Environmental Biotechnology Lecture Note-220110

LECTURE NOTE
ENVIRONMENTAL BIOTECHNOLOGY
Dr. Tran Thi My Dieu
Ho Chi Minh, January 2010

ENVIRONMENTAL BIOTECHNOLOGY
Chapter 1
INTRODUCTION
Dr. Tran Thi My Dieu
VAN LANG UNIVERSITY Jan. 2010 DENTEMA
CONTENTS
The roles of bioprocesses in

environmental technology
Applications of bioprocesses in
environmental technology
VAN LANG UNIVERSITY DENTEMA
THE ROLES OF BIOPROCESSES
Is it important? Why?

APPLICATIONS OF BIOPROCESSES
IN ENVIRONMENTAL TECHNOLOGY
Biological wastewater treatment

and reuse
Biological solid waste treatment
and reuse
Biological air pollution treatment

and reuse
IN ENVIRONMENTAL TECHNOLOGY Lan can baûo veä
OÁng thu nöôùc sau xöû lyù
Biological WWT Saøn coâng taùc
Maùng thu nöôùc

daïng raêng cöa
Thieát bò taùch pha

OÁng daãn khí
khí – loûng - raén
Vaùch höôùng
doøng hình coâ n
Caàu thang
Voû thieát bò
OÁng thoaùt
khí
Hoã n hôïp
Bình haáp nöôùc thaûi
thuï khí
Lôùp buøn kî khí
Boït khí
OÁng bôm nöôùc
vaøo thieát bò UASB
UASB Dung dòch

NaOH 5%
Boä phaän phaân phoái
ñeàu löu löôïng nöôùc
thaûi
Biological wastewater treatment & reuse
VAN LANG UNIVERSITY Granular sludge DENTEMA

India
VAN LANG UNIVERSITY UASB DENTEMA
692 days
752
A B C D E
752 days
832
A B C D E
832 days
852
A B C D E
A shift in population is noticeable (Dominated by Methanosaeta like organism)-
organism)- 832
days
Granules – B reactor ( Bulk reetha seed ) showed increased methongenic
population, Which is reflected in the IAS studies for the same period
period
UASB
Suspended growth AS Attached growth AS

400m 3
400m/D3/D
Trickling filter

RBC
RBC
Nitrogen
removal

Biodegradable constituents removal

Nutrient removal (N & P)
Specific trace organic compound removal
WHY?
Biological solid waste treatment & reuse
Digester
BTA PROCESS
Digester

Biological solid waste treatment
DRANCO PROCESS, BELGIUM, 1984
composting plant Open heap composting

Drum, with artificial

Box/container composting aeration system
Anaerobic digester Anaerobic digestion plant


Reuse biowaste to produce compost

Reuse biowaste to produce biogas
Increase biodegradable efficiency of
organic materials in landfill
WHY?
Odor removal by EM
Methan gas (landfill gas) removal
WHY?
THE ROLES OF BIOPROCESSES
Environmental friendly treatment

technologies of biodegradable wastes
To transform biodegradable constituents
into acceptable end products
Reduce harm to environment & public
health
Reduce treatment cost

Solid wastes Gas recovery Wastewater

Cassava harvesting Market
Livestock feed Composting WWTS

processing
Fish culturing
Breeding
Resource
Cassava cultivation
QUESTION?
Your Roles?

CHAPTER 2
FUNDAMENTALS OF BIOLOGICAL
WASTEWATER TREATMENT
2.1 OVERVIEW OF BIOLOGICAL WASTEWATER TREATMENT
2.1.1 Objectives of biological treatment
The overall objectives of the biological treatment of domestic wastewater are to (1) transform
dissolved and particulate biodegradable constituents into acceptable end products, (2) capture
and incorporate suspended and nonsettleable colloidal solids into a biological flocs or biofilm,
(3) transform or remove nutrients such as nitrogen and phosphorus, and (4) in some cases,
remove specific trace organic constituents and compounds.
For industrial wastewater, the objective is to remove or reduce the concentration of organic and
inorganic compounds. Because some of the constituents and compounds found in industrial
wastewater are toxic to microorganisms, pretreatment may be required before the industrial
wastewater can be discharged to a municipal collection system.
For agricultural irrigation return wastewater, the objective is to remove nutrients, specifically
nitrogen and phosphorus that are capable of stimulating the growth of aquatic plants.
2.1.2 Role of microorganisms in wastewater treatment
The removal of dissolved and particulate carbonaceous BOD and the stabilization of organic
matter found in wastewater is accomplished biologically using a variety of microorganisms,
principally bacteria. Microorganisms are used to oxidize (or convert) the dissolved and
particulate carbonaceous organic matter into simple end products and additional biomass, as
represented by the following equation for the aerobic biological oxidation of organic matter:
Organic material + xO2 + yNH3 + zPO43- microorganisms new cells + tCO2 + uH2O (2-1)
where x, y, z, t, u = the stoichiometric coefficient.
In the Eq (2-1), oxygen, ammonia and phosphate are used to represent the nutrients needed for
the conversion of the organic matter to simple end products (carbon dioxide and water). The
term shown over the directional arrow is used to denote the fact that microorganisms are needed
to carry out the oxidation process. The term new cells is used to represent the biomass produced
as a results of the oxidation of the organic matter. Microorganisms are also used to remove
nitrogen and phosphorus in wastewater treatment processes. Specific bacteria are capable of
oxidizing ammonia (nitrification) to nitrite and nitrate, while other bacteria can reduce the
oxidized nitrogen to gaseous nitrogen. For phosphorus removal, biological processes are
configured to encourage the growth of bacteria with the ability to take up and store large
amounts of inorganic phosphorus.
Because the biomass has a specific gravity slightly greater than that of water, the biomass can be
removed from the treated liquid by gravity settling. It is important to note that unless the biomass
produced from the organic matter is removed on a periodic basis, complete treatment has not
Environmental Biotechnology Handout
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been accomplished because the biomass, which itself is organic, will be measured as BOD in the
effluent. Without the removal of biomass from the treated liquid, the only treatment achieved is
that associated with the bacterial oxidation of a portion of the organic matter originally present.
2.1.3 Types of biological processes for wastewater treatment
The principal biological processes used for wastewater treatment can be divided into two main
categories: suspended growth and attached growth (or biofilm) processes.
Suspended growth processes
In suspended growth processes, the microorganisms responsible for treatment are maintained in
liquid suspension by appropriate mixing methods. Many suspended growth processes used in
municipal and industrial wastewater treatment are operated with a positive dissolved oxygen
concentration (aerobic), but applications exist where suspended growth anaerobic (no oxygen
present) reactors are used, such as for high organic concentration industrial wastewaters and
organic sludges. The most common suspended growth process used for municipal wastewater
treatment is the activated sludge process.
The activated sludge process was developed around 1913 Lawrence Experiment Station in
Massachusetts by Clark and Gage (Metcalf and Eddy, 1930), and by Arden and Lockett (1914) at
the Manchester Sewage Works in Manchester, England. The activated sludge process was so
named because it involved the production of an activated mass of microorganisms capable of
stabilizing a waste under aerobic conditions. In the aeration tank, contact time is provided for
mixing and aerating influent wastewater with the microbial suspension, generally referred to as
the mixed liquor suspended solids (MLSS) or mixed liquor volatile suspended solids (MLVSS).
Mechanical equipment is used to provide the mixing and transfer of oxygen into the process. The
mixed liquor then flows to a clarifier where the microbial suspension is settled and thickened.
The settled biomass, described as activated sludge because of the presence of active
microorganisms, is returned to the aeration tank to continue biodegradation of the influent
organic material. A portion of the thickened solids is removed daily or periodically as the
process produces excess biomass that would accumulate along with the non-biodegradable solids
contained in the influent wastewater. If the accumulated solids are not removed, they will
eventually find their way to the system effluent.
An important feature of the activated sludge process is the formation of floc particles, ranging in
size from 50 to 200 µm, which can be removed by gravity settling, leaving a relatively clear
liquid as the treatment effluent. Typically, greater than 99 percent of the suspended solids can be
removed in the clarification step.
Attached growth processes
In attached growth processes, the microorganisms responsible for the conversion of organic
material or nutrients are attached to an inert packing material. The organic material and nutrients
are removed from the wastewater flowing past the attached growth also known as a biofilm.
Packing materials used in attached growth processes include rock, gravel, slag, sand, redwood,
and a wide range of plastic and other synthetic materials. Attached growth processes can also be
operated as aerobic or anaerobic processes. The packing can be submerged completely in liquid
or not submerged, with air or gas space above biofilm liquid layer.
The most common aerobic attached growth process used is the trickling filter in which
wastewater is distributed over the top area of a vessel containing non-submerged packing
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material. Historically, rock was used most commonly as the packing material for trickling filters,
typical depths ranging from 1.25 to 2.0 m. Most modern trickling filters vary in height from 5 to
10 m and are filled with a plastic packing material for biofim attachment. The plastic packing
material is designed such that about 90 to 95 percent of the volume in the tower consists of void
space. Air circulation in the void space, by either natural draft or blowers, provides oxygen for
the microorganisms growing as an attached biofilm. Influents wastewater is distributed over the
packing and flows as a nonuniform liquid film over the attached biofilm. Excess biomass sloughs
from the attached growth periodically and clarification is required for liquid/solids separation to
provide an effluent with an acceptable suspended solids concentration. The solids are collected at
the bottom of the clarifier and removed for waste-sludge processing.
2.2 COMPOSITION AND CLASSIFICATION OF MICROORGANISMS
Biological processes for wastewater treatment consist of mixed communities with a wide variety
of microorganisms, including bacteria, protozoa, fungi, rotifers and possibly algae. To provide a
basic understanding of the nature of microorganisms, the topics introduced in this section are: (1)
cell composition and (2) environmental factors affect microbial activity.
2.2.1 Cell composition
To support microbial growth in biological system, appropriate nutrients must be available.

Reviewing the composition of a typical microbial cell will provide a basis for understanding the
nutrients needed for growth. Prokaryotes are composed of about 80 percent water and 20 percent
dry material, of which 90 percent is organic and 10 percent is inorganic. The most widely used
empirical formula for the organic fraction of cells is C5H7O2N first proposed by Hoover and
Porges (1952). About 53 percent by weight of the organic fraction is carbon. The formulation
C60H87O23N12P can be used when phosphorus is also considered. It should be noted that both
formulation are approximations and may vary with time and species, but they are used for
practical purposes. Nitrogen and phosphorus are considered macronutrients because they are
required in comparatively large amounts. Prokaryotes also require trace amount of metallic ions,
or micronutrients, such as zinc, manganese, copper, molybdenum, iron and cobalt. Because all of
these elements and compounds must be derived from the environment, a shortage of any of these
substances would limit and in some case, alter growth.
2.2.2 Environmental factors affect microbial activity
Environmental conditions of temperature and pH have an important effect on the selection,

survival, and growth of microorganisms. In general, optimal growth for a particulate
microorganism occurs within a fairly narrow range of temperature and pH, although most
microorganisms can survive within much broader limits. Temperatures below the optimum
typically have a more significant effect on growth rate than temperature above the optimum; it
has been observed that growth rate double with approximately every 10oC increase in
temperature until the optimum temperature is reached. According to the temperature range in
which they function best, bacteria may be classified a psychophilic, mesophilic or thermophilic.
Table 2.1 Temperature classification of biological processes
Type Temperature range (oC) Optimum range (oC)

Psychrophilic 10-30 12-18
Mesophilic 20-50 25-40
Thermalphilic 35-75 55-65
Source: Metcalf and Eddy, 2003.
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The pH of the environment is also a key factor in the growth of organisms. Most bacteria cannot
tolerate pH levels above 9.5 or below 4.0. Generally, the optimum pH for bacterial growth lies
between 6.5 and 7.5. Different archaea are able to grow at thermophilic and ultrathermophilic
(60 to 80oC) temperatures, extremely low pH and high salinity.
2.3 INTRODUCTION TO MICROBIAL METABOLISM
Basic to the design of a biological treatment process, or to the selection of the type of biological
process to be used, is an understanding of the biochemical activities of microorganisms.
Different microorganisms can use a wide range of electron acceptors, including oxygen, nitrite,
nitrate, iron, sulfate, organic compounds and carbon dioxide.
2.3.1 Carbon and energy sources for microbial growth
To continue to reproduce and function properly, an organism must have sources of energy,
carbon for the synthesis of new cellular material, and inorganic elements (nutrients) such as
nitrogen, phosphorus, sulfur, potassium, calcium, and magnesium. Organic nutrients (growth
factors) may also be required for cell synthesis. Carbon and energy sources, usually referred to as
substrates, and nutrient and growth factor requirements for various types of organisms are
considered in the following discussions.
Carbon sources. Microorganisms obtain their carbon for cell growth from with either organic
matter or carbon dioxide. Organisms that use organic carbon for the formation of new biomass
are called heterotrophs, while organisms that derive cell carbon from carbon dioxide are called
autotrophs. The conversion of carbon dioxide to cellular carbon compounds requires a reductive
process, which requires a net input of energy. Autotrophic organisms must therefore spend more
of their energy for synthesis than do heterotrophts, resulting in generally lower yields of cell
mass and growth rates.
Energy sources. The energy needed for cell synthesis may be supplied by light or by a chemical
oxidation reaction. Bacteria can oxidize organic or inorganic compounds to gain energy. Those
organisms that are able to use light as an energy sources are called phototrophs. Phototrophic
organism may be either heterotrophic (certain sulfur-reducing bacteria) or autotrophic (algae and
photosynthetic bacteria). Organisms phototrophs, chemotrophs may be either heterotrophic
(protozoa, fungi, and most bacteria) or autotrophic (nitrifying bacteria). Chemoautotrophs obtain
energy from the oxidation of reduced inorganic compounds, such as ammonia, nitriate, ferrous
iron, and sulfile. Chemoheterotrophs usually derive their energy from the oxidation of organic
compounds.
The energy-producing chemical reactions by chemotrophs are oxidation-reduction reactions that

involve the transfer of electrons from an electron donor to an electron acceptor. The electron
donor is oxidized and the electron acceptor is reduced. The electron donors and acceptors can be
either organic or inorganic compounds, depending on the microorganism. The electron acceptor
may be available within the cell during metabolism (endogenous) or it may be obtained from
outside the cell (i.e. dissolved oxygen). Organisms that generate energy by enzyme-mediated
electron transport to an external electron acceptor are said to have a respiratory metabolism. The
use of an internal electron acceptor is termed fermentative metabolism and is less efficient
energy-yielding process that respiration. Heterotrophic organisms that are strictly fermentative
are characterized by lower growth rates and cell yields that respiratory heterotrophs.
When oxygen is used for the electron acceptor the reaction is termed aerobic, and reactions
involving other electron acceptors are considered anaerobic. The term anoxic is used to
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distinguish the use of nitrate or nitrate for electron acceptors from the others under anaerobic
conditions. Under anoxic condition, nitrate or nitrate reduction to gaseous nitrogen occurs, and
this reaction is also referred to as biological denitrification. Organism that can only meet their
energy needs with oxygen are called obligate aerobic microorganisms. Some bacteria can use
oxygen or nitrate/nitrite as electron acceptors when oxygen is not available. These bacteria are
called facultative aerobic bacteria.
Organism that generate energy by fermentation and that can exist only in an environment that is
devoid of oxygen are obligate anaerobes. Facultative anaerobes have the ability to grow in
either the presence or absence of molecular oxygen and fall into two subgroups, based on their
metabolic abilities. True facultative anaerobes can shift from fermentative to aerobic respiratory
metabolism, depending on the presence or absence of molecular oxygen. Aerotolerant anaerobes
have a strictly fermentative metabolism but are relatively insensitive to the presence of molecular
oxygen.
2.3.2 Nutrient and growth factor requirement
Nutrient, rather than carbon or energy sources, may at times be the limiting material for
microbial cell synthesis and growth. The principal inorganic nutrients needed by microorganisms
are N, S, O, K, Mg, Ca, Fe, Na and Cl. Minor nutrients of importance include Zn, Mn, Mo, Se,
Co, Cu and Ni. Required organic nutrients, known as growth factor, are compounds needed by
an organism as precursors or constituents of organic cell material, which cannot be synthesized
from other carbon sources. Although growth factor requirements differ from one organism to
another, the major growth factors fall into the following three classes: (1) amino acids, (2)
nitrogen bases (i.e. purines and pyrimidines) and (3) vitamins.
For municipal wastewater treatment sufficient nutrients are generally present, but for industrial
wastewaters nutrients may need to be added to the biological treatment processes. The lack of
sufficient nitrogen and phosphorus is common especially in the treatment of food – processing
wastewaters or wastewater high in organic content. Using the formula C60H87O23N12P, it is
possible to estimate amount of nitrogen and phosphorus needed per 100 g of cell biomass.
2.4 BACTERIAL GROWTH
2.4.1 Bacterial reproduction
Bacteria can reproduce by binary fission, by asexual mode or by budding. Generally, they
reproduce by binary fission, in which the original cell becomes two new organisms. The time
required for each division, which is termed the generation time can vary from days to less than
20 min. This rapid change in biomass with time is a hypothetical example, for in biological
treatment systems, bacteria would not continue to divide indefinitely because of environmental
limitations, such as substrate and nutrient availability.
2.4.2 Bacterial growth patterns in a batch reactor
Bacterial growth in a batch reactor is characterized by identifiable phases as illustrated on Fig.

2.1. The curves shown on Fig. 2.1 represent what occurs in a batch reactor in which, at time zero,
substrate and nutrients are present in excess an only a very small populations of biomass exists.
As substrate is consume, four distinct growth phases develop sequentially.
1. The lag phase. Upon addition of the biomass, the lag phase represents the time required for
the organism to acclimate to their new environment before significant cell division and
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biomass production occur. During the lag phase enzyme induction may be occurring and/or
the cells may be acclimating to changes in salinity, pH, or temperature. The apparent extent
of the lag phase may also be affected by ability to measure the low biomass concentration
during the initial batch phase.
2. The exponential-growth phase. During the exponential-growth phase, bacterial cells are
multiplying at their maximum rate, as there is no limitation due to substrate or nutrients. The
biomass growth curve increase exponentially during this period. With unlimited substrate and
nutrients the only factor that affects the rate of exponential growth is temperature.
3. The stationary phase. During this phase, the biomass concentration remains relatively
constant with time. In this phase, bacterial growth is no longer exponential and the amount of
growth is offset by the death of cells.
4. The death phase. In the death phase, the substrate bas been depleted so that no growth is
occurring, and the change in biomass concentration is often observed as an approximate
constant fraction of the biomass remaining that is lost each day.
2.4.3 Bacterial growth and biomass yield
In biological treatment processes, cell growth occurs concurrent with the oxidation of organic or
inorganic compounds, as described above. The ratio of the amount of biomass produced to the
amount of substrate consumed (g biomass/g substrate) is defined as the biomass yield, and
typically is defined relative to the electron donor used.
g biomass produced
Biomass yield Y = --------------------------------------------- (2-2)
g substrate utilized (i.e. consumed)
For example, for aerobic heterotrophic reactions with organic substrates, the yield is expressed as
g biomass/g organic substrate; for nitrification the yield is expressed as g biomass/g NH4-N
oxidized and for the anaerobic degradation of volatile fatty acids (VFAs) to produce methane,
the yield is expressed as g biomass/g VFAs used. Where specific compounds are measured and
known such as ammonia, the yield is quantified relative to the amount of compound used. For
aerobic or anaerobic treatment of municipal and industrial wastewater containing a large number
of organic compounds, the yield is based on a measureable parameter reflecting the overall
organic compounds consumption, such as COD or BOD. Thus, the yield would be g biomass/g
COD removed or g biomass/g BOD removed.
2.4.4 Measuring biomass growth
Because biomass is mostly organic material, an increase in biomass can be measured by volatile
suspended solids (VSS) or particulate COD (total COD minus soluble COD). Other parameters
that are used to indicate biomass growth are protein content, DNA and ATP, a cellular
compound involved in energy transfer. Of these growth measurement parameters, VSS is the
parameter used most commonly to follow biomass growth in full-scale biological wastewater
treatment systems because its measurement is simple and minimal time is required for analysis.
It should be noted that the VSS measured includes other particulate organic matter in addition to
biomass. Most wastewater contain some amount of non-biodegradable VSS and possibly influent
VSS that may be slowly degraded in the biological reactor. These solids are included with
biomass in the VSS measurement. Nevertheless the VSS measurement is used as an apparent
indicator of biomass production and also provides as useful measurement of reactor solids in
general.
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For laboratory research on biological treatment processes, growth parameters that can be related
to true microbial mass are often used. Of these, protein is the most popular growth parameter due
to the relative ease of measurement and the fact that about 50 percent of biomass dry weight is
protein. Both ATP and DNA have also been used, especially where the reactor solids contain
proteins and other solids that are not associated with biomass. Where very low biomass
concentrations are involved, turbidity measurement may be used to provide a rapid and simple
means of observing cell growth. Bacterial cell counts have also been used to enumerate the
biomass population. A portion of a diluted sample is applied to an agar growth plate, and after
incubation, the number of colonies formed are counted and used to determine the number of
bacterial cells in the culture. It should be noted, however, that not all bacteria are culturable.
2.4.5 Estimating biomass yield and oxygen requirements
As given by Eq. (2-1), a definite stoichiometric relationship exists between the substrate
removed, the amount of oxygen consumed during aerobic heterotrophic biodegradation, and the
observed biomass yield. The most common approach used to define the fate of the substrate is to
prepare a COD mass balance. The COD is used because the substrate concentration in the
wastewater can be defined in terms of its oxygen equivalence, which can be accounted for being
conserved in the biomass or oxidized. In general, the exact stoichiometry involved in the
biological oxidation of a mixture of wastewater compounds is never known. However, for the
purpose of illustration, assume organic matter can be represented as C6H12O6 (glucose) and new
cells can be represented as C5H7O2N. Thus neglecting nutrients other than nitrogen, Eq. (2-1) can
be written as:
3C6H12O6 + 8O2 + 2NH3 Æ 2C5H7O2N + 8CO2 + 12H2O (2-3)

