digester) for 30 Minutes.
6. When the timer expires, carefully remove the vial from the reactor. Set the vial in the
attest tube rack. Let the vial cool to room temperature.
7. Add 2ml of 1.54N Sodium Hydroxide Standard Solution to the vial
8. Put the cap on the vial. Invert to mix.
9. Clean the vial
10. Insert the vial into the 16mm cell holder
11. Push ZERO; The display shows 0.00 mg/L PO43-.
12. Add the contents of one Phosver 3 powder pillow to the vial.
13. Put the cap on the vial; shake to mix for 20-30 seconds. The powder will not dissolve
completely.
14. Clean the vial
15. Insert the vial into the 16-mm Cell holder
16. Start the instrument timer. A 2-minute reaction time starts, Measure the sample within 8
minutes after the timer expires.
17. Push READ, Result show in mg/L PO43-.

Method #02: Procedure17: -

Since phosphorus exists in several distinct forms in wastewater samples and the approved
test method measures only the orthophosphate form, pretreatment methods have been
20
developed to convert the various forms of phosphate-phosphorus to the orthophosphate form.
If the only determination to be made is Total Phosphate-Phosphorus, the sample is digested
to convert both the polyphosphate and the organic phosphate to the ortho form at the same
time. If the analyst must test for the various types of phosphate, an acid-hydrolysis must be
performed. Since this is not required for most facilities, it is not included in this text.
Equipment: -
1. Hot plate
2. Tongs or gloves
3. Scoop (0.4 g capacity)
4. 125 mL Erlenmeyer flasks (acid washed)
5. 50 mL graduated cylinders (acid washed)
Reagents: -
1. Phenolphthalein indicator
2. Strong Acid
3. Ammonium persulfate, crystal or Potassium persulfate
4. Sodium hydroxide, 1 N
Laboratory procedure: -
18
1. Measure 50 mL or an appropriate amount of sample diluted to 50 ml with distilled water.
2. Add one drop phenolphthalein indicator. If a red color develops, add sulfuric acid solution
until the color disappears.
3. Add 1 ml of the sulfuric acid solution and 0.4 g of ammonium persulfate (or 0.5 g
potassium persulfate K2S2O8).
4. Boil gently for 30 to 40 minutes or until the total volume is 10 mL.
5. Cool, add one drop of phenolphthalein, and neutralize to a faint pink color with 1 N
sodium hydroxide.
6. Makeup to 50 mL with distilled water. The digested sample is then tested for total
phosphate.
Note: if precipitate forms, do not filter.
7. Take 10ml Sample to dilute it with 90 mL distilled water.
8. Add 4ml Ammonium molybdate Solution

21
 9. Add 0.5ml or ten drops of Stannous chloride Solution (Sncl2)
(Blue color Appear)
10. Pour sample on Quartz Cuvette glass
11. Measure Reading on Spectrophotometer (Calibrate the Device first)

Phosphorus Reagents:-
Making of Sodium hydroxide 1 N
Dissolve 40 g of Sodium hydroxide pellets in 1 L distilled water.
Making Strong Acid
300 ml H2SO4 to 600 mL DW cool it and then add 4ml HNO3 and dilute it to 1 liter.
Note: - Basically, in this method for measuring Total phosphorus, we‟re converting the
“combined” phosphate to the Ortho form for analysis. Which finally gives the total
phosphorus result.

Ortho-P (Po4-P)

Forms of Phosphorus: -
1. Orthophosphates (Reactive Phosphates)
2. Condensed Phosphates (Pyro, Meta, and Polyphosphates)
3. Organic Phosphates

Orthophosphates, also known as Reactive Phosphates, is the main constituent in fertilizers

used for agriculture and residential purposes.
Orthophosphates found in natural water provide a good estimation of the amount of
phosphorus available for algae and plant growth. This is the form of phosphorus that is most
readily utilized by biota.
Orthophosphates can be carried into streams and lakes through runoff.
Hold Time: -
Total phosphorus: 28 days
Orthophosphates: 48 hours
Procedure: -
12. 100ml (10ml Sample + 90ml D.W)

22
 13. Add 4ml Ammonium molybdate Solution
14. Add 0.5ml or ten drops of Stannous chloride Solution (Sncl2)
(Blue color Appear)
15. Pour sample on Quartz Cuvette glass
16. Measure Reading on Spectrophotometer (Calibrate the Device first)

Making Molybdate Reagent: -

1. 25 gm Ammonia in 175 ml DW
2. 400ml Distilled water + 280ml H2SO4 (98%) Glass bottle
3. Add Ammonia Solution and dilute it to 1 liter.

Making Stannous chloride solution: -

1.2.5 gm sncl2.2H2O in 100 L glycerol.

Alkalinity

Alkalinity is the capacity of the sample to neutralize standard acid (N/50 H2SO4), It is
measured volumetrically by titrating the sample with (N/50 H2SO4) and is reported in terms of
equivalent caco3 (Acid-base titration method).
Reagents: -
1. N/50 H2SO4 (0.02 N H2SO4)
2. Bromocresol green mixed indicator

Procedure for Total Alkalinity: -

1. 100 ml (50ml Sample +50ml D.W)
2. bromocresol green (Indicator) 7-8 drops
(Green Color Appears)
3. Titrate with N/50 H2SO4 (0.02 N H2SO4)
(Yellow Color Appears)
4. Calculate it by ml of Titrate * 20 mg/L

Or:
1. Take a 100 ml sample (unfiltered) in a conical flask.

