Wastewater Treatment

Jae K. (Jim) Park

Department of Civil and Environmental Engineering
University of Wisconsin-Madison

1
 Microorganisms – Classifications
Energy
 Solar radiation: Photo-synthetic autotrophs, e.g., algae
 Organics: Heterotrophs, e.g., activated sludge biomass,
denitrifiers, etc.
 Inorganics: Chemoautotrophs, e.g., nitrifiers
Oxygen use
 Obligate (strict): use only one condition for growth
 Facultative: use either dissolved oxygen or chemically
derived oxygen (from nitrate, sulfate or carbonate) for
respiration and use organic materials for energy and
growth
Temperature
 Psychrophiles: < 20°C., opt. 13°C
 Mesophiles: 20~45°C, opt. 35°C
 Thermophiles: 45~60°C, opt. 55°C
2
 Organic Matter  Energy for
Mircoorganisms

 Carbonaceous Energy: Carbon as energy source

Heterotrophs

 Nitrogenous Energy: Nitrogen as energy source

Chemoautotrophs

3
 Energy Measurement (1)
 Theoretical Oxygen Demand (ThOD)
 Chemical Oxygen Demand (COD)
 Biochemical (Biological) Oxygen Demand
(BOD)
 Carbonaceous BOD (C)

 Nitrogenous BOD (N)

 Total Organic Carbon (TOC)

4
 Energy Measurement (2)
 Theoretical Oxygen Demand (ThOD)
1. Carbonaceous demand: C  CO2; N  NH3
2. Nitrogenous demand: NH3  HNO2; HNO2  HNO3
3. ThOD = O2 req. in steps 1& 2
Ex. Glycine (10 mg/L) [CH2(NH2)COOH] (MW = 75 g/mol)
1. Carbonaceous demand
CH2(NH2)COOH + 1.5O2  2CO2 + H2O + NH3
2. Nitrogenous demand
NH3 + 1.5O2  HNO2 + H2O; HNO2 + 0.5O2  HNO3
3. ThOD = [1.5 + (1.5+0.5)] mol O2/mol glycine
= 3.5 × 32 g O2/mol = 112 g O2/mol
= 112  75 g/mol = 1.49 g O2/g glycine
Thus, ThOD = 1.49 x 10 mg/L = 14.9 mg/L
Cannot be used if chemical composition is not known. 5
 Energy Measurement (3)
 Chemical Oxygen Demand (COD)
 O2 req. for oxidation of organics
 Oxidize carbonaceous matter with a strong oxidant

(e.g., hot dichromate sol. with sulfuric acid)

2  Heat a  8c
CnHaOb  cCr2O7  8cH Catalyst nCO2  H2O  2cCr 3
silver sulfate 2
Dichromate
H2SO4 2n a b
c  
3 6 3
 Reduction of O2
4e- + 4H+ + O2  2H2O
1 mole of O2 (32 g)  4e- equivalents
1 g COD  1 g O2  1/8 electron equiv.
 NH not oxidized (carbonaceous energy only)
3
 Aromatic hydrocarbons (benzene and toluene) and

pyridines are not oxidized 6

 Domestic Wastewater
COD Fractionation
Influent COD
(Sti)
100%

Biodegradable Unbiodegradable
COD (Sbi) COD (Sui)
~80% ~20%

Sol. readily Partic. slowly Soluble Particulate

biodegradable biodegradable unbiodeg. unbiodeg.
COD (Sbsi) COD (Sbpi) COD (Susii) COD (Supi)
~20% ~60% ~7% ~13%

7
 Energy Measurement (4)
 Biochemical (Biological) Oxygen Demand (BOD)
 O2 required for microbial decomposition
 Oxygen consumption by microorganisms

BODu
DO consumed, mg/L

Nitrogenous
energy

BOD5
Inadequate to assess the electron
Carbonaceous
donor capacity; after 5 days, still
energy
some biodegradable matters exist.