3 (180) 8 (32) 2 (17) 2 (113)
As given by the above equation, the substrate used (glucose in this case) is divided between that
found in new cells and that oxidized. The yield based on the glucose consumed can be obtained
as follows:
⎛ g ⎞
2⎜⎜113 ⎟
∆(C5 H 7O2 N ) ⎝ mole ⎟⎠
Y= = = 0.42 g cells/g glucose used
∆ (C6 H12O6 ) ⎛ g ⎞
3⎜⎜180 ⎟
⎝ mole ⎟⎠
In practice, COD and VSS are used to represent the organic matter and the new cells,
respectively. To express the yield on a COD basis, the COD of glucose must be determined. The
COD of glucose can be determined by writing a balanced stoichiometric reaction for the
oxidation of glucose to carbon dioxide as follows:
C6H12O6 + 6O2 Æ 6CO2 + 6H2O (2-4)

(180) 6 (32)
The COD of glucose is

⎛ g ⎞
6⎜⎜ 32 ⎟
∆(O2 ) ⎝ mole ⎟⎠
COD = = = 1.07 g O2/ g glucose
∆(C6 H12O6 ) ⎛ g ⎞
⎜⎜180 ⎟
⎝ mole ⎟⎠
The theoretical yield expressed in terms of COD, accounting for the portion of the substrate
converted to new cells, is
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⎛ g ⎞
2⎜⎜113 ⎟
∆(C5 H 7O2 N ) ⎝ mole ⎟⎠
Y= = = 0.39 g cells/g COD used.
∆(C6 H12O6 asCOD ) ⎛ g ⎞⎛ gCOD ⎞
3⎜⎜180 ⎟⎜1.07
⎝ mole ⎟⎠⎝ gglu cos e ⎟⎠
It should be noted that the actual observed yield in a biological treatment process will be less
than the value given above, because a portion of the substrate incorporated into the cell mass will
be oxidized with time by the bacteria to obtain energy for cell maintenance.
The quantity of oxygen utilized can be accounted for by considering: (1) the oxygen used for
substrate oxidation to CO2 and H2O, (2) the COD of the biomass, and (3) the COD of any
substrate not degraded. Based on the formula C5H7O2N, the oxygen equivalent of the biomass
(typically measured as VSS) is approximately 1.42 g COD/g biomass VSS, as given below.
C5H7O2N + 5O2 Æ 5CO2 + NH3 + 2H2O (2-5)

(113) 5 (32)
The COD of cell tissue is

⎛ g ⎞
5⎜⎜ 32 ⎟
∆(O2 ) ⎝ mole ⎟⎠
COD = = = 1.42 g O2/g cells
∆(C5 H 7O2 N ) ⎛ g ⎞
⎜⎜113 ⎟
⎝ mole ⎟⎠
Based on the above relationships, the oxygen consumed per unit of COD utilized for the reaction
given by Eq. (2-3) can be determined from a mass balance on COD as follows:
COD utilized = COD cells + COD of oxidized substrate (2-6)
The COD of the oxidized substrate is equal to oxygen consumed; thus:
Oxygen consumed = COD utilized – COD cells (2-7)
Oxygen consumed = 1.07 g O2/g glucose x 3 mole x 180 g glucose/mole – 1.42 g O2/g cells x 2
moles x 113 g cells/mole = 577.8 – 320.9 = 256.9 g O2
Thus, the oxygen consumed per unit of COD used is:
Oxygen consumed 256.9 g O2

---------------------- =-------------------------------------------------------------- = 0.44 g O2/g COD used
Glucose as COD 3 mole (1.07 g COD/g glucose) (180 g glucose/mol)
The amount of oxygen required based on the COD balance as given above is in agreement with
the oxygen used based on the stoichiometry as defined by Eq. (2-3) in which 8 moles of oxygen
are required for 3 mole of glucose.
Oxygen used 8 (32 g O2/mole)

--------------------- = ----------------------------------------------- = 0.44 g O2/g COD used
Glucose as COD 3 (180 g/mol) (1.07 g COD/g glucose)
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2.4.6 Observed versus synthesis yield
In the evaluation and modeling of biological treatment systems as distinction is made between
the observed yield and the synthesis yield (or true yield). The observed biomass yield is based on
the actual measurements of biomass production and substrate consumption and is actually less
than the synthesis yield, because of cell loss concurrent with cell growth. In full-scale
wastewater treatment processes the term solid production (or solid yield) is also used to describe
the amount of VSS generated in the treatment process. The terms is different from the synthesis
biomass yield values because it contains other organic solids from the wastewater that are
measured as VSS, but are not biological.
The synthesis yield is the amount of biomass produced immediately upon consumption of the
growth substrate or oxidation of the electron donor in the case of autotrophic bacteria. The
synthesis yield is seldom measure directly and is often interpreted from evaluating biomass
production data for reactors operating under different conditions. Synthesis yield values for
bacterial growth are affected by the energy that can be derived from the oxidation-reduction
reaction, by the growth characteristics of the carbon source, by the nitrogen source, and by
environmental factors such as temperature, pH and osmotic pressure. As illustrated in this
section, the synthesis yield can be estimated if the stoichiometry or the amount of energy
produced in the oxidation-reduction reaction is known.
2.5 MICROBIAL GROWTH KINETICS
The performance of biological processes used for wastewater treatment depends on the dynamics
of substrate utilization and microbial growth. Effective design and operation of such systems
requires an understanding of the biological reactions occurring and an understanding of the basic
principles governing the growth of microorganisms. Further, the need to understand all of the
environmental conditions that affect the substrate utilization and microbial growth rate cannot be
overemphasized, and it may be necessary to control such conditions as pH and nutrients to
provide effective treatment. The purpose of this section include (1) microbial growth kinetics
terminology, (2) rate of utilization of soluble substrate, (3) other rate expressions for the
utilization of soluble substrate, (4) rate of soluble substrate production from biodegradable
particulate organic matter, (5) the rate of biomass growth with soluble substrates, (6) kinetic
coefficients for substrate utilization and biomass growth, (7) the rate of oxygen uptake, (8)
effects of temperature, (9) total volatile suspended solids and active biomass, and (19) net
biomass and observed yield.
2.5.1 Microbial growth kinetics terminology
The kinetics of microbial growth govern the oxidation (i.e. utilization) of substrate and the
production of biomass, which contributes to the total suspended solids concentration in a
biological reactor. Because municipal and industrial wastewater contain numerous subsrates, the
concentration of organic compounds is defined, most commonly, by the biodegradable COD
(bCOD) or UBOD, both of which are comprised of soluble (dissolved), colloidal and particulate
biodegradable components. Both bCOD and UBOD represent measurable quantities that apply to
all of the compounds. In the formulation of kinetic expressions in this section biodegradable
soluble COD (bsCOD) will be used to quantify the fate of biodegradable organic compounds
because it easily relates to the stoichiometry of substrate oxidized or used in cell growth.
The biomass solids in a bioreactor are commonly measured as total suspended solid (TSS) and
volatile suspended solid (VSS). The mixture of solids resulting from combining recycled sludge
with influent wastewater in the bioreactor is termed mixed liquor suspended solids (MLSS) and
9
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mixed liquor volatile suspended solids (MLVSS). The solids are comprised of biomass, non-
biodegradable volatile suspended solids (nbVSS), and inert inorganic total suspended solids
(iTSS). The nbVSS is derived from the influent wastewater and is also produced as cell debris
from endogenous respiration. The ITSS originates in the influent wastewater.
2.5.2 Rate of utilization of soluble substrate
The goal in biological wastewater treatment is, in most cases, to deplete the electron donor (i.e.
organic compounds in aerobic oxidation). For heterotrophic bacteria the electron donors are the
organic substances being degraded; for autotrophic nitrifying bacteria it is ammonia or nitrate or
nitrite or other reduced inorganic compounds. The substrate utilization rate in biological systems
can be modeled with the following expression for soluble substrates. Because the mass of
substrate is decreasing with time due to substrate utilization and Eq. (2-8) is used in substrate
mass balances, a negative value is shown.
kXS
rsu = − (2-8)
Ks + S
Where rsu = rate of substrate concentration change due to utilization, g/m3.d

k = maximum specific substrate utilization rate, g substrate/g microorganisms.d
X = biomass (microorganism) concentration, g/m3
S = growth-limiting substrate concentration in solution, g/m3
Ks = half-velocity constant, substrate concentration at one-half the maximum specific
substrate utilization rate, g/m3
Equation (2-8) will be recognized as a saturation-type equation. For substrate removal, Eq. (2-8)
has been referred to as the Michaelis-Menten equation (Bailey and Ollis, 1986), Equation (2-8) is
also of the form proposed by Monod for the specific growth rate of bacteria in which the limiting
substrate is available to the microorganisms in a dissolved form (Monod, 1942; 1949).
When the substrate is being used at its maximum rate, the bacteria are also growing at their
maximum rate. The maximum specific growth rate of the bacteria is thus related to the maximum
specific substrate utilization rate as follows.
µ m = kY (2-9)
and
µm
k= (2-10)
Y
Where µm = maximum specific bacterial growth rate, g new cells/g cells.d
k = maximum specific substrate utilization rate, g/g.d
Y = true yield coefficient, g/g (defined earlier)
Using the definition for the maximum specific substrate utilization rate given by Eq. (2-8), the
substrate utilization rate is also reported in the literature as:
µ m XS
rsu = − (2-11)
Y (K s + S )
10
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2.5.3 Other rate expressions for the utilization of soluble substrate
In reviewing kinetic expressions used to describe substrate utilization and biomass growth rates,
it is very important to remember that the expression used to model biological processes are all
empirical, based on experimentally determined coefficient values. Besides the substrate limited
relationship presented above, other expressions that have been used to describe substrate
utilization rates include the following:
rsu = -k (2-12)
rsu = -kS (2-13)
rsu = - kXS (2-14)
rsu = -kX.(S/So) (2-15)
The particular rate expression used to define kinetics of substrate utilization depends mainly on
the experimental data available to if the kinetic equations and the application of the kinetic
model. In some cases, the first-order model shown in Eq. (2-14) is satisfactory for describing
substrate utilization rates when the biological treatment process will be operated at relatively low
substrate concentrations. Fundamental in the use of any rate expression is its application in a
mass-balance analysis to be discussed in the following section. Also with regard to modeling
biological treatment processes, kinetic models should not be applied outside of the range of the
conditions used to develop model coefficients.
2.5.4 Rate of soluble substrate production from biodegradable particulate organic matter
The rate expressions for substrate utilization and biomass growth presented thus far are based on
the utilization of soluble substrates. In municipal wastewater treatment only about 20 to 50
percent of the degradable organic material enters as soluble compounds, and for some industrial
wastewaters the soluble organic material may be a low to moderate fraction of the total
degradable organic substrates. Bacteria cannot consume the particulate substrates directly and
employ extracellular enzymes to hydrolyze the particulate organics to soluble substrates, which
can be according to the rate expressions described above. The particulate substrate conversion
rate is also a rate-limiting process that is dependent on the particulate substrate and biomass
concentrations. A rate expression for particulate substrate conversion is shown as follows (Grady
et al., 1999):
kp P ( X )X
rsc , p = −
(K x +P
X
) (2-16)
Where rsc,P = rate of change of particulate substrate concentration due to conversion to soluble
substrate, g/m3/d
KP = maximum specific particulate conversion rate, g P/g X.d
P = particulate substrate concentration, g/m3
X = biomass concentration, g/m3
Kx = half-velocity degradation coefficient, g/g
The particulate degradation concentration is expressed relative to the biomass concentration,

because the particulate hydrolysis is related to the relative contact area between the non-soluble
organic material and the biomass.
11
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2.5.5 Rate of biomass growth with soluble substrates
The following relationship between the rate of growth and the rate of substrate utilization is
applicable in both batch and continuous culture systems.
kXS
rg = −Yrsu − kd X = Y − kd X (2-17)
Ks + S
Where rg = net biomass production rate, g VSS/m3.d

Y = synthesis yield coefficient, g VSS/g bs COD
kd = endogenous decay coefficient, g VSS/g VSS.d
Other terms are as defined above.
If both side of Eq. (2-17) are divided by the biomass concentration X, the specific growth rate is
defined as follows:
rg kS
µ= =Y − kd (2-18)
X KS + S
Where µ = specific biomass growth rate, g VSS/g VSS.d
As shown, the specific growth rate corresponds to the change in biomass per day relative to the
amount of biomass present, and is a function of the substrate concentration and the endogenous
decay coefficient.
The endogenous decay coefficient accounts for the loss in cell mass due to oxidation of internal
storage products for energy for cell maintenance, cell death, and predation by organisms higher
in the food chain. It should be noted that micoorganisms in all growth phases require energy for
cell maintenance; however, it appears that the decay coefficient most probably changes with cell
age. Usually, these factor are lumped together, and it is assumed that the decrease in cell mass
caused by them is proportional to the concentration of organisms present. The decrease in mass
is often indentified in the literature as the endogenous decay. In Eq. (2-17) the coefficient kd is
the endogenous decay rate coefficient.
In biological treatment processes, both the substrate utilization and biomass growth rates are
controlled by some limiting substrate, as given by Eqs. (2-11) and (2-17). The growth limiting
substrate can be any of the essential requirements for cell growth (i.e., electron donor, electron
acceptor, or nutrients), but often it is the electron donor that is limiting, as other requirements are
usually available in excess. Thus, when the term substrate is used to described growth kinetics, it
generally refers to the electron donor.
2.5.6 Kinetic coefficients for substrate utilization and biomass growth
The coefficient values (k, Ks, Y and kd) used to predict the rate of substrate utilization and
biomass growth can vary as a function of the wastewater source, microbial population, and
temperature. Kinetic coefficient values are determined from bench-scale testing or full-scale
plant test results. For municipal and industrial wastewater the coefficient values represent the net
effect of microbial kinetics on the simultaneous degradation of variety of different wastewater
constituents. Typical kinetics coefficient values are reported in Table 2.2 for the aerobic
oxidation of BOD in domestic wastewater.
12
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Table 2.2 Typical kinetic coefficients for the activated sludge process for the removal of organic matter
from domestic wastewater
Coefficient Unit Value*

Range Typical
k g bsCOD/g VSS.d 2-10 5
Ks mg/L BOD 25-100 60
mg/L bsCOD 10-60 40
Y mg VSS/mg BOD 0.4-0.8 0.6
mg VSS/mg bsCOD 0.3-0.6 0.4
kd g VSS/g VSS.d 0.06-0.15 0.10
*
values reported are for 20oC
Source: Metcalf and Eddy, 2003.
2.5.7 Rate of oxygen uptake
The rate of oxygen uptake is related stoichiometrically to the organic utilization rate and growth
rate. Thus, the oxygen uptake rate can be defined as:
ro = - rsu – 1.42rg (2-19)
where ro = oxygen uptake rate, g O2/m3.d

rsu = rate of substrate utilization, g bsCOD, m3.d
1.42 = the COD of cell tissue, g bsCOD/g VSS
rg = rate of biomass growth, g VSS/m3.d
A negative sign is required in front of the term rsu in Eq. (2-19), because the rate of substrate
utilization as given by Eq. (2-11) is negative (i.e., the substrate concentration decreases with
time). The factor 1.42 represents the COD of cell tissue as defined previously by Eq. (2-5).
2.5.8 Effects of temperature
The temperature dependence of the biological reaction-rate constants is very important in

assessing the overall efficiency of a biological treatment process. Temperature not only
influences the metabolic activities of the microbial population but also has a profound effect on
such factors as gas-transfer rates and the settling characteristics of the biological solids. The
effect of temperature on the reaction rate of a biological process is expressed as follows:
kT = k20θ (T − 20 ) (2-20)
Where kT = reaction-rate coefficient at temperature T, oC

k20 = reaction-rate coefficient at 20oC;
θ = temperature-activity coefficient
T = temperature, oC
Value for θ in biological systems can vary from 1.02 to 1.25.
2.5.9 Total volatile suspended solids and active biomass
The kinetic expressions used to describe biological kinetics and growth rate are related to the
active biomass concentration X in the treatment reactor. In reality the VSS in a reactor consists
of more than active biomass, and the fraction of active biomass can vary depending on the
wastewater characteristics an operating conditions. The other components that contribute to the
13
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VSS concentration are cell debris, following endogenous decay, and non-biodegradable VSS
(nbVSS) in the influent wastewater fed to the biological reactor.
During cell death, cell lysis occurs with the release of cellular materials into the liquid for
consumption by other bacteria. A portion of the cell mass (cell wall) is not dissolved and remains
as non-biodegradable particulate matter in the system. The remaining non-biodegradable
material is referred to as cell debris and represents about 10 to 15 percent of the original cell
weight. Cell debris is also measured as VSS and contributes to the total VSS concentration
measured in the reactor mixed liquor. The rate of production of cell debris is directly
proportional to the endogenous decay rate.
rxd = fd.kd.X (2-21)
where rxd = rate of cell debris production, g VSS/m3.d

fd = fraction of biomass that remains as cell debris, 0.10-0.15 g VSS/g VSS
The nbVSS concentration resulting from cell debris is typically a relatively small fraction of the
VSS in a bioreactor used to treat municipal and some industrial wastewaters. As noted above, a
variable amount of MLVSS that is not biomass originates from the nbVSS in the influent
wastewater. For typical untreated municipal wastewater the nbVSS concentration may be in the
range from 60 to 100 mg/L, and following primary treatment may range from 10 to 40 mg/L.
Total volatile suspended solids. The VSS production rate in the aeration tank can be defined as
the sum of the biomass production given by Eq. (2-17), the nbVSS production viven by Eq (2-
21) and the nbVSS in he influent:
rXT,VSS = -Yrsu – kdX + fdkdX + Q.Xo,i/V (2-22)

Net nbVSS nbVSS from nbVSS in
from soluble cells influent
bCOD
where rXT,VSS = total VSS production rate, g/m3.d
Q = influent flow-rate, m3/d
Xo.i = influent nbVSS concentration, g/m3
V = volume of reactor, m3
Other terms are as defined respectively.
Active biomass
From Eq. (2-22), the fraction of active biomass in the mixed-liquor VSS (MLVSS) is the ratio of
the sum of the growth and decay term divided by the total MLVSS production:
− Yrsu − kd X
FX , act = (2-23)
rX T ,VSS
Where Fx, act = active fraction of biomass in MLVSS, g/g.
2.5.10 Net biomass yield and observed yield
Net biomass yield and observed yield
The term true yield was defined as the amount of biomass produced during cell synthesis relative
to the amount of substrate degraded. In the design and analysis of biological treatment processes,
14
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two other yield terms are important: (1) the net biomass yield and (2) the observed solid yield.
The first is used as an estimate of the amount of active microorganisms in the system, and the
second as the amount of sludge production.
Net biomass yield. The net biomass yield is the ratio of the net biomass growth rate to the
substrate utilization rate:
Ybio = - rg/rsu (2-24)
Where Ybio = net biomass yield, g biomass/g substrate used.
Observed yield. The observed yield accounts for the actual solids production that would be
measured for the system and is shown as follows:
Yobs = - rxT,VSS/rsu (2-25)
Where Yobs = observed yield, g VSS produced/g substrate removed
2.6 MODELING SUSPENDED GROWTH TREATMENT PROCESSES
2.6.1 Description of suspended growth treatment processes
The complete-mix reactor with recycle will be considered in the following discussion as a model
for suspended growth processes. The schematic flow diagram shown on Fig. 2.1 Include the
nomenclature used in the following mass-balance equations. A similar complete-mix reactor may
be used in laboratory studies to asses wastewater treatability and to obtain model kinetic
coeffients.
All biological treatment reactor designs are based on using mass balances across a define volume
for each specific constituent of interest (i. e. biomass, substrate, etc.). The mass balance includes
the flow-rates for the mass of the constituent entering and/or leaving the system and appropriate
reaction rate terms for the depletion or production of the constituent within the system. The units
for a mass balance are usually given in mass per volume per time. For all mass balance a check
of the units is recommended to assure that the mass-balance equations are correct.
Fig. 2.1 Schematic diagram of activated-sludge process with model nomenclature: (1) with wasting from
the sludge return line and (b) with wasting from the aeration tank (Metcalf and Eddy, 2003).
15
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2.6.2 Biomass mass balance
A mass balance for the mass of microorganisms in the complete-mix reactor shown on Fig. 2.1a
can be written as follows:
- General word statement:
Rate of accumulation Rate of flow of Rate of flow of Net growth of

of microorganism microorganism microorganism microorganism (2-26)
= - +
within the system into the system out of the system within the
boundary boundary boundary boundary
- Simplified word statement:
Accumulation = inflow – outflow + net growth (2-27)
- Symbolic representation:
V = QX o − [(Q − QW )X e + QW X R ] + rg V
dX
(2-28)
dt
Where dX/dt = rate of change of biomass concentration in reactor measured as g VSS/m3.d