23
 2. Add 0.2 ml bromocresol green or mixed indicator (5 drops)
3. Titrate with N/50 H2SO4 (0.02 N H2SO4) to a yellow endpoint
Green at pH > 4.5 – Yellow at pH < 4.5
4. Record ml of acid used (T ml)
5. Total alkalinity = T * 10 mg/L as CaCo3

Note: - Minimum 100 mg/L residual Alkalinity is required in treated water.

Acidity

Acidity is the capacity of the sample to neutralize the standard base (N/50 NaOH), Mineral
Acidity is present in discharge from mines and industrial wastewater (Samples of pH < 4).
Carbon dioxide acidity is present in samples having a pH of 3.5 – 8.5 and the sources of carbon-
dioxide acidity in natural water are:
I. Atmospheric carbon dioxide dissolved in water
II. Aqueous carbon dioxide from bacterial decomposition of organics in soil and water.
III. It is measured volumetrically by titrating the sample with N/50 NaOH using
phenolphthalein indicator. The method, however, applies to the sample having acidity >
2mg/L.
Reagents: -
1. N/50 NaOH
2. Phenolphthalein Indicator
Procedure: -
1. Take 100 ml sample to add 1 ml phenolphthalein indicator (colorless; pH <8.5)
2. Titrate with N/50 NaOH until the appearance of pink color (pH =8.5)
3. Record ml of base used (V ml)
4. Calculation: Acidity = V *10 mg/L CaCO3

TS (Total Solids) or Solids in Wastewater

19
The residue remaining after a wastewater sample has been evaporated and dried at a
specified temperature (103 to 105° C).

24
 Proteins (50%)

Carbohydrates
70% Organic
(25%)

Solids
Fats (10%)

Grit

30% Inorganic Salts

Metals

TS = TSS + TDS
TS = TFS + TVS
TFS = FSS + FDS
TVS = VSS + VDS
TS = FSS + FDS + VSS + VDS
[ ]

[ ]

[ ]

25
 TSS Test

20
The portion of the TS retained on a filter with a specific pore size, measured after being
dried at a specified temperature (105°C). The filter used most commonly for the determination
of TSS is the Whatman glass fiber filter, which has a nominal pore size of about 1.58 µm.
Apparatus & Reagents used: -
1. Pre weighed filter papers (1.58 µm)
Note: - Filters with standard pore sizes varying from 0.45 µm to about 2.0 µm have been
used for the TSS test.
2. Aluminum dish (Almonium Paper)
3. Marker
26
 4. Vacuum Flask and Vacuum
5. Filtration apparatus (Filter Support)
6. Tweezers
7. Pipette (5ml)
8. D.W
9. Oven
10. Timer
11. Desiccator
12. Analytical Balance

Procedure: -
1. Mark the Dry filter papers
2. Put it on oven dry 105° for 1 hour
3. Weigh the filter papers (W1)
4. Take Samples
a. Inlet: 20 – 50ml
b. Outlet: 100 – 200ml
5. Filter the sample on Filter Papers

27
 6. Put the filter papers on oven dry 105° for 6 hours
7. Weigh the filter papers (W2)

[ ]

TDS (Total Dissolved Solids)

21
Those Solids that pass through the filter, and are then evaporated and dried at a
specified temperature. It should be noted that what is measured as TDS is comprised of colloidal
and dissolved solids. Colloids are typically in size range from 0.001 to 1 µm.
TDS = TS – TSS
Measuring:-
1. Put the Crucible glasses on the oven for 1 hour
2. Cool the Crucible glasses to room temperature and weigh the weight of them (W1)
3. Pour 100 ml Filtered sample on glasses for TDS measuring
4. Put them on the oven for 24 hours
5. Putting out the crucible glasses and weighing the weight (W2)

[ ]

For Improving the TDS removal, it is recommended to go for RO (Reverse Osmosis) since TDS
cannot be removed by the biological treatment system.
Note: we can also use Digital TDS meter for TDS measuring.

MLSS (Mixed Liquor Suspended Solids) & MLVSS (Mixed Liquor Volatile Suspended
Solids)

MLSS is used to indicate the concentration of suspended solids in activated sludge.

MLVSS represents the concentration of biomass in activated sludge. To measure the MLVSS,
you can filtrate the activated sludge through 0.45 microfilters. Then, heating at 550 degrees C.
22
Mixed liquor is a combination of sludge and water removed from the clarifier in the wastewater
treatment process and reintroduced into an earlier phase of the treatment process. The mixed

28
liquor contains microorganisms that digest the wastes in the raw water.
Mixed Liquor Suspended Solids (MLSS) is a test for the total suspended solids in a sample of
mixed liquor. This test is essentially the same as the test we perform for TSS, except for the use
of mixed liquor as the water sample. Besides, the concentration of suspended solids found in the
mixed liquor is typically much higher than that found in the raw or treated water. MLSS
concentrations are often higher than 1,000 mg/L, but should not exceed 4,000 mg/L.
MLVSS, or Mixed Liquor Volatile Suspended Solids, is a test for the amount of volatile
suspended solids found in a sample of mixed liquor. Volatile solids are those solids that are
burnt up when a sample is heated to 550°C. Most of the volatile solids in a sample of mixed
liquor will consist of microorganisms and organic matter. As a result, the volatile solids
concentration of mixed liquor is approximately equal to the amount of microbes in the water. It
can be used to determine whether there are enough microorganisms present to digest the sludge.
In a wastewater treatment plant, operators should test for MLSS three times per week and
MLVSS once per week. Both tests should use grab samples taken from the same location in the
treatment plant.
Equipments: -
 Desiccator
 Drying oven, for operation at 103 to 105°C
 Analytical balance, capable of weighing to 0.1 mg
 Graduated cylinder
 Low-form beaker
 Filtration apparatus, which can be any one of the following:
o Membrane filter funnel
o Gooch crucible, 50 mL to 1000 mL capacity, with Gooch crucible adapter
 Filter flasks, of sufficient capacity for sample size
 Vacuum pump
 Tubing
 Crucible glasses
 Muffle furnace