5 Time, days ~30

8
 Energy Measurement (5)
 Biochemical (Biological) Oxygen Demand (BOD)
 Carbonaceous BOD: aerobic heterotrophs
 Decompose organic molecules to minerals (CO )
2
and residues
 Obtain their cell carbon from the organic material

 Nitrogenous BOD: obligate aerobic chemoautotrophs

 Characteristics of nitrifiers (chemoautotrophs)

 DO < 2 mg/L action slow

 DO < 0.5 mg/L action ceases
 Optimum pH: 8.0; pH < 7.2: slows down
 More sensitive than heterotrophs to toxins
 Slow growers (longer sludge age required)
9
 Inert Organic Matter
 Measured with COD
 Not biodegraded, thus not measured with BOD5
 Polymerized waste product
 Inert material from lysed cells
 Refractory organics: humic acid (M.W. –
5,000~100,000); fulvic acid (2,000~10,000)
 Certain high M.W. carbohydrates alone or in
combination with humic material are resistant to
microbial attack.
 High M.W. carbohydrates are excreted at the end of
the logarithmic growth phase and help forming
flocs by bridging of bacterial cells.
10
 Acclimated Culture

 Selection of populations by controlling

environmental factors to encourage
only the desired species.
 An increase in the biodegradation rate of
a chemical after exposure of the microbial
community to the chemical for
some period of time.

11
 Example of Acclimation
Lag phase Result of acclimation
Acclimation
25
Chemical concentration
20
Concentration, mg/L

CO 2 production, vol.
Biomass conc., mg/L
15

10
CO2 production
5
Microbial biomass
0
0 5 10 15 20 25 30 35 40
Days

12
 Example Acclimation
 Hazardous Industrial Wastewater

NBW

NBW

NBW
Feed

NBW
RBW

ratio
RBW

RBW

RB
W
Biological Treatment
Day 1 Day 2 Day 3 Day 4 Day 5

RBW: Readily biodegradable wastewater, e.g., glucose, methanol,

domestic wastewater, etc.
NBW: Not readily biodegradable wastewater, e.g., industrial
wastewater, hazardous wastewater, polychlorinated biphenyls
(PCBs), pentachlorophenol, etc.
13
Influence of Acclimated Biomass
on COD of Treated Wastewaters
BOD5 equivalents
Total COD – BOD5
COD, mg/L

Not readily
Biodegradable Non-biodegradable
COD COD

Untreated Treated with Treated with

Raw unacclimated acclimated
biomass biomass
BOD: Not affected by acclimation
COD: Significantly affected by acclimation 14
 BOD, CODCr, CODMn, TOC
Organic matter
Biodegradable Unbiodegradable

TOC
CODCr Cl-, H2S

CODMn Cl-, H2S

BOD5 Nitrification

15
 Energy Measurement (6)
 Total Organic Carbon (TOC) O2 + 4H+ + 4 e- = 2 H2O
 Oxidize in a combustion chamber with O 2
 Easy to measure
• TOC values are very
Glucose, C6H12O6 (M.W. = 180) similar for both glucose
C6H12O6 + 6 O2  6 CO2 + 6 H2O and glycerol; however,
COD values are quite
6 moles O2  6  4 = 24 e- different.
24/6 = 4 e- available per unit organic C • Thus, waste specific;
Ex. 100 mg/L of glucose: TOC and COD = ? cannot apply the result
TOC: (6  12)/180  100 = 40 mg/L C to other WWTPs.
COD: (6  32)/180  100 = 107 mg/L O • Good as an operational
Glycerol, C3H8O3 (M.W. = 92) tool with previous
C3H8O3 + 7/2 O2  3 CO2 + 4 H2O historical data.
7/2 moles O2  7/2  4 = 14 e-
14/3 = 4.67 e- available per unit organic C Similar
Ex. 100 mg/L of glycerol: TOC and COD = ?
TOC: (3  12)/92  100 = 39 mg/L C Different
COD: (3.5  32)/92  100 = 122 mg/L O 16
 Energy Measurement (7)
 BOD5/COD ratio: a good indicator for
biodegradability of a specific wastewater
 Domestic wastewater
 BOD5/COD ­0.4 ~ 0.8
 BOD5/TOC ­1.0 ~ 1.6