V = reactor volume (i.e., aeration tank), m3
Q = influent flow-rate, m3/d
Xo = concentration of biomass in influent, g VSS/m3
Qw = waste sludge flow-rate, m3/d
Xe = concentration of biomass in influent, g VSS/m3
XR = concentration of biomass in return line from clarifier, g VSS/m3
rg = net rate of biomass production, g VSS/m3.d
It it is assumed that the concentration of microorganisms in the influent can be neglected and that
steady-state conditions prevail (dX/dt = 0), Eq. (2-29) can be simplified to yield:
(Q – QW)Xe + QwXR = rgV (2-29)
If Eq. (2-29) is combined with Eq. (2- 17), the result is:
(Q − QW )X e + QW X R = −Y
rsu
− kd (2-30)
VX X
Where X = concentration of the biomass in the reactor, g/m3.
The inverse of the term on the left-hand side of Eq. (2-30) is defined as the average solids
retention time (SRT) as given below:
VX
SRT = (2-31)
(Q − QW )X e + QW X R
Note that the numerator in Eq. (2-31) represents the total mass of solids in the aeration tank and
the denominator corresponds to the amount of solids lost per day in the effluent and by
16
………………………………………………………………………………………………………………………………………………………….
intentional wasting (QW). By definition the SRT is the solids in the system divided by the mass
of solids removed per day. Using the above definition of STR, Eq. (2-30) can be written as:
1 r
= −Y su − k d (2-32)
SRT X
The term of 1/SRT is also related to µ, the specific biomass growth rate, as given by the Eq. (2-
33):
1
=µ (2-33)
SRT
Thus, for a complete-mix activated-sludge process, the SRT (which can be controlled by solids
wasting) is the inverse of the average specific rate that relates to the process biokinetics.
In Eq. (2-32), the term (-rsu/X) is known as the specific subsrtate utilization rate U. The specfic
substrate utilization rate U is calculated as follows:
rsu Q(S o − S ) So − S
U= = = (2-34)
X VX τX
Where U = specific substrate utilization rate, g BOD or g COD/g VSS.d

Q = wastewater flow-rate, m3/d
So = influent soluble substrate concentration, g BOD or bsCOD/m3
S = effluent soluble substrate concentration, g BOD or bsCOD/m3
V = volume of aeration tank, m3
X = biomass concentration, g/m3
τ = hydraulic detention time, V/Q, d
Other terms as defined above
Substituting Eq. (2-8) into Eq. (2-32) yields:
1 YkS
= − kd (2-35)
SRT K s + S
The solids retention time (SRT) is an important design and operating parameter for the activated-
sludge process. The SRT is the average time the activated-sludge solids are in the system.
Assuming that the solids inventory in the clarifier shown on Fig. 2.1a is nefligicle compared to
that in the aeration tank, the SRT is determined by dividing the the mass of solids in the aeration
tank by the solids removed daily via the effluent and by wasting for process control. For many
activated-sludge processes, where good flocculation occurs and the clarifier is designed properly,
the effluent VSS is typically less than 15 g/m3. Where the effluent VSS is low, excess solids
must be removed from the system by wasting. Wasting is accomplished most commonly by
removing biomass (sludge) from the clarifier underflow recycle line as shown as Fig. 2.1a.
Alternatively, wasting can be accomplished from the aeration tank as shown on Fig. 2.1b.
Solving Eq. (2-35) for the effluent dissolved substrate concentration S yields:
K s [1 + k d × SRT ]
S= (2-36)
SRT (Yk − k d ) − 1
17
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It should be noted that Eq. (2-36), the effluent soluble substrate concentration for a complete-mix
activated-sludge process is only a function of the SRT and kinetic coefficients for growth and
decay. The effluent substrate concentration is not related to the influent soluble substrate
concentration, but as will be shown in other mass balances, the influent concentration affects the
biomass concentration.
2.6.3 Substrate mass balance
The mass balance for substrate utilization in the aeration tank (Fig. 2.1a) is:
Accumulation = inflow – outflow + generation
dS
V = QSo − QS + rsuV (2-37)
dt
Where So = influent soluble substrate concentration, g/m3
Substituting the value for rsu (Eq. 2-8) and assuming steady-state condition (dS/dt = 0), Eq. (2-
37) can be rewritten as:
⎛ V ⎞⎛ kXS ⎞
S o − S = ⎜⎜ ⎟⎟⎜⎜ ⎟⎟ (2-38)
⎝ Q ⎠⎝ K s + S ⎠
The volume of the aeration tank divided by the influent flow-rate is τ, the hydraulic retention
time.
If Eq. (2-35) is solved for the term S/(Ks + S) and substituted into Eq. (2-38), the following
expression is obtained for the biomass concentration in the aeration tank:
⎛ SRT ⎞ ⎡ Y (S o − S ) ⎤
X =⎜ ⎟⎢ ⎥ (2-39)
⎝ τ ⎠ ⎣1 + k d SRT ⎦
As given by Eq. (2-39), the reactor biomass concentration is a function of the system SRT, the
aerobic tank τ, the synthesis yield coefficient, the amount of substrate removed (So –S), and the
endogenous decay coefficient.
The same equations can be applied to describe an activate-sludge process with no clarifier and
thus no return sludge flow. For the case with no return sludge, all of the solid produced are
present in the effluent from the aeration tank and the SRT equals the τ.
VX
SRT = =τ (2-40)
QX
The importance of the system SRT in determining the effluent soluble substrate concentration
and aeration tank biomass concentration is clear from an examination of Eqs. (2-36) and (2-39).
As indicated by the waste sludge flow term (Qw) is Eq. (2-31), a selected SRT value can be
maintained by the amount of solids wasted per day to control the process performance. Similarly,
it can be shown that by wasting from the aeration tank, the SRT can be controlled by wasting a
given percentage of the aeration tank volume each day.
18
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2.6.4 Mixed liquor solids concentration and solids production
The solids production from a biological reactor represents the mass of material that must be
removed each day to maintain the process. It is of interest to quantify the solids production in
terms of TSS, VSS, and biomass. By definition, the SRT also provides a convenient expression
to calculate the total sludge produced daily from the activated-sludge process:
X TV
PX T ,VSS = (2-41)
SRT
Where PXT, VSS = total solids wastes daily, g VSS/d

XT = total MLVSS concentration in aeration tank, g VSS/m3
V = volume of rector, m3
SRT = solid retention time, d
Because the 1/SRT in Eq. (2-41) represents the fraction of solids wasted per day and the mixed
liquor can be assumed to be a homogeneous mixture of biomass and other solids, Eq. (2-41) can
be used to calculate the amount of solids wasted for any of the mixed liquor components. For the
amount of biomass wasted per day (Px), the biomass concentration X can be used in place of XT
in Eq. (2-41).
Mixed liquor solids concentration
The total MLVSS in the aeration tank equals the biomass concentration X plus the nbVSS
concentration Xi:
X T = X + Xi (2-42)
A mass balance is needed to determine the nbVSS concentration in addition to the active
biomass VSS concentration. The MLVSS nbVSS concentration is affected by the amount of
nbVSS in the influent wastewater, the amount of nbVSS wasted per day, and the amount of cell
debris produced from cell decay. A materials balance on the inert material is as follows:
Accumulation = inflow – outflow + generation
⎛ dX i ⎞ XV
⎜ ⎟V = QX o ,i − i + rx ,iV (2-43)
⎝ dt ⎠ SRT
Where Xo,i = nbVSS concentration in influent, g/m3

Xi = nbVSS concentration in aeration tank, g/m3
rX,i = rate of nbVSS production from cell debris, g/m3.d
At steady-state (dXi/dt = 0) and substituting Eq. (2-21) for rX,i in Eq. (2-43) yields:
X iV
0 = QX o ,i − + rX ,iV (2-44)
SRT
SRT
X i = X o,i × + f d kd X × SRT (2-45)
τ
Combining Eq. (2-39) and Eq. (2-45) for X and Xi produce the following equation that can be
used to determine the total MLVSS concentration:
19
………………………………………………………………………………………………………………………………………………………….
SRT Y × (S o − S ) X × SRT
XT = × + f d kd X × SRT + o ,i (2-46)
τ 1 + kd × SRT τ
Heterotrophic Cell Non-biodegradable
biomass debris VSS in influent
Solids production
By substituting Eq. (2-46) for XT in Eq. (2-41) and replacing τ with V/Q, the amount of VSS
produced and wasted daily is determined as follows:
QY (S o − S )
PX ,VSS = + f d k d XV + QX o ,i (2-47)
1 + k d × SRT
Fig. 2.2 Biodegradable soluble COD, biomass, and MLVSS concentrations versus SRT for complete-mix
activated-sludge process (Metcalf and Eddy, 2003).
Eq. (2-39) is substituted for the biomass concentration (X) in Eq. (2-47) to show the daily VSS
production rate in terms of the substrate removed, influent nbVSS, and kinetic coefficient as
follows:
QY (S o − S ) f d k d YQ(S o − S )SRT
PX ,VSS = + + QX o ,i (2-48)
1 + k d SRT 1 + k d SRT
A B C
The effect of SRT on the performance of an activated-sludge system for soluble substrate
removal is illustrated on Fig. 2.2. In addition to the soluble substrate concentration, the total VSS
concentration which includes nabs is also shown. As the SRT increases, more biomass decays
and thus more cell debris accumulates, so that the difference between MLVSS and biomass VSS
concentration increases with SRT. Also illustrated on Fig. 2.2 is the fact that the soluble
substrate concentration is very low (< 5 mg/L) at SRTs above 2 d. The low substrate
concentration is typical of the activated-sludge process when used for the treatment of municipal
wastewaters, and illustrates how effectively the organic compounds are degraded in the
activated-sludge process.
20
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The total mass of dry solids wasted per day is based on the TSS, which includes the VSS plus
inorganic solids. Inorganic solids are in the influent wastewater (TSS-VSS) and the biomass
contains 10 to 15 percent inorganic solids by dry weight. The influent inorganic solids are not
soluble, and are assumed captured in the mixed liquor solids and removed in the wasted solids.
Eq. (2-48) is modified to calculate the solids production in terms of TSS by adding the influent
inorganic solids and by calculating the biomass in terms of TSS by assuming a typical biomass
VSS/TSS ratio of 0.85. The ratio of VSS/TSS may vary from 0.80 to 0.90.
+ C + Q(TSS o − VSSo )
A B
PX ,TSS = + (2-49)
0.85 0.85
Where PX,TSS = net waste activated sludge produced each day, measured in terms of total
suspended solids, kg/d
TSSo = influent wastewater TSS concentration, g/m3
VSSo = influent wastewater VSS concentration, g/m3
The mass of MLVSS and MLSS can be ontained by using Eqs. (2-48) and (2-49), respectively,
with Eq. (2-41) as follows:
Mass of MLVSS = XVSS.V = PX,VSS.SRT (2-50)
Mass of MLSS = XTSS.V = PX,TSS.SRT (2-51)
By selecting an appropriate MLSS concentration, the aeration volume can be determined from
Eq. (2-51). MLSS concentration in the range of 2000 to 4000 mg/L may be selected and must be
compatible with the sludge settling characteristics and clarifier design.
2.6.5 The observed yield
The observed yield Yobs is based on the amount of solids production measured relative to the
substrate removal, and may be calculated in terms of g TSS/g bsCOD or g BOD, or relative to
VSS as g VSS/g bsCOD or gBOD. The measured solids production is the sum of the solids in the
system effluent flow and the solids intentionally wasted, which equals the term Px defined in
Eqs. (2-41), (2-48) and (2-49). The observed yield for VSS can be calculated by dividing Eq. (2-
48) by the substrate removal rate, which is Q(So-S):
Y f k Y × SRT X o,i
Yobs = + d d + (2-52)
1 + kd × SRT 1 + kd × SRT So − S

Where Yobs = g VSS/g substrate removed
For wastewaters with no nbVSS in the influent the solids production consists of only active
biomass and cell debris, and the observed yield for VSS is as follows:
Y f k Y × SRT
Yobs = + d d (2-53)
1 + kd × SRT 1 + kd × SRT
21
………………………………………………………………………………………………………………………………………………………….
The impact of non-biodegradable influent VSS in Eq. (2-52) on the observed yield depends on
the wastewater characteristics and the type of pretreatment. The effluent substrate concentration
is generally very low compared to So and the term Xo,i/(So –S) can be approximated by Xo,i/So
values range from 0.10 to 0.30 g/g with primary treatment and 0.30 to 0.50 without primary
treatment.
2.6.6 Oxygen requirement
The oxygen required for the biodegradation of carbonaceous material is determined from a mass
balance using the bCOD concentration of the wastewater treated and the amount of biomass
wasted from the system per day. If all of the bCOD were oxidized to CO2 and H2O, the oxygen
demand would equal the bCOD concentration, but bacteria only oxidize a portion of the bCOD
to provide energy and use a portion of the bCOD for cell growth. Oxygen is also consumed for
endogenous respiration, and the amount will depend on the system SRT. For a given SRT, a
mass balance on the system can be done where the bCOD removal equals the oxygen used plus
the biomass VSS remaining (in terms of an oxygen equivalent), as was shown by Eq. (2-7).
Thus, for a suspended growth process, the oxygen used is:
Oxygen used = bCOD removed – COD of waste sludge (2-54)
Ro = Q(So – S) – 1.42 Px,bio (2-55)
Where Ro = oxygen required, kg/d

Px,bio = biomass as VSS wasted per day, kg/d
It is important to note that Px,bio includes active biomass and cell debris derived from cell growth
and is thus the sum of terms A and B in Eq. (2-48).
2.6.7 Design and operating parameters
In the mass balance for the complete-mix reactor, presented above, the SRT was introduced as
the fundamental process parameter that affects the treatment efficient and general performance
for the activated-sludge process. Two other activated-sludge process parameters used for the
design and operation of the activated-sludge process, process parameters used for the design and
operation of the activated-sludge process, the food to microorganism ratio and the volumetric
loading rate, are introduce below.
Food to microorganism (F/M) ratio
The F/M ratio is defined as the rate of BOD or COD applied per unit volume of mixed liquor:
Total applied substrate rate QSo

F/M = ----------------------------------- = ------ (2-56)
Total microbial biomass VX
and
So
F/M = ---- (2-57)
τX
Where F/M = food to biomass ratio, g BOD or bsCOD/g VSS.d

Q = influent wastewater flow-rate, m3/d
22
………………………………………………………………………………………………………………………………………………………….
So = influent BOD or bsCOD concentration, g/m3
V = aeration tank volume, m3
X = mixed liquor biomass concentration in the aeration tank, g/m3
τ = hydraulic retention time of aeration tank, V/Q, d
Specific substrate utilization rate
The F/M ratio can be related to the specific substrate utilization rate U, defined earlier, by the
process efficiency:
(F/M)E
U = --------- (2-58)
100
Where E = BOD or bsCOD process removal efficiency as defined by Eq. (2-59):
So - S
E, % = --------- x 100 (2-59)
So
Substituting Eq. (2-56) for the F/M ratio and the term [(So-S)/So]100 for the process efficiency
yields the specific substrate utilization rate U, as given previously:
So - S
U = -------
τX
The value of U can also be calculated by dividing –rsu in Eq. (2-8) by X:
kS
U = --------- (2-61)
Ks + S
By combining Eqs. (2-61) and (2-35):
1
----- = YU - kd (2-62)
SRT
Again, the net specific growth rate µ is also equal to 1/SRT. In turn, as given by Eq. (2-62), the
specific growth rate is equal t the synthesis yield times the specific substrate utilization rate
minus the endogenous decay rate. By substituting Eq. (2-58) into Eq. (2-62) the SRT can be
related to the F/M ratio as follows:
1 E
----- = Y(F/M) ------ - kd (2-63)
SRT 100
For systems designed for the treatment of municipal wastewater with activated-sludge SRT
values in the 20- to 30- d range, the F/M value may range from 0.10 to 0.05 g BOD/g VSS.d,
respectively. At SRT in the range of 5 to 7 d, the value may range from 0.3 to 0.5 g BOD/g
VSS.d, respectively.,
23
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Organic volumetric loading rate
The organic volumetric loading rate, defined as the amount of BOD or COD applied to the
aeration tank volume per day, is:
QSo
Lorg = (2-64)
V × 103 g / kg
Where Lorg = volumetric organic loading, kg BOD/m3.d

Q = influent wastewater flow-rate, m3/d
So = influent BOD concentration, g/m3
V = aeration tank volume, m3
2.6.8 Process performance and stability
The effects of the kinetics considered above on the performance and stability of the system
shown on Fig. 2.3 will now be examined further. It was shown in Eqs. (2-61) and (2-33) that
1/SRT, the net microorganism specific growth rate, and U, the specific substrate utilization ratio,
are related directly. For a specified waste, a given biological community, and a particular set of
environmental conditions, the kinetic coefficients Y, k, Ks and kd, are fixed. It is important to
note that domestic wastewater may have significant variability in its composition and may not
always be treated as a single waste type in evaluating the kinetic coefficients. For given values of
the coefficients, the effluent substrate concentration from the reactor is a direct function of the
SRT, as shown on Eq (2-36). Setting the SRT value fixes the values of U and µ and also defines
the efficiency of biological waste stabilization. Equation (2-36) for substrate is plotted on Fig.
2.3 for a growth-specified complete-mix system with recycle. As shown, the treatment efficiency
and the substrate concentration are related directly to the SRT, and the reactor hydraulics (i.e.,
complete-mix or plus-flow).
Fig. 2.3 Effluent substrate concentration and removal efficiency for complete-mix and plus-flow reactors
with recycle versus SRT (Metcalf and Eddy, 2003).
It can also be seen from Fig. 2.3 that there is a certain value of SRT below which waste
stabilization does not occur. The critical SRT value is called the minimum solids retention
residence time SRTmin. Physically, SRTmin is the residence time at which the cells are washed out
or wasted from the system faster than they can reproduce. The minimum SRT can be calculated
using Eq. (2-35), in which S = So. When washout occurs, the influent concentration So is equal to
the effluent waste concentration S.
24
………………………………………………………………………………………………………………………………………………………….
1 YkSo
= − kd (2-65)
SRTmin K s + So
In many situations encountered in waste treatment, So is much greater than Ks so that Eq. (2-64)
can be rewritten to yield:
1 1
~ Yk − kd (2-66) or ~ µ m − kd (2-67)
SRTmin SRTmin
Equations (2-66) and (2-67) can be used to determine the SRTmin. Biological treatment processes
should not be designed with SRT values equal to SRTmin. To ensure adequate waste treatment,
biological treatment processes are usually designed and operated with a design SRT value from 2
to 20 times SRTmin. In effect, the ratio of the design SRT (SRTdes) to SRT min can be considered
to be a process safety factor SF against system failure.
SRTdes
SF = (2-68)
SRTmin
2.7 AEROBIC BIOLOGICAL OXIDATION
2.7.1 Process description
The removal of BOD can be accomplished in a number of aerobic suspended growth or attached
growth treatment processes. Both require sufficient contact time between the wastewater and
heterotrophic microorganisms, and sufficient oxygen and nutrients. During the initial biological
uptake of the organic material, more than half of it is oxidized and the remainder is assimilated
as new biomass, which may by further oxidized by endogenous respiration. For both suspended
and attached growth processes, the excess biomass produced each day is removed and processed
to maintain proper operation and performance. The biomass is separated from the treated effluent
by gravity separation, and more recent designs using membrane separation are finding
applications.
2.7.2 Microbiology
A wide variety of microorganisms are found in aerobic suspended and attached growth treatment
processes used for the removal of organic material. Aerobic heterotrophic bacteria found in these
processes are able to produce extra-cellular biopolymers that result in the formation of biological
flocs (or biofilms for attached growth processes) that can be separated from the treated liquid by
gravity settling with relatively low concentrations of free bacteria and suspended solids.
Protozoa also play an important role in aerobic biological treatment processes. By consuming
free bacteria and colloidal particulates, protozoa aid effluent clarification. Protozoa require a
longer SRT than aerobic heterotrophic bacteria, prefer dissolved oxygen concentration above 1.0
mg/L, and are sensitive to toxic materials. Thus, their presence is a good indicator of a trouble-
free stable process operation. Because of their size, protozoa can easily be observed with a light
microscope at 10 to 200 magnifications. Rotifers can also be found in activated sludge and in
biofilms, as well as nematodes and other multi-cellular microorganism. These organisms occur at
longer biomass retention times, and their importance has not been well defined.
25
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Aerobic attached growth processes depending on the biofilm thickness, generally have a much
more complex microbial ecology that activated sludge with films containing bacteria, fungi,
protozoan, rotifers, and possibly annelid worms, flatworms, and nematodes (WEF, 2000).
Depending on process loadings and environmental conditions, a number of nuisance organisms

can also develop in the activated sludge process. The principal problem caused by nuisance
organism is a condition known as bulking sludge, in which the biological floc has poor settling
characteristics. In the extreme, bulking sludge can result in high effluent suspended solids
concentrations and poor treatment performance. Another nuisance condition, foaming, has been
related to the development of two bacteria genera Nocardia and Microthrix (Pitt and Jenkins,
1990), which have hydrophobic cell surfaces and attach to air bubble surfaces, where they
stabilize the bubbles to cause foam (see Fig. 2.4). The organisms can be found at high
concentrations in the foam above the activated-sludge liquid.
(a) (b)
Fig. 2.4 Examples of foam caused by Nocardia accumulated on the surface of activated sludge aeration
tanks (Metcalf and Eddy, 2003).
2.7.3 Stoichometry of aerobic biological oxidation
The stoichiometry for aerobic oxidation was discussed previously but is repeated here for
completeness. In aerobic oxidation, the conversion of organic mater is carried out by mixed
bacterial cultures in general accordance with the stoichiometry shown below.
Oxidation and synthesis:

Bacteria
COHNS + O2 + nutrients CO2 + NH3 + C5H7NO2 + other end products (2-69)
Organic new cell
Matter
Endogenous respiration:
Bacteria
C5H7NO2 + 5O2 5CO2 + 2H2O + NH3 + energy (2-70)
113 160
1 1.42
I Eq. (2-69), COHNS is used to represent the organic matter in wastewater, which serve as the
electron donor while the oxygen serves as the electron acceptors. Although the endogenous
respiration reaction (2-70) is shown as resulting in relatively simple end products and energy,
stable organic end products are also formed. From Eq. (2-5), it was shown that if all of the cells
26
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(i. e. the electron donor) were oxidized completely, the UBOD or COD of the cells is equal to
1.42 times the concentration of cells as VSS. At longer SRT values, a greater portion of the cells
will be oxidized.
2.7.4 Growth kinetics
The form of the rate expressions for substrate utilization and biomass growth for the
heterotrophic oxidation of organic substrates, based on the stoichiometry given above, were
presented previously but are repeated below for ease of reference.
kXS
rsu = −
Ks + S
kSX
rg = −Yrsu − kd X = Y − kd X
Ks + S
Both of the above expressions are of the saturation type. As noted previously, these expressions
are similar to the saturation equation proposed by Monod (1942) for growth ad the Michaelis-
Menten equation for substrate utilization (Bailey and Ollis, 1986). Typical k and Ks values at
20oC vary from 8.0 to 12.0 g COD/g VSS.d and 10 to 40 g bsCOD/m3, respectively. The Ks
value can vary depending on the nature and complexity of the bsCOD components. For easily
biodegradable single substrates, Ks values of less than 1.0 mg bsCOD/L have been measured
(Bielefeldt and Stensel, 1999).
Applying the above expressions for substrate utilization and biomass growth leads to the
development of a series of design parameters including the solids retention time (SRT), the food
to microorganism ratio (F/M), and the specific utilization rate (U). These design parameters are
applied to the design of a variety of activated-sludge processes. With the exception of some
difficult-to-degrade constituents in industrial wastewaters, the kinetics for aerobic oxidation of
organic substrates seldom control the SRT design value for the activated-sludge process. For
good floc formation, sufficient time is needed for the biomass in the activated-sludge aeration
tank to develop extracellular polymers and a floc structure. More optimal flocculation and TSS
removal in clarification occur typically at SRT values greater than 2.5 to 3.0 d at 20oC and 3 to 5
d at 10oC. However, some wastewater-treatment plants in warmer climates operate at SRT values
varying from less than 1.0 to 1.5 d. Excessively long SRTs (> 20 d( may lead to floc
deterioration with the development of small pinpoint floc particles that produce a more turbid
effluent. However, even with pinpoint floc, effluent suspended solids concentrations of less than
30 g/m3 are generally achieved. The SRT may be varied in treatment plant operations to find the
most optimal settling condition.
2.7.5 Environmental factors
For carbonaceous removal, pH in the range of 6.0 to 9.0 is tolerate, while optimal performance
occurs near a neutral pH. A reactor DO concentration of 2.0 mg/L is commonly used, and at
concentrations above 0.50 mg/L there is little effect of the DO concentration on the degradation
rate. For industrial wastewaters care must be taken to assure that sufficient nutrients (N and P)
are available for the amount of bsCOD to be treated. Heterotrophic bacteria responsible for BOD
removal can tolerate higher concentrations of toxic substances as compared to the bacteria
responsible for ammonia oxidation or the production of methane.
27
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2.8 BIOLOGICAL NITRIFICATION
Nitrification is the term used to describe the two-step biological process in which ammonia
(NH4-N) is oxidized to nitrite (NO2-N) and nitrite is oxidized to nitrate (NO3-N). The need for
nitrification in wastewater treatment arises from water quality concerns over: (1) the effect of
ammonia on receiving water with respect to DO concentrations and fish toxicity, (2) the need to
provide nitrogen removal to control eutrophication, and (3) the need to provide nitrogen control
for water-reuse applications including groundwater recharge. For reference, the current (2001)
drinking water maximum contaminant level (MCL) for nitrate nitrogen is 45 mg/L as nitrate or
10 mg/L as nitrogen. The total concentration of organic and ammonia nitrogen concentration in
municipal wastewaters is typically in the range from 25 to 45 mg/L as nitrogen based on flow-
rate of 450 L/capita.d (120 gal/capita.d). In many parts of the world with limited water supplies,
total nitrogen concentrations in excess of 200 mg/L as N have been measured in domestic
wastewater.
As with BOD removal, nitrification can be accomplished in both suspended growth and attached
growth biological processes. For suspended growth processes, a more common approach is to
achieve nitrification along with BOD removal in the same single-sludge process, consisting of an
aeration tank, clarifier and sludge recycle system (see Fig. 2.5a). In cases where there is a
significant potential for toxic and inhibitory substances in the wastewater, a two-sludge
suspended growth system may be considered (see Fig. 2.5b). The two-sludge system consists of
two aeration tanks and two clarifiers in series with the first aeration tank/clarifier unit operated at
a short SRT for BOD removal. The BOD and toxic substance are removed in the first unit, so
that nitrification can proceed unhindered in the second. A portion of influent wastewater usually
has efficient solid flocculation and clarification. Because the bacteria responsible for nitrification
grow much more slowly that heterotrophic bacteria, systems designed for nitrification generally
have much longer hydraulic and solid retention times than those for systems deigned only for
BOD removal.
In attached growth systems used for nitrification, most of the BOD must be removed before
nitrifying organisms can be established. The heterotrophic bacteria have a higher biomass yield
and thus can dominate the surface are of fixed-film systems over nitrifying bacteria. Nitrification
is accomplished in an attached growth reactor after BOD removal or in a separate attached
growth system designed specifically for nitrification.
2.8.2 Microbiology
Aerobic autotrophic bacteria are responsible for nitrification in activated sludge and biofilm
processes. Nitrification, as noted above, is a two-step process involving two groups of bacteria.
In the first state, ammonia is oxidized to nitrate by one group of autotrophic bacteria. In the
second state, nitrite is oxidized to nitrate by another group of autotrophic bacteria. It should be
noted that the two groups of autotrophic bacteria are distinctly different. Starting with classical
experiments on nitrification by Winogradsky (1981), the bacteria genera commonly noted for
nitrification in wastewater treatment are the autotrophic bacteria Nitrosomonass and Nitrobacter,
which oxidize ammonia to nitrate and then to nitrate, respectively. Other autotrophic bateria
genera capable of obtaining energy from the oxidation of ammonia to nitrite (prefix with
Nitroso-) are Nitrosococcus, Nitrispira, Nitrosolobus, and Nitrisorobrio (Painter, 1970). It
should be noted that during the 1990s, many more autotrophic bacteria were identified as being
capable of oxidizing ammonia.
28
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Besides Nitrobacter, nitrite can also be oxidized by other autotrophic bacteria (Nitro-) genera:
Nitricoccus, Nitrospira, Nitrospina and Nitroeystis. Using oligo-nucleotide probes for ammonia-
oxidizing bacteria, Wagner et al. (1995) showed that Nitrosomonas was common in activated
sludge systems. For nitrite oxidation in activated sludge, Teske et al. (1994) found that
Nitrococcus was quite prevalent. Whether different growth conditions can select for different
genera of nitrifying bacteria or if heir nitrification kinetics are significantly different is unknown
at present.
Fig. 2.5 Process configuration used for biological nitrification: (a) single-sludge suspended growth
system and (b) two-sludge suspended growth system (Metcalf and Eddy, 2003).
2.8.3 Stoichiometry of biological nitrification
The energy-yielding two-step oxidation of ammonia to nitrate is as follows:
Nitroso-bacteria:
2NH4+ + 3O2 Æ 2NO2- + 4H+ + 2H2O (2-71)
Nitro-bacteria:
2NO2- + O2 Æ 2NO3- (2-72)
Total oxidation reaction:
NH4+ + 2O2 Æ NO2 + 2H+ + H2O (2-73)
Based on the above total oxidation reaction, the oxygen required for complete oxidation of
ammonia is 4.57 g O2/g N oxidized with 3.43 g O2/g used for nitrate production and 1.14 g O2/g
NO2 oxidized. When synthesis is considered, the amount of oxygen required is less than 4.57 g
O2/ g N. In addition to oxidation, oxygen is obtained from fixation of carbon dioxide and
nitrogen into cell mass.
29
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Neglecting cell tissue, the amount of alkalinity required to carry out the reaction given in Eq. (2-
73) can be estimated by writing Eq. (2-73) as follows:
NH4+ + 2HCO3- + 2O2 Æ NO3- + 2CO2 + 3H2O (2-74)
In the above equation, for each g of ammonia nitrogen (as N) converted, 7.14 g of alkalinity as
CaCO3 will be required [2 x 50 g CaCO3/eq)/14].
Along with obtaining energy, a portion of the ammonium ion is assimilated into cell tissue. The
biomass synthesis reaction can be represented as follows:
4CO2 + HCO3- + NH4+ + H2O --> C5H7O2N + 5O2 (2-75)
As noted previously, the chemical formula C5H7O2N is used to represent the synthesized bacteria
cells.
The wastewater nitrogen concentration, BOD concentration, alkalinity, temperature, and

potential for toxic compounds are major issues in the design of biological nitrification processes.
Nitrifying bacteria need CO2 and phosphorus for cell growth, as well as trace elements. With
such a low cell yield, the CO2 in air is adequate and phosphorus is seldom limiting. Trace
element concentrations that have been found to stimulate the growth of nitrifying bacteria in pure
culture work are Ca = 0.5, Cu = 0.01, Mg = 0.03, Mo = 0.001, Ni = 0.10 and Zn = 1.0 mg/L
(Poduska, 1973).
For nitrification systems operated at temperatures below 28oC, ammonia-oxidation kinetics

versus nitrite-oxidation kinetics are rate-limiting, so that designs are based on saturation versus
nitrite-oxidation s given below, assuming excess DO is available:
⎛ µ N ⎞
µ n = ⎜⎜ nm ⎟⎟ − kdn (2-76)
⎝ Kn + N ⎠
Where µn = specific growth rate of nitrifying bacteria, g new cells/g cells.d

µnm = maximum specific growth rate of nitrifying bacteria, g new cells/g cells.d
N = nitrogen concentration, g/m3
kn = half-velocity constant, substrate concentration at one-half the maximum specific
substrate utilization rate, g/m3
kdn = endogenous decay coefficient for nitrifying organisms, g VSS/g VSS.d
A wide range of maximum specific growth rates have been reported as a function of temperature
(Randall et al., 1992). At 20oC, reported µnm varies from 0.25 to 0.77 g VSS/ g VSS/ The wide
range of nitrification growth rates may be due to the presence of inhibitory substances in the
wastewater and/or variations in experimental techniques and methods of analysis. In any event,
the µnm values for nitrifying organisms are much lower than the corresponding values for
heterotrophic organisms, requiring much longer SRT values for nitrifying activated – sludge
systems. Typical design SRT values may range from 10 to 20 d at 10oC to 4-7 d at 20oC. Above
28oC, both ammonia and nitrite oxidation kinetics should be considered. At elevated
temperatures, the relative kinetics of NH4-N and NO2--N oxidation change, and NO2-N will
accumulate at lower SRT values.
30
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For fully acclimated complete-mix activated sludge nitrification systems, at temperatures below
25oC with sufficient Do present, the NO2--N concentration may be less than 0.10 mg/L as
compared to NH4-N concentrations in the range of 0.50 to 1.0 mg/L. However, during the
initiation of nitrification, NO2-N concentrations will be greater than NH4-N concentrations, as
the growth of nitrite-oxidizing bacteria cannot occur until the ammonia-oxidizing bacteria
generate nitrite. Under transient conditions, NO2-N concentrations of 5 to 20 mg/L are possible.
Nitrification rates are affected by the liquid DO concentration in activated sludge. In contrast to
what has been observed for aerobic heterotrophic bacteria degradation of organic compounds,
nitrification rates increase up to DO concentrations of 3 to 4 mg/L. To account for the effects of
DO, the expression for the specific growth rate (Eq. 2-76) is modified as follows:
⎛ µ N ⎞⎛ DO ⎞
µ n = ⎜⎜ nm ⎟⎟⎜⎜ ⎟⎟ − k m (2-77)
⎝ K n + N ⎠⎝ K o + DO ⎠
Where DO = dissolved oxygen concentration, g/m3

K0 = half-saturation coefficient for DO, g/m3
Other terms as defined previously.
While the above kinetic model and coefficients, based on observed results, can be used to
describe nitrification in system at low modest organic loadings, the use of these models will
generally overpredict nitrification rates in system with high organic loading rate. Stenstrom and
Song (1991) have shown experimentally that the effect of DO on nitrification is affected by the
activated-sludge floc size and density, and total oxygen demand of the mixed liquor. Nitrifying
bacteria are distributed within a floc containing heterotrophic bacteria and other solids, with floc
diameter ranging from 100 to 400 µm. Oxygen from the bulk liquid diffused in to floc particles
and bacteria deeper within the floc are exposed to lower DO concentration. At higher organic
loadings, there is a greater substrate in the mixed liquor, which cause a high oxygen consumption
rate within the floc. Therefore, a higher bulk liquid DO concentration is need maintain the same
internal floc DO concentration and subsequence nitrification rates.
At low DO concentration (<0.50 mg/L) where nitrification rate are greatly inhibited, the low DO
inhibition effect has been shown to be greater for Nitrobacter than for Nitrosomonas. In such
case, incomplete nitrification will occur with increase NO2-N concentration in the effluent. The
presence of nitrite in the effluent is particularly troublesome for plants that use chlorination for
disinfection, as nitrite is readily oxidized by chlorine requiring 4g chlorine/g NO2-N.
Environment factors
Nitrification is affected by a number of environmental factor including pH, toxicity, metals, and
unionized ammonia.
Hydrogen-Ion Concentration (pH). Nitrification is pH sensitive and rates decline significantly

at pH values below 6.8. At pH values near 5.8 to 6.0, the rates may be 10 to 20 percent of the
rate at pH 7.0 (U.S. EPA, 1993). Optimal nitrification rates occur at pH values in the 7.5 to 8.0
range. A pH of 7.0 to 7.2 is normally used to maintain reasonable nitrification rate, and for
locations with low-alkalinity water, alkalinity is added at the wastewater-treatment plant to
maintain acceptable pH values. The amount of alkalinity added depend on the initial alkalinity
concentration and the amount of NH4-N to be oxidized. Alkalinity may be added in the form of
lime, soda ash, sodium bicarbonate, or magnesium hydroxide depending on cost and chemical
handling issues.
31
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Toxicity. Nitrifying organism are sensitive to wide range of organic and inorganic compounds
and at concentration well below those concentrations that would affect aerobic heterotrophic
organisms. In many case, nitrification rate are inhibited even thought bacteria continue to grow
and oxidize ammonia and nitrite, but at significantly reduced rates. In some case, toxicity may be
sufficient to kill the nitrifying bacteria.
Nitrifiers have been shown to be good indicators of the presence of organic toxic compounds at
low concentration (Blum and Speece, 1991). Toxic organic compounds have been listed by
Hockenbury and Grady (1977) and Sharma and Ahlert (1977). Compounds that are toxic include
solvent organic chemicals, amines, proteins, tannins, phenolic compounds, alcohols, cyanates,
ethers, carbamates, and benzene. Because of the numerous compounds that can inhibit
nitrification, it is difficult to pinpoint the source of nitrification toxicity for wastewater plants
whit inhibition, and extensive sampling of the collection system is normally needed to find the
source.
Metals. Metals are also of concern for nitrifiers and Skinner and Walker (1961) have shown
complete inhibition of ammonia oxidation at 0.25 mg/L nickel, 0.25 mg/L chromium, and 0.10
mg/L copper.
Un-ionized ammonia. Nitrification is also inhibited by un-ionized ammonia (NH3) or free

ammonia, and un-ionized nitrous acid (HNO2). The inhibition effects are dependent on the total
nitrogen species concentration, temperature, and pH. At 20oC and pH 7.0, the NH4
concentrations at 100 mg/L and 20 mg/L may initiate inhibition of NH4-N and NO2-N oxidation,
respectively, and NO2-N concentrations at 280 mg/L may initiate inhibition of NO2-N oxidation
(US.EPA, 1993).
2.9 BIOLOGICAL DENITRIFICATION
The biological reduction of nitrate to nitric oxide, nitrous oxide, and nitrogen gas is termed
denitrification. Biological denitrification is an integral part of biological nitrogen removal, which
involves both nitrification and denitrification. Compared to alternatives of ammonia stripping,
breakpoint chlorination, and ion exchange, biological nitrogen removal is generally more cost-
effective and used more often. Biological nitrogen removal is used in wastewater treatment
where there are concerns for eutrophication and where ground water must be protected against
elevated NO3—N concentrations where wastewater treatment plant effluent used for groundwater
recharge and other reclaimed water applications.
Two modes of nitrogen removal can occur in biological processes, and these are termed
assimilating and dissimilating nitrate reduction (see Fig. 2.6). Assimilating nitrate reduction
involves the reduction of nitrate to ammonia for use in cell synthesis. Assimilation occurs when
NH4-N is not available and is independent of DO concentration. On the other hand, dissimilating
nitrate reduction or biological denitrification is coupled to the respiratory electron transport
chain, and nitrate and nitrite is used as an electron acceptor for the oxidation of a variety of
organic or inorganic electron donors.
Two basic flow diagrams for activated-sludge denitrification and the conditions that drive the
denitrification reaction rates are illustrated in Fig. 2.7. The first flow diagram (Fig. 2.7a) is for
the Modified Ludzak-Ettinger (MLE) process (U.S. EPA, 1993), the most common process used
for biological nitrogen removal in municipal wastewater treatment. The process consists of an
anoxic tank followed by the aeration tank where nitrification occurs. Nitrate produced in the
32
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aeration tank is recycled back to the anoxic tank. Because the organic substrate in the influent
wastewater provides the electron donor for oxidation reduction reactions using nitrate, the
process is termed substrate denitrification. Further, because the anoxic process precedes the
aeration tank, the process is known as a preanoxic denitrification.
Fig. 2.6 Nitrogen transformations in biological treatment processes (Metcalf and Eddy, 2003).
Fig. 2.7 Type of denitrification processes and the reactors used for their implementation: (a) substrate
driven (preanoxic denitrification) and (b) endogenous driven (postanoxic denitrification)
(Metcalf and Eddy, 2003).
In the second process shown on Fig. 2.7b, denitrification occurs after nitrification and the
electron donor source is from endogenous decay. The process illustrated on Fig. 2.7b is generally
termed a postanoxic denitrification as BOD removal has occurred first and is not available to
drive the nitrate reduction reaction. When a postanoxic denitrification process depends solely on
endogenous respiration for energy, it has much slower rate of reaction than for the preanoxic
processes using wastewater BOD. Often an exogenous carbon source such as methanol or acetate
is added to postanoxic processes to provide sufficient BOD for nitrate reduction and to increase
the rate of denitrification. Postanixic processes include both suspended and attached growth
33
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granular-medium filtration process, both nitrate reduction and effluent suspended solid removal
occurs in the same reactor.
The denitrification preanoxic and postanoxic processes described employ heterotrophic bacteria
for nitrate reduction, but other pathways for biological nitrogen removal exist. Ammonia can be
converted to nitrogen gas by novel autotrophic bacteria under anaerobic conditions and by
heterotrophic-nitrifying bacteria under aerobic conditions. The organisms identified with these
reactions are presented in the Microbiology section below. Littleton et al. (2000) looked for
possible influence of the autotrophic denitrifying bacteria or to heterotrophic nitrifying bacteria
for nitrogen removal in aerated full-scale, long SRT, single-sludge, activated-sludge systems but
could not find any significant activity for them. However, the use of novel autotrophic
denitrifying bacteria has been demonstrated in a process for the treatment of high ammonia
strength aerobic digestion centrate in a fluidized bed reactor at 30 to 35oC (Strous et al. 1997,
Jetten et al. 1999). The fluidized bed reactor is effective in maintaining these relatively slow
growing bacteria. Under anaerobic conditions NH4+ is oxidized by NO2- to produce nitrogen gas
and small amount of nitrate. About 1.3 moles of NO2- are used per mole of NH4+ (Strous et al.
1999a). A portion of the centrate stream must be nitrified to nitrite, but no carbon source is
needed for nitrogen removal. The process is termed the Annamox process, which stands for
anaerobic ammonium oxidation. The oxidation of ammonia to nitrite in anaerobic digester
centrate has been done in a process termed the SHARON process, which can be used with the
Annamox process (Jetten et al. 1999).
2.9.2 Microbiology
A wide range of bacteria has been shown capable of denitrification, but similar microbial
capability has not been found in algae or fungi. Bacteria capable of denittrification are both
autotrophic and heterotrophic. The heterotrophic organisms include the following genera:
Achromobacter, Acinetobacter, Agrobacterium, Alcaligenes, Arthrobacter, Bacillus,
Chromobacterium, Corynebacterium, Flavobacterium, Hypomicrobium, Moraxella, Neisseria,
Paracoccus, Propionibacterium, Pseudomonas, Rhizobium,Rhodopseudomonas, Spirillum, and
Vibrio (Payne, 1981). In addition, Gayle (1989) list Halobacterium and Methanomonas,
Pseudomonas species are the most common and widely distributed of all the denitrifiers, and
have been shown to use a wide array of organic compounds including hydrogen, methanol,
carbonhydrates, organic acids, alcohols, benzoates, and other aromatic compounds (Payne,
1981). Most of these bacteria are facultative aerobic organisms with the ability to use oxygen as
well as nitrate or nitrite, and some can also carry out fermentation in the absence of nitrate or
oxygen. Other autotrophic bacteria that can denitrify use hydrogen and reduced sulfur
compounds as electron donors during denitrification. Both groups of organisms can grow
heterotrophically if an organic carbon source is present (Gayle, 1989).
Nitrogen removal can also be accomplished by the heterotrophic and autotrophic-nitrifying