Laboratory Procedure: -
29
 1. Collect a grab sample of mixed liquor.
2. Put the Crucible glasses on an oven for an hour for moisture absorption.
3. Put the Cruiciclbe glasses on a desiccator for cooling.
4. Mark the Crucible glasses and weigh them.
5. Pour 100 mL Mixed liquor sample on Cruiclbe for TS and TDS (After Filtering Sample)
for each.
6. Put them on the oven for operation at 103 to 105°C.
7. Measure the Cruiebles after cooling.
8. Calculate the Mixed Liquor solids in the sample, as follows:
[ ]

[ ]

Mixed Liquor Solids, mg/L = TS - TDS

9. Ignite the Crucibles from step 1 in a muffle furnace at 550°C. An ignition time of 15 to
20 minutes is usually sufficient for 200 mg residue. However, when igniting more than
one sample or when igniting heavier samples, the ignition time may need to be
increased.
4. Let the crucibles cool partially in the air until most of the heat has dissipated. Then
transfer the filter to a desiccator to cool the rest of the way to air temperature.
5. Weigh the Crucibles and record the weight in the Data section.
7. Calculate the volatile solids in the sample, as follows:

[ ]

[ ]

Volatile Solids, mg/L = TS – TDS

Note: - Mixed Liquor = Wastewater + Suspended Solids + Biofilm

TC (Total Coliform) & FC (Fecal Coliform)

30
 Water carries solids (TS, SS, Turbidity…etc.) besides water also carries living
organisms/microorganisms. Analysis of water for all the known pathogen is time-consuming, an
expensive exercise, and requires expertise, that‟s why we consider here Indicator Organisms.
Indicator Organisms
Rather than testing for each pathogen, it is easier to test for only one group of
microorganisms whose presence is an assured evidence/ indication that the wastewater has been
polluted by feces of humans or warm-blooded animals this microorganism may be called an
indicator organism. And Coliform bacteria are Indicator organisms.
23
Total coliforms include bacteria that are found in the soil, in water that has been influenced by
surface water and inhuman or animal waste. Incubation at 35oC @24h
Procedure:-
1. Preparing broths
a. Lauryl Sulphate broth
For 500ml: 17.8 gm Lauryl Sulphate powder +500 ml DW +0.5 gm Bromocresol
Purple powder indicator
b. Tryptone Water (Tryptone broth)
For 1000 ml: 15 gm Tryptone Water + 1000 ml DW
2. Place sterilized fermentation test tubes containing 10 ml Lauryl tryptose broth in three-
tube stand
3. Sterilize Micro tips and Stands of Tryptone water and Lauryl Sulphate broth
4. Add 1 ml of sample into a test tube containing 9 ml Water Tryptone
Repeat it with 8 Water Tryptone tubes
5. Now take 1ml of Micropipette and 1ml of sterilized microtip
6. Add 1ml of a sample into a test tube containing 10 ml of Lauryl Sulphate broth
Repeat it with 24 Lauryl Sulphate broth tubes
7. Now set incubator Temperature at 35 oC
8. Put Sample inside Incubator
9. Allow it to incubate in the next 24-48 hours
10. Growth gas and/or acid produced (shades of yellow color) shows a positive test.
Coliform group present.

31
 After reading, we use the following Table:

From here, FC test starts:

Fecal coliforms are the group of the total coliforms that are considered to be present specifically
in the gut and feces of warm-blooded animals. Incubation at 45oC @24h
Fecal coliforms are considered a more accurate indication of animal or human waste than the
total coliforms.
11. Gently shake or rotate tubes, using a sterile 3 or 3.5 mm dia. Loop, transfer growth from
each presumptive fermentation tube to EC broth.
12. Incubate inoculated EC broth tubes in an incubator at 44.5 ± 0.2 oC for 24 ± 2h.
13. Gas produced. Positive test. The fecal coliform group present
From here E.coli test starts:
14. Transfer growth from each presumptive fermentation tube to EC-MUG
15. Presence of bright blue fluorescence is considered a positive response for E.coli

Note: - Untreated domestic sewage contains upwards of 3 million coliforms per 100 mL.

32
 SV30 & SVI

The sludge volume index (SVI) is the volume in mL occupied by one gram of MLSS after 30
minutes of settling in a 1,000 mL graduated cylinder and has units of mL/g. The SVI is a
measure of the settleability of the activated sludge in a secondary or final clarifier. Lower values
of the SVI indicate better sludge settleability. For good settleability, SVI should be in the range
of 50-150. SVI outside this range indicates poor settling and may require optimization of the
upstream biological treatment process. Specifically, a high SVI may indicate what is called
„Sludge bulking”, which is the prevalence of slow-settling filamentous bacteria which thrive in
severe growing conditions such as low F/M, low oxygen, low nutrients, low pH, or due to the
nature of the organics in the wastewater (e.g., for industrial wastewater effluent with
contaminants that are difficult to degrade).

SVI =

Note: - The main sludge stabilization processes are alkaline stabilization (usually with lime),
Anaerobic digestion, Aerobic digestion, and composting.
Anaerobic digestion stabilizes sludge, reduce the amount of it, and produces biogas.