 BOD5/COD  0.6: can be decomposed

completely, biological treatment feasible
 BOD5/COD  0.2: cannot be decomposed
easily, chemical or physical treatment desired
 BOD5/COD  0: has toxic materials
17
 TOC Analyzer
Measure the amount of total organic
carbon present in a liquid sample;
Convert inorganic carbon in the sample to
CO2 after adding acid and strip CO2 by a
sparge carrier gas;
Oxidize organic carbon by either
combustion, UV persulfate oxidation,
ozone promoted, or UV fluorescence; and
Measure CO2 stripped using the
conductivity or non-dispersive infrared
(NDIR) detection system.

On-Line TOC Analyzer: a reagentless

analyzer designed for continuous
monitoring of organics. 18
 Use of BOD5, COD, and TOC
BOD5
 Good for regulating organic loading to a receiving water
body for DO depletion by heterotrophs
 Not good for design since some organics biodegrade
slowly or after acclimation
COD
 Not good for regulation since it does not reflect true
organic loading impact to aqua systems
 Good for design if the input and output within a
biological system is monitored; true energy count for
carbonaceous energy only
TOC
 Good for operating a wastewater treatment plant due
to real time monitoring capability
 Values cannot be transferred to other wastewater due
to specificity of carbon in the wastewater in terms of
electro donor capability
19
 Priority Pollutants
 Designated by EPA in 1979
 A list of 126 specific pollutants that includes 14 heavy
metals and 112 specific organic chemicals
 Heavy Metals (Total and Dissolved): heavy, dense,
metallic elements that occur only at trace levels in
water, but are very toxic and tend to accumulate
 Pesticides: DDT, Aldrin, Chlordane, Endosulfan, Endrin,
Heptachlor, and Diazinon
 Polycyclic Aromatic Hydrocarbons (PAHs):
naphthalene, anthracene, pyrene, and benzo(a)pyrene
 Polychlorinated biphenyls (PCBs): organic chemicals
that formerly had widespread use in electrical
transformers and hydraulic equipment
20
 Fats, Oils, and Grease (FOG)
 Sewage backups and overflows can lead to costly
clean ups and repairs, as well as public health
concerns.
 Many utilities acknowledge fat, oil, and grease
(FOG) as the main cause of sewer clogging.
 EPA estimated that utilities spend on average
$33,000 per mile of sewer per year on capital project
and $8,000 per mile for O&M (2004).
 The capital investment in wastewater infrastructure
is over $13 billion annually (EPA, 2002).
 Local government and utilities pay up to 90% of
capital expenditures on wastewater infrastructure
(AMSA and WEF, 1999).
21
Causes of Sanitary Sewer Overflow

Line breaks Misc. (5%)

(10%) Blockages (48%)
Mechanical or
power
failures (11%)

Wet weather I/I (26%)

EPA, 2004
22
 Causes of Sewer Clogging
Roots and FOG
(4%) FOG (47%)
Roots (22%)

Grit, rock, and other debris (27%)

EPA, 2004
23
 Sewer Clogging

FOG

Wastewater

24
Consequences of Sewer Clogging
Sewer overflow

25
Consequences of Sewer Clogging
Odor

Crown corrosion of
H2S + 2O2  H2SO4
Bacteria
concrete pipes
26
Consequences of Sewer Clogging
Odor