bacteria under certain conditions, and by a unique bacteria associated with the Annamox process,
which was discovered in the mid-1990s. Denitrification can occur under anaerobic condition by
heterotrophic nitrifying bacteria (Robertson and Kuenen, 1990, and Patureau et al. 1994), so that
simultaneous nitrification and denitrification exist with the conversion of ammonia to gaseous
nitrogen products. The heterotrophic bacteria, Paracocus pantotropha, have been studied
extensively for simultaneous oxidation and nitrate reduction. The oxidation of ammonia by
heterotrophic bacteria requires energy, which can be obtained by nitrate or nitrite reduction by
P.pantotropha under aerobic conditions. A readily available substrate, such as acetate, is also
needed. Due to the need for the carbon substrate, which is in limited supply in aerobic activated-
sludge systems, little growth of heterotrophic nitrifiers is expected (van Loosdrech and Jetten,
1998) in aerobic wastewater treatment systems.
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Autotrophic nitrifying bacteria, such as Nitrosomonas europaea, can use nitrite to oxidize
ammonia, with production of nitrogen gas, when dissolved oxygen is not present (Boc et al.
1995). With oxygen present, these bacteria oxidize the ammonia with oxygen as electron
acceptors.
Ammonia oxidation with the reduction of nitrite under anaerobic conditions has also been shown
at temperatures above 20oC in the Annamox process (Strous et al. 1997). The bacteria in
Annamox process are different than the autotrophic nitrifying bacteria described above, in that it
cannot use oxygen for ammonia oxidation (Jetten et al. 1999). The Annamox bacteria could not
be isolated and grow in pure culture (Strous et al. 1999b), but an enrichment was obtained by
density purification for 16S rRNA extraction and analysis. Phylogenetic analysis showed that the
bacteria is in the order Planctomycetales, a division with domain Bacteria. Under anaerobic
conditions the ammonia oxidation rate by the Annamox bacteria was shown to be 6 to 10 times
faster than that for N. Europaea (Jetten et al. 1999).
2.9.3 Stoichiometry of Biological Denitrification
Biological denitrification involves the biological oxidation of many organic substrates in

wastewater treatment using nitrate or nitrite as the electron acceptors instead of oxygen. In the
absent of DO or under limited DO concentrations, the nitrate reductase enzyme in the electron
transport respiratory chain is induced, and helps to transfer hydrogen and electrons to nitrate as
the terminal electron acceptor. The nitrate reduction reactions involves the following reduction
steps from nitrate to nitrite, to nitric oxide, to nitrous oxide, and to nitrogen gas:
NO3- ÆNO2- Æ NO Æ N2O Æ N2 (2-78)
In biological nitrogen removal process, the electron donor is typically one of three sources: (1)
the bsCOD in the influent wastewater, (2) the bsCOD produced during endogenous decay, and
(3) an exogenous source such as methanol or acetate. The latter has been added in separate
treatment units, such as polishing filters, after nitrification where almost no bsCOD remains. The
term C10H19O3N is often used to represent the biodegradable organic matter in wastewater (U.S.
EPA, 1993).
Wastewater:
C10H19O3N + 10NO3- Æ 5N2 + 10CO2 + 3H2O + NH3 + 10OH- (2-79)
Methanol:
5CH3OH + 6 NO3- Æ 3N2 + 5 CO2 + 7 H2O + 6 OH- (2-80)
Acetate:
5 CH3COOH + 8 NO3- Æ 4N2 + 10 CO2 + 6 H2O +8 OH- (2-81)
In all the above heterotrophic denitrification reactions, one equivalent of alkalinity is produced
per equivalent of NO3-N reduced, which equates to 3.57g of alkalinity (as CaCO3) production
per g of nitrate nitrogen reduced. Recall from nitrification that 7.14 alkalinity (as CaCO3) was
consumed per g of NH4-N oxidized, so that by denitrification about one-half of the amount
destroyed by nitrification can be recovered.
35
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For biological denitrification, the biokinetic equations used to describe bacteria growth and
substrate utilization are similar to those described previously in this chapter for aerobic
heterotrophic bacteria. The rate of soluble substrate utilization is also controlled by the soluble
substrate concentration, with nitrate serving as the electron acceptor in lieu of oxygen. The
nitrate concentration controls the substrate utilization kinetics only at very low NOrN
concentrations, near 0.1 mg/L. Nitrate serves as an electron acceptor in the same way as oxygen
from a biokinetics perspective, and thus the nitrate utilization rate (denitrification rate) is
proportional to the substrate utilization rate.
There are two general cases where the substrate utilization rate controls the denitrification rate.
The first is for anoxic/aerobic processes where the organic substrate (electron donor) is from the
influent wastewater fed into the anoxic reactor. The second is for postanoxic denitrification,
where nitrate reduction is done after secondary treatment in a reactor receiving another carbon
source. Because the BOD is depleted from secondary treatment, an exogenous carbon source is
used to drive the nitrate reduction reaction. In most cases methanol is supplied to create the
demand for the electron acceptor in suspended growth or attached growth processes. Endogenous
respiration creates a demand for nitrate in addition to that caused by substrate utilization and
oxidation. This reaction occurs in the mixed liquor of anoxic tanks and is at a much lower rate
than the denitrification rate caused by substrate utilization.
In the case where wastewater provides the electron donor, an anoxic reactor receives the influent
wastewater and it is followed in the treatment system by an aerobic reactor where nitrification
occurs. Heterotrophic bacterial growth occurs in both the anoxic and aerobic zones with nitrate
and oxygen consumption, respectively. The mixed liquor biomass concentration can be
calculated based on the total amount of BOD removed, but only a portion of that biomass can
use both nitrate and oxygen as electron acceptors. The microorganisms in the other portion are
strict aerobes and can only use oxygen as the electron acceptor. To apply biokinetic expressions
for denitrification, the substrate utilization rate expression is modified to account for the fact that
only a portion of the biomass is active in the anoxic zone.
The substrate utilization rate rsu expression is modified by a term to show a lower utilization rate
in the anoxic zone as follows:
kXSη
rsu = − (2-82)
KS + S
Where η = fraction of denitrifying bacteria in the biomass, g VSS/gVSS

K, X, S, Ks are as defined previously.
When nitrate is used as an electron acceptor instead of oxygen, the maximum specific substrate
utilization rate (k) may be lower than the rate with oxygen as the electron acceptor. In Eq. (2-82),
the k value used is the same as that used for oxygen, and any effect of lower kinetic rate is
incorporated into the η term. The Ks value has been found to be similar, whether nitrate or
oxygen is the electron acceptor (Stensel and Home, 2000).
The value for η has been found to vary from 0.20 to 0.80 for preanoxic denitrification reactors
fed domestic wastewaters (Stensel and Home, 2000). The activated sludge configuration, the
system SRT, and the fraction of influent BOD removed with nitrate appear to affect the η value.
For anoxic/aerobic processes with substantial substrate and nitrate removal in the preanoxic zone
η may be close to 0.80. In spite of the fact that only a portion of the mixed liquor biomass can
36
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use nitrate, the anoxic reactor volumes used for anoxic/aerobic processes range from only 10 to
30 percent of the total volume (anoxic plus aerobic) for treating domestic wastewater.
For postanoxic suspended growth or attached growth processes the biomass is developed under
mainly anoxic conditions and with a selected single organic substrate. In this case the η term is
not necessary because the biomass consists of mainly denitrifying bacteria. The biokinetic
equations presented previously can then be used with the appropriate kinetic coefficient values
(k, Ks, Y, kd) to design a postanoxic complete-mix suspended growth process. The kinetic
coefficient values for growth using methanol have been developed at 10°C and 20°C in
laboratory studies (Randall et aI., 1992). The kinetics for methanol utilization are such that the
SRTs required for a denitrification suspended growth process are in the same range as SRTs for
aerobic systems designed for BOD removal only, about 3 to 6 d.
Effect of dissolved oxygen concentration
Dissolved oxygen can inhibit nitrate reduction by repressing the nitrate reduction enzyme. In
activated-sludge flocs and biofilms, denitrification can proceed in the presence of low bulk liquid
DO concentrations. A dissolved oxygen concentration of 0.2 mg/L and above has been reported
to inhibit denitrification for a Pseudomonas culture (Skerman and MacRae, 1957; Terai and
Mori, 1975) and by Dawson and Murphy (1972) for activated-sludge treating domestic
wastewater. Nelson and Knowles (1978) reported that denitrification ceased in a highly dispersed
growth at a DO concentration of 0.13 mg/L. The effect of nitrate and DO concentration on the
biokinetics is accounted for by two correction factors to Eq. (2-82) expressed in the form of two
saturation terms as follows:
⎛ kXS ⎞⎛⎜ ⎞⎛ K o' ⎞

⎟⎟(η )
NO3 ⎟⎜
rsu = −⎜⎜ ⎟⎟ ⎜ (2-83)
⎝ S
K + S ⎜
⎠⎝ S , NO3
K + NO ⎟
3 ⎠⎝ K '
o + DO ⎠
Where Ko’ = DO inhibition coefficient for nitrate reduction, mg/L

KS,NO3 = half velocity coefficient for nitrate limited reaction, mg/L
Other terms are as defined previously.
The value of Ko’ is system-specific. Values in the range from 0.1 to 0.2 mg/L have been
proposed for Ko’ and 0.1 mg/L for Ks.NO3 (Barker and Dold, 1997). Assuming a Ko value of 0.1
mg/L the rate of substrate utilization with nitrate as the electron acceptor at DO concentrations of
0.10, 0.20, and 0.50 mg/L would be at 50, 33, and 17 percent of the maximum rate, respectively.
Effect of simultaneous nitrification-denitrification
In activated-sludge systems, the issue of DO concentration is confounded by the fact that the
measured bulk liquid DO concentration does not represent the actual DO concentration within
the activated-sludge floc. Under low DO concentration conditions, denitrification can occur in
the floc interior, while nitrification is occurring at the floc exterior. Also in activated sludge
tanks operated at low DO concentrations, both aerobic and anaerobic zones exist depending on
mixing conditions and distance from the aeration point, so that nitrification and denitrification
can be occurring in the same tank. Under these conditions, nitrogen removal that occurs in a
single aeration tank is referred to as simultaneous nitrification and denitrification. Although both
nitrification and denitrification are occurring at reduced rates as indicated by the DO effects
described for both processes, if a sufficient SRT and HRT exist, the overall nitrogen removal can
be significant. Rittman and Langeland (1985) reported greater than 90 percent nitrogen removal
by nitrification and denitrification in an activated-sludge system used to treat municipal
37
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wastewater at DO concentrations below 0.50 mg/L and with values of HRT greater than 25
hours.
Environmental factors
Alkalinity is produced in denitrification reactions and the pH is generally elevated, instead of

being depressed as in nitrification reactions. In contrast to nitrifying organisms, there has been
less concern about pH influences on denitrification rates. No significant effect on the
denitrification rate has been reported for pH between 7.0 and 8.0, while Dawson and Murphy
(1972) showed a decrease in the denitrification rate as the pH was decreased from 7.0 to 6.0 in
batch un acclimated tests.
2.10 BIOLOGICAL PHOSPHORUS REMOVAL
The removal of phosphorus by biological means is known as biological phosphorus removal.

Phosphorus removal is generally done to control eutrophication because phosphorus is a limiting
nutrient in most freshwater systems. Treatment plant effluent discharge limits have ranged from
0.10 to 2.0 mg/L of phosphorus depending on plant location and potential impact on receiving
waters. Chemical treatment using alum or iron salts is the most commonly used technology for
phosphorus removal, but since the early 1980s success in full-scale plant biological phosphorus
removal has encouraged further use of the technology. The principal advantages of biological
phosphorus removal are reduced chemical costs and less sludge production as compared to
chemical precipitation.
In the biological removal of phosphorus, the phosphorus in the influent wastewater is

incorporated into cell biomass, which subsequently is removed from the process as a result of
sludge wasting. Phosphorus accumulating organisms (PAOs) are encouraged to grow and
consume phosphorus in systems that use a reactor configuration that provides PAOs with a
competitive advantage over other bacteria. The reactor configuration utilized for phosphorus
removal is comprised of an anaerobic tank having a HRT value of 0.50 to 1.0 h that is placed
ahead of the activated-sludge aeration tank (see Fig. 2.8). The contents of the anaerobic tank are
mixed to provide contact with the return activated sludge and influent wastewater. Anaerobic
contact tanks have been placed in front of many different types of suspended growth processes,
with aerobic SRT values ranging from 2 to 40 d.
Phosphorus removal in biological systems is based on the following observations (Sedlak, 1991):
1. Numerous bacteria are capable of storing excess amounts of phosphorus as polyphosphates in

their cells.
2. Under anaerobic conditions, PAOs will assimilate fermentation products (e.g., volatile fatty
acids) into storage products within the cells with the concomitant release of phosphorus from
stored polyphosphates.
3. Under aerobic conditions, energy is produced by the oxidation of storage products and
polyphosphate storage within the cell increases.
38
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Fig. 2.8 Biological phosphorus removal: (a) typical reactor configuration. Photos below flow diagram are
of (b) transmission electron microscope images of polyhydroxybutyrate and (c) polyphosphate
storage granules (Metcalf and Eddy, 2003).
A simplified version of the processes occurring in the anaerobic and aerobic/anoxic reactors or
zones is presented below. In many applications for phosphorus removal, an anoxic reactor
follows the anaerobic reactor and precedes the aerobic reactor. Most PAOs can use nitrite in
place of oxygen to oxidize their stored carbon source. A more comprehensive description of the
biochemistry and intracellular transformations can be found in Wentzel et al. (1991).
Processes occurring in the anaerobic zone
- Acetate is produced by fermentation of bsCOD which, as defined earlier, is dissolved

degradable organic material that can be assimilated easily by the biomass. Depending on the
value of T for the anaerobic zone, some colloidal and particulate COD is also hydrolyzed and
converted to acetate, but the amount is generally small compared to that from the bsCOD
conversion.
- Using energy available from stored polyphosphates, the PAOs assimilate acetate and produce
intracellular polyhydroxybutyrate (PHB) storage products. Some glycogen contained in the
cell is also used. Concurrent with the acetate uptake is the release of orthophosphate (O-P04),
as well as magnesium, potassium, and calcium cations.
- The PHB content in the PAOs increases while the polyphosphate decreases.
Processes occurring in the aerobic/anoxic zone
- Stored PHB is metabolized, providing energy from oxidation and carbon for new cell growth.
- Some glycogen is produced from PHB metabolism.
39
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- The energy released from PHB oxidation is used to form polyphosphate bonds in cell storage
so that soluble orthophosphate (O-P04) is removed from solution and incorporated into
polyphosphates within the bacterial cell. Cell growth also occurs due to PHB utilization and
the new biomass with high polyphosphate storage accounts for phosphorus removal.
- As a portion of the biomass is wasted, stored phosphorus is removed from the biotreatment
reactor for ultimate disposal with the waste sludge.
The events occurring in the anaerobic and aerobic zones are illustrated graphically on Fig. 2.9.
Fig. 2.9 Fate of soluble BOD and phosphorus in nutrient removal reactor (adapted from Sedlak, 1991;
Metcalf and Eddy, 2003).
2.10.2 Microbiology
Phosphorus is important in cellular energy transfer mechanisms via adenosine triphosphate

(ATP) and polyphosphates. As energy is produced in oxidation reduction reactions, adenosine
diphosphate (ADP) is converted to ATP with 7.4 kcal/mole of energy captured in the phosphate
bond. As the cell uses energy, ATP is converted to ADP with phosphorus release. For common
heterotrophic bacteria in activated-sludge treatment the typical phosphorus composition is 1.5 to
2.0 percent. However, many bacteria are able to store phosphorus in their cells in the form of
energy-rich polyphosphates, resulting in phosphorus content as high as 20 to 30 percent by dry
weight. The polyphosphates are contained in volutin granules within the cell along with Mg2+,
Ca2+, and K+ cations.
In the anaerobic zone, concentrations of O-PO4 as high as 40 mg/L can be measured in the
liquid, as compared to wastewater influent concentrations of 5 to 8 mg/L. The high concentration
of O-PO4 can be taken as an indication that phosphorus release by the bacteria has occurred in
this zone. Also in this zone, significant amounts of poly-bhydroxybutyrate (PHB) are found
stored in bacteria cells, but the PHB concentration declines appreciably in the subsequent anoxic
and/or aerobic zones and can be measured and quantified. The O-PO4 is taken up from solution
in the aerobic and anoxic zones, generally leading to very low remaining concentrations. Based
on investigations of biological phosphorus removal, it was found that acetate was essential to
forming the PHB under anaerobic conditions, which provided a competitive advantage for the
PAOs.
The anaerobic zone in the anaerobic/aerobic treatment process is termed a "selector," because it
provides conditions that favor the proliferation of the PAOs, by the fact that a portion of the
influent bCOD is consumed by the PAOs instead of other heterotrophic bacteria. Because the
PAOs prefer low-molecular-weight fermentation product substrates, the preferred food source
would not be available without the anaerobic zone that provides for the fermentation of the
influent bsCOD to acetate. Because of the polyphosphate storage ability, the PAOs have energy
available to assimilate the acetate in the anaerobic zone. Other aerobic heterotrophic bacteria
40
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have no such mechanism for acetate uptake, and they are starved while the PAOs assimilate
COD in the anaerobic zone. It should also be noted that the PAOs form very dense, good settling
floc in the activated sludge, which is an added benefit. In some facilities, the anaerobic/aerobic
process sequence has been used because of the sludge settling benefits, even though biological
phosphorus removal was not required.
Care must be taken in the handling of the waste sludge from biological phosphorus removal
systems. When the sludge is held under anaerobic conditions, phosphorus release will occur.
Release of O-PO4 is possible even without acetate addition as the bacteria use the stored
polyphosphate for an energy source. The release of O-PO4 can also occur after extended contact
time in the anaerobic zone of the biological phosphorus treatment system. In that case the
released phosphorus may not be taken up in the aerobic zone because the release was not
associated with acetate uptake and PHB storage for later oxidation. The release of O-PO4 under
these conditions is termed secondary release (Barnard, 1984), which can lead to a lower
phosphorus removal efficiency for the biological process.
2.10.3 Stoichiometry of biological Phosphorus removal
Based on the description of the phosphorus removal mechanism, acetate uptake in the anaerobic
zone is critical in determining the amount of PAOs that can be produced and, thus, the amount of
phosphorus that can be removed by this pathway. If significant amounts of dissolved oxygen or
nitrate enter the anaerobic zone, the acetate can be depleted before it is taken up by the PAOs,
and treatment performance will be hindered.
Biological phosphorus removal is not used in systems that are designed with nitrification without
including a means for denitrification to minimize the amount of nitrate in the return sludge flow
to the anaerobic zone.
The amount of phosphorus removed by biological storage can be estimated from the amount of
bsCOD that is available in the wastewater influent as most of the bsCOD will be converted to
acetate in the short anaerobic hydraulic detention time HRT. The following assumptions are used
to evaluate the stoichiometry of biological phosphorus removal: (1) 1.06 g acetate/g bsCOD will
be produced as most of the COD fermented will be converted to VFAs due to the low cell yield
of the fermentation process, (2) a cell yield of 0.30 g VSS/g acetate, and (3) a cell phosphorus
content 0.3 g Pig VSS. Using these assumptions, about 10 g of bsCOD will be required to
remove I g of phosphorus by the biological storage mechanism. Other bCOD removal in the
activated-sludge system will result in additional phosphorus removal by normal cell synthesis.
Better performance for biological phosphorus removal systems is achieved when bsCOD or
acetate is available at a steady rate. Periods of starvation or low bsCOD concentrations result in
changes in the intracellular storage reserves of glycogen, PHB, and polyphosphates and rapidly
lead to decreased phosphorus removal efficiency (Stephens and Stensel, 1998).
Biological phosphorus growth kinetics are within the same order of magnitude of other
heterotrophic bacteria. Mamais and Jenkins (1992) showed that biological phosphorus removal
could be maintained in anaerobic/aerobic systems at SRTs greater than 2.5 d at 20°C. A
maximum specific growth rate at 20°C is given as 0.95 g/god (Barker and Dold, 1997).
41
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System performance is not affected by DO as long as the aerobic zone DO concentration is

above 1.0 mg/L. At pH values below 6.5, phosphorus removal efficiency is greatly reduced
(Sedlak, 1991).
In biological phosphorus removal systems, sufficient cations associated with polyphosphate