SOUR (Specific Oxygen Uptake Rate) of activated Sludge

24
The specific oxygen uptake rate (SOUR) is a measure of the quantity of oxygen consumed by
microorganisms and is a relative measure of the rate of biological activity. As microorganisms
become more active, the SOUR increases and vice versa.
Why Is It Used?
 Research has shown that the SOUR and final effluent COD can be correlated. Therefore,
changes in the SOUR can be used to predict final effluent quality. If SOUR increases, it
is an indication of an increase in the MLSS respiration rate and may require additional
oxygen to stabilize.
 Furthermore, toxic or high organic loads can often be detected before severe deterioration
of effluent quality occurs. Changes in the SOUR on effluent samples will indicate
changes in loading.
33
  SOUR is also used in conjunction with COD determinations of influent waste strength to
predict operating problems and alert plant operators of potential treatment interruptions
and potential effluent permit violations. A comparison of SOUR to influent COD and
control charts of SOUR can help operators to determine if the process is under control or
requires modification of operational parameters.
Procedure: -
 For analyzing OUR, the sample of the mixed liquor is filled into a 1000 mL capacity
flask containing a magnetic stirring device. The sample is being aerated for several
minutes to reach the saturation DO concentration in the order of 6.8 mg/L. Then
immediately, a DO probe is being inserted into the flask. Enough samples were displaced
with the probe containing the electrode to fill the flared top of the flask to isolate its
contents from the atmosphere. The sample then stirred to provide adequate mixing, and
the DO is being monitored for 10 minutes at a 1-minute interval.
The specific oxygen uptake rate value was 17mgO2 g-1VSS.h, which is good and indicates
healthy biomass in the aeration tank as well as excellent oxidation of the substrate.
Typically more than 20-30 mg O2 g-1VSS.h suggests the rapid uptake of the substrate under a
higher F/M ratio and lower SRT and Values less than 10 mg O2 g-1VSS.h indicates slower uptake
of BOD under lower F/M ratio and high SRT.

FOOD TO MICROORGANISM (F/M) RATIO

What is F/M Ratio?

The food-to-microorganism (F/M) ratio is a measurement of the amount of incoming food (kg of
Influent BOD) divided by the kilograms of microorganisms in the system.
The higher the F/M ratio, the more food there is relative to the quantity of microorganisms. If the
F/M ratio is too high, the bacteria will not form a good floc.
A low F/M ratio means there are many microorganisms, but the food supply is limited. This is
good, up to a point. With a limited food supply, the bacteria lose their motility and clump
together to form a good floc for settling. However, caution is needed as too low of an F/M ratio
can be deleterious and lead to the formation of filamentous bacteria and a reduction in clarifier
performance due to sludge bulking.
34
The F/M ratio is a process control number that helps to determine the proper number of
microorganisms for the system. This is typically controlled by increasing or decreasing the
sludge wasting rate.
For calculating F/M, the following information is needed
• Influent flow into the aeration tank (MLD)
• Influent BOD (mg/l) concentration to the aeration tank.
• Mixed Liquor Volatile Suspended Solids Concentration (mg/l)
• The volume of aeration tank (in m3).
F= Influent Flow (MLD) X Influent BOD Concentration (mg/L)
M= Aeration System Volume (in millions of Liters) x MLVSS or MLSS (mg/L)

F/M =

Note - The Food/Microorganisms ratio, which is to be set for a given STP. It can be in the range
of 0.05 to 0.40 (5% to 40%).

SRT (Sludge Retention Time)

Calculation of SRT: -

SRT = (days)

a = Total Volume of Aeration tank (m3)

b = MLSS of Aeration Tank (mg/L)
c = Sludge generated (kg/day)
Bacteriological Analysis

Microbial enumeration was performed in the influent and Effluent samples with or without
chlorination to analyze the pathogen removal efficacy of the PVA gel beads based WWTP. A
coliform analysis was performed by the MPN tube method. The cell counts for heterotrophic
plate count, E.coli sp., Staphylococci species, Pseudomonas species, Shigella, and Salmonella
species was performed by the culture plate method, according to Bhatia et al.
FESEM characterization of PVA gel beads

35
The analysing the characteristics of the microorganisms in the PVA process, the change in the
PVA-gel beads and the enrichment of microorganisms was investigated using the FESEM at
different magnifications 50 X, 100X, 500 X and 1000X.
16S rRNA analysis of PVA gel beads

DNA extraction

Microbial diversity of the biomass inside the PVA gel beads was identified using the 16S rRNA
analysis. PVA gel beads were microtomed (10 no.) DNA extraction is done using commercially
available kits such as QIAGEN, ZYMO RESEARCH, Thermo Fisher, as per the manufacturer‟s
recommendation.
PCR amplification and cloning

40 ng of extracted DNA was used for amplification along with 10 PM of each primer. Initial
denaturation was carried out at 95ºC. The aforementioned condition, denaturation at 95 ºC for 15
sec, annealing at 60 ºC for 15 sec, elongation at 72 ºC for 2 mins, final extension at 72 ºC for 10
mins and hold at 4 ºC was repeated for 25 Cycles. Universal bacterial primers, 16F- 5‟
AGAGTTTGATCMTGGCTCAG 3‟ 16R- 5‟ TACGGYTACCTTGTTACGACTT 3‟ were used
for the study.
Table 1. PCR Primers used in the present study
Primers Sequence (5_ to 3_) Specificity References
11f GTT TGA TCC TGG CTC AG Bacteria 16S rDNA Kane et al., 1993
1492r TAC CTT GTT ACG ACT T Bacteria 16S rDNA Lin and Stahl, 1995
Nitrobacter16S
NIT3 CCT GTG CTC CAT GCT CCG Wagner et al., 1996
rDNA
Nitrospira16S
Ntspa0685 CGG GAA TTC CGC GCT C Hovanec et al., 1998
rDNA
Rotthauwe wt al. ,
AmoA-1F GGG GTT TCT ACT GGT GGT amoA
1997
CCC CTC KGS AAA GCC TTC Rotthauwe wt al. ,
AmoA-2R amoA
TTC 1997