27
 Trans Fatty Acids (TFA)
 Created in an industrial process that adds hydrogen
to liquid vegetable oils to make them more solid
 Easy to use, inexpensive to produce, and last a long
time
 Give foods a desirable taste and texture
 Use trans fats to deep-fry foods because oils with
trans fats can be used many times in commercial
fryers
 Raise bad LDL (low density lipoproteins ) cholesterol
levels and lower your good HDL (high density
lipoproteins) cholesterol levels
28
 Use of Zero Trans Fatty Acids
 Inefficient removal in conventional grease
removal systems
 Potential foaming in wastewater treatment
plant aeration basins
 No knowledge on the fate of zero trans fatty
acids in sewers and wastewater treatment
plants

29
 Prevention of Sewer Clogging (1)
 Grease trap or interceptors,
exhaust hood filters, and
floor mats
 Proprietary grease removal
devices

30
Prevention of Sewer Clogging (2)
 Chemicals and additives (emulsifiers, detergents or caustic
substances) that claim to dissolve grease
 Prohibited for use as an additive because these substances
reduce the efficiency of the interceptor or trap
 Best Management Practices (BMP) during daily operations to
keep FOG out of drains leading to the sewer
 Enzymes
 Prohibited as additives due to the same effect as
emulsifiers
 Microorganisms
 Not prohibited as an additive
 Education

31
 Nitrogen
 Main species
Organic nitrogen
NH4+: Ionized ammonia, nutrient to algae
NH3: Free (unionized) ammonia, toxic to fish
NO2-: Intermediate byproduct of nitirification, < 1
mg/L, causes the hemoglobin in the blood to
change to methemoglobin, cause
methemoglobinemia (‘blue baby’ syndrome)
NO3-: Final product of nitrification, undeveloped
digestive tracts of an infant possess bacteria
that convert nitrate into nitrite, < 10 mg/L
32
 Nitrogen Transformation
in Biological Treatment Processes
Organic nitrogen
(proteins, urea, etc.)

Bacterial decomposition
and hydrolysis
Ammonia nitrogen Assimilation Organic nitrogen Organic nitrogen
(NH3-N) (bacterial cells) (net growth)
O Lysis and autooxidation
Nitrification

Nitrite (NO2-)
O2
Denitrification
Nitrate (NO )
3
-
Nitrogen gas (N2)

Organic carbon
(substrate)
33
 Subdivision of Total Influent TKN
Influent TKN
(Nti)
100%

- Organically
NH 3 & NH 4 (Nai) bound N (Nti - Nai)
~75% ~25%

Unbiodegrad. Unbiodegrad. Biodegrad.

soluble N Particulate N N
(Nui) (Npi) (Nai)
~3% ~10% ~12%

Total Kjeldahl Nitrogen (TKN): sum of organic nitrogen, ammonia

(NH3), and ammonium (NH4+) in biological wastewater treatment
34
 Free Ammonia (Unionized, NH3) and
Ionized (NH4+) Ammonia

More
toxic
to fish
pH
eff
ect

More toxic
Temperature effect
to fish

Ammonia not
regulated in winter 35
Nitrification: Chemoautotrophs (1)
 Nitrification: Conversion from ammonia to NO2- / NO3-

NH4+ + 1.5O2Nitrosomonas NO2- + H2O + 2H+ + New biomass

Nitrobactor
NO2- + 0.5O2 NO3- + New biomass
 Oxygen demand
Conversion of NH4 to NO2 Conversion of NO2- to NO3
g g
1.5 mol  16 2O 0.5 mol  16 2O
mol gO mol gO
 3.43  1.14
g gN g gN
1 mol  14 N 1 mol  14 N
mol mol
 Total oxygen demand for nitrification: 4.57 g O/g N
36
Nitrification: Chemoautotrophs (2)
 CO2 (carbonate): carbon source
 Ammonia: energy transfer source in a non-
assimilative way so only a small amount of biomass
(sludge) is produced
NH4+ + 1.5O2Nitrosomonas NO2- + H2O + 2H+ + New biomass
H+ +CO32- → HCO3-; H+ +HCO3- → H2CO3
 Alkalinity
 2H+  1 mol alkalinity [CaCO3 (40+12+16×3=100
g/mol)]
 100 g Alk/14 g N = 7.14 g Alk consumed/g N nitrified