storage must also be available. The recommended molar ratios of Mg, K, and Ca to phosphorus
are 0.71, 0.50, and 0.25, respectively (Wentzel et aI., 1989).
Thus, for an influent soluble phosphorus concentration of 10 mg/L, 5.6, 6.3, and 3.2 mg/L of
Mg, K, and Ca, respectively, would be required. The relative amounts of these cations associated
with phosphate storage are 0.28, 0.26, and 0.09 mole/mole of phosphorus, respectively (Sedlak,
1991). Most municipal wastewaters have sufficient amounts of these inorganic elements, but
care must be taken to assure sufficient amounts in industrial applications or laboratory
experiments.
2.11 ANAEROBIC FERMENTATION AND OXIDATION
Anaerobic fermentation and oxidation processes are used primarily for the treatment of waste
sludge (see Fig. 2.10) and high-strength organic wastes. However, applications for dilute waste
streams have also been demonstrated and are becoming more common. Anaerobic fermentation
processes are advantageous because of the lower biomass yields and because energy, in the form
of methane, can be recovered from the biological conversion of organic substrates. Although
most fermentation processes are operated in the mesophilic temperature range (30 to 35°C), there
is increased interest in thermophilic fermentation alone or before mesophilic fermentation. The
latter is termed temperature phased anaerobic digestion (TPAD) and is typically designed with a
sludge SRT of 3 to 7 d in the first thermophilic phase at 50 to 60°C and 7 to 15 d in the final
mesophilic phase (Han and Dague, 1997). Thermophilic anaerobic digestion processes, are used
to accomplish high pathogen kill to produce Class A biosolids, which can be used for
unrestricted reuse applications.
For treating high-strength industrial wastewaters, anaerobic treatment has been shown to provide
a very cost-effective alternative to aerobic processes with savings in energy, nutrient addition,
and reactor volume. Because the effluent quality is not as good as that obtained with aerobic
treatment, anaerobic treatment is commonly used as a pretreatment step prior to discharge to a
municipal collection system or is followed by an aerobic process.
Fig. 2.10 Views of anaerobic digesters: (a) Kuwait City, Kuwait and (b) egg-shaped digester at Okinawa,
Japan (Metcalf and Eddy, 2003).
42
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Three basic steps are involved in the overall anaerobic oxidation of a waste: (1) hydrolysis, (2)
fermentation (also known as acidogenesis), and (3) methanogenesis. The three steps are
illustrated schematically on Fig. 2.11. The starting point on the schematic for a particular
application depends on the nature of the waste to be processed.
Fig. 2.11 Anaerobic process schematic of hydrolysis, fermentation, and methanogenesis (Metcalf and
Eddy, 2003).
Hydrolysis. The first step for most fermentation processes, in which particulate material is
converted to soluble compounds that can then be hydrolyzed further to simple monomers that are
used by bacteria that perform fermentation, is termed hydrolysis. For some industrial
wastewaters, fermentation may be the first step in the anaerobic process.
Fermentation. The second step is fermentation (also referred to as acidogenesis). In the

fermentation process, amino acids, sugars, and some fatty acids are degraded further, as shown
on Fig. 2.11. Organic substrates serve as both the electron donors and acceptors. The principal
products of fermentation are acetate, hydrogen, CO2 and propionate and butyrate. The propionate
and butyrate are fermented further to also produce hydrogen, CO2 and acetate. Thus, the final
products of fermentation (acetate, hydrogen, and CO2) are the precursors of methane formation
(methanogenesis). The free energy change associated with the conversion of propionate and
butyrate to acetate and hydrogen requires that hydrogen be at low concentrations in the system
(H2 < 10-4 atm), or the reaction will not proceed (McCarty and Smith, 1986).
Methanogenesis. The third step, methanogenesis, is carried out by a group of organisms known
collectively as methanogens. Two groups of methanogenic organisms are involved in methane
production. One group, termed aceticlastic methanogens, split acetate into methane and carbon
dioxide. The second group, termed hydrogen-utilizing methanogens, use hydrogen as the
electron donor and CO2 as the electron acceptor to produce methane. Bacteria within anaerobic
processes, termed acetogens, are also able to use CO2 to oxidize hydrogen and form acetic acid.
However, the acetic acid will be converted to methane, so the impact of this reaction is minor.
43
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As shown on Fig. 2.12, about 72 percent of the methane produced in anaerobic digestion is from
acetate formation.
Fig. 2.12 Carbon and hydrogen flow in anaerobic digestion process (Metcalf and Eddy, 2003).
2.11.2 Microbiology
The group of nonmethanogenic microorganisms responsible for hydrolysis and fermentation

consists of facultative and obligate anaerobic bacteria. Organisms isolated from anaerobic
digesters include Clostridium spp., Peptococcus anaerobus, Bifidobacterium spp.,
Desulphovibrio spp., Corynebacterium spp., Lactobacillus, Actinomyces, Staphylococcus, and
Escherichia coli. Other physiological groups present include those producing proteolytic,
lipolytic, ureolytic, or cellulytic enzymes.
The microorganisms responsible for methane production, classified as archaea, are strict obligate
anaerobes. Many of the methanogenic organisms identified in anaerobic digesters are similar to
those found in the stomachs of ruminant animals and in organic sediments taken from lakes and
rivers. The principal genera of microorganisms that have been identified at mesophilic conditions
include the rods (Methanobacterium, Methanobacillus) and spheres (Methanococcus,
Methanothrix, and Methanosarcina).
Methanosarcina and Methanothrix (also termed Methanosaeta) are the only organisms able to
use acetate to produce methane and carbon dioxide. The other organisms oxidize hydrogen with
carbon dioxide as the electron acceptor to produce methane. The acetate-utilizing methanogens
were also observed in thermophilic reactors (van Lier, 1996; Zinder and Koch, 1984; and
Ahring, 1995). Some species of Methanosarcina were inhibited by temperature at 65°C, while
others were not, but no inhibition of Methanothrix was shown. For hydrogen-utilizing
methanogens at temperatures above 60°C, Methanobacterium was found to be very abundant.
Syntrophic relationships in fermentation
The methanogens and the acidogens form a syntrophic (mutually beneficial) relationship in
which the methanogens convert fermentation end products such as hydrogen, formate, and
acetate to methane and carbon dioxide. Because the methanogens are able to maintain an
extremely low partial pressure of H2, the equilibrium of the fermentation reactions is shifted
toward the formation of more oxidized end products (e.g., formate and acetate). The utilization
of the hydrogen produced by the acidogens and other anaerobes by the methanogens is termed
interspecies hydrogen transfer. In effect, the methanogenic organisms serve as a hydrogen sink
that allows the fermentation reactions to proceed. If process upsets occur and the methanogenic
organisms do not utilize the hydrogen produced fast enough, the propionate and butyrate
44
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fermentation will be slowed with the accumulation of volatile fatty acids in the anaerobic reactor
and a possible reduction in pH.
Nuisance organisms
Nuisance organisms in anaerobic operations are the sulfate-reducing bacteria, which can be a
problem when the wastewater contains significant concentrations of sulfate. These organisms
can reduce sulfate to sulfide, which can be toxic to methanogenic bacteria at high enough
concentrations. Where high sulfide concentrations occur, one solution is to add iron at controlled
amounts to form iron sulfide precipitate. Sulfate-reducing bacteria, obligate anaerobes of the
domain Bacteria, are morphologically diverse, but share the common characteristic of being able
to use sulfate as an electron acceptor and are divided into one of two groups depending on
whether they produce fatty acids or use acetate. Group I sulfate reducers can use a diverse array
of organic compounds as their electron donor, oxidizing them to acetate and reducing sulfate to
sulfide. A common genus found in anaerobic biochemical operations is Desulfovibrio. Group II
sulfate reducers oxidize fatty acids, particularly acetate, to carbon dioxide, while reducing sulfate
to sulfide. A bacteria commonly found in this group is in the genus Desulfobacter.
2.11.3 Stoichiometry of anaerobic fermentation and oxidation
A limited number of substrates are used by the methanogenic organisms and reactions defined as
CO2 and methyl group type reactions are shown as follows (Madigan et al., 1997), involving the
oxidation of hydrogen, formic acid, carbon monoxide, methanol, methylamine, and acetate,
respectively.
4H2 + CO2 Æ CH4 + 2H2O (2-84)

4HCOO- + 4H+ Æ CH4 + 3CO2 + 2H2O (2-85)
4CO + 2H2O Æ CH4 + 3CO2 (2-86)
4CH3OH Æ 3CH4 + CO2 + 2H2O (2-87)
4(CH3)3N + H2O Æ 9CH4 + 3CO2 + 6H2O + 4NH3 (2-88)
CH3COOH Æ CH4 + CO2 (2-89)
In the reaction for the aceticlastic methanogens as given by Eq. (2-89), the acetate is cleaved to
form methane and carbon dioxide.
A COD balance can be used to account for the changes in COD during fermentation. Instead of
oxygen accounting for the change in COD, the COD loss in the anaerobic reactor is accounted
for by the methane production. By stoichiometry the COD equivalent of methane can be
determined. The COD of methane is the amount of oxygen needed to oxidize methane to carbon
dioxide and water.
CH4 + 2O2 Æ CO2 + 2H2O (2-90)
From the above, the COD per mole of methane is 2(32 gO2/mole) = 64 g O2/mole CH4. The
volume of methane per mole at standard conditions (O°C and 1 atm) is 22.414 L, so the CH4
equivalent of COD converted under anaerobic conditions is 22.414/64 = 0.35 L CH4/g COD.
In anaerobic processes two rate-limiting concepts are important: (1) the hydrolysis conversion
rate and (2) the soluble substrate utilization rate for fermentation and methanogenesis. The
hydrolysis of colloidal and solid particles does not affect the process operation and stability but
45
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does affect the total amount of solids converted. In anaerobic digestion processes used for
municipal waste sludges, greater than 30 days detention time is needed to approach full
conversion of solids. The soluble substrate utilization kinetics are of great concern to develop a
stable anaerobic process.
Because of the relatively low free energy change for anaerobic reactions, growth yield
coefficients are considerably lower than the corresponding values for aerobic oxidation. TYpical
synthesis yield and endogenous decay coefficients for fermentation and methanogenic anaerobic
reactions are Y = 0.10 and 0.04 g VSS/g VSS and kd = 0.04 and 0.02 g VSS/g VSS.d,
respectively.
The process is more stable when the volatile fatty acid (VFA) concentrations approach a minimal
level, which can be taken as an indication that a sufficient methanogenic population exists and
sufficient time is available to minimize hydrogen and VFA concentrations. The rate-limiting step
is the conversion of VFAs by the methanogenic organisms and not the fermentation of soluble
substrates by the fermenting bacteria. Thus, the methanogenic growth kinetics are of most
interest in anaerobic process designs. Appropriate system SRTs are selected based on kinetics
and treatment goals. At 20,25, and 35°C, the washout or SRTmin values for methanogenesis are
7.8, 5.9, and 3.2 d, respectively (Lawrence and McCarty, 1970). Thus, with a factor of safety of
5, design SRT values would be about 40, 30, and 15 days, respectively, for a suspended growth
process. Safety factors higher than 5 have been used to provide a more stable process (Parker and
Owen, 1986).
Anaerobic processes are sensitive to pH and inhibitory substances. A pH value near neutral is
preferred and below 6.8 the methanogenic activity is inhibited. Because of the high CO2 content
in the gases developed in anaerobic processes (30 to 35 percent CO2)' a high alkalinity is needed
to assure pH near neutrality. An alkalinity concentration in the range of 3000 to 5000 mg/L as
CaCO3 is often found. For sludge digestion sufficient alkalinity is produced by the breakdown of
protein and amino acids to produce NH3, which combines with CO2 and H2O to form alkalinity
as NH4(HCO3) For industrial wastewater applications, especially for waste containing mainly
carbohydrates, it is necessary to add alkalinity for pH control. Substances inhibitory to anaerobic
processes are NH3, H2S, and various other inorganic and organic compounds.
2.12 BIOLOGICAL REMOVAL OF TOXIC AND RECALCITRANT ORGANIC

COMPOUNDS
Most of the organic compounds in domestic wastewater and some in industrial wastewaters are
of natural origin and can be degraded by common bacteria in aerobic or anaerobic processes.
However, currently there are over 70,000 synthetic organic chemicals, termed xenobiotic
compounds, in general use (Schwarzenbach et al., 1993).
Unfortunately, some of these organic compounds pose unique problems in wastewater treatment,
due to their resistance to biodegradation and potential toxicity to the environment and human
health. Organic compounds that are difficult to treat in conventional biological treatment
processes are termed refractory. In addition, there are naturally occurring substances, such as
those found in petroleum products, that are of similar concern.
46
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2.12.1 Development of biological treatment methods
Since the early 1970s, information and knowledge related to the biodegradation of toxic and
refractory compounds has increased significantly, based on work with specific industrial
wastewaters (i.e., petrochemical, textile, pesticide, pulp and paper, and pharmaceutical
industries). In addition, since the 1980s, significant progress has also been made on the
biodegradation of organic substances found at hazardous waste sites. Work in both of these fields
has expanded knowledge on the capabilities and limitations of biodegradation. With a few
exceptions most organic compounds can be biodegraded eventually, but in some cases the rates
may be slow, unique environmental conditions may be required (i.e., redox potential, pH,
temperature), fungi may be needed instead of prokaryotes, or specific bacteria capable of
degrading the xenobiotic compounds may be needed. For example, anaerobic degradation of
polychlorinated biphenyls (PCB) occurred using bacteria seed from sediment in the Hudson
River where PCB had accumulated over decades, but after 1.5 years of exposure in a laboratory
anaerobic digester used to treat municipal wastewater plant sludge, bacteria could not be
developed to degrade PCB (Ballapragada et al., 1998).
Importance of specific microorganisms
The ability to degrade toxic and recalcitrant compounds will depend primarily on the presence of
appropriate microorganism(s) and acclimation time. In some cases, special seed sources are
needed to provide the necessary microorganisms. Once the critical microorganism is present,
long-term exposure to the organic compound may be needed to induce and sustain the enzymes
and bacteria required for degradation. Acclimation times can vary from hours to weeks
depending on the microorganism population and organic compound. Melcer et al. (1994) found
that a period of 3 weeks was required before complete removal of dichlorobenzene (DCB)
occurred in a municipal activated-sludge plant. They noted that intermittent addition of DCB
resulted in much lower treatment efficiencies. Strand et al. (1999) showed that after 4 weeks of
constant exposure to dinitrophenol in a laboratory activated-sludge process, seeded from a
municipal wastewater plant, dinitrophenol degradation increased from 0 to 98 percent. When
dinitrophenol was not added to the process, the ability to degrade dinitrophenol was eventually
lost. Thus, it appears that a relatively constant supply of toxic and recalcitrant organic
compounds can lead to better biodegradation performance than intermittent additions.
Biodegradation pathways
The three principal types of degradation pathways that have been observed are (l) the compound
serves as a growth substrate; (2) the organic compound provides an electron acceptor; and (3) the
organic compound is degraded by cometabolic degradation. In cometabolic degradation, the
compound that is degraded is not part of the microorganism's metabolism. Degradation of the
compound is brought about by a nonspecific enzyme and provides no benefit to the cell growth.
Complete biodegradation of toxic and recalcitrant organic compounds to harmless end products
such as CO2 and H20 or methane may not always occur, and instead biotransformation to a
different organic compound is possible. Care must be taken to determine if the organic
compound produced is innocuous, or just as harmful as or more harmful than the initial
compound.
2.12.2 Anaerobic degradation
Many toxic and recalcitrant organic compounds are degraded under anaerobic conditions, with
the compound serving as a growth substrate with fermentation and ultimate methane production.
Typical examples include nonhalogenated aromatic and aliphatic compounds such as phenol,
47
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toluene, alcohols, and ketones. However, most chlorinated organic compounds are not attacked
easily under anaerobic conditions and do not serve as growth substrates. Fortuitously, many of
these compounds also serve as electron acceptors in anaerobic oxidation reduction reactions.
Most of the work and application for anaerobic degradation of chlorinated organic compounds
have been related to subsurface contamination of chlorinated solvents at hazardous waste sites
(McCarty, 1999).
Examples of chlorinated compounds degraded under anaerobic conditions include

tetrachloroethene, trichloroethene, carbon tetrachloride, trichlorobenzene, pentachlorophenol,
chlorohydrocarbons, and PCBs. The chlorinated compound serves as the electron acceptor, and
hydrogen produced from fermentation reactions provides the main electron donor. Hydrogen
replaces chlorine in the molecule, and such reactions have generally been referred to as
anaerobic dehalogenation or anaerobic dechlorination. For example, dechlorination of
tetrachloroethene proceeds sequentially with a loss of chlorine in each step via trichloroethene to
dichloroethene to vinyl chloride and finally to ethene.
As the number of chlorine molecules on the organic molecule decreases, the reactions tend to be
slower and less complete. Dechlorination of tetrachloroethene, trichlorobenzene, and
pentachlorophenol has been demonstrated in lab-scale anaerobic digesters (Ballapragada et aI.,
1998) treating municipal primary and secondary sludge.
However, the reaction rates were slow with mono- and dichlorophenol and mono-I
dichlorobenzenes remaining. Conversion of tetrachloroethene to vinyl chloride and ethene
occurred in the digesters after one year of acclimatization and constant exposure of the
chloroethenes.
2.12.3 Aerobic biodegradation
With proper environmental conditions, seed source, and acclimation time, a wide range of toxic
and recalcitrant organic compounds have been found to serve as growth substrates for
heterotrophic bacteria. Such compounds include phenol, benzene, toluene, poly aromatic
hydrocarbons, pesticides, gasoline, alcohols, ketones, methylene chloride, vinyl chloride,
munitions compounds, and chlorinated phenols. However, many chlorinated organic compounds
cannot be attacked readily by aerobic heterotrophic bacteria and thus do not serve as growth
substrates. Some of the lesser chlorinated compounds, such as dichloromethane, 1,2-
dichloroethane, and vinyl chloride can be used as growth substrates by aerobic bacteria.
Fortunately, a number of chlorinated organic compounds are degradable by cometabolic
degradation. It should be noted that organic compounds that are saturated fully with chlorine are
degraded only by anaerobic dechlorination (Stensel and Bielefeldt, 1997).
Chlorinated organic compounds that have been degraded by cometabolic degradation include
trichloroethene, dichloroethene, vinyl chloride, chloroform, dichloromethane, and
trichloroethane. Cometabolic degradation is possible by bacteria that produce nonspecific mono-
oxygenase or dioxygenase enzymes. These enzymes mediate a reaction with oxygen and
hydrogen and change the structure of the chlorinated compound. Bacteria that produce
oxygenase enzymes oxidize certain substrates that induce the enzyme. Oxygenase-producing
bacteria include methanotrophic bacteria that oxidize methane, a number of bacteria that can
oxidize phenol or toluene, a number of bacteria that can oxidize propane, and nitrifying bacteria
that oxidize ammonia to nitrite.
The reaction of the nonspecific oxygenase enzyme with the organic chlorinated compound
typically produces an intermediate compound that is degraded by other aerobic heterotrophic
48
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bacteria in the biological consortia. Various reactor designs have been developed to apply this
biological process for treatment of contaminated groundwater or vapor extraction gas streams
(Lee et al., 2000). While such reactions are possible in municipal and industrial biological
wastewater treatment processes, a large amount of the chlorinated organic compounds that may
be present are more likely lost from the process by volatilization during aeration, because of their
high volatility and the minimal potential for cometabolic bacteria to be present.
2.13 BIOLOGICAL REMOVAL OF HEAVY METALS
Metal removal in biological treatment processes is mainly by adsorption and complexation of the
metals with the microorganisms. In addition, processes that result in transformations and
precipitation of metals are possible. Microorganisms combine with metals and adsorb them to
cell surfaces because of interactions between the metal ions and the negatively charged microbial
surfaces. Metals may also be complexed by carboxyl groups found in microbial polysaccharides
and other polymers, or absorbed by protein materials in the biological cell.
The removal of metals in biological processes has been found to fit adsorption characteristics
displayed by the Freundlich isotherm model (Mullen et al., 1989; Kunz et al., 1976). A
significant amount of soluble metal removal has been observed in biological processes, with
removals ranging from 50 to 98 percent depending on the initial metal concentration, the
biological reactor solids concentrations, and system SRT. In anaerobic processes the reduction of
sulfate to hydrogen sulfide can promote the precipitation of metal sulfides. A classic example is
the addition of ferric or ferrous chloride to anaerobic digesters to remove sulfide toxicity by the
formation of iron sulfide precipitates.
49
CHAPTER 3
FUNDAMENTALS OF BIOLOGICAL SOLID