36
Sequencing and Analysis

Nanopore sequencing was performed by using 1µg of DNA template. Alpha and Beta diversity
was analyzed for the tested sequences. OTUs obtained were tested based on similarity and
consistency.
Abundance

Abundance percentage of the prevalent bacterial species and their metabolic pathways was
studied. Data were presented for the taxonomic plot analysis up to the phylum level.
Support material for biomass attachment

PVA-gel biofilm has a spherical shape and 3 to 4 mm size (diameter). These biofilms are
incredibly porous, having a pore size of 10-20 microns, and such high porosity favors a better
supply of air/oxygen and carbon to the residing bacteria inside the biofilm, which results in
stable and efficient treatment Figure 3. All the PVA-gel biofilm characteristics are shown in
Table.
Table: Beads characteristic
BEADS CHARACTERISTICS VALUES
Beads diameter 3-4 mm
Effective surface area ~2500 m2/m3
Specific gravity 1.025
Pore size 10-20 microns
Weight(g/100 gel 3.84
Volume(ml/100 gels) practically 3.9

37
Biomass determination on PVA gel beads
One hundred beads were weighed after air drying for 20-24 hours, and their volume was
measured by using the buoyancy principle, that is, 100 beads were immersed in 10 ml of distilled
water, and the volumetric increase was noted as 3.9 mL was observed. Thus, the weight and
volume of a single bead were ascertained as 0.038 g and 0.039 mL.
MLSS and MLVSS determination

[ ]

W1 = weight of clean/unused 100 PVA gel beads after oven dry at 105oC for 24 h
W0 = weight of biomass containing/used 100 PVA gel beads after oven dry at 105oC for 24 h
V = Volume of PVA gel reactor

3.4.6 MICROBIOTA OF ACTIVATED SLUDGE

Activated Sludge as Biological Process
The activated sludge process relies on many different types of microorganisms to treat the
wastewater. Therefore, the activated sludge process is a living process that requires knowledge
of the microorganisms involved. We should know which microorganisms are desirable and
undesirable and how these microorganisms respond to the environment in the aeration tanks.

Tools for Process Control

 Since it is a biological process, the activated sludge process is complex and
continuously changing. Microbiology is a tool can be used to control the process. It can
be challenging to control the process based on lab data, calculated process parameters,
and visual observations alone, especially when we are confronted with conflicting data.
At times like this, a visual observation of the numbers and types of
microorganisms in a sample of activated sludge using a microscope can be
beneficial in figuring out what is happening and deciding which process change
to make.
 Another benefit of the “microbiology tool” is that it can be used to forecast potential plant
upsets. Plant operators who react to plant upsets instead of preventing them often find
that their effluent quality degrades before their process change takes effect.
Incorporating microscopic observations of the activated sludge into a routine process

38
 control strategy will enable us to “see” the beginning stages of deteriorating conditions
before any significant impact on the final effluent quality.

Activated Sludge Organisms

95 % of microorganisms in activated sludge are bacteria, and 4 % are higher organisms such as
protozoa, rotifers, nematodes, etc.,
Bacteria:
 Bacteria are single-celled organisms and constitute the majority of organisms in
activated sludge
 Bacteria have the following designations based on their shape:
– round or spherical shape
– cylindrical or rod shape
– spiral shape
 Bacteria can‟t be visible under a 100 X microscope. In order to see their shape, it is
necessary to use a magnification of about 400x to 1000x. The optics must be useful in
order to resolve them correctly at this magnification.
 A special kind of bacterial growth significant in activated sludge systems is called the
filament. Filaments are formed by filamentous bacteria, which attach themselves to each
other, forming multi-cellular chains.

Protozoa
 Protozoa are single-celled organisms ranging in size from 10 microns to over 300
microns. They are easily visible under the microscope at 100X magnification.
 Their primary function in the treatment process is to remove non-flocculent bacteria and
very small floc that would not settle
 Protozoa contribute very little to the removal of organic nutrients; however, their
presence greatly enhances the clarity of the water. The numbers of the different types of
protozoa will give an indication of treatment system performance
 The presence or absence of protozoa is an indicator of the amount of bacteria in the
sludge and the degree of treatment.
 Like bacteria, protozoa must have oxygen to survive. Lack of oxygen will severely limit
the kind and number of protozoa present.

39
  Protozoa are more sensitive to pH than floc-forming bacteria are. They have an optimum
range of 7.2 - 7.4 but can tolerate 6.0 - 6.8.
 They can be further classified into an amoeba, flagellates, free-swimming ciliates, and
stalked ciliates. The ciliates are a group of protozoans characterized by the presence of
hair-like organelles called cilia,
Metazoa (Rotifers and Nematodes)
 Metazoan animals are multi-cellular, mitochondrial eukaryotes. Today Metazoa
encompasses all animals with differentiated tissues, including nerves and muscles It
includes rotifers and nematodes
 Rotifers are multi-cellular animals with rotating cilia on the head and a forked tail.
They are an indication of an old activated sludge with a high MCRT and are usually
associated with a turbid effluent.
 Nematodes are strict aerobes and can metabolize solid organic matter that is not
easily metabolized by other microorganisms. They are usually found in sludge from
extended aeration plants

For the sake of studying their behavior in activated sludge, we will classify Activated sludge
microbiota in the following five categories:
1. Amoebae
2. Flagellates
3. Free-swimming ciliates
4. Stalked ciliates
5. Rotifers
6. Nematodes

Different species will thrive under different conditions. Some are more efficient at gathering food
and will out-compete less efficient species. The relative proportions of microbiota types are
highly dependent on the sludge age and F/M ratio (Figure 14).
The succession of activated sludge microbiota during the establishment of activated sludge can
be depicted in figure 15.