37
Nitrification: Chemoautotrophs (3)
Example
 Influent TKN = 42 mg N/L; Effluent TKN  2 mg/L
 Alkalinity = 200 mg/L as CaCO3
Oxygen demand?
 4.57 g O/g N × (42 – 2) mg N/L = 182.8 mg O/L
Alkalinity after nitrification?
 7.14 g Alk/g N × (42 – 2) mg N/L = 285.6 mg/L as CaCO 3
 Unless additional alkalinity (CaO, Na2CO3, NaOH, etc.)
is added, nitrification will stop (see the next slide).
 Since the influent is 200 mg/L, 85.6 mg/L + 10~15 mg/L
(residual) = 95.6~100.6 mg/L as CaCO3 required
38
 Effect of pH on Nitrification

Operational
range

Nitrifiers: very sensitive to pH

Thus, buffer capacity (alkalinity) of wastewater important
39
Denitrification: Heterotrophs (1)
2NO3- + 10e- + 12H+ → N2  + 6H2O
gN
2 mol  14
mol  2.8 g N/e 
10 e  gO
8 
O2 + 4e- + 4H+ → 2H2O e  2.86 g O
gO gN gN
1 mol  16 2 2.8 
mol 
e
 8 g O/e
4 e
gO
2.86 (denitrification)
gN
 100  63% O saved
gO
4.57 (nitrification)
gN
40
 Denitrification : Heterotrophs (2)
 ~1 mol of H+ is recovered from denitrification

 Thus, Alk of 3.57 g/g N recovered

 For low alkalinity water, denitrification is

recommended.
 Denitrification conditions
No O2
Readily biodegradable soluble substrate (COD)

 For complete removal of nitrogen species from

wastewater: nitrification followed by
denitrification
41
 Substrate Requirement
for Denitrification
0.67 0.33
CODutilized = CODbiomass + O2 utilized = fcv  X + O2 utilized
= YCOD CODutilized + O2 utilized
fcv = COD/VSS = CODbiomass/X (mg COD/mg VSS)
YCOD = CODbiomass/CODutilized (mg COD/mg COD)
Biomass
empirical O2 utilized = (1 - YCOD) CODutilized
formula
YCOD = fcv Yh (mg VSS/mg COD)
C5H7O2N + 5O2  5CO2 + 2H2O + NH3
(5 × 16 × 2 g)  (1 × 113 g) = 1.42 mg COD/mg VSS
COD/VSS = 1.42 mg COD/mg VSS 0.67
O2 = (1 - fcvYh) CODutilized
Nitrate consumption per mg COD utilized
2.86 mg O2/mg NO3- N  (1 - 1.42·0.47) mg O2/mg COD
42
 COD Requirement for
Denitrification
Example
 CODinf = 400 mg/L, CODeff = 50 mg/L, TKNinf = 55 mg
N/L, TKNeff = 5 mg N/L, Q = 10 MGD
 COD (methanol) required for denitrification?
Solution
 (55-5) mg N/L × 8.6 mg COD/mg N = 430 mg COD/L
req.
 [430-(400-50)] mg/L = 80 mg COD/L req.
 Methanol (CH3OH) (MW = 32 g/mol)
 CH3OH + 1.5O2 → CO2 + 2H2O
 80 mg COD/L×10 MGD  1.5×16×2 g COD/32 g MeOH =
202 kg/day = 445 lb of MeOH/day 43
 Phosphorus
Source: human body waste, food
waste, various household detergents

Subdivision of Total Influent P

Influent TP
(Pti)
100%

Organically
Sol. PO (Psi)
4
-
bound P (Pti - Pbi)
70 ~ 90% 10 ~ 20% 10 ~ 30%
in the activated
sludge process
44
 Forms of Phosphate
mg/L
Old Now Forms
5 4 Orthophosphate
3 0 Tripolyphosphate (detergents)
1 0 Pyrophosphate (breakdown of tri-P)
1 1 Organic phosphates
0 ? Hexametaphosphate (corrosion inhibitor)
10 5 Total