WASTE TREATMENT TECHNOLOGY
3.1 OVERVIEW OF BIOLOGICAL SOLID WASTE TREATMENT
Before considering the specific processes employed for the biological conversion of wastes, it
will be helpful to review (1) the general nutritional requirements of the microorganisms
commonly encountered in solid waste conversion facilities, (2) the type of microbial metabolism
based on the need for molecular oxygen, (3) the types of microorganisms of importance in the
conversion of solid waste, (4) environmental requirements, (5) aerobic and anaerobic
transformations, and (6) process selection.
3.1.1 Nutritional requirements for microbial growth
To continue to reproduce and function properly, an organism must have a sources of energy;
carbon for the synthesis of new cell tissue, and inorganic elements (nutrients) such as nitrogen,
phosphorus, sulfur, potassium, calcium, and magnesium. Organic nutrients (growth factors) may
also be required for cell synthesis. Carbon and energy sources, usually referred to as substrates,
and nutrient and growth factor requirements for various types of organisms are considered in the
following discussion.
Nutrient and growth factor requirements
Nutrients, rather than carbon or an energy source, may at times be the limiting material for
microbial cell synthesis and growth. The principal inorganic nutrients needed by microorganisms
are nitrogen, sulfur, phosphorus, potassium, magnesium, calcium, iron, sodium, and chlorine.
Minor nutrients of importance include zinc, manganese, molybdenum, selenium, cobalt, copper,
nickel, and tungsten.
In addition to the inorganic nutrients just cited, some organisms may also need organic nutrients.
Required organic nutrients, known as growth factors, are compounds needed by an organism as
precursors or constituents of organic cell material that cannot be synthesized from other carbon
sources. Although growth factor requirements differ from one organism to another, the major
growth factors fall into the following three classes: (1) amino acids, (2) purines and pyrimidines,
and (3) vitamins.
Microbial nutrition and biological conversion processes
The major objective in most biological conversion processes is the conversion of the organic
matter in the waste to a stable end product. In accomplishing this type of treatment, the
chemoheterotrophic organisms are of primary importance because of their requirement for
organic compounds as both carbon and energy source. The organic fraction of municipal solid
waste typically contains adequate amounts of nutrients (both inorganic and organic) to support
the biological conversion of the waste. With some commercial wastes, however, nutrients may
not be present in sufficient quantities. In these cases, nutrient addition is necessary for the proper
bacterial growth and for the subsequent degradation of the organic waste.
50
3.1.2 Types of microbial metabolism
Chemoheterotrophic organisms may be further grouped according to their metabolic type and
their requirement for molecular oxygen. Organisms that generate energy by enzyme-mediated
electron transport from an electron donor to an external electron acceptor (such as oxygen) are
said to have a respiratory metabolism. In contrast, fermentative metabolism does not involve the
participation of an external electron acceptor. Fermentation is a less efficient energy-yielding
process than respiration; as a consequence, heterotrophic organisms that are strictly fermentative
are characterized by lower growth rates and cell yields than respiratory heterotrophs.
When molecular oxygen is used as the electron acceptor in respiratory metabolism, the process is
known as aerobic respiration. Organisms that are dependent on aerobic respiration to meet their
energetic needs can exist only when there is a supply of molecular oxygen. These organisms are
called obligate aerobic. Oxidized inorganic compounds such as nitrate and sulfate can function
as electron acceptors for some respiratory organisms in the absence of molecular oxygen. In
environmental engineering, processes that make use of these organisms are often referred to as
anoxic.
Organisms that generate energy by fermentation and that can exist only in an environment that is
devoid of oxygen are obligate anaerobic. There is another group of microorganisms, which has
the ability to grow in either the presence or the absence of molecular oxygen. These organisms
are called facultative anaerobes.
The facultative organisms fall into two subgroups, based on their metabolic abilities. True
facultative anaerobes can shift from fermentative to aerobic respiratory metabolism, depending
upon the presence or absence of molecular oxygen. Aero-tolerant anaerobes have a strictly
fermentative metabolism but are relatively insensitive to the presence of molecular oxygen.
3.1.3 Types of microorganisms
Microorganisms are commonly classified, on the basis of cell structure and function, as
eukaryotes, eubacteria, and archaebacteria. The prokaryotic groups (eubacteria and
archaebacteria) are of primary importance in biological conversion of the organic faction of solid
wastes and are generally referred to simply as bacteria.
Bacteria
Bacteria are single cells-spheres, rods, or spirals. Spherical forms (cocci) vary from 0.5 to 4 µm
in diameter; rods (bacilli) are from 0.5 to 20 µm long and 0.5 to 4 µm wide; spirals (spirilla) may
be more than 10 p.m long and about 0.5 µm wide. Bacteria are ubiquitous in nature and are
found in aerobic (in the presence of oxygen) and anaerobic (in the absence of oxygen)
environments. Because of the wide variety of inorganic and organic compounds that can be used
by bacteria to sustain growth, bacteria are used extensively in a variety of industrial operations to
accumulate intermediate and end products of metabolism. Tests on a number of different
bacterial species indicate that they are about 80 percent water and 20 percent dry material, of
which 90 percent is organic and 10 percent is inorganic. An approximate empirical formula for
the organic fraction is C5H7NO2. On the basis of this formula, about 53 percent by weight of the
organic fraction is carbon. Compounds that make up the inorganic portion include P2O5 (50
percent), CaO (9 percent), Na2O (11 percent), MgO (8 percent), K2O (6 percent), and Fe2O3 (1
percent). Because all these elements and compounds must be derived from the environment, a
shortage of these substances would limit, and in some cases alter, the growth of bacteria.
51
Fungi
Fungi are considered to be multicellular, nonphotosynthetic, heterotrophic protists. Most fungi

have the ability to grow under low-moisture conditions, which do not favor the growth of
bacteria. In addition, fungi can tolerate relatively low pH values. The optimum pH value for
most fungal species appears to be about 5.6, but the viable range is from 2 to 9. The metabolism
of these organisms is essentially aerobic, and they grow in long filaments, called hyphae,
composed of nucleated cell units and varying in width from 4 to 20 µm. Because of their ability
to degrade a wide variety of organic compounds over a broad range of environmental conditions,
fungi have been used extensively in industry for the production of valuable compounds, such as
organic acids (e.g., citric, gluconic), various antibiotics (e.g., penicillin, griseofulvin), and
enzymes (e.g., cellulase, protease, amylase).
Yeasts
Yeasts are fungi that cannot form filaments (mycelium) and are therefore unicellular. Some
yeasts form elliptical cells 8 to 15 µm by 3 to 5 µm, whereas others are spherical, varying in size
from 8 to 12 µm in diameter. In terms of industrial processing operations, yeasts may be
classified as "wild" and "cultured." In general, wild yeasts are of little value, but cultured yeasts
are used extensively to ferment sugars to alcohol and carbon dioxide.
Actinomycetes
The actinomycetes are a group of organisms with intermediate properties between bacteria and
fungi. They are similar in form to fungi, except that the width of the cell is only 0.5- to 1.4 µm.
In industry, this group of microorganisms is used extensively for the production of antibiotics.
Because their growth characteristics are similar, actinomycetes are often grouped with fungi for
discussion purposes.
3.1.4 Environmental requirements
Environmental conditions of temperature and pH have an important effect on the survival and
growth of microorganisms. In general, optimal growth occurs within a fairly narrow range of
temperature and pH values, although the microorganism may be able to survive within much
broader limits. For instance, temperatures below the optimum typically have a more significant
effect on the bacterial growth rate than temperatures above the optimum. It has been observed
that growth rates double with approximately every woe increase in temperature until the
optimum temperature is reached. According to the temperature range in which they function
best, bacteria may be classified as psychrophilic, mesophilic, or thermophilic.
The hydrogen ion concentration, expressed as pH, is not a significant factor in the growth of
microorganisms, in and of itself, within the range from 6 to 9 (which represents a thousandfold
difference in the hydrogen ion concentration). Generally, the optimum pH for bacterial growth
lies between 6.5 and 7.5. However, when the pH goes above 9.0 or below 4.5, it appears that the
undissociated molecules of weak acids or bases can enter the cell more easily than hydrogen and
hydroxide ions and, by altering the internal pH, damage the cell.
Moisture content is another essential environmental requirement for the growth of

microorganisms. The moisture content of the organic wastes to be converted must be known,
especially if a dry process such as composting is to be used. In many composting operations, it
has been necessary to add water to obtain optimum bacterial activity. The addition of water in
52
anaerobic fermentation processes will depend on the characteristics of the organic waste and the
type of anaerobic process that is used.
The biological conversion of an organic waste requires the biological system to be in a state of
dynamic equilibrium. To establish and maintain dynamic equilibrium, the environment must be
free of inhibitory concentrations of heavy metals, ammonia, sulfides, and other toxic
constituents.
3.1.5 Aerobic biological transformations
The general aerobic transfonnation of solid waste can be described by means of the following
equation:
Bacteria
Organic matter + O2 + nutrients Æ new cells + resistant organic matter + CO2 + H2O + NH3
+ SO42- + … + heat
If the organic matter in solid waste is represented (on a molar basis) as CaHbOcNd, the production
of new cells and sulfate is not considered, and the composition of the resistant material is
represented (on a molar basis) as CwHxOyNz, then the amount of oxygen required for the aerobic
stabilization of the biodegradable organic fraction of MSW can be estimated by using the
following equation:
CaHbOcNd + 0.5 (ny + 2s + r – c)O2 Æ nCwHxOyNz + sCO2 + rH2O + (d-nx)NH3
The tenns CaHbOcNd and CwHxOyNz represent the empirical mole composition of the organic
material initially present and at the conclusion of the process. If complete conversion is
accomplished, the corresponding expression is
4a + b – 2c – 3d b - 3d
CaHbOcNd + ---------------------- O2 Æ aCO2 + -------- +H2O + dNH3
4 2
In many cases the ammonia, NH3, produced from the carbonaceous oxidation of organic matter
is oxidized further to nitrate, NO3 - (a process known as nitrification). The amount of oxygen
required for the oxidation of ammonia to nitrate can be computed by the following equations:
NH3 + 3/2O2 Æ HNO2 + H2O

HNO2 + 1/2O2 Æ JNO3
------------------------------------
NH3 + 2O2 Æ H2O + HNO3
3.1.6 Anaerobic biological transformations
The production of methane from solid wastes by anaerobic digestion, or anaerobic fermentation
as it is often called, is described in the following discussion.
Process microbiology. The biological conversion of the organic fraction of municipal solid
waste under anaerobic conditions is thought to occur in three steps. The first step in the process
involves the enzyme-mediated transfonnation (hydrolysis) of higher-molecular-mass compounds
into compounds suitable for use as a source of energy and cell tissue. The second step involves
the bacterial conversion of the compounds resulting from the first step into identifiable lower-
53
molecular-mass intennediate compounds. The third step involves the bacterial conversion of the
intennediate compounds into simpler end products, principally methane and carbon dioxide.
In the anaerobic decomposition of wastes, a number of anaerobic organisms work together to

bring about the conversion of organic portion of the wastes to a stable end product. One group of
organisms is responsible for hydrolyzing organic polymers and lipids to basic structural building
blocks such as fatty acids, monosaccharides, amino acids, and related compounds. A second
group of anaerobic bacteria fennents the breakdown products from the first group to simple
organic acids, the most common of which in anaerobic digestion is acetic acid. This second
group of microorganisms, described as nonmethanogenic, consists of facultative and obligate
anaerobic bacteria that are often identified in the literature as "acidogens" or "acid fonners." A
third group of microorganisms converts the hydrogen and acetic acid fonned by the acid fonners
to methane gas and carbon dioxide. The bacteria responsible for this conversion are strict
anaerobes, called methanogenic, and are identified in the literature as "methanogens" or
"methane fonners." Many of the methanogenic organisms identified in landfills and anaerobic
digesters are similar to those found in the stomachs of ruminant animals and in organic
sediments taken from lakes and river. The most important bacteria of the methanogenic group
are the ones that utilize hydrogen and acetic acid. They have very slow growth rates; as a result,
their metabolism is usually considered rate-limiting in the anaerobic treatment of an organic
waste. Waste stabilization in anaerobic digestion is accomplished when methane and carbon
dioxide are produced. Methane gas is highly insoluble, and its departure from a landfill or
solution represents actual waste stabilization.
Biochemical pathways. It is important to note that methane bacteria can only use a limited
number of substrates for the formation of methane. Currently, it is known that methanogens use
the following substrates: CO2 + H2, formate, acetate, methanol, methylamines, and carbon
monoxide. Jypical energy-yielding conversion reactions involving these compounds are as
follows:
4H2 + CO2 Æ CH4 + 2H2O

4HCOOH Æ CH4 + 3CO2 + 2H2O
CH3COOH Æ CH4 + CO2
4CH3OH Æ 3CH4 + CO2 + 2H2O
4(CH3)3N + 6H2O Æ 9CH4 + 3CO2 + 4NH3
4CO + 2H2O Æ CH4 + 3CO2
In anaerobic fermentation, the two principal pathways involved in the formation of methane are
(1) the conversion of carbon dioxide and hydrogen to methane and water and (2) the conversion
of formate and acetate to methane, carbon dioxide, and water. The methanogens and the
acidogens form a syntrophic (mutually beneficial) relationship in which the methanogens convert
fermentation end products such as hydrogen, formate, and acetate to methane and carbon
dioxide. The methanogens are able to utilize the hydrogen produced by the acidogens, because of
their efficient hydrogenase. Because the methanogens are able to maintain an extremely low
partial pressure of H2, the equilibrium of the fermentation reactions is shifted towards the
formation of more oxidized end products (e.g., formate and acetate). The utilization of the
hydrogen, produced by the acidogens and other anaerobes, by the methanogens is termed
interspecies hydrogen transfer. In effect, the methanogenic bacteria remove compounds that
would inhibit the growth of acidogens.
Environmental factors. To maintain an anaerobic treatment system that will stabilize an organic
waste efficiently, the nonmethanogenic and methanogenic bacteria must be in a state of dynamic
equilibrium. To establish and maintain such a state, the reactor contents should be void of
54
dissolved oxygen and free from inhibitory concentrations of free ammonia and such constituents
as heavy metals and sulfides. Also, the pH of the aqueous environment should range from 6.5 to
7.5. Sufficient alkalinity should be present to ensure that the pH will not drop below 6.2, because
the methane bacteria cannot function below this point. When digestion is proceeding
satisfactorily, the alkalinity will normally range from 1000 to 5000 mg/L and the volatile fatty
acids will be less than 250 mg/L. Values for alkalinity and volatile fatty acids in the high-solids
anaerobic digestion process can be as high as 12,000 and 700 mg/L, respectively. A sufficient
amount of nutrients, such as nitrogen and phosphorus, must also be available to ensure the proper
growth of the biological community. Depending on the nature of the sludges or waste to be
digested, growth factors may also be required. Temperature is another important environmental
parameter. The optimum temperature ranges are the mesophilic, 30 to 38°C (85 to 100°F), and
the thermophilic, 55 to 60°C (131 to 140°F).
Gas production. The general anaerobic transformation of solid waste can be described by means
of the following equation.
Organic matter + H2O + nutrients Æ new cells + resistant organic matter + CO2 + CH4 + NH3 +
H2S + heat
For practical purposes, the overall conversion of the organic fraction of solid waste to methane,
carbon dioxide, and ammonia can be represented with the following equation:
CaHbOcNd Æ nCwHxOyNz + mCH4 + sCO2 + rH2O + (d – nx)NH3
The terms CaHbOcNd and CwHxOyNz are used to represent (on a molar basis) the composition of
the organic material present at the start and the end of the process, respectively.
In operations where solid wastes have been mixed with wastewater sludge, it has been found that
the gas collected from the digesters contains between 50 and 60 percent methane. It has also
been found that about 10 to 16 ft3 of gas is produced per pound of biodegradable volatile solids
destroyed.
3.1.7 Biological process selection
Aerobic and anaerobic processes both have a place in solid waste management. Each process
offers different advantages. In general, the operation of anaerobic processes is more complex
than that of aerobic processes. However, anaerobic processes offer the benefit of energy recovery
in the form of methane gas and thus are net energy producers. Aerobic processes, on the other
hand, are net energy users because oxygen must be supplied for waste conversion, but they offer
the advantage of relatively simple operation and, if properly operated, can significantly reduce
the volume of the organic portion of MSW. The operational characteristics of both aerobic and
anaerobic solid waste-processing systems are described in the following sections.
3.2 Aerobic composting
Aerobic composting is the most commonly used biological process for the conversion of the
organic portion of municipal solid waste (MSW) to a stable humus-like material known as
compost. Applications of aerobic composting include (1) yard waste, (2) separated MSW, (3)
commingled MSW, and (4) co-compo sting with wastewater sludge. Process descriptions and
design guidelines for aerobic composting are presented in this section.
55
All aerobic composting processes are similar in that they all incorporate three basic steps: (1)
preprocessing of the MSW, (2) aerobic decomposition of the organic fraction of the MSW, and
(3) product preparation and marketing. Windrow, aerated static pile, and in-vessel are the three
principal methods used for the composting of the organic fraction of MSW. While these
processes differ primarily in the method used to aerate the organic fraction of solid waste, the
biological principles remain the same, and, when designed and operated properly, all produce a
similar-quality compost in approximately the same time period.
3.2.2 Process microbiology
During the aerobic composting process a succession of facultative and obligate aerobic
microorganisms is active. In the beginning phases of the composting process, mesophilic
bacteria are the most prevalent. After the temperatures in the compost rise, thermophilic bacteria
predominate, leading to thermophilic fungi, which appear after 5 to 10 days. In the final stages,
or curing period as it is sometimes known, actinomycetes and molds appear. Because significant
concentrations of these microorganisms may not be present in some types of biodegradable
waste (e.g., newspaper), it may be necessary to add them to the composting material as an
additive or inoculum.
The microbiology of all aerobic composting processes is similar. Critical parameters in the
control of aerobic composting processes include moisture content, C/N ratio, and temperature.
For most biodegradable organic wastes, once the moisture content is brought to a suitable level
(50 to 60 percent) and the mass aerated, microbial metabolism speeds up. The aerobic
microorganisms, which utilize oxygen, feed upon the organic matter and develop cell tissue from
nitrogen, phosphorus, some of the carbon, and other required nutrients. Much of the carbon
serves as a source of energy for the organisms and is burned up and respired as carbon dioxide.
Because organic carbon can serve both as a source of energy and cell carbon, more carbon is
required than, for example, nitrogen.
3.2.3 Design and operational considerations
Particle size
Most materials that comprise the organic fraction of MSW tend to be irregular in shape. This
irregularity can be reduced substantially by shredding the organic materials before they are
composted. Particle size influences the bulk density, internal friction and flow characteristics,
and drag forces of the materials. Most important of all, a reduced particle size increases the
biochemical reaction rate during aerobic composting process. The most desirable particle size for
compo sting is less than 2 inches (5 cm), but larger particles can be composted. The particle size
of the material being composted is governed to some extent by the finished-product requirements
and by economic considerations.
Carbon-to-nitrogen ratio
The most critical environmental factor for composting is the carbon-to-nitrogen ratio (C/N ratio).
The optimum range for most organic wastes is from 20 to 25 to 1. As shown in Table 3.1,
sludges have low C/N ratios, whereas yard wastes, such as leaves and newspaper, have relatively
high C/N ratios. It should be noted that the C/N ratios given in Table 3.1 are based on the total
dry weights of carbon and nitrogen, not on the dry weight of the biodegradable fraction of the
organic material. In general, all of the organic nitrogen present in most organic compounds will
become available, whereas not all of the organic carbon will be biodegradable. Depending on the
56
particular waste material, the C/N ratio computed on the basis of total weights of carbon and
nitrogen could be quite misleading, especially in those cases where all of the available nitrogen
is biodegradable, but only a portion of organic carbon is biodegradable, (e.g., lignin in waste
paper). For example, assuming that all of the nitrogen is available, the C/N ratio for the organic
fraction of MSW can vary from about 34 to 60 depending on whether it is assumed that the
available carbon is partially or totally biodegradable. Blending of a waste high in carbon and low
in nitrogen (e.g., newsprint) with a waste that is high in nitrogen (e.g., yard wastes) is used to
achieve optimum C/N ratios for composting.
Blending and seeding
Two design factors that may affect the blending of organic fraction of municipal solids waste for
composting are C/N ratio and moisture content. Laboratory analyses are usually required to
determine how the various organic materials should be blended for aerobic composting. If the
organic fraction of MSW contains significant amounts of paper or other substrates rich in carbon,
other organic materials such as yard wastes, manure, or sludge from wastewater treatment plants
can be blended to provide a near optimum C/N ratio. Similarly, materials too wet and too dry for
good composting can be blended in proper proportion to achieve an optimum moisture content.
Seeding involves the addition of a volume of microbial culture sufficiently large to effect the
decomposition of the receiving material at a faster rate.
Table 3.1 Nitrogen content and nominal C/N ratios of selected compostable material (dry weight)
Source: Tchobanoglous et al., 1993.
57
Moisture content
The optimum moisture content for aerobic compo sting is in the range of 50 to 60 percent.
Moisture can be adjusted by blending of components or by addition of water. When the moisture
content of compost falls below 40 percent, the rate of composting will be slowed.
Mixing/turning
Initial mixing of organic wastes is essential to increase or decrease the moisture content to an
optimum level. Mixing can be used to achieve a more uniform distribution of nutrients and
microorganisms. Thrning of the organic material during the composting process is a very
important operational factor in maintaining aerobic activity. Because turning can be specified by
moisture content, waste characteristics, or air requirements, it is impossible to specify a
minimum frequency of turning or number of turns in a general terms. For an organic waste
having a maximum moisture of 55 to 60 percent and a compo sting period of 15 days, the first
turn has been suggested at the third day. Thereafter, it should be turned every other day for a
total of four to five turns.
Temperature
Aerobic composting systems can be operated in either the mesophilic, 30 to 38°C (85 to 100°F),
or the thermophilic, 55 to 60°C (131 to 140°F), temperature regions. The temperature rise
observed in actively composting wastes is caused by the exothermic reactions associated with
respiratory metabolism. In aerated static pile and in-vessel composting systems, the temperature
can be regulated by monitoring the temperature and controlling the airflow. In windrow
composting, temperature can only be controlled indirectly, by varying the frequency of turning
based on temperature measurements. In general, pile temperature will drop 5 to 10°C after
turning, but will return to its previous level within several hours. Windrow temperatures decrease
after 10 to 15 days as the readily biodegradable organic material is oxidized.
Control of pathogens
The destruction of pathogenic organisms is an important design element in the compost process,
as it will affect the temperature profile and aeration process. Data on the thermal death points for
a number of pathogenic organisms are summarized in Table 3.2. As shown in Table 3.2, the die-
off of pathogens is a function of time and temperature. For example, the Salmonella species of
bacteria can be destroyed in 15 to 20 minutes when exposed to a temperature of 60°C, or in one
hour at 55°C. From Table 14-8, it is apparent that most pathogens will be destroyed rapidly when
all parts of the compost pile are subjected to a temperature of about 55°C. Only a few can
survive at temperatures up to 67°C for a short period of time. Elimination of all pathogenic
microorganisms can be accomplished by allowing the composting waste to reach a temperature
of 70°C for 1 to 2 hours. The U.S. Environmental Protection Agency has required specific time-
temperature standards for pathogen control in composting systems (see Table 3.3). These
conditions are easily met in properly operating composting systems.
Air requirements
In processes with forced aeration, such as the aerated static pile and the in-vessel systems, the
total air requirement and air flow rate are essential design parameters.
pH control
58
Control of pH is another important parameter in evaluating the microbial environment and waste
stabilization. The pH value, like the temperature, of the compost varies with time during the
composting process. The initial pH of the organic fraction of MSW is typically between 5 and 7.
The pH of compo sting material will vary according to the pH-time profile shown in Fig. 14-3a.
In the first few days of composting, the pH drops to 5 or less. At this stage, the organic mass is at
ambient temperature, the multiplication of indigenous mesophilic organisms begin, and the
temperature rises rapidly. Among the products of this initial stage are simple organic acids,
which cause the drop in pH. After about three days, the temperature reaches a thermophilic stage,
and the pH begins to rise to approximately 8 or 8.5 for the remainder of the aerobic process. The
pH value falls slightly during the cooling stage and reaches to a value in the range of 7 to 8 in the
mature compost. If the degree of aeration is not adequate, anaerobic conditions will occur, the
pH will drop to about 4.5, and the composting process will be retarded.
Table 3.2 Temperature and time of exposure required for destruction of some common pathogens and
parasites
Table 3.3 EPA requirements for pathogen control in compost processes
59
Degree of decomposition
A suitable methodology for the measurement of the degree of decomposition is not available.
However, several methodologies have been proposed. The proposed methods are (a) final drop in
temperature, (b) degree of self-heating capacity, (c) amount of decomposable and resistant
organic matter in the composted material, (d) rise in the redox potential, (e) oxygen uptake, (f)
growth of the fungus Chaetomium gracilis, and (g) the starch-iodine test. The laboratory analysis
of chemical oxygen demand (COD) and the lignin test provide a quick check for determining the
degree of decomposition. A low COD value and a high lignin content (greater than 30 percent) is
indicative of a stable compost.
Control of odor
The majority of the odor problems in aerobic composting processes are associated with the
development of anaerobic conditions within the compost pile. In many large-scale aerobic
composting systems, it is common to find pieces of magazines or books, plastics (especially
plastic films), or similar materials in the organic material being composted. These materials
normally cannot be decomposed in a relatively short time in a compost pile. Furthermore,
because sufficient oxygen is often not available in the center of such materials, anaerobic
conditions can develop. Under anaerobic conditions, organic acids will be produced, many of
which are extremely odorous. To minimize the potential odor problems, it is important to reduce
the particle size, remove plastics and other nonbiodegradable materials from the organic material
to be composted, or use source-separated or uncontaminated feedstocks.
Land requirements
Land area requirements are another important element which must be considered in the aerobic
composting processes. For example, in windrow compo sting for a plant with a capacity of 50
ton/d, about 2.5 acres of land would be required. Of this total, 1.5 acres would be devoted to
buildings, plant equipment, and roads. For each additional 50 tons, it is estimated that 1.0 acre
would be required for the composting operation and that 0.25 acre would be required for
buildings and roads. The land requirement for highly mechanized systems varies with the
process. An estimate of 1.5 to 2.0 acres for a plant with a capacity of 50 ton/d is not
unreasonable; for larger plants, the unit area requirements would be less.
Processing compost for market
The economics of compost systems are greatly enhanced if the compost can be sold. To be
marketable, compost must be of a consistent size; free from contaminants such as glass, plastic,
and metals; and free of objectionable odors. The type of processing used to prepare the compost
for marketing will depend on the specifications for the compost. Shredding and screening are
commonly used to produce a more uniform product. In some cases, additives may be added to
enhance the value of the final product.
3.2.3 Selection of aerobic composting processes
Because the performance of properly operating windrow, aerated static pile, and in-vessel
composting processes is essentially the same, the selection among alternative processes is based
on capital and operating costs, land availability, operational complexity, and potential for
nuisance problems.
An additional factor to consider is that the windrow and aerated static pile processes utilize
standard components and have been designed and constructed successfully "in-house" by many
60
municipalities and private firms. The in-vessel processes employ proprietary designs and
custom-made equipment and are usually procured on a turnkey basis from a single vendor. Many
in-vessel compost systems are procured on full-service contracts and are owned and operated by
the vendor or a third party. The economics of these arrangements should be compared carefully
to ownership and operation of a simpler windrow or aerated static pile system by the
municipality itself. A successful composting operation is highly dependent on proper operation
and maintenance as well as design.
3.3 LOW-SOLIDS ANAEROBIC DIGESTION
Low-solids anaerobic digestion is a biological process in which organic wastes are fermented at
solids concentrations equal to or less than 4 to 8 percent. The low-solids anaerobic fermentation
process is used in many parts of the world to generate methane gas from human, animal, and
agricultural wastes, and from the organic fraction of MSW. One of the disadvantages of the low-
solids anaerobic digestion process as applied to solid wastes is that considerable water must be
added to wastes to bring the solids content to the required range of 4 to 8 percent.
The addition of water results in a very dilute digested sludge, which must be dewatered prior to
disposal. The disposal of the liquid stream resulting from the dewatering step is an important
consideration in the selection of the low-solids digestion process.
There are three basic steps involved whenever the low-solids anaerobic digestion process is used
to produce methane from the organic fraction of MSW. The first step involves the preparation of
the organic fraction of the MSW. Typically, for commingled solid waste the first step involves
receiving; sorting and separation; and size reduction. Size reduction is also required for source-
separated materials.
The second step involves the addition of moisture and nutrients, blending, pH adjustment to
about 6.8, and heating of the slurry to between 55 and 60°C, and the anaerobic digestion is
carried out in a continuous-flow reactor whose contents are mixed completely. In some
operations, a series of batch reactors have been used instead of one or more continuous-flow
completemix reactors.
In most operations, the required moisture content and nutrients are added to the wastes to be
processed, in the form of wastewater sludge or cow manure. Depending on the chemical
characteristics of the sludge or manure, additional nutrients may also have to be added. Because
foaming and the formation of surface crusts have caused problems in the digestion of solid
wastes, adequate mixing is of fundamental importance in the design and operation of such
systems.
The third step in the process involves the capture, storage, and, if necessary, separation of the gas
components. The dewatering and disposal of the digested sludge is an additional task that must
be accomplished. In general, the processing of the digested sludge produced from low-solids
anaerobic digestion is so expensive that the process has seldom been used.
Carried out in the absence of oxygen, the anaerobic stabilization process or conversion of the
organic materials in MSW occurs in three steps. As discussed previously, the first step in the
process involves the enzyme-mediated transformation (hydrolysis) of higher-molecularmass
compounds into compounds suitable for use as a source of energy and cell tissue. The second
61
step involves the bacterial conversion of the compounds resulting from the first step into
identifiable lower-molecular-mass intermediate compounds. The third step involves the bacterial
conversion of the intermediate compounds into simpler end products, principally methane and
carbon dioxide.
3.3.3 Process design considerations
Although the process of anaerobic digestion of the organic fraction of MSW is not developed
fully, some of the important design considerations are summarized in Table 3.4. In operations
where solid wastes have been mixed with wastewater sludge, it has been found that the gas
collected from the digesters contains between 50 and 60 percent methane. It has also been found
that about 10 ft3 of gas is produced per pound of biodegradable volatile solids destroyed.
Because of the variability of the results reported in the literature, it is recommended that pilot
plant studies be conducted if the digestion process is to be used for the conversion of MSW or
other organic wastes.
Process selection
Process selection between anaerobic processes is typically between the low-solids process and
the high-solids process, described in the following section. Selection of equipment and facilities
for the low-solids anaerobic digestion process usually involves the type of mixing equipment
(internal mixers, internal gas mixing, and external pump mixing), the general shape of the
digester (e.g., circular or egg-shaped), the control systems, and the ancillary facilities needed for
mixing the incoming wastes and dewatering the digested sludge.
Table 3.4 Important design considerations for the low-solids anaerobic digestion of the organic
fraction of MSW
62
3.4 HIGH-SOLIDS ANAEROBIC DIGESTION
High-solids anaerobic digestion is a biological process in which the fermentation occur at a total
solids content of about 22 percent or higher. The high-solids anaerobic digestion is a relatively
new technology and its application for energy recovery from the organic fraction of MSW has
not been developed fully. Two important advantages of the high-solids anaerobic digestion
process are lower water requirements and higher gas production per unit volume of the reactor
size. The major disadvantage of this process is that at present (1992), limited full-scale operating
experience is available.