40
 Figure 14. Relative Proportion of Microbiota with SVI, F/M ratio and sludge Age

Figure 15. The success of microorganisms during the establishment of activated sludge
41
Microbiota enumeration in aeration sludge
Protozoa are single-celled organisms ranging in size from 10 microns to over 300
microns. They are easily visible under the microscope at 40 X and 100X magnification.
Their main function in the treatment process is to remove non-flocculent bacteria and
very small floc that would not settle. Protozoans are responsible for the flocculation
process, which results in the biosorption phenomenon of organic particles. These
processes are essential in the treatment of conventional pollutants and micro-
contaminant degradation. Hence, the presence or absence of protozoa is an indicator of
the amount of bacteria in the sludge and the degree of treatment.

Methodology:
Qualitative microscopic observations were carried out in mixed liquor sample of aeration
tank. 25 µL sub-samples of sludge were examined under phase contrast (Radison
RXLr5) illumination at 40 X and 100X magnifications. Four replicates of final volume 1
mL were tested. Enumeration of microbiota was done according to scale where, (+) = 5-
10 protozoans/mL; (++) = 10-100 protozoans/mL; (+++) = 100-1000 protozoans/mL;
(++++) = >1000 protozoans/mL

Table 13. Microbiota condition in aeration sludge of 50 MLD Activated Sludge Plant

S.No. Microbiota Enumeration Microscopic Images Remarks

Amoeba

42
 Occurs under High
organic Load or
1. Arcella +
high F/M ratio

Flagellates

The indicator of
satisfactory effluent
quality appears
2. Peranema + when the sludge is
in the recovering
state.

Free Swimming Ciliates

The species
indicates the poor
3. Colpidium ++ settling condition of
sludge

43
 Appears when the
load is high, and its
number increased
when sludge is in
good condition.
4. Litonotus + However, the
number is not
adequate, so it
cannot direct its
function.

Sessile Ciliates

Treatment is
satisfactory if found
the inadequate
number. However,
6. Vorticella + the number is not
adequate, so it
cannot direct its
function.

Rotifers/Rotaria

Indicator of good
quality effluent
when found in
Worms
NIL abundance.
8. (Juvenile)
However, they are
absent from the
plant.

Filamentous organisms

44
 Multiplies in the
aerobic system
Filaments ++
9. leading to sludge
bulking

Where, (+) = 5-10 protozoans/mL;

(++) = 10-100 protozoans/mL;
(+++) = 100-1000 protozoans/mL;
(++++) = >1000 protozoans/mL
Condition of sludge:
 Pin point flocs but sludge condition was not satisfactory

The fewer number of microbiota species enumerated during the microscopic examination of the
aeration sludge depicted the unsatisfactory condition of the treatment process. High organic
load, coupled with low DO operation, lowers the count of stalked and free-swimming ciliated
protozoan species. It results in turbid effluent with elevated BOD and TSS concentrations. Thus,
plant performance is directly proportional to the presence of an adequate number of microbiota
species within the well-aggregated sludge flocs.

45
3.5 SUMMARY OF PERFORMANCE EVALUATION

The performance evaluation of STP was conducted by grab as well as composite sampling. In
addition, 24-h time variable concentrations of significant water quality parameters such as pH,
ORP, Conductivity, and COD were also analyzed and discussed.
 Based on the analysis of grab and composite samples, it was observed that the BOD
and TSS of secondary and tertiary treated effluent exceeded the discharge standards.
 The concentration of primary, secondary, and thickened sludge was found to be very
low. It indicates that the primary, secondary, and thickeners are not functioning properly.
 The concentration of digested sludge is 86714 mg/L, i.e., 8.764 % consistency. The
consistency assumed in the design is 6%. Hence, it shows that thickening takes place in
a digester; however, VSS degradation and gas production is much lower.
 The minimum and maximum pH, ORP, DO, and conductivity in the raw sewage, PST
outlet, CCT outlet, and tertiary varied from 7.2-8.3, -300 – (-550) mV, 0-5.1 mg/L and
1197-1265 µS/cm respectively. No large variations in pH values were observed, which
indicates that there is no mixing of industrial wastewater in the sewer system.
 Most of the heavy metals in treated effluent were lower than the prescribed standards.
Hence, the treated sewage can be applied for irrigation.
 Heavy metals in dewatered sludge were observed lower than the ceiling concentration
recommended by US EPA, 1995 Part 503 Rules.
 The actual operating MLSS and MLVSS are 2031 and 1114 mg/L, respectively, which
can be acceptable.
 DO in aeration tanks found to be varying between 0.16-0.75 mg/L. It indicates that the
aeration system is not functioning properly. As discussed minimum of 2 mg/L DO must
be maintained in the aeration tank for satisfactory removal of organic matter.
 The observed SVI is 139 ml/L, which is more than the targeted value of 125 ml/g. It
indicates slight sludge bulking in the aeration tanks system
 The specific oxygen uptake rate (SOUR) value was observed as 27.8 mg O2 g-1VSS.h,
which is acceptable.
 Operating F/M based on MLVSS is estimated as 0.68 d-1, which is very high. The system
should operate at the F/M ratio of 0.3-0.4 d-1. Hence, operating MLVSS concentration
needs to be increased to 2000 mg/L.
46
  Operating SRT of the system is estimated at 6.32 days, which is acceptable.
 Unit Sludge production is estimated at 1.07 kg TSS/kg-BOD removed, which is quite
higher than the literature value of 0.7 kg TSS/kg- BOD removed for conventional
activated sludge plants in the USA.
 A fewer number of microbiota species enumerated during the microscopic examination
of the aeration sludge depicted the unsatisfactory condition of the treatment process.
High organic load, coupled with low DO operation, lowers the count of stalked and free-
swimming ciliated protozoan species. It results in turbid effluent with elevated BOD and
TSS concentrations.