Why? Ban of phosphate-based detergents

45
 Phosphorus in Wastewater
 Addition to water
Corrosion (and scale) control in drinking
water
Industrial water softening
Boiler waters
Cleaning compounds

 Sewage
1.2 lb/capita/yr from human and food waste

46
 Mechanisms of Polyphosphate-
Accumulating Organisms (PAOs)
Short chain
fatty acids
(SCFAs) New Cell
Organic (Acetate)
substrate NADH, ATP
PHA
Glycogen
ATP Glycogen
ATP PHA
Facultative
microbes Poly-P Poly-P

PAOs PO4 3- PAOs PO4 3-

Anaerobic condition Aerobic condition

PHA: Polyhydroxyalkanoates 47
 Observations in Biological Phosphorus
Removal (BPR) Systems
AN O
Ortho-P
Bulk
Acetate Liquid

PHA

Glycogen Biomass
Poly-P
Reaction Time
PHA: Polyhydroxyalkanoates 48
 BPR Mechanism

Anaerobic Aerobic

Ortho-P
mg/L

Acetate
Time
Biomass Biomass
Poly-β-
hydroxybutyrate
(PHB)
(Storage) Poly-P PHB Poly-P
49
 Anaerobic/Oxic Process
Readily
biodegradable Vital for P Uptake
soluble COD

Excess sludge
Better SRT control
SRT: Solid retention time, sludge age, or mean cell residence
time (MCRT); total biomass in the system/biomass wasted/loss 50
Whole Effluent Toxicity
Bioassay

51
 Bioassay
 Part of whole effluent toxicity (WET) tests for
NPDES permit
 Use of a biological organism to test for
chemical toxicity

Mysidopsis bahia, female, approx. 6 mm in length

Ceriodaphnia dubia 52
 Use of Toxicity Testing in Water
Quality Based Toxics Control
 To characterize and measure the aggregate
toxicity of an effluent or ambient waters
 To measure compliance with whole effluent
toxicity limits
 As an investigative tool and to measure
progress in a toxicity reduction program
 As an ambient instream measure of toxicity to
identify pollution sources

53
 Bioassay
 Tested sample: most commonly, effluent from
industrial or municipal wastewater discharges
 Sample holding time: max. 36 hrs stored at 4°C
 Test organisms
Ceriodaphnia dubia (water flea)
Pimephales promelas (fathead minnow)
Cyprinella leedsi (bannerfin shiner)
Mysidopsis bahia (mysid shrimp)
Menidia beryllina (tidewater silverside)
 Acute test: 24, 48, or 96 hrs (species specific)
 Chronic test (short-term): 4~10 (7) days

54
Rules for Conducting Toxicity Tests
 40 CFR 136.3 -Table 1A (List of Approved
Biological Methods)
 Effective November 15, 1995

 Amended November 19, 2002 and effective

December 19, 2002
 Methods must be followed as they are
written
 Incorporate by Reference
 Methods for Measuring the Acute Toxicity of Effluents
to Freshwater and Marine Organisms. 5th Edition,
USEPA, Office of Water, October 2002, EPA 821-R-02-
012
 Short-term Methods for Estimating the Chronic
Toxicity of Effluents and Receiving Waters to
Freshwater Organisms. 4th Edition, USEPA, Office of
Water, October 2002, October 2002, EPA 821-R-02-
013
 Short-term Methods for Estimating the Chronic
Toxicity of Effluents and Receiving Waters to Marine
and Estuarine Organisms. 3rd Edition. USEPA, Office
of Water, October 2002, EPA 821-R-02-014
56
 USEPA Methods Documents
 Health and safety
 Quality assurance
 Facilities, equipment and supplies
 Test organisms and culture methods
 Dilution water
 Effluent sampling and handling
 Endpoints and data analysis
 Individual test methods
 Report preparation and test review
57
 Test Types
 Acute and Short-term Chronic Tests
Static non-renewal
Static renewal
Flow through
 Test Species dependent
 Use dependent