The three steps described for the low-solids anaerobic digestion are also applied in high-solids
anaerobic digestion process. The principal difference is at the end of the process, where less
effort is required to dewater and dispose of the digested sludge.
The process microbiology for the high-solids anaerobic digestion is as described previously for
the low-solids anaerobic digestion process. However, because of the high solids concentration,
the effects of many environmental parameters on microbial population are more severe. For
example, ammonia toxicity can affect the methanogenic bacteria, which will have an adverse
effect on system stability and methane production. In most cases, the ammonia toxicity can be
prevented by a proper adjustment of the C/N ratio of the input feedstock.
3.4.3 Process design considerations
Although the high-solids anaerobic digestion process is not developed fully, some of the
important design considerations are summarized in Table 3.5. In general, the high-solids
anaerobic digestion process is capable of stabilizing more organic waste and producing more gas
per unit of volume of reactor than is the low-solids process considered previously.
Table 3.5 Important design considerations for the high-solids anaerobic digestion of the organic fraction
of MSW
Item Comment
Size of material Wastes to be digested should be shredded to a size that will not
interfere with the efficient functioning of feeding and discharging
mechanisms
Mixing equipment The mixing equipment will depend on the type of reactor to be used
Percentage of sold wastes Depends on the characteristics of the sludge
mixed with sludge
Mass retention time Use 20 to 30 for design, or base design on results of pilot plant studies
Loading rate based on 6 to 7 kg/m3.d. Not well defined at present time. Significantly higher
biodegradable volatile solids rates have been reported
Solid concentration Between 20 and 35% (22 to 28% typical)
Temperature Between 30 and 38oC for mesophilic and between 55 and 60oC for
thermophilic reactor
Gas production 0.625 to 1.0 m3/kg of biodegradable volatile solids destroyed (CH4 = 50
percent; CO2 = 50 percent)
63
3.4.4 Process selection
At the present time, as described in the following section, there are no full scale high-solids
digestion processes in operation in the United States. However, there are several different high-
solids processes under development in the United States and Europe, and at least three are
operational in Europe. As these processes become more fully developed and tested, it is
anticipated that a number of anaerobic processes will be available commercially.
3.5 DEVELOPMENT OF ANAEROBIC DIGESTION PROCESSES AND

TECHNOLOGIES FOR TREATMENT OF THE ORGANIC FRACTION OF MSW
The purpose of this section is to introduce the reader to the emerging technologies involving the
use of the anaerobic digestion process for the production of methane and a humus product from
the organic fraction of MSW and other organic materials.
3.5.1 Anaerobic digestion technologies
In recent years, there has been a great interest in applying the anaerobic digestion process for the
processing of the organic fraction of MSW because of the opportunity to recover methane and
the fact that the digested material is similar to compost produced aerobically. Most of the work
with the anaerobic digestion process is going on in Europe. The combined high-solids anaerobic
digestion/aerobic composting process under development in the United States is considered
below.
3.5.2 Combined high-solids anaerobic digestion/aerobic composting
The high-solids anaerobic digestion/aerobic composting process, developed by Professor Bill

Jewell at Cornell University, combines the high-solids anaerobic digestion and aerobic
composting processes. The major advantage of this process is the complete stabilization of the
organic waste with a net energy recovery and without the need for major dewatering equipment.
Other advantages include pathogen control and volume reduction.
Process description
The high-solids anaerobic digestion/aerobic composting process is a two-stage process. The first
stage of the two-stage process involves the high-solids (25 to 30 percent) anaerobic digestion of
the organic fraction of MSW to produce a gas composed of methane and carbon dioxide. The
anaerobic reactor operates under thermophilic conditions 129 to 133°F (54 to 56°C) with a
nominal hydraulic retention time of 30 days.
The second stage involves the aerobic composting of the anaerobically digested solids to
increase the solids content from 25 to 65 percent or more, depending on the final use. The output
from the second stage is a fine humus-like material with a thermal content of about 6000 to 6400
Btu/lb and a specific weight of about 35 lb/ft3. Because the final humus that is produced is
combustible, it appears that it can be fired directly in a boiler when mixed with other fuels or
pelletized for use as a fuel source. Alternatively, the humus-like material can be used as a soil
amendment.
64
Process applications
The combined high-solids anaerobic digestion/aerobic composting process is in the early stages
of development. Design considerations for the first stage of this process are the same as for the
high-solids anaerobic digestion process. The two major design parameters for the second stage
are the aeration and thermal energy requirement for the destruction of pathogens.
This process can be used to process the combined organic fraction of MSW and wastewater
treatment plant sludge. Because the highsolids anaerobic digestion/aerobic compostion process
can be used to process both the organic fraction of MSW along with wastewater treatment plant
sludge, the use of costly sludge dewatering facilities and the need to treat the liquid resulting
from the dewatering of the sludge can be eliminated. Biogas produced from this process can be
used for methanol production. The heat required for the anaerobic and aerobic reactors will be
recovered from the thermal energy of the fluidized bed combustion reactor.
3.6 OTHER BIOLOGICAL TRANSFORMATION PROCESSES
The principal biological processes used for the transformation of the organic fraction of MSW
are summarized in Table 3.6. Apart from the aerobic composting and the anaerobic digestion
processes, enzymatic hydrolysis and fermentation following acid or enzymatic hydrolysis are the
biological processes that have received most attention. With the national focus on solid waste
management and increasing landfill disposal costs, it is anticipated that a number of new
processes will become available in the future.
Table 3.6 Biological processes for the recovery of conversion products from the organic fraction of MSW
Process Conversion product Preprocessing

Aerobic conversion Compost (soil conditioner)Separation of organic fraction,
particle size reduction
Anaerobic digestion (in landfill) Methane and carbon dioxide None, other than placement in
containment cells
Anaerobic digestion (low- Methane and carbon dioxide, Separation of organic fraction,
solids, 4 to 8 percent solids) digested solids particle size reduction
Anaerobic digestion (high- Methane and carbon dioxide, Separation of organic fraction,
solids, 22 to 35 percent solids) digested solids particle size reduction
Enzymatic hydrolysis Glucose from cellulose Separation of cellulose-containing
materials
Fermentation (following acid or Ethanol, single-cell protein Separation of organic fraction,
enzymatic hydrolysis) particle size reduction, acid or
enzymatic hydrolysis to produce
glucose
3.7 CHEMICAL TRANSFORMATION PROCESSES
Chemical transformation processes include a number of hydrolysis processes, which are used to
recover compounds such as glucose and furfural, and a variety of other chemical conversion
processes used to recover compounds such as synthetic oil, gas, and cellulose acetate. Methanol,
an alternative liquid fuel, can also be produced. These chemical processes are not used routinely
for the transformation of the organic fraction of MSW, because these compounds can also be
manufactured from other cellulose-containing wastes, such as wheat straw, sugar cane bagasse,
and corncobs. The economic viability of these processes is closely linked to the cost of
alternative feedstocks. For example, agricultural wastes are currently cheaper to procure than
either source-separated or machineprocessed MSW.
65
Acid hydrolysis
The cellulose molecule is comprised of about 3000 glucose units, is soluble in water and many
organic solvents, and is relatively immune to attack by most microorganisms. If the cellulose
molecule is hydrolyzed, the glucose can be recovered. Acid hydrolysis, used to recover glucose
from cellulose, involves treating a finely divided suspension of cellulose-containing waste (e.g.,
newsprint) with a weak acid. The suspension is then heated to between 180 and 230°C and slight
pressure is applied. Under these conditions, the cellulose in the waste is converted into glucose
and other sugars. The amount of glucose recovered depends on the characteristics of the waste. It
is estimated that upwards of 80 percent of the weight of kraft paper may be recovered as sugar.
acid
(C6H10O5)n + H2O Æ nC6H1206
cellulose glucose
Lignin is not affected by the process. The sugar and glucose extracted from the cellulose can be
converted by other chemical and biological processes into alcohols and other industrial
chemicals.
Methanol production from methane
The methane produced by the anaerobic digestion of the organic portion of MSW can be
converted to methanol, a liquid fuel. The conversion process involves the following two
reactions, which are carried out in series.
catalyst
CH4 + H2O Æ CO + 3H2
catalyst
CO + 2H2 Æ CH3OH
In the first reaction, which is endothermic, biogas, containing methane, is reacted with steam in a
catalyst filled reactor to form carbon monoxide and hydrogen gas. In the second reaction which
is exothermic, the products of the first reaction are converted catalytically to form methanol. The
principal advantage of producing methanol from biogas that contains methane is that the
resulting fuel is both storable and transportable. Methanol is currently manufactured from natural
gas at a lower cost than it could be from biogas produced from the anaerobic digestion of MSW.
Because the cost of fossil fuels is highly sensitive to political trends, the relative economics of
methanol production from biogas could change in the future.
3.8 ENERGY PRODUCTION FROM BIOLOGICAL CONVERSION PRODUCTS
Once conversion products have been derived from solid wastes by either anaerobic digestion
(methane), or chemical transformation (methanol), the next step involves their uses and/or
storage. If energy is to be produced from these products, an additional conversion step is
required. Biogas can be used directly with internal combustion (IC) engines and gas turbines to
generate electricity.
Where IC engines are used, they should be started with propane or natural gas and operated until
the engine is at its operating tempeature, at which point they can be switched to biogas operation.
Before an engine is shut down, it again should be operated with propane or natural gas for about
20 minutes or so. By starting and stopping the engine with propane or natural gas, corrosion
problems associated with hydrogen sulfide can be avoided. Biogas from landfills is being used to
fuel IC engines in a wide range of power outputs from 50 kW to 5 MW.
66
With gas turbines, the biogas is compressed under high pressure so that it can be used more
effectively in the turbine. Such systems are in widespread use fueled with biogas recovered from
landfills. Gas turbine systems are generally used in the 1 to 5 MW power range.
In large installations, the most common flow diagram for the production of electric energy
involves the use of a steam turbine-generator combination. Steam can be produced in a boiler
fired with either biogas (methane) or MSW derived liquid fuel (methanol). A number of steam
turbine-generator installations are in operation thoughout the United States, fueled with biogas
recovered from landfills. The largest such installation in the United States generates 50 MW of
electricity at the Puente Hills landfill near Whittier, California.
67

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LECTURE NOTE

Ho Chi Minh, January 2010

Dr. Tran Thi My Dieu

VAN LANG UNIVERSITY Jan. 2010 DENTEMA

The roles of bioprocesses in

VAN LANG UNIVERSITY DENTEMA

THE ROLES OF BIOPROCESSES

VAN LANG UNIVERSITY DENTEMA

Biological wastewater treatment

Biological air pollution treatment

Biological WWT Saøn coâng taùc

Maùng thu nöôùc

Thieát bò taùch pha

Lôùp buøn kî khí

UASB Dung dòch

VAN LANG UNIVERSITY Granular sludge DENTEMA

Suspended growth AS Attached growth AS

VAN LANG UNIVERSITY DENTEMA

VAN LANG UNIVERSITY DENTEMA

VAN LANG UNIVERSITY DENTEMA

VAN LANG UNIVERSITY DENTEMA

Biodegradable constituents removal

VAN LANG UNIVERSITY DENTEMA

VAN LANG UNIVERSITY DENTEMA

DRANCO PROCESS, BELGIUM, 1984

VAN LANG UNIVERSITY DENTEMA

composting plant Open heap composting

VAN LANG UNIVERSITY DENTEMA

Drum, with artificial

VAN LANG UNIVERSITY DENTEMA

Anaerobic digester Anaerobic digestion plant

VAN LANG UNIVERSITY DENTEMA

VAN LANG UNIVERSITY DENTEMA

Reuse biowaste to produce compost

Methan gas (landfill gas) removal

Environmental friendly treatment

Reduce treatment cost

Solid wastes Gas recovery Wastewater

Livestock feed Composting WWTS

VAN LANG UNIVERSITY DENTEMA

VAN LANG UNIVERSITY DENTEMA

2.1 OVERVIEW OF BIOLOGICAL WASTEWATER TREATMENT

2.1.1 Objectives of biological treatment

2.1.2 Role of microorganisms in wastewater treatment

where x, y, z, t, u = the stoichiometric coefficient.

2.1.3 Types of biological processes for wastewater treatment

Suspended growth processes

Attached growth processes

2.2 COMPOSITION AND CLASSIFICATION OF MICROORGANISMS

2.2.1 Cell composition

To support microbial growth in biological system, appropriate nutrients must be available.

2.2.2 Environmental factors affect microbial activity

Environmental conditions of temperature and pH have an important effect on the selection,

Table 2.1 Temperature classification of biological processes

Type Temperature range (oC) Optimum range (oC)

2.3 INTRODUCTION TO MICROBIAL METABOLISM

2.3.1 Carbon and energy sources for microbial growth

The energy-producing chemical reactions by chemotrophs are oxidation-reduction reactions that

2.3.2 Nutrient and growth factor requirement

2.4 BACTERIAL GROWTH

2.4.1 Bacterial reproduction

2.4.2 Bacterial growth patterns in a batch reactor