CHARACTERISTICS OF THE DEWATERED SLUDGE

As shown in figure 8 and Table 7, the dewatered sludge sample was collected in recycled plastic
bags from the centrifuge unit of the C-Tech plant.

8(a) 8(b)
47
 Figure 8(a) & 8(b): Dewatered sludge sample at 25 MLD Abohar, Punjab

Table7. Physico-chemical characteristics of the Dewatered Sludge

Organic Compost
Parameters Units Mean
(FCO 2009)

pH - 7.64 6.5-7.5

EC (µS/cm) 730 -

Moisture content (%) 62.71 15-25

Organic matter (%) 26.31 -

Total Organic Carbon, percent

(%) 14.62 12
by weight

Total Nitrogen (as N), percent

(%) 1.414 0.8
by weight

Total Phosphate (as P2O5),

(%) 0.58 0.4
percent by weight

Carbon Nitrogen Ratio (C/N) - 10.34 <20

 pH
the pH of the dewatered sludge range about 7.64. The desired limit of the Organic
compost (FCO 2009) standard is between 6.5 -7.5.
 Organic Carbon
Organic carbon of the dewatered sludge was 14.62 % (wt). The values were higher than
the desired limits of 2.62 % of the Organic compost (FCO 2009) standards.
 Total Nitrogen

48
 Total Nitrogen values of the dewatered sludge were 1.414 % (wt) as compared to the
desired limits of 0.8 % of the Organic compost (FCO 2009) standards.
 C/N ratio
The C/N ratio of the dewatered sludge was calculated about 10.34. The values were
within the desired limits of the Organic compost (FCO 2009) standards.
Most of the heavy metals in effluent were lower than the prescribed standards (Table 8). Hence,
the treated sewage can be applied for irrigation.

Table 8. Heavy Metal Concentration in Composite samples

50 MLD Standards
Heavy Metal Unit
Inlet Outlet (MOEFCC, 2016)

Total Cr mg/L 0.17 0.12 2

Pb mg/L 017 0.03 0.1

Co mg/L 0.01 0.01 -

Al mg/L 4.09 3.98 -

Mn mg/L 0.17 0.10 2

B mg/L 2.65 2.84 -

Cu mg/L 0.11 0.06 3

Cd mg/L 0.21 0.05 0.05

Ni mg/L 0.14 0.06 3

Zn mg/L 3.84 2.97 5

Heavy metals in dewatered sludge were observed lower than the ceiling concentration
recommended by US EPA, 1995 Part 503 Rules (Table 9).

Table 9. Heavy Metals in Dewatered Sludge

Organic Compost
Heavy Metal Unit Dewatered US EPA Ceiling
(FCO 2009)

49
 Sludge Concentration for Land

application of Biosolids

Total Cr mg/kg 105.78 420 50

Pb mg/kg 14.24 300-840 100

Co mg/kg 4.54 - -

Al mg/kg 4966.96 - -

Mn mg/kg 171.56 - -

B mg/kg 482.28 - -

Cu mg/kg 104.72 1500-4300 300

Cd mg/kg 21.56 39-85 5.0

Ni mg/kg 81.80 420 50

Zn mg/kg 1924.22 2800-7500 1000

SETTLING CHARACTERISTICS OF SLUDGE

The Characteristics of good sludge quality are:
1. Settles fairly rapidly as contrasted to a sludge that settles very fast of very slowly.
2. Concentrates uniformly in 30 to 60 minutes as contrasted to a sludge that concentrates
very rapidly is less than 30 minutes or very slowly needing 2 hours or more.
3. Produces flocculent solids that form large, strong flow particles that settle well, resist shear in
the aeration tank and filter the supernatant to remove stray particles.
4. Produces a clear supernatant.
5. It is a normally deep tan to light brown.
6. Normally has a pleasant musk or earthy odor.

Settling characteristics are generally quantified by SVI (Sludge Volume Index) measurement

Sludge Volume Index (SVI)

The sludge volume index (SVI) is the volume in mL occupied by one gram of MLSS after 30
minutes of settling in a 1,000 mL graduated cylinder and has units of mL/g. The SVI is a
50
measure of the settleability of the activated sludge in a secondary or final clarifier. Lower values
of the SVI indicate better sludge settleability.

Typical Values of SVI shows the quality of sludge (Figure 10)

Figure 10. Typical values of SVI and Quality of sludge

A bulking sludge is defined as one that settles and compacts slowly with a sludge volume index
(SVI) of >150 ml/g. An optimum “sludge age” exists, which provides adequate separation of the
cell mass from the liquid. For a specific system the optimum sludge age (SRT or sludge
wastage rate can be determined by
plotting SVI vs. Sludge age
(SRT) Figure 11

51
 Figure 11: Sludge Age vs. SVI

Procedure of SVI
 1L mixed liquor sludge was taken into a measuring cylinder
 Sludge was allowed to settle for 30 min to determine its SV30
 A proper settling of sludge was observed as after 30 minutes of settling, the sludge level
reached 200 ml in a one-liter cylinder (figure 6).