58
 Test Design
 5 Concentrations + Control
Serial dilution’s of effluent and “control
water” (also termed “dilution water”)
Dilution series of 0.5 or greater
Single concentration test

 Replicates

 Randomization (organisms/chambers)
 Acute Toxicity Tests
 Test Procedures
96 hours or less (species specific)
Mortality is the measured endpoint
For daphnia mortality determined by
immobilization
 Advantages
Less expensive and time consuming than chronic
Endpoint is easy to quantify
 Disadvantages
Indicates only lethal concentrations
Only the effects of fast acting chemicals are
exhibited 60
 Acute Test Acceptability Criteria
 Minimum control survival at least 90%

 Temperature maintained at 20±1o C

 Maximum test organism age at start:

14 days for fish
5 days for Mysid shrimp
24 hours for daphnids (Ceriodaphnia dubia
and Daphnia magna)

61
 Short-term Chronic Toxicity Tests
 Test Procedures
Typically 4-10 days
Mortality, growth, fecundity, reproduction
 Advantages
More sensitive than acute, assess parameters
other than lethality
May better reflect real world
 Limitations
More costly and time intensive than acute
More sensitive to low level contamination

62
 Chronic Test Acceptability Criteria
 Minimum control survival 80%
 Minimum control dry weight (average):
0.25 mg for fish
0.20 mg for Mysid shrimp
 Minimum of 15 young (average) for control C. dubia
 Temperature maintained @ 25 +/- 1o C
 Maximum test organism age at start:
48 hours for fish
7 days for Mysid shrimp
24 hours for daphnids

63
 Selection of Dilution Water

 May be either a standard laboratory water or the

receiving water
 Choice of water is dependent on the objectives of
the test
Absolute toxicity use standard water
Estimate of toxicity in uncontaminated
receiving water, use receiving water
Contaminated receiving water, use laboratory
water

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 Acute Test Endpoints
 LC50 - Concentration of effluent that is lethal
to 50 percent of the exposed organisms at a
specific time of observation (e.g. 96 hr LC50),
(expressed as % effluent)
 NOAEC - No Observed Adverse Effect
Concentration
Lowest concentration at which survival is
not significantly different from the control
always set equal to 100% effluent
 EC - Effect Concentration

65
 Test Data
 Typical dose response where mortality
increases as the concentration of effluent
in the mixture increases.
 LC50 would be somewhere between 25%
effluent and 50% effluent.

6.25 % 12.5 % 25.0% 50.0% 100.0%

Control Effluent Effluent Effluent Effluent Effluent

0% Mortality 0% mortality 20 % Mortality 40% Mortality 80% Mortality 100% Mortality

66
 Chronic Test Endpoints
 IC25 - Inhibition Concentration- Concentration
of effluent which has an inhibitory effect on 25%
of the test organisms for the monitored effect,
as compared to the control (expressed as %
effluent).

 NOEC - No Observable Effect Concentration -

Highest concentration of effluent tested which
shows no statistically significant effect on the
organisms as compared to the control
(expressed as % effluent).

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 Toxicity Values
 NPDES permits in the past used a “no observable
effect concentration” (NOEC) to measure chronic
toxicity and a 96-hour lethal concentration 50
(LC50) to measure acute toxicity.
 Permits are now being issued with an inhibition
concentration 25% (IC25).