Figure 12: Sludge settling in 1000 ml measuring cylinder for SV30 at site
Before and after 30 min

52
 Parameters Unit Values

SV30 mL/L 280

SVI mL/g 139

The observed SVI is 139, which is more than the target of 125. It indicates that filaments are
higher in number than desired.
8.6 SLUDGE GENERATION & WASTING (ACTUAL)
PRIMARY SLUDGE
The sludge produced in the bottom of the primary clarifier is the primary sludge

Solids leaving as primary sludge (kg/d) = primary sludge SS mg/l x primary sludge flow
MLD
 Primary Sludge SS = 5644 mg/L (Table 6.0)
 Primary sludge Flow (m3/day) = Number of Pumps x Pump Capacity x operating
hours/day= 2 x 40 m3/h x 18.8 h/d =1504 m3/d or 1.5 MLD
 Solids leaving as primary sludge = 5644 mg/L x 1.5 MLD = 8466 kg/d

SECONDARY SLUDGE
The sludge needs to wasted from the bottom of the secondary clarifier to maintain the desired
mass of microorganisms in the tank. It is typically wasted when the actual MCRT or sludge age
is higher than the targeted value
Solids leaving as WAS (kg/d) = WASSS mg/l x WAS flow MLD
 WASSS mg/l = 3250 mg/L (Table 6.0)
 WAS flow Million (m3/d) = Number of Pumps x Pump Capacity x operating hours/day = 2
x 40 x 3.8 h = 304 m3/d or 0.3 MLD
 Soilds leaving as secondary sludge = 3250 mg/L x 0.3 MLD = 975 kg/d

TOTAL SLUDGE PRODUCED (DRY SOLIDS) = 8466 + 975 = 9441 Kg/d

Total BOD removed in the system (kg/d) = Flow (MLD) x (inlet BOD-outlet BOD) mg/L
= 50 MLD x (208-33) mg/L = 8750 kg/d (Table 7.0)

53
Unit Sludge production = kg TSS/kg BOD removed = 9441/8750 = 1.07.
The unit sludge production is on the higher side. It is 0.7 kg TSS/kg BOD removed for
conventional activated sludge plants in the USA (Table 12). The higher sludge production is due
to the higher percentage of inert or fixed solids in Indian sewage.

Table 12: Typical Unit Sludge Production in Suspended Growth STPs in the USA
Process Type Kg TSS/kg BOD removed
Primary Clarification 1.7
Activated sludge with primary clarification 0.7
Activated sludge without primary clarification – 0.85
Conventional
Extended Aeration 0.65

3.4.5 SLUDGE RETENTION TIME (SRT) OR MEAN CELL RESIDENCE TIME (MCRT)
The Mean Cell Residence Time (MCRT) or solids retention time is an average measure of how
long the microorganisms remain in contact with the substrate (food source). MCRT is also
known as solids retention time (SRT).

MCRT is not a mass balance, but rather a measure of how many days microorganisms are kept
in the activated sludge process before being wasted. Activated sludge treatment systems are
constantly generating new solids. In fact, as a rule of thumb, about 0.5 kg of new solids are
produced per kg of BOD removed. So if we remove 100 kg of BOD, we will produce about 50 kg
of new solids. If we fail to remove the new solids produced, the treatment system will suffer
deteriorated performance.

The amount of solids needed in aeration system depends on a number of factors, including
design, weather, and effluent limits. Typically, any system with ammonia-nitrogen limits will
require a higher MCRT, more air, and a corresponding higher MLSS concentration in the
aeration basin

MCRT = Solids in the aeration system (kg)

Solids leaving the system, (kg/day)

54
Solids in aeration system = MLSS (mg/l) x aeration tank volume (ML)

„Solids leaving the system‟ refers to the solids lost to waste activated sludge (WAS) and the
solids leaving the clarifier through the effluent.

Solids leaving as WAS (kg/d) = WASSS mg/l x WAS flow MLD

Solids leaving the clarifier as ESS kg/day = ESS mg/l x discharge flow, MLD

Putting the parts of the MCRT equation together

MCRT = solids in the aeration system, kg
solids in the effluent kg/day + solids wasted kg/d
Why is MCRT Used?
Control of the plant using the MCRT is accomplished by adjusting the sludge wasting and return
rates to achieve the desired MCRT.
Let‟s look at the equation again:

MCRT = Solids in the aeration system (kg)

Solids leaving the system, (kg/day)
We can see that increasing the “solids leaving the system” (wasting), will decrease the MCRT
(or shorten the solids retention time).

In general:
o Increasing the wasting rate will decrease the MCRT
o Decreasing the wasting rate will increase the MCRT.

NOTE:
A longer sludge age (MCRT) yields less sludge production than a younger sludge age (MCRT).
This is because BOD (food) is used for both maintenance energy (staying alive) and growth
(using excess food beyond that needed for maintenance requirements).

Calculation:
Solids in aeration system = MLSS (mg/l) x aeration tank volume (Million Liters)
= 2031 mg/L x 10.64 ML =21609 kg (MLSS) (Table 10)

55
Solids in the effluent kg/day = 49 mg/L x 50 MLD = 2450 kg/d (Table 7)
Solids wasted kg/d = 3250 mg/L x 0.3 MLD = 975 kg/d
SRT (days) = 21609 kg (MLSS) = 6.31 days
2450 + 975

Typical Values
MCRTs ranging from 3 to 15 days are typical for conventional activated sludge plants. MCRTs
less than three days will produce a sludge that is young with and slow settling and will reflect in
turbid effluent quality.

56
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