Limit: more stringent LC50, IC25, NOAEC

LC50, IC25 Toxicity

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 Toxic Units (TU’s)
 Reciprocal of the fractional LC50, NOEC, IC25
value

 Calculated by dividing the value into 100

TUa = 100/LC50
TUc = 100/IC25

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Methodology for Setting Limits (1)
 IC25 is a calculation based on the design flow of the
POTW and the seven-day low flow over 10 years in
the receiving stream (7Q10) as follows:
IC25 = design flow/(7Q10 + design flow) × 100
Example:
 The low flow for the receiving stream (7Q10) is 23
MGD. The design flow for the POTW is 4 MGD.
IC25 = 4/(23+4) × 100
IC25 = 14.8%
 The POTW demonstrates toxicity if the test value is
less than or equal to the calculated value of 14.8%.
This constitutes a violation of the NPDES permit.
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 Methodology for Setting Limits (2)
 A serial dilution that the laboratory: 59.2, 29.6,
14.8, 7.4, 3.7 and a control with 0% effluent.
 Toxicity is demonstrated if there is a statistical
significant difference in any dilution from the
control set.
 The difference can be in any of the three
parameters: survival, reproduction, or growth.
 In the example, the effluent fails if toxicity
appears in the 14.8% or 7.4% or 3.7% dilutions.

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 Redox Reaction

Organic molecule (C+H+O+N+S+P) Electron

(wastewater, hazardous chemicals, donor
etc.)

Oxidized

CO2, H+, and e-

Reduced
Electron
A molecule acceptor
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 Aerobic Condition O

 Aerobic respiration
 O2 present
 Electron acceptor: O2 (→ H2O)
 Good for large volumes of dilute
wastewater (< 500 mg BOD5/L)
 High growth rates, thus high sludge
production (0.3~1 lb VSS/lb BOD5)
 Produce a more stable end product
73
 Anoxic Condition AX

 Anaerobic respiration (denitrification)

 No dissolved oxygen
 NO3- and NO2- present
 Electron acceptor: NO2- and NO3- (→ N2 + H2O)
 Relatively high sludge production
 Should be avoided in the clarifier

74
 Anaerobic Condition AN

 Fermentation
 No O2, NO3-, NO2-, or SO42- present
 Electron acceptor: endogenously generated by the
microorganism
 Good for concentrated wastes (> 1000 mg BOD5/L)
 Low sludge production

Complex organic compounds

Low molecular weight fatty acids

CH4, CO2, and H2O

75
 Microbial Classification
Carbon
Energy CO2 Organics
Chemical Chemoautotroph Chemoheterotroph
H2 bacteria Animals
Sulfur bacteria Most bacteria
Nitrifiers Fungi
Iron bacteria Protozoa
Electron donor H2, S, H2S, Fe2+, NH3, NO3- Organics (reduced)
Light Photoautotroph Photoheterotroph
Green plants Few algae
Algae Cyanobactor
Purple bacteria Some purple & green
Green bacteria bacteria
Electron donor H2O, S, H2S Organics (reduced)

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 Oxidation-Reduction Potential (ORP)
ORP Process Electron Conditions
mV acceptors
3
+300 2 Oxic
1
O2 or
+200
aerobic
+100 4
0 NO3- Anoxic
-100
SO42-
-200 5 Fermentative
6 7
-300 anaerobic
8 Carbonaceous
-400 organics

1. Organic carbon oxidation 4. Denitrification 7. Acid formation

2. Polyphosphate release 5. Polyphosphate uptake 8. Methane formation
3. Nitrification 6. Sulfide formation
77
Three Origins of Life
Phylogenetic Tree
Bacteria
Origin of life
Archaea

Eukarya

The phylogenetic tree

shows that the Eukarya are
more closely related to the
Archaea than they are to
the Bacteria.

PROCARYOTA
78
 Archaea
 A group of single-celled microorganisms
 Requires neither sunlight for photosynthesis as do
plants, nor oxygen.
 Absorbs CO2, N2, or H2S and gives off methane gas as
a waste product.

79
 Bacteria
 Single-celled microorganisms which can exist either
as independent (free-living) organisms or as
parasites, typically 0.5–5.0 µm length
Classification
 Shape

 Ability to form spores

 Method of energy production
(glycolysis for anaerobes,
cellular respiration for aerobes)
 Nutritional requirements
E. Coli  Reaction to the Gram stain